Molecular detection and species identification of dermatophytes by SYBR-Green real-time PCR in-house methodology using hair samples obtained from dogs and cats

Lay Summary The aim of this work was to develop a molecular detection strategy for dermatophytes by SYBR-Green real-time PCR of hair samples from animals. The melting curve analysis of the CHS1 fragment revealed to be reproducible, showing a single distinct peak for distinct dermatophyte species and allowed the diagnosis of dermatophytosis in dogs and cats caused mainly by Trichophyton mentagrophytes, Microsporum sp., and Nannizzia gypsea). The classical dermatophytes diagnosis is based on mycological culture and microscopy observation both human and animal hair, skin, and nail samples. The aim of this work was to develop the new in-house real-time PCR with pan-dematophyte reaction for detection and identification of the main dermatophytes directly from hair samples, providing a simple and rapid diagnosis of dermatophytosis in dogs and cats. An in-house SYBR-Green real-time PCR was designed and used for detecting a DNA fragment encoding chitin synthase 1 (CHS1). A total of 287 samples were processed by culture, microscopic examination with KOH 10%, and real-time PCR (qPCR) analysis. Melting curve analysis of the CHS1 fragment revealed to be reproducible, showing a single distinct peak for each species of dermatophyte, namely Trichophyton mentagrophytes, T. verrucosum, Microsporum canis, and Nannizzia gypsea (formerly M. gypseum). Then, out of the 287 clinically suspected cases of dermatophytosis, 50% were positive for dermatophytes by qPCR, 44% by mycological culture, and 25% by microscopic examination. Microsporum canis was identified in 117 samples tested by culture and 134 samples tested by qPCR, followed by N. gypsea in 5 samples (either tested by culture or qPCR) and T. mentagrophytes detected in 4 and 5 samples when tested by culture or qPCR, respectively. Overall, qPCR allowed the diagnosis of dermatophytosis in clinical samples. The results suggest this newly proposed in-house real-time PCR assay can be used as alternative diagnosis and rapid identification of dermatophytes frequently associated to clinical hair samples of dogs and cats.