Rapid genotypic detection of rpoB and katG gene mutations in Mycobacterium tuberculosis clinical isolates from Northern India as determined by MAS-PCR

Abstract

Background: There is a growing need to develop rapid laboratory research methods to counter the menace of drug resistant tuberculosis (MDR-TB) cases worldwide especially in developing countries. The present study was undertaken to investigate the type and frequency of rpoB and katG mutations in rifampicin (RIF) and isoniazid (INH) resistant strains respectively of Mycobacterium tuberculosis (MTB) circulating in Northern India and to explore the utility of multiplex-allele-specific (MAS)-PCR assay for detection of drug-resistant MTB isolates in low resource set up.

Methods: Phenotypic and genotypic drug susceptibility testing (DST) was performed on 354 MTB isolates.

Results: Mutation in rpoB gene was found most frequently at codons 531, 526 and 516 (59.83%, 45.29% and 22.22%, respectively). Further, combinations of 2-3 point mutations were also observed in 19.66% of RIF-resistant MTB strains. The frequency of mutations in katG gene was found at codon 315 among 82.95% of the INH-resistant MTB isolates. MAS-PCR detected rpoB and katG mutations in phenotypically resistant isolates with sensitivities of 93% and 83% respectively.

Conclusion: MAS-PCR assays can be used for rapid detection of drug-resistant TB strains in routine diagnostic practice, enabling early administration of appropriate treatment regimens to the affected patients.

Figure 1

Agarose gel electrophoresis patterns of MAS–PCR assays: (A) rpoB526 analysis: Lanes: 3‐5, and 1,2,6 strains with rpoB526 mutant and susceptible alleles respectively; Lane 7: H37Rv. (B) rpoB516 analysis: Lanes: 1, 3, 4, and 2,5,6 strains with rpoB516 mutant and susceptible alleles respectively; Lane 7: H37 Rv. (C) rpoB531 analysis: Lanes: 1, 2, 6,7, and 3,4 strains with rpoB531 mutant and susceptible alleles respectively; Lane 5: H37Rv. (D) katG315 analysis: Lanes 3,4,5, and 1,2,6 strains with katG315 mutant and susceptible alleles respectively; Lane 7: H37Rv. M (Molecular marker): (A & B) 50‐bp, (C & D) 100‐bp DNA ladder (Bangalore Gene, Bangalore).