Rapid and specific identification of emerging Trichophyton mentagrophytes genotype VII using an in-house developed and validated real-time PCR assay.

Trichophyton mentagrophytes genotype VII (TmVII) is an emerging dermatophyte, primarily associated with sexual transmission, that causes persistent, painful, and inflammatory skin lesions. TmVII is part of the T. interdigitale/T. mentagrophytes species complex, which comprises 28 genotypes. To facilitate rapid and specific identification of TmVII, a real-time PCR (rt-PCR) assay was developed targeting three unique single-nucleotide polymorphisms in the internal transcribed spacer 1 (ITS1) region. Assay specificity was enhanced by introducing a mismatch at the 3' ends of both primers. Using genomic DNA (gDNA) from cultured TmVII isolates, the assay demonstrated high sensitivity, detecting as little as 0.0002 ng gDNA per rt-PCR. The assay was highly specific, with no cross-reactivity observed against other fungal pathogens when a cycle threshold cutoff of 37 was used. Among the 497 mold isolates tested, 47 were confirmed as TmVII by rt-PCR, fully concordant with results from the standard ITS-PCR/Sanger sequencing method. The rt-PCR assay demonstrated high sensitivity, specificity, reproducibility, and speed, with a turnaround time of 1 day after DNA extraction compared with 2 to 7 days for Sanger sequencing. Future studies planned with primary skin samples in collaboration with healthcare providers are expected to further enhance the diagnostic utility of the TmVII rt-PCR assay currently developed.IMPORTANCEWe developed a TaqMan probe-based real-time PCR (rt-PCR) assay to accurately identify the emerging and rapidly spreading Trichophyton mentagrophytes genotype VII (TmVII) isolate. Timely identification and appropriate treatment of TmVII infections are essential to limit their spread and improve patient outcomes. Planned studies using primary skin samples, in collaboration with healthcare providers, are expected to further enhance the diagnostic utility of the TmVII rt-PCR assay.