In-Depth Molecular Characterization of Mycobacterium tuberculosis from New Delhi – Predominance of Drug Resistant Isolates of the ‘Modern’ (TbD1−) Type

Abstract

BackgroundIndia has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of Mycobacterium tuberculosis in India is limited.Methodology/Principal FindingsThus, 65 M. tuberculosis isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP). The Central Asian (CAS) 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30) of all isolates, with shared-type (ST) 26 being the most dominant genotype comprising 24.6% (n = 16) of all isolates. Other sub-lineages observed were: East-African Indian (EAI)-5 (9.2%, n = 6), EAI6_BGD1 (6.2%, n = 4), EAI3_IND, CAS and T1 with 6.2% each (n = 4 each), Beijing (4.6%, n = 3), CAS2 (3.1%, n = 2), and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7%) did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the pncA gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates) and in 1 orphan. Mutations in the pncA gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the rpoB gene, the katG gene and in both rpoB and katG genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045). The increased frequency of drug-resistance could not be linked to the patients' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36], P = 0.79). Fifty-six percent of all new tuberculosis patients had mutations in either the katG gene or the rpoB gene, or in both katG and rpoB genes.ConclusionCAS1_DELHI isolates circulating in New Delhi, India have a high frequency of mutations in the rpoB and katG genes. A silent mutation (S65S) in the pncA gene can be used as a putative genetic marker for CAS-lineage isolates.

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

“…In our study, both EAI and CAS lineage strains occurred more frequently (75%) in patients between the ages of 18-44 years of age, reflecting ongoing transmission in North India. Our observations of the prevalence and proportion of CAS1-DEL and EAI3-IND corroborate recent findings from India, that the bulk of TB infections in North India is caused by the TbD1-negative/PGG1 genogroup , Stavrum et al 2009) as opposed to infections in South India, where the TbD1-positive/PGG1 EAI lineage predominates (Narayanan et al 2008). Lastly, the high diversity of M. tuberculosis strains in our study, despite the fact that they were isolated from patients attending health facilities within a 10 km radius in a country with a high level of TB endemicity, was remarkable.…”

“…Other recent studies from North India have also failed to find isolates belonging to SIT54 (Mathuria et al 2008. Thus, even though the majority of the isolates in our study (76/101) belonged to M. tuberculosis complex lineages, such as CAS, EAI and Manu that are frequently reported in the Indian subcontinent (Bhanu et al 2002, Singh et al 2004, Kulkarni et al 2005, Brudey et al 2006, Suresh et al 2006, Mathuria et al 2008, Stavrum et al 2009), changes occurring within clades reemphasise the need for continuous cluster investigation to detect any change in the circulating spoligotypes.…”

“…The most common spoligotype found in our study was SIT26 (CAS1-Delhi, n = 21, 20.8% of all isolates), followed by SIT11 (EAI3-IND lineage, n = 11 strains, 10.9% of all isolates). Reportedly a predominant spoligotype in Northern India (Bhanu et al 2002, Singh et al 2004, Suresh et al 2006, Mathuria et al 2008, Stavrum et al 2009), SIT26 was found at a lower frequency (7.4% of all strains) in a study from Mumbai (Kulkarni et al 2005) and represents only 1.7% of strains in the SITVIT2 database. SIT26 is limited mainly to the Middle East and to Central Asia.…”

“…Although various studies have been performed in India using spoligotyping of M. tuberculosis (Mistry et al 2002, Singh et al 2004, Kulkarni et al 2005, Mathuria et al 2008), very few studies have reported the use of MIRU-VNTRs for molecular typing of M. tuberculosis isolates , Stavrum et al 2009, Narayanan et al 2010. Therefore, the present study validates the use of repetitive elements for molecular typing of M. tuberculosis in Indian isolates and further underlines the usefulness of MIRU-VNTRs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.…”

“…MIRU-VN-TR typing shows a discriminatory power close to that of IS6110 fingerprinting and can be used to differentiate between M. tuberculosis isolates that lack or have low copy numbers of IS6110 elements (Supply et al 2000). Very few studies in India have reported use of MIRU-VNTRs for molecular typing of M. tuberculosis isolates , Stavrum et al 2009, Narayanan et al 2010). …”