Clinical performance of two commercial PCR assays for the detection of macrolide resistance in <i>Mycoplasma pneumoniae</i>

Macrolides are the first-line treatment for <i>Mycoplasma pneumoniae</i> infections; however, macrolide resistance has been reported. We evaluated the clinical performance of two commercial assays for detecting macrolide-resistant <i>M. pneumoniae</i>: the LightMix Modular <i>Mycoplasma Macrolide</i> (TIB Molbiol) and the <i>Mycoplasma Pneumoniae</i> and Macrolides-Resistant Strain Nucleic Acid Test Kit (Mole Bioscience), using 23S rRNA Sanger sequencing as the reference method. Eight <i>M. pneumoniae</i> strains, 10 non-<i>M</i>. <i>pneumoniae Mycoplasma</i> strains, and 237 clinical samples were tested. Overall, the Mole Bioscience kit failed to detect <i>M. pneumoniae</i> in 27.7% of positive samples, whereas the TIB Molbiol kit missed only 5.1%. The clinical sensitivities for detecting macrolide resistance-associated mutations in clinical samples were 90.9% for the TIB Molbiol kit and 81.5% for the Mole Bioscience kit. All false wild-type results corresponded to samples harboring a mutation at position 2067 (<i>M. pneumoniae</i> numbering) of the 23S rRNA gene. The clinical specificities were 97.9% and 94.4% for the TIB Molbiol and Mole Bioscience kits, respectively. Notably, the TIB Molbiol kit also detected 23S rRNA mutations in <i>Mycoplasma genitalium</i> and <i>Mycoplasma amphoriforme</i>, whereas the Mole Bioscience kit yielded false-resistant results in high-load specimens. In conclusion, these commercial kits offer a convenient approach for detecting macrolide resistance in routine clinical practice. The Mole Bioscience kit demonstrated limited sensitivity for <i>M. pneumoniae</i> detection, restricting its utility for resistance identification, and neither assay reliably detected resistance-associated mutations at position 2067. The global <i>M. pneumoniae</i> reemergence highlights the need for advanced assays detecting all macrolide resistance-associated mutations.IMPORTANCE<i>Mycoplasma pneumoniae</i> is a leading cause of bacterial respiratory tract infections, and a marked resurgence was reported worldwide in autumn 2023. Macrolides are the recommended first-line treatment for <i>M. pneumoniae</i> infections; however, resistance rates vary widely across regions and may compromise therapeutic efficacy. Rapid identification of macrolide resistance is therefore critical to ensure appropriate patient management. Because culture-based methods are slow and have limited sensitivity, molecular assays have become central to both pathogen detection and resistance determination. However, few simple and reliable commercial assays have been developed and evaluated for the detection of macrolide resistance in routine diagnostic laboratories. In this study, we evaluated the clinical performance of two real-time PCR-based commercial assays for detecting macrolide resistance-associated mutations in <i>M. pneumoniae</i>. Our findings support the implementation of rapid molecular tools to guide timely therapeutic decisions, including the early use of second-line antimicrobials when resistance is detected.