AbstractBackgroundDuring the past 5 years, an outbreak of recalcitrant dermatophytosis due to a novel Trichophyton species generally resistant to terbinafine, T. indotineae, has spread out from South Asia to many countries around the World. These isolates cannot be reliably differentiated from other Trichophyton spp. on the basis of morphological traits and the current laboratory diagnostics relies on sequencing of ribosomal DNA ITS region.ObjectivesIn this study, we aimed to introduce two inexpensive and rapid PCR‐based assays for differentiation between T. indotineae and other dermatophytes.MethodsThe first introduced assay is based on PCR‐restriction fragment length polymorphism (PCR‐RFLP) analysis, involving the amplification of TOP2 sequences and the digestion of PCR products by Cfr13I restriction enzyme. The second assay is proposed as conventional endpoint species‐specific PCR amplification of the C120‐287 intergenic locus. To validate the assays, a total of 191 Trichophyton spp. and 2 Microsporum canis strains with known ITS region sequences were used. From the T. mentagrophytes / T. interdigitale species complex (TMTISC), strains with 18 different ITS genotypes were tested. The sample of TMTISC isolates included 41 T. indotineae strains.ResultsTOP2 PCR‐RFLP and T. indotineae‐specific PCR were positive with testing on DNA of all 41 T. indotineae isolates and two strains of T. mentagrophytes belonging to ITS Types XIII and XVI, but negative with other species and other TMTISC ITS genotypes (n = 152). Therefore, the specificity of both new assays was 99%.ConclusionThe two developed diagnostic assays provide accurate and cost‐effective means of identifying cultured T. indotineae isolates.