Improved rapid molecular diagnosis of multidrug-resistant tuberculosis using a new reverse hybridization assay, REBA...

Rapid diagnosis of multidrug-resistant tuberculosis (MDR-TB) is essential for the prompt initiation of effective second-line therapy to improve treatment outcome and limit transmission of this obstinate disease. A variety of molecular methods that enable the rapid detection of mutations implicated in MDR-TB have been developed. The sensitivity of the methods is dependent, in principle, on the repertoire of mutations being detected, which is typically limited to mutations in the genes rpoB, katG and the promoter region of inhA. In this study, a new reverse hybridization assay, REBA MTB-MDR (M&D), that probes mutations in the oxyR-ahpC intergenic region, in addition to those in rpoB, katG and the inhA promoter region, was evaluated. A set of 240 Mycobacterium tuberculosis clinical isolates from patients receiving retreatment regimens was subjected to conventional phenotypic drug-susceptibility testing (DST) and the REBA MTB-MDR assay. The nucleotide sequences of the loci known to be involved in drug resistance were determined for comparison. In brief, the results showed that the REBA MTB-MDR assay efficiently recognized nucleotide changes in the oxyR-ahpC intergenic region as well as those in rpoB, katG and the inhA promoter region with higher sensitivity, resulting in an 81.0 % detection rate for isoniazid resistance. Inclusion of the oxyR-ahpC intergenic region in the REBA MTB-MDR assay improved the overall sensitivity of molecular DST for MDR-TB from 73.1 to 79.9 %.