Usanee Wattananandkul, Sukanya Saikaew, Sirikwan Sangboonruang, Rodjana Pongsararuk, Prapaporn Srilohasin, Bordin Butr-Indr, Sorasak Intorasoot, Chayada Sitthidet Tharinjaroen, Natthawat Semakul, Surachet Arunothong, Angkana Chaiprasert, Khajornsak Tragoolpua
Drug-resistant tuberculosis remains a major challenge to tuberculosis control, particularly in settings with limited access to conventional molecular testing. This study developed and preliminarily evaluated RR-TB GoldDx and Hr-TB GoldDx, rapid visual assays for detecting rifampicin- and isoniazid-resistance-associated mutations in Mycobacterium tuberculosis. The assays combine recombinase polymerase amplification with unmodified gold nanoparticles (AuNPs) for amplification confirmation and thiol-probe-modified AuNPs for detecting resistance-associated mutations at rpoB codons 516, 526, and 531, katG codon 315, and position -15 of the fabG1-inhA promoter. Evaluation using DNA from 50 M. tuberculosis isolates yielded positive internal-control results for all samples. RR-TB GoldDx achieved accuracies of 84% and 86% against phenotypic drug-susceptibility testing and DNA sequencing, respectively, with Cohen's kappa values of 0.68 and 0.72. Hr-TB GoldDx achieved 80% accuracy and a kappa value of 0.60 against phenotypic testing, while the inhA promoter and katG assays achieved sequencing-based accuracies of 84% and 86%, respectively. Results were available within 1 h at an estimated reagent cost of THB 500-550 (approximately USD 15-16) per test without conventional thermocycling, real-time fluorescence detection, or gel electrophoresis. These instrument-minimal assays show potential for rapid drug-resistance screening but require validation in larger, genetically diverse cohorts and direct respiratory specimens.