qPCR-Based Molecular Detection of Trichophyton indotineae by Targeting Divergent Sequences.

Trichophyton indotineae, formerly known as T. mentagrophytes internal transcribed spacer (ITS) genotype VIII, has been recognized over the last decade due to its high virulence and resistance to treatment. Its accurate identification in routine mycology laboratories remains challenging, as it shares phenotypic traits and substantial rDNA ITS similarity with T. mentagrophytes and T. interdigitale. This study aimed to identify more divergent and stable sequences via whole-genome comparisons between T. indotineae and T. interdigitale to facilitate highly specific targeting of T. indotineae using a validated quantitative polymerase chain reaction (qPCR)-based method. Our whole-genome comparison revealed at least 22 unique sequences of T. indotineae compared to T. interdigitale and revealed the divergence of the former from the reference genomes of other Trichophyton species. Among these, a 499 bp segment was identified as the most genetically distinct sequence within the T. indotineae genome. Seventy-three dermatophyte strains [T. indotineae (n = 66), non-T. indotineae (n = 7)], were tested using our qPCR assay targeting the above-mentioned stable 499-bp region. Regarding analytical performance, our T. indotineae-specific qPCR assay exhibited high sensitivity (93.3%) and specificity (100%), with a detection limit of ~ 15 genomic copies. Our approach has the potential to establish highly sensitive and specific qPCR assays without relying on specialized assay designs for single nucleotide polymorphisms in the ITS or other loci. This approach offers a practical solution for updating molecular diagnostics, particularly for novel taxa such as T. indotineae, for which limited gene data are available in public databases.