ABSTRACT
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.
Subject(s)
Adult , Female , Humans , Male , Young Adult , DNA, Bacterial , Genetic Variation , Minisatellite Repeats , Mycobacterium tuberculosis , Bacterial Typing Techniques , Cluster Analysis , Genotype , India , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Repetitive Sequences, Nucleic AcidABSTRACT
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.