Incidence and detection of M.tb multi-drug resistance in Hyderabad population using the PCR–sequence method

ABSTRACT Introduction: Tuberculosis (TB) is a multifaceted disease caused by Mycobacterium tuberculosis ( M.tb ) is a pathogenic bacterium, which poses to public health problem in both industrialized and developing countries. According to the World Health Organization, 8.8 million active cases of TB are diagnosed each year, and of these, almost 2 million die. Once thought to be under control, but TB infection levels are rising, and the threat is compounded by new, virulent, and drug-resistant strains. So we determine the Incidence and Detection of M.tb multi-drug resistance (MDR) in the Hyderabad population using the polymerase chain reaction (PCR)–Sequencing Method. Methodology: A prospective hospital-based study involving 60 patients was conducted for assessment of phenotypic conventional drug susceptibility testing combined with sequencing-based mutation screening, for increased sensitivity and identification of novel mutations in first-line M.tb drugs. Participants’ age ranged from >18 to <60 years; both male and female patients positive for M.tb were included. A total of 60 samples (40 were non-responders and 20 were responders) were collected and tested for M.tb by Multiplex PCR, as well as confirmed by acid-fast bacilli Culture. To know the MDR status rpoB and katG genes were amplified by using the designated primers and were sequenced to know the mutations and analyzed with reference sequences. Results: It has now been established that mutations in the rpoB gene and katG gene of M. tuberculosis are associated with resistance to Rifampicin and Isonazid, respectively. Present study, 67.5% of the non-responder group had a mutation at rpoB531 and katG315 . 22.5% of the responder group had an Insertional mutation at rpoB513 and katG315. However, 4% of the non-responder group had a mutation at both rpoB codons 513 and 531 and katG codons 315 and 299. There are no mutations found in the control strain. Moreover, a novel mutation was also identified in this research where an extra Arginine was inserted at codon 513. Conclusion: We have shown the usefulness of our DNA sequencing strategy for drug susceptibility screening. Most MDR TB strains, which are resistant to both RIF and INH, are resistant to other antitubercular drugs.