High-resolution melting analysis for the rapid detection of fluoroquinolone and streptomycin resistance in Mycobacterium tuberculosis

Molecular methods for the detection of drug-resistant tuberculosis are potentially more rapid than conventional culture-based drug susceptibility testing, facilitating the commencement of appropriate treatment for patients with drug resistant tuberculosis. We aimed to develop and evaluate high-resolution melting (HRM) assays for the detection of mutations within gyrA, rpsL, and rrs, for the determination of fluoroquinolone and streptomycin resistance in Mycobacterium tuberculosis (MTB). A blinded series of DNA samples extracted from a total of 92 clinical isolates of MTB were analyzed by HRM analysis, and the results were verified using DNA sequencing. The sensitivity and specificity of the HRM assays in comparison with drug susceptibility testing were 74.1% and 100.0% for the detection of fluoroquinolone resistance, and 87.5% and 100.0% for streptomycin resistance. Five isolates with low level resistance to ofloxacin had no mutations detected in gyrA, possibly due to the action of efflux pumps, or false negativity due to mixed infections. One fluoroquinolone-resistant isolate had a mutation in a region of gyrA not encompassed by our assay. Six streptomycin-resistant strains had undetectable mutations by HRM and DNA sequencing, which may be explained by the fact that not all streptomycin-resistant isolates have mutations within rpsL and rrs, and suggesting that other targets may be involved. The HRM assays described here are potentially useful adjunct tests for the efficient determination of fluoroquinolone and streptomycin resistance in MTB, and could facilitate the timely administration of appropriate treatment for patients infected with drug-resistant TB.