Dual PCR-Sanger Sequencing Assay for Simultaneous sqle-Based Identification and Resistance Detection in Trichophyton Species.

Trichophyton fungal infections are common and increasingly complicated by terbinafine resistance. To support accurate molecular identification and resistance profiling, we provide a validated dataset of full-length squalene epoxidase (sqle) gene sequences from 493 Trichophyton isolates representing 18 species, addressing limited availability and misannotation of sqle sequences in public databases, particularly within the T. mentagrophytes complex. On the basis of that dataset, we designed 2 Sanger sequencing assays for simultaneously identifying species and detecting resistance-associated mutations. One targets the full-length gene, whereas the other amplifies a short region capturing all key resistance hotspots and species-specific single-nucleotide polymorphisms. The partial assay was evaluated on 66 clinical specimens and enabled clinically relevant species identification without internal transcribed spacer sequencing, supporting culture-independent detection. Of note, this approach differentiates T. indotineae from closely related species, overcoming a key diagnostic limitation and supporting clinical decision-making, surveillance, and management of resistant infections, despite limited resolution within certain species complexes.