Combined Locked Nucleic Acid Probes and High-Resolution Melting Curve Analysis for Detection of Rifampicin-Resistant Tuberculosis in Northern Thailand
Yee Mon Thant,
Sukanya Saikaew,
Chayada Sitthidet Tharinjaroen,
Ponrut Phunpae,
Rodjana Pongsararuk,
Kanya Preechasuth,
Bordin Butr-Indr,
Sorasak Intorasoot,
Khajornsak Tragoolpua,
Angkana Chaiprasert,
Usanee Wattananandkul
Affiliations
Yee Mon Thant
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Sukanya Saikaew
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Chayada Sitthidet Tharinjaroen
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Ponrut Phunpae
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Rodjana Pongsararuk
Office of Disease Prevention and Control Region, 1 Chiang Mai, Department of Disease Control, the Ministry of Public Health, Muang District, Chiang Mai 50000, Thailand
Kanya Preechasuth
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Bordin Butr-Indr
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Sorasak Intorasoot
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Khajornsak Tragoolpua
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Angkana Chaiprasert
Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
Usanee Wattananandkul
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Rifampicin-resistant tuberculosis (RR-TB) has become a major threat globally. This study aims to develop a new assay, RIF-RDp, to enhance the detection of RR-TB based on combined locked nucleic acid (LNA) probes with high-resolution melting curve analysis (HRM). Two new LNA probes were designed to target the class-III and IV mutations of rpoB, H526D, and D516V. LNA probes showed 100% specificity in the 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 kit. This assay showed sensitivity and specificity of 94.55% and 98.18% compared to DST, and 96.36% and 100% compared to DNA sequencing. The efficacy of RIF-RDp was comparable to the commercial kit and DNA sequencing. The Cohen’s Kappa statistic showed almost perfect agreement between RIF-RDp and the commercial kit (κ = 0.95), and RIF-RDp and DNA sequencing (κ = 0.96). Furthermore, this is the first report of the rare mutation profiles, S531W, and a triple codon deletion (510–512) in northern Thailand. According to high accuracy, the RIF-RDp assay may render an easy-to-use, low-cost, and promising diagnostics of RR-TB in the future.
