Introduction: Knowledge of the prevalence and distribution of multidrug-resistant tuberculosis (MDR-TB) genotypes in northern Thailand is still limited. An accurate, rapid, and cost-effective diagnostic of MDR-TB is crucial to improve treatment and control of increased MDR-TB. Materials and Methods: The molecular diagnostic assays named "RIF-RD" and "INH-RD" were designed to detect rifampicin (RIF) and isoniazid (INH) resistance based on real-time PCR and high-resolution melting curve analysis. Applying the ∆T m cutoff values, the RIF-RD and INH-RD were evaluated against the standard drug susceptibility testing (DST) using 107 and 103 clinical Mycobacterium tuberculosis (Mtb) isolates from northern Thailand. DNA sequence analysis of partial rpoB, katG, and inhA promoter of 73 Mtb isolates, which included 30 MDR-TB, was performed to elucidate the mutations involved with RIF and INH resistance. Results: When compared with the phenotypic DST, RIF-RD targeting rpoB showed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 83.9, 98.6, 96.9, and 92.0%, respectively. The multiplex reaction of the INH-RD targeted both katG and inhA promoter showed high sensitivity, specificity, PPV, and NPV of 97.1, 94.2, 89.2, and 98.5%, respectively. Six patterns of rpoB mutation, predominately at codons 531 (50%) and 526 (40%) along with a rare S522L (3.33%) and D516V (3.33%), were detected. A single pattern of katG mutation (S315T) (63.3%) and four patterns of inhA promoter mutation, predominately −15 (C>T), were found. Approximately, 17% of MDR-TB strains possessed double mutations within the katG and inhA promoter. Conclusion: Up to 86.7% and 96.7% of MDR-TB could be accurately detected by RIF-RD and INH-RD, emphasizing its usefulness as a low unit price assay for rapid screening of MDR-TB, with confirmation of INH resistance in low and middle-income countries. The MDR-TB genotypes provided will be beneficial for TB control and the development of drugresistant TB diagnostic technology in the future.

Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) remains a global health concern because of the development of drug resistance. The adaptability of MTB in response to a variety of environmental stresses is a crucial strategy that supports their survival and evades host defense mechanisms. Stress regulates gene expression, particularly virulence genes, leading to the development of drug tolerance. Mannose-capped lipoarabinomannan (ManLAM) is a critical component of the cell wall, functions as a virulence factor and influences host defense mechanisms.Purpose: This study focuses on the effect of isoniazid (INH) stress on the regulation of ManLAM-related genes, to improve our understanding of virulence and drug resistance development in MTB. Materials and Methods: MTB with distinct drug resistance profiles were used for gene expression analysis. Multiplex-real time PCR assay was performed to monitor stress-related genes (hspX, tgs1, and sigE). The expression levels of ManLAM-related genes (pimB, mptA, mptC, dprE1, dprE2, and embC) were quantified by qRT-PCR. Sequence analysis of drug resistance-associated genes (inhA, katG, and rpoB) and ManLAM-related genes were performed to establish a correlation between genetic variation and gene expression. Results: INH treatment activates the stress response mechanism in MTB, resulting in a distinct gene expression pattern between drug resistance and drug-sensitive TB. In response to INH, hspX was up-regulated in RIF-R and MDR. tgs1 was strongly up-regulated in MDR, whereas sigE was dramatically up-regulated in the drug-sensitive TB. Interestingly, ManLAM-related genes were most upregulated in drug resistance, notably MDR (pimB, mptA, dprE1, and embC), implying a role for drug resistance and adaptability of MTB via ManLAM modulation. Conclusion:This study establishes a relationship between the antibiotic stress response mechanism and the expression of ManLAMrelated genes in MTB samples with diverse drug resistance profiles. The novel gene expression pattern in this work is valuable knowledge that can be applied for TB monitoring and treatment 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.

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.

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.