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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.

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A silicon nitride Ion Sensitive Field Effect Transistor (ISFET) based immunosensor was developed as a low-cost and label-free electrical detection for the detection of antigen 85 complex B (Ag85B). The sensing membrane of the ISFET was modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA), yielding an aldehyde-terminated surface. This group is available for immobilization of a monoclonal antibody against a recombinant Ag85B protein (anti-Ag85B antibody). The optimal concentration for anti-Ag85B antibody immobilization onto the modified ISFET was 100 μg ml. This optimal condition provided the maximal binding capability and minimal non-specific background signal. The binding event between the recombinant Ag85B antigen and anti-Ag85B antibody on the ISFET surface is presented by monitoring the gate potential change at a constant drain current. The dose response for the recombinant Ag85B protein showed a linear response between 0.12 and 1 μg ml without significant interference from other recombinant proteins. The analytical imprecision (CV%) and accuracy of this Ag85B protein biosensor were 9.73-10.99% and 95.29%, respectively. In addition, an irrelevant antibody and other recombinant proteins were employed as a negative control to demonstrate the non-specific interaction of the antigen and antibody. The success of this immunosensor system for Ag85B protein detection facilitates the construction of a promising device which can shorten the turnaround time for the diagnosis of tuberculosis compared to a standard culture method. Furthermore, this device could also be applied for real-time growth monitoring of Mycobacterium tuberculosis in a mycobacterial culture system.

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