Objectives: Fungal infections of nails, skin and hair (tineas) caused by dermatophytes such as Trichophyton (T.) spp., Microsporum (M.) spp. or Epidermophyton (E.) spp. are one of the most common human infections in the world. As the epidemiology varies between different dermatophyte species a specific diagnosis plays an important role to ensure a targeted therapy. In order to evaluate the DermaGenius® multiplex real-time PCR assays (PathoNostics), qPCR results were compared to routine diagnostic methods including fungal culture detection, microscopy of native specimens and a standard PCR-ELISA assay. Specimens that could not be identified by the routine diagnostic methods were further analysed by Sanger sequencing.	
Material and methods: The DNA of 31 clinically isolated skin specimens and 18 fungal cultures was isolated using the QIAamp DNA Mini Kit (Qiagen). Dermatophytes were identified macroscopically by cultural detection, partly microscopically by fluorescence staining of the native specimen and molecular biologically using an uniplex PCR-ELISA assay. Sanger sequencing of both, the ITS region of the  rDNA and TEF1α gene was used to further analyse dermatophytes when routine diagnostics could not give a definite result. Quantitative real-time polymerase chain reaction (qPCR) was performed with the DermaGenius® complete multiplex kits (PathoNostics) using the qPCR instrument LightCycler  480 II (Roche).	
Results: In total, all (n = 49) clinical specimens and cultures could be detected with the DermaGenius® complete multiplex assay (PathoNostics). Out of 49 samples, DermaGenius® complete assay could identify 37 (76%) correctly by their specific melting temperature. Fourteen samples were detected, but not differentiated including T. soudanense (n = 1) which was identified as T. rubrum, T. erinacei (n = 2) as T. benhamiae, T. equinum (n = 1) as T. tonsurans and M. ferrugineum (n = 1) as M. canis. Although different variants of T. mentagrophytes (n = 4), T. quinckeanum (n = 2) and T. schoenleinii (n = 1) species were detected by qPCR, they all showed similar melting temperatures to either T. interdigitale or T. mentagrophytes.
Conclusion: The DermaGenius® multiplex qPCR assays (PathoNostics) are able to identify the most frequently isolated clinically prevalent dermatophytes and are suitable for routine molecular diagnostic laboratories as they enable high samples throughput with limited hands-on time. However, the identification and differentiation of less common dermatophytes as well as variants of T. mentagrophytes is still complicated since they are phylogenetically very closely related. After taking the anamnesis of patients or a fungal culture into consideration, only sequencing enables an identification of dermatophytes that could not be detected during routine diagnostics.

Detection and identification of pathogenic dermatophytes using multiplex real-time PCR assays