ABSTRACT
Background & objectives: One-fifth of the world’s new tuberculosis (TB) cases and two-thirds of cases in the South East Asian region occur in India. Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated to understand the transmission of TB. This study was aimed to investigate the molecular epidemiology of M. tuberculosis genotypes in Varanasi, north India, and their association with clinical presentation among patients with pulmonary TB. Methods: M. tuberculosis isolates from 104 TB patients attending a tertiary referral hospital of north India were screened for susceptibility to isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STR) by proportion method and multiplex-allele-specific-polymerase chain reaction (MAS-PCR). These were genotyped by spoligotyping. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Results: Eighty three of 104 isolates were distributed in 38 SITs, of which SIT3366 was newly created within the present study. The mass of ongoing transmission with MDR-TB isolates in Varanasi, northern India, was linked to Beijing genotype followed by the CAS1_Delhi lineage. HIV-seropositive patients had a significantly higher proportion of clustered isolates than HIV-seronegative patients and compared with the wild type(wt) isolates, the isolates with katG315Thr mutation were considerably more likely to be clustered. Interpretation & conclusions: This study gives an insight into the M. tuberculosis genetic biodiversity in Varanasi, north India, the predominant spoligotypes and their impact on disease transmission. In this region of north India, TB is caused by a wide diversity of spoligotypes with predominance of four genotype lineages: Beijing, CAS, EAI and T. The Beijing genotype was the most frequent single spoligotype and strongly associated with multi drug resistant (MDR)-TB isolates. These findings may have important implications for control and prevention of TB in north India.
ABSTRACT
Background & objectives: The conventional techniques used in TB diagnosis like AFB (acid fast bacilli) smear microscopy lack sensitivity and the gold standard, culture test takes time. A test based on multiplex polymerase chain reaction (PCR) targeting the 38 kDa gene and IS6110 insertion sequence, specific to Mycobacterium tuberculosis was developed to further increase the sensitivity of a TB-PCR kit targeting only 38 kDa gene developed earlier in the same laboratory. The multiplex test was validated using sputum samples from pulmonary TB (PTB) cases. The sensitivity and specificity were compared with AFB smear examination and Lowenstein-Jensen (LJ) culture test. Methods: Multiplex PCR amplifying 340 and 245 bp sequence of 38 kDa gene and IS6110, respectively was standardized and analytical sensitivity was verified. Sputum samples (n=120) obtained from PTB cases were subjected to AFB smear examination, LJ culture and a multiplex as well as single target PCR test. Additionally, 72 non-TB respiratory samples were included in the study as negative controls. Results: Analytical sensitivity of multiplex PCR was found to be 100 fg for 38 kDa gene and 1 fg for IS6110. Multiplex PCR, using both the targets, showed highest sensitivity of 81.7 per cent, followed by 69.2 per cent for L-J culture test and 53.3 per cent for AFB smear when clinical diagnosis was considered as a gold standard. The sensitivity of detection of M. tuberculosis in AFB smear positive and negative samples by multiplex PCR was 93.7 and 67.9 per cent, respectively. Sensitivity of 77.1 per cent observed for the detection of M. tuberculosis with single target PCR increased to 89.2 per cent with multiplex PCR in culture positive samples. Four samples showed positive PCR results only with primers for 38 kDa gene. Interpretation & conclusions: Multiplex PCR increased the sensitivity of single target PCR and will be useful in diagnosing paucibacillary smear negative samples. Further, it can also be used to detect samples with M. tuberculosis strains lacking IS6110.