Identification and quantification of <i>gyrA</i> variants in fluoroquinolone-resistant <i>Mycobacterium tuberculosis</i> in a MeltArray reaction

Fluoroquinolones (FQs) resistance in Mycobacterium tuberculosis (MTB), primarily driven by mutations in the quinolone resistance-determining region (QRDR) of gyrA , poses a major concern in treating for multidrug-resistant tuberculosis (MDR-TB). These QRDR mutations are known to confer varying levels of resistance, leading to differences in treatment outcomes. Here, we introduced the MeltArray MTB/FQs assay, a multiplex PCR method that detected 11 gyrA -QRDR mutations and quantified their fractions via a polynomial regression algorithm-based formula, enabling mutation identification and quantification within 3 h in one reaction. This assay, with a limit of detection (LOD) of 50 copies/reaction, could identify mixtures containing 5% mutant gDNA across 50 to 50,000 copies/reaction. Clinical evaluation of 442 culture samples displayed 95.23% sensitivity and 99.32% specificity compared with phenotypic antimicrobial susceptibility testing (pAST). Evaluation of 121 paired sputum-culture samples revealed sensitivities of 90.32% in sputum samples and 95.24% in culture samples, both with specificities of 100%, when compared with pAST. Further evaluation of 285 sputum samples showed 93.75% positive percent agreement (PPA) and 98.10% negative percent agreement (NPA) in comparison with the MeltPro MTB/FQs kit (Zeesan Biotech, China). All mutant samples identified by MeltArray MTB/FQs but classified as susceptible by pAST or as wild type by MeltPro MTB/FQs were confirmed through Sanger sequencing and droplet digital PCR (ddPCR). The formula for predicting mutation fraction (MUT%) showed accuracy rates of 88.00%, 88.89%, and 83.33% in the training, validation, and test data sets, respectively, when compared with ddPCR results. Overall, MeltArray MTB/FQs assay offers an upgraded alternative for routine FQs resistance monitoring.IMPORTANCERising FQs resistance has driven the spread of pre-extensively drug-resistant tuberculosis (pre-XDR-TB), challenging global tuberculosis (TB) control efforts. Conventional molecular assays for FQs resistance often cannot distinguish between low-level and high-level resistance mutations or detect low-fraction heteroresistant populations. In this study, we established a MeltArray MTB/FQs assay that can identify all the 11 critical mutations in the gyrA -QRDR with a LOD of 50 copies/reaction, enabling direct, culture-independent analysis of sputum samples. By using an algorithm to quantify mutations at levels as low as 5% in mixtures, MeltArray achieved both mutation identification and quantification within 3 h in a reaction, thus providing a powerful tool for early detection and precise management of pre-XDR-TB.