Development and evaluation of rifampicin-resistant tuberculosis diagnostic assay using high-resolution melting curve analysis combined with fluorophore probes

Abstract

Rifampicin-resistant tuberculosis (RR-TB) has become a global major public health threat. This study aims to develop a new assay, RIF-RDp, to enhance the detection of RR-TB based on combined fluorescence-labelled locked nucleic acid (LNA) probes with high-resolution melting curve analysis (HRM). Two new LNA probes were designed to target the common class-III and IV mutations of rpoB, H526D, and D516V. LNA probes showed 100% specificity in detection of mutant targets among characterized and blinded Mycobacterium tuberculosis (Mtb) isolates. The performance of RIF-RDp was evaluated using 110 blinded clinical Mtb isolates in northern Thailand against drug- susceptibility testing (DST), DNA sequencing, and a commercial real time PCR test kit. This assay showed sensitivity and specificity of 94.55% and 98.18% compared to DST, and 96.36% and 100% compared DNA sequencing method. The efficacy of RIF-RDp was comparable to the commercial kit and DNA sequencing method. The Cohen’s Kappa statistic showed almost perfect agreement between RIF-RDp and the commercial kit (κ = 0.95, 95%CI 0.89-1), RIF-RDp and DNA sequencing (κ = 0.96, 95%CI 0.92-1). Furthermore, this is the first report of the rare mutation profiles, S531W and a triple codon deletion (510-512) in northern Thailand. According to its high accuracy, the RIF-RDp assay may render an easy-to-use, low-cost, and promising diagnostics of RR-TB in the future.