Clinico-pathological profile, radiological presentation and drug susceptibility pattern of new smear positive (category I) pulmonary tuberculosis: a single centre experience in Delhi, India.

Background: Aim of current study was to determine the clinical characteristics, radiological, laboratory features and anti-tubercular drug sensitivity in new smear positive (category I) pulmonary tuberculosis cases in a tertiary care dedicated TB OPD, Delhi. Methods: The study was a cross-sectional observational study and consists of 100 cases of new smear positive pulmonary tuberculosis cases (category I) irrespective of age and sex. The sputum were collected, stained with Ziehl-Nielsen (Z-N) staining and ultimately inoculated on Lowenstein-Jensen (L-J) media for six weeks. All sputum smear positive cases were subjected to culture and drug-susceptibility testing by 1% proportion method on Lowenstein-Jensen (LJ) medium. The Drug-Susceptibility Testing (DST) for isoniazid (INH), rifampicin (R-cin), ethambutol (EMB) and streptomycin (SM) were performed. Results: The age & sex distribution of 100 patients showed that majority of the patients (79%) belonged to 2nd, 3rd and 4th decades & 60 % were males and 40% were female with male to female ratio 3:2 respectively. Cough (83%), fever (77%) and weight loss (76%) were the most common presenting clinical features. The chest X-ray of 100 smear positive patients showed that 53% of patients had evidence of 35% unilateral and 18% bilateral consolidation and 46% had cavitary lesions on chest X-ray (PA view) with 37% and 9% of patients having unilateral and bilateral cavities respectively. Of these 82 culture positives, 56.1% (n=46) were susceptible to all first-line anti-tubercular drugs, while 43.9% (n=36) were resistant to mostly one or other anti-tubercular drugs (INH, R-cin, SM or EMB). Conclusion: We stressed the importance of early diagnosis of new cases by clinico-pathological features, identifying of drug resistance trends in anti-tubercular treatment naïve patients, in order to assess the efficacy of current interventions. Overall, these findings emphasize the importance of early diagnosis of drug resistance pattern of M. tuberculosis isolates to anti-tubercular in category I patients as well as its association with HIV across the country to timely modify and strengthen the national programs in order to prevent the emergence of MDR-TB strains and avert the threat of XDR-TB.

Footprints of genetic susceptibility to pulmonary tuberculosis: Cytokine gene variants in north Indians.

Background & objectives: Tuberculosis is (TB) responsible for high morbidity and mortality worldwide. Cytokines play a major role in defense against Mycobacterium tuberculosis infection. Polymorphisms in the genes encoding the various pro- and anti-inflammatory cytokines have been associated with tuberculosis susceptibility. In this study we examined association of 25 sequence polymorphisms in six candidate cytokine genes namely IFNG, TNFB, IL4, IL1RA, IL1B and IL12 and their related haplotypes with risk of developing pulmonary tuberculosis (PTB) among north Indians. Methods: Pulmonary TB (n=110) patients and 215 healthy controls (HC) from north India were genotyped. Purified multiplex PCR products were subjected to mass spectrometry using Sequenom MassARRAY platform to generate the genotypes in a population-based case-control study. Results: Using multiple corrections, significant overall risk against PTB was observed at seven loci which included variants in IFNG at rs1861493 and rs1861494; IL1RA at rs4252019, IL4 variant rs2070874, IL12 variants rs3212220, rs2853694 and TNFB variant rs1041981. Analysis of gene structure revealed two haplotype blocks formed by IFNG variants rs1861493 and rs1861494. The TA haplotype was significantly over-represented (P=0.011) in the cases showing a two-fold risk in the current population (Odds ratio=1.59 CI=1.101 to 2.297) and TNFB variants at rs2229094 and rs1041981 contributed to two haplotypes which were in strong linkage disequilibrium (LD) with AT haplotype showing a three-fold risk (P=0.0011, Odds ratio=3, CI=0.1939 to 0.7445) of developing PTB in north Indians. Interpretation & conclusions: Our study showed six novel associations of cytokine gene variants with susceptibility to PTB in north Indians. Variants of IFNG and TNFB emerged as factors imposing a significant risk of developing PTB in north Indians apart from risk indicated by IL1RA, IL4 and IL12.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75 percent) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

A simple method to differentiate between Mycobacterium tuberculosis and non-tuberculous mycobacteria directly on clinical specimens.

We report the applicability of testing susceptibility to paranitrobenzoic acid (PNB) directly on clinical samples as a rapid screening assay, to detect M. tuberculosis and differentiate it from non-tuberculous mycobacteria (NTM). One hundred smear positive sputum samples from patients with pulmonary tuberculosis attending the Department of Respiratory Medicine at VP Chest Institute, Delhi, were cultured on Löwenstein Jensen medium with and without 0.5 mg/ml paranitrobenzoic acid. Serial concentrations of known cultures of H37Rv, M. fortuitum, M. scrofulaceum and M. avium were used as controls in the study. After 3 weeks of incubation, growth was observed on all the drug free Löwenstein Jensen slants but none of the slants containing PNB, which inhibited the growth of M. tuberculosis. The cultures were further confirmed to be M. tuberculosis by niacin, nitrate and catalase tests. Direct susceptibility to PNB was thus found to be a simple, cheap and technically feasible method of preliminary identification of M. tuberculosis and its effective differentiation from NTM, which may be adapted for use at Level II laboratories, especially in developing countries.

Genotype analysis of human Mycobacterium avium isolates from India.

BACKGROUND & OBJECTIVE: Mycobacterium avium has emerged as a major opportunistic pathogen, infecting nearly 50 per cent of HIV/AIDS patients in the western world. There is no report from India regarding the typing profile of M. avium, a potential pathogen, the present study was undertaken to assess the genotypic diversity of Indian M. avium isolates of human origin. METHODS: A total of 65 biochemically identified M. avium isolates from sputum samples of patients with chronic pulmonary illness were subjected to IS1245 based restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) typing. RESULTS: IS1245 insertion sequence based RFLP demonstrated polymorphism in 84.6 per cent isolates, while 15.4 per cent isolates did not hybridize on Southern blot and therefore were RFLP negative. Among the 55 RFLP positive isolates, 8 showed 1-3 bands, 19 had bands ranging between 4-9, and 28 isolates had >10 bands each. Although the isolates could be clubbed on the basis of number of bands, the banding profile was highly polymorphic. Among the 55 isolates typeable by RFLP, four clusters and 40 unique types of polymorphism were observed. Application of IS1245 based PCR typing on the same isolates showed that 87.7 per cent isolates were typeable. Interestingly the 10 isolates that were not typeable by IS1245 RFLP were typeable by IS1245 based PCR typing. Among the 57 PCR typed isolates a cluster of 14 isolates with identical 3-banded pattern was observed. Notably, 5 of the ten IS1245 RFLP negative isolates were within this cluster. INTERPRETATION & CONCLUSION: Our results demonstrated that M. avium isolates from India were highly polymorphic with remarkable genetic diversity and heterogeneous RFLP profile. We observed that 47 per cent (n=27) isolates had RFLP profile suggestive of bird and animal origin indicating a strong association with the environment. By applying two typing methods based on IS1245 on the isolates 100 per cent typeability could be achieved.

Paucibacillary tuberculosis--a retrospective study.

Paucibacillary tuberculosis poses difficulty in the treatment and control of tuberculosis. The present study aims at understanding the extent of such cases among the patients, with respiratory symptoms, attending the outpatient department of the VP Chest Institute, Delhi. The analysis is based on the smear and culture examination of the sputum specimens, submitted from such patients during 1992-1998. Present results demonstrate that 34.8% were paucibacillary (direct smear negative; culture positive cases). Sex has no influence on smear examination results. The frequency of smear negative cases varied in the different age groups; the largest number belonged to the > or = 45 years age group (p < 0.05). Culture examination elicited positive result in these cases.