Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay

Abstract

Background & objectives: Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens. Methods: Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB , codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST). Results: The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR). Interpretation & conclusions: The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.