Evaluation of simple molecular methods for distinction of the newly emerging dermatophyte <i>Trichophyton indotineae</i>

Trichophyton indotineae has emerged as a significant global public health concern due to its role in recalcitrant dermatophytosis and antifungal treatment failure. Precise identification of T. indotineae is essential for timely and effective therapy, and for curbing the spread of antifungal resistance. However, current routine diagnostic methods are limited to reliably distinguish T. indotineae from other closely related dermatophytes. This study aimed to develop and evaluate three simple, cost-effective molecular methods for the accurate differentiation of T. indotineae. In silico analyses were performed to identify specific restriction enzyme cut sites within the internal transcribed spacer (ITS) region and the topoisomerase II gene of T. indotineae. A total of 430 dermatophyte isolates, including 267 previously identified by ITS sequencing and 163 clinical isolates of unknown identity, were subjected to PCR amplification of ITS and topoisomerase II followed by restriction fragment length polymorphism (PCR-RFLP) analysis using EarI (Eam11041) and BsuRI (HaeIII), respectively. Additionally, a previously described T. indotineae-specific PCR assay was evaluated. The enzyme EarI digested the ITS region of T. indotineae, producing a distinct PCR-RFLP pattern; likewise, BsuRI digested the topoisomerase II gene, enabling accurate differentiation of T. indotineae. The isolates previously identified by ITS sequencing were correctly classified by both methods, achieving high sensitivity and specificity. The T. indotineae-specific PCR assay demonstrated high sensitivity, although faint cross-reactivity was observed with T. tonsurans isolates. The ITS-PCR-RFLP and topoisomerase II-PCR-RFLP methods demonstrated high accuracy, affordability, and speed for the reliable identification of T. indotineae, making them suitable for routine use in clinical laboratories, especially in resource-limited settings. Although the T. indotineae-specific PCR assay showed high sensitivity, occasional cross-reactivity with T. tonsurans suggests that it should be interpreted with caution and ideally used alongside confirmatory tests.