Multiplex real-time PCR assays for subspecies identification and macrolide resistance genotyping within the Mycobacterium abscessus species

ABSTRACT Rapid and accurate subspecies-level identification and macrolide resistance genotyping of the Mycobacterium abscessus (MAB) species are essential for guiding effective therapy. We developed and clinically validated a closed-tube, two-reaction MAB-Multiplex Real-Time PCR assay for this purpose. Assay I targets conserved loci identified from 348 MAB genomes for subspecies-level differentiation, while Assay II targets common macrolide resistance mutations in erm(41) and 23S rRNA ( rrl ). Reference standards were whole-genome sequencing, reference strains, and targeted next-generation sequencing for Assay I, and sequencing for Assay II. Assay I achieved 100% sensitivity and specificity across 135 isolates and 202 clinical specimens, with a limit of detection (LOD) of 17.5 fg–140 fg per reaction (genomic DNA) and approximately 10³ CFU/mL (sputum). Assay II was evaluated on 77 isolates and 21 clinical MAB-positive specimens, yielding 100% genotyping accuracy in isolates and showing >93% sensitivity, with 100% specificity for erm(41) variants in clinical specimens. The LOD was 112.5 fg for erm(41) and 15 pg–150 pg for rrl per reaction (genomic DNA). Delivering comprehensive results within 180 min at approximately $1.00 per test, this culture-independent diagnostic tool offers a rapid, affordable, and highly accurate alternative to conventional methods. Its integration into routine clinical workflows promises to significantly advance the precision management and epidemiological surveillance of MAB infections. IMPORTANCE Infections caused by Mycobacterium abscessus are exceptionally challenging because different subspecies dictate distinct treatment outcomes and therapeutic strategies. While conventional diagnostics require weeks of bacterial culture, existing rapid molecular alternatives remain limited by high costs, complex workflows, or insufficient resolution to distinguish closely related subspecies. To address these clinical gaps, we developed, to our knowledge, the first rapid, affordable, culture-independent multiplex PCR system that integrates subspecies identification with macrolide resistance genotyping. This closed-tube system operates directly on clinical specimens or bacterial isolates, simultaneously identifying subspecies and key genetic determinants of macrolide resistance. Delivering comprehensive results within 180 min for approximately one dollar per test, this method provides an accessible and robust solution for same-day therapy guidance and may improve patient management and epidemiological surveillance of these severe infections.