Molecular characterization of hepatitis A virus from children hospitalized at a tertiary care centre in northwest India.

Background & objectives: Hepatitis A virus (HAV) infection is a major cause of childhood hepatitis, prevalent worldwide. HAV is classified into seven genotypes I-VII; genotypes III and I are the most common among humans. The present work was carried out to identify the genotypes prevalent in children suspected to have acute viral hepatitis (AVH), hospitalized at a tertiary care centre in northwest India. Methods: A total of 1269 blood samples from children (0-15 yr of age) clinically suspected of viral hepatitis were screened for anti-HAV IgM. Acute phase serum was processed for RNA extraction and amplified by nested polymerase chain reaction (PCR) followed by sequencing of representative samples. Results: Among the 1269 samples tested, 642 (50.59%) were positive for anti-HAV IgM; among the positive samples, 171 patients having a history of less than seven days were tested by PCR, of whom 141 (82.45%) were found to be PCR positive. Nucleotide sequencing of a representative 44 samples showed high homology; all the samples were found to be of genotype IIIA. Interpretation & conclusions: Hepatitis A was prevalent during July to September and in predominantly children less than five years age. Only genotype IIIA was detected in all the samples.

Aetiological study of viruses causing acute encephalitis syndrome in North West India.

CONTEXT: Acute encephalitis syndrome (AES) is a serious public health problem, caused mainly by viruses. However, the profile of viruses causing AES in Rajasthan is not well characterised. AIMS: The present study was undertaken to identify the viruses causing AES and develop diagnostic algorithm so as to help in improved diagnosis, treatment, prevention and control. SETTINGS AND DESIGN: The present study is a hospital-based descriptive, observational study. Samples were processed at Grade-1 DHR/ICMR Viral Research and Diagnostic Laboratory at SMS, Jaipur. SUBJECTS AND METHODS: Cerebrospinal fluid (CSF) samples were processed for IgM antibody detection by enzyme-linked immunosorbent assay (ELISA) for mumps virus (MPV), measles virus (MV), Rubella virus (RV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and Dengue virus using commercial kits. Nucleic acid was extracted from CSF using automated extraction system. Real-time polymerase chain reaction was done using specific primers and probes for Herpes simplex virus (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and enterovirus (EV). STATISTICAL ANALYSIS USED: Statistical analysis was done using ANOVA. RESULTS: Among 3088 patients, 702 (22.7%) patients were positive for one or more viruses. HSV (261;8.45%) was the most common followed by EBV (173;5.6%), VZV (97;3.1%), CMV (68;2.2%), EV (32;1.03%), MPV (27;0.9%), DV (28;0.9%), MV (19;0.6%) and RV (6;0.2%). CONCLUSIONS: AES occurred sporadically in Rajasthan, samples should be tested first for herpes group of viruses followed by EV or/and for arboviruses depending on season or measles, mumps and RVs in children.

Prevalence of Mycobacterium tuberculosis with multiple copies of IS6110 elements in Gulbarga, South India.

This study was carried out to ascertain the prevalence of Mycobacterium tuberculosis by insertion sequence 6110 (IS6110) based DNA fingerprinting method in Gulbarga district belonging to the southern part of India. Results showed that among the 52 M. tuberculosis isolates studied, 57.7% exhibited more than 5 copies of IS6110 showing the prevalence of M. tuberculosis with multiple copies of IS6110 elements.

Molecular typing of Mycobacterium tuberculosis isolates from different parts of India based on IS6110 element polymorphism using RFLP analysis.

BACKGROUND & OBJECTIVE: IS 6110 based typing remains the internationally accepted standard and continues to provide new insights into the epidemiology of Mycobacterium tuberculosis. The aim of the study was to characterize M. tuberculosis isolates obtained from different parts of India based on IS6110 element polymorphism using restriction fragment length polymorphism (RFLP) analysis. METHODS: RFLP was analyzed among 308 isolates of M. tuberculosis deposited in the Mycobacterial Repository Centre, Agra, from different parts of India. DNAs isolated from these strains were restricted with Pvu II, transferred on to nylon membrane and hybridized with a PCR amplified DIG-labeled 245 bp IS6110 probe. RESULTS: Based on the copy number, M. tuberculosis isolates were classified into four groups, (i) lacking IS6110 element; (ii) low copy number (1-2); (iii) intermediate copy number (3-5); and (iv) high copy number (6-19). Copy number higher than 19 however was not observed in any of the isolates studied. At the national level, 56 per cent of the isolates showed high copy number of IS6110, 13 per cent showed intermediate copy number, 20 per cent showed low copy number, whereas 11 per cent isolates lacked IS6110 element. At the regional level, there was not much difference in the RFLP profiles of isolates (IS6110 copy numbers/patterns) from different parts of the country. INTERPRETATION & CONCLUSION: IS6110 DNA based fingerprinting could be a potentially useful tool for investigating the epidemiology of tuberculosis in India.

Simultaneous ethambutol & isoniazid resistance in clinical isolates of Mycobacterium tuberculosis.

BACKGROUND & OBJECTIVE: There is a need to understand the nature of drug resistance patterns and predictors of emergence of drug resistance in Mycobacterium tuberculosis. There could be common factors/mechanisms for resistance to the drugs, isoniazid and ethambutol, both acting on cell wall. The present study was conducted to analyze the antimycobacterial susceptibility patterns of M. tuberculosis isolates to determine the minimum inhibitory concentrations (MICs) of ethambutol for M. tuberculosis; and to find out possible association of ethambutol resistance with isoniazid resistance. METHODS: A total of 380 M. tuberculosis isolates were tested for their susceptibilities to ethambutol at 2, 4, 6 microg/ml, isoniazid at 1 microg/ml and rifampicin at 64 microg/ml using MIC method. RESULTS: 44.21, 24.73 and 14.21 per cent isolates were resistant to ethambutol at concentrations of 2, 4 and 6 microg/ml respectively. At 6 microg/ml of ethambutol concentration, 85.18 per cent ethambutol resistant isolates were resistant to isoniazid also. At the same ethambutol concentration a fraction of 28.75 per cent isoniazid resistant isolates were ethambutol resistant. INTERPRETATION & CONCLUSION: Ethambutol resistance was accompanied with isoniazid resistance in a large percentage of isolates whereas ethambutol resistance was weakly linked with multidrug resistance. On the other hand, association between isoniazid and ethambutol resistance was weak showing one way linkage.

Characterization of mycobacteria isolated from bovines by PRA-targetting hsp 65 gene region.

Bovine tuberculosis caused by the bacterium Mycobacterium bovis is a major infectious disease of animals and has zoonotic importance for humans. Even though the incidence is believed to be very low in India, human tuberculosis caused by M. bovis has been increasingly recognized in many other countries of the world. As differentiation of mycobacterial species take long time, a method for the rapid identification of mycobacteria isolated from bovine samples to the species level was used, which is based on polymerase chain reaction (PCR) of the gene encoding for the 65-kD protein followed by restriction analysis. The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria and generate M. tuberculosis complex specific pattern. PRA was performed on 33 bovine isolates of which 90.9% (30/33) isolates were identified clearly as M. tuberculosis complex, M. fortuitum, M. phlei and M. smegmatis using restriction enzyme Hae III.

Optimization of procedures for isolation of mycobacteria from soil and water samples obtained in northern India.

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.