Detection of diarrheagenic Escherichia coli by multiplex PCR.

Background: Diarrheagenic E.coli (DEC) are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus) forEnteropathogenic E.coli (EPEC), hlyA for Enterohemorrhagic E.coli (EHEC), elt and stla for Enterotoxigenic E.coli (ETEC), CVD432 for Enteroaggregative E.coli (EAEC) and ial for Enteroinvasive E.coli (EIEC). Results: In 200 children with diarrhea 52 (26%) DEC infections were found. Among 100 controls 8 (8%) DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%)and controls (5,5%), followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5%) and 3 (1.5%) of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.

Bacteriological spectrum of cholecystitis and its antibiogram.

Bile Cultures for aerobic and anaerobic bacteria were carried out on 125 samples from patients with chronic cholecystitis with cholelithiasis. Cultures grew 71(56.8%) aerobes and 17(13.6%) anaerobic microorganisms. Polymicrobial infection was seen in 7(16.2%) cases. E. coli (45.07%) and Klebsiella (25.35%) were predominant among the aerobes and Bacteroides fragilis (58.82%) was predominant among the anaerobes. Highest incidence of the disease was observed in the fourth decade of life and females predominated in this study.

Correlation of the suicide phenomenon in Aeromonas species with virulence and enteropathogenicity.

Certain strains of mesophilic Aeromonads like A. hydrophila, A. veronii biotype sobria and A. caviae when grown in broth containing 0.5% glucose, undergo growth inhibition concomitant with acetate accumulation. As these strains become nonviable after 24 h, this phenomenon is termed suicide. We investigated suicidal strains of Aeromonas species as means of understanding animal virulence and enteropathogenicity. Non suicidal strains of A. Hydrophila showed and overall 88.8% lethality rate and non suicidal strains of A. veronii biotype sobria showed 83.3% lethality rate and was nil for its suicidal part. Of the two suicidal A. caviae strains tested, none were lethal. The present data suggest that the suicide phenomenon may explain strain specific [A. veronii biotype sobria, A. hydrophila] and species specific [A. caviae] virulence and enteropathogenicity.

Activity of Ocimum sanctum (the traditional Indian medicinal plant) against the enteric pathogens.

Aqueous & alcoholic extracts of O. sanctum were prepared. Two concentrations of these extracts (30 mg & 60 mg) were tried against the enteric pathogens & candida albicans by Agar diffusion method. Wide zones of inhibition were observed at 60 mg concentration of extract. Aqeous extract showed wider zone of inhibition when compared to alcoholic extract. Aqueous extract showed wider zones of inhibition for Klebisella, E. Coil, Proteus & Staphylococcus aureus. Alcoholic extract showed wider zone for vibrio cholerae.

Chromobacterium violaceum diarrhea.

This is the first case of Chromobacterium violaceum diarrhoea from coastal Karnataka reported in a 2 year 10 months old girl. Stool culture yielded Chromobacterium violaceum and was sensitive to ampicillin, gentamicin, chloramphenicol, erythromycin and septran. Patient completely recovered with ampicillin and gentamicin.

Cryptosporidium and isospora belli diarrhoea in immunocompromised hosts.

Cryptosporidium and isospora, two of the intestinal coccidian parasites known to be the causative agents of acute diarrhoea in animals, have now emerged as one of the main causes of prolonged life threatening diarrhoea in immunocompromised patients particularly so in patients with AIDS. Between June 1996 and December 1997, a total of 75 immunocompromised patients presenting with diarrhoea were investigated both for Cryptosporidium and Isospora. The study group consisted of cancer and AIDS patients with history of diarrhoea. Cryptosporidium oocysts were detected in 35 patients (46.7%). One of the faecal samples from an AIDS patient with diarrhoea showed the presence of both Cryptosporidium and Isospora oocysts. To the best of our knowledge, this is the second documented report of Isospora associated diarrhoea in an AIDS patient from India. The various techniques used for demonstration of these parasites were modified acid fast staining, Safranine Methylene-blue staining and direct immunofluorescence test.

Shigella and the fluorinated quinolones.

Between July 1996 to June 1997 a total of 531 Stool samples from cases of acute diarrhoea, dysentry, colitis admitted to Kasturba Medical College Hospital, Manipal were investigated. Shigella were isolated from 29 samples (5.46%). Out of them, Shigella flexneri were 16 (55.17%), Shigella boydii 8 (27.58%), Shigella dysentriae 3 (10.34%) and Shigella sonnei 2 (6.89%). Five strains of Shigella flexneri and one strain of Shigella dysentriae were found to show resistance to Nalidixic acid and the newer fluoroquinolones viz., Ciprofloxacin, Norfloxacin and Ofloxacin. The in vitro 'R' factor transfer study showed that the resistance was not transferable.

Prevalence of rubella virus in suspected cases of congenital infections.

This study includes a total of 342 infants suspected of having congenital infections from January 1991-December 1993. Serum samples of these infants were tested for rubella specific IgM antibodies by mu ELISA. Of the total 342 infants, 52 (15.2%) were found to be positive for IgM antibodies to rubella virus. The commonest clinical presentation in infants with IgM antibodies to rubella virus was bilateral congenital cataract and hepatosplenomegaly.

Rotavirus and bacterial enteropathogens causing acute diarrhea.

The etiology of rotavirus in acute diarrhoeal illness in children 0-5 years of age, admitted to the Pediatric wards of Kasturba Medical College Hospital, Manipal was studied, over a period of one year. Rotavirus in the faecal samples detected by the slide latex agglutination test accounted for 14.9% of the diarrheas with maximum incidence in the 7-12 months of age group (57.5%). Bacterial enteropathogens continued to play a significant role in diarrheal diseases. Salmonella enteritis was found more in the age group 0-6 months and shigellosis in 37-60 months. In a control study of 100 children who had no diarrhea, 2 were found positive for rotaviruses.

Mycotic keratitis: a study in coastal Karnataka.

Fungi were isolated from 67 cases out of the 295 cases of corneal ulcers investigated. Aspergillus species and species of Candida were the major fungal members isolated. Allescheria boydii was isolated from 3 cases, having no previous history of injury to the eye or infection with bacterial or viral agents. A boydii corneal infection is a rare occurrence. Higher incidence of mystic keratitis was seen among females than males. No relationship to seasonal changes could be established. Bacterial infection was associated in 46. 27% of the cases of mycotic keratitis and Staphylococcus was the predominant bacterial pathogen observed.