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.

Isoniazid (INH) is an antibiotic that is widely used to treat tuberculosis (TB). Adaptation to environmental stress is a survival strategy for Mycobacterium tuberculosis and is associated with antibiotic resistance development. Here, mycobacterial adaptation following INH treatment was studied using a multi-stress system (MS), which mimics host-derived stress. Mtb H37Rv (drug-susceptible), mono-isoniazid resistant (INH-R), mono-rifampicin resistant (RIF-R), and multidrug-resistant (MDR) strains were cultivated in the MS with or without INH. The expression of stress-response genes (hspX, tgs1, icl1, and sigE) and lipoarabinomannan (LAM)-related genes (pimB, mptA, mptC, dprE1, dprE2, and embC), which play important roles in the host–pathogen interaction, were measured using real-time PCR. The different adaptations of the drug-resistant (DR) and drug-susceptible (DS) strains were presented in this work. icl1 and dprE1 were up-regulated in the DR strains in the MS, implying their roles as markers of virulence and potential drug targets. In the presence of INH, hspX, tgs1, and sigE were up-regulated in the INH-R and RIF-R strains, while icl1 and LAM-related genes were up-regulated in the H37Rv strain. This study demonstrates the complexity of mycobacterial adaptation through stress response regulation and LAM expression in response to INH under the MS, which could potentially be applied for TB treatment and monitoring in the future.

Tuberculosis (TB) remains a major global health challenge, particularly in resource-limited settings where access to rapid and reliable diagnostics is limited. Conventional diagnostic methods, such as smear microscopy and culture, are either time-consuming or lack adequate sensitivity. This study optimized recombinase polymerase amplification (RPA) using 16 primer combinations targeting IS6110 highly specific to the Mycobacterium tuberculosis complex (MTC). A novel naked-eye assay, TB-GoldDx, was developed by integrating RPA combined with gold nanoparticles (AuNPs), enabling equipment-free diagnostics. TB-GoldDx demonstrated a detection limit of 0.001 ng of MTB H37Rv DNA (~210 bacilli) per 25 µL reaction. Among 100 bacterial strains, it achieved 95.83% sensitivity and 100% specificity among 100 bacterial strains, comprising 72 MTB isolates and 28 nontuberculous bacterial species. In 140 sputum samples, the assay showed 81.43% sensitivity and 58.57% specificity versus acid-fast bacilli (AFB) smear microscopy, with sensitivity improving to 95.45% in high-load AFB 3+ specimens. Compared to a commercial line probe assay (LPA), TB-GoldDx exhibited slightly higher sensitivity (84.78% vs. 82.61%) but lower specificity (54.05% vs. 78.38%). Delivering rapid, visual results in under an hour, TB-GoldDx offers a low-cost, easily deployable solution for point-of-care tuberculosis detection, especially in underserved regions, reinforcing global End TB efforts.

The epidemiology and genotypes of multidrug-resistant tuberculosis (MDR-TB), a global public health threat, remain limited. The genotypic distribution and factors associated with MDR-TB in upper northern Thailand between 2015 and 2019 were investigated. The DNA sequencing of rpoB, katG, and inhA promoter of 51 multidrug-resistant Mycobacterium tuberculosis isolates revealed nine patterns of the rpoB gene mutation distributed in seven provinces. The S531L mutation was the most common mutation in all provinces. The rpoB mutation in Chiang Rai, Chiang Mai, and Lampang was highly diverse compared to other areas. Here, the mutation profiles that have yet to be reported in northern Thailand (H526P, Q513P, and H526C) were detected in Chiang Rai province. The S315T katG mutation was the most common genotype associated with INH resistance, especially in Chiang Mai and Lampang. Further analysis of data from 110 TB patients (42 MDR-TB and 68 drug-susceptible TB) revealed that <60 years of age was a significant factor associated with MDR-TB (OR = 0.316, 95% CI 0.128–0.784, p = 0.011) and ≥60 years of age was a significant factor associated with the S315T katG-mutation (OR = 8.867, 95% CI 0.981–80.177, p = 0.047). This study highlighted the necessity for continuous surveillance and risk factor monitoring for effective control of MDR-TB.

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.