The Use of Polymerase Chain Reaction in Laboratory Diagnosis of Dermatophytosis

Dermatophytes are among the common causes of fungal infections in the community. Classical diagnostic tests for dermatophytosis have some disadvantages such as failure of direct microscopy in species differentiation and culture methods being time consuming and having low sensitivity. The aim of this study was to investigate the performance of polymerase chain reaction (PCR) in the identification of dermatophytes directly from the clinical samples and the cultures. A total of 123 samples that comprise 63 skin and 60 nail scrapings obtained from 110 patients (69 female, 41 male; age range: 4-82 years) who were prediagnosed as dermatophytosis, were included in the study. Samples were examined with routine direct microscopy, culture and two different nested PCR (nPCR) protocols. The first was a pandermatophyte nPCR protocol targeting chitin synthase gene (CHS-1) of dermatophytes and the second was a nPCR protocol which targets specific ITS-1 genes of Trichophyton rubrum and T.mentagrophytes. Similar PCR methods were also applied to cultivated strains. Sequence analysis was performed for the samples that yielded positive results in pan-dermatophyte nPCR and negative results in T.rubrum/T. mentagrophytes - specific nPCR. Hyphae and/or spore structures were observed in 62 (50%) samples with direct microscopic examination and dermatophytes were isolated in 30 (24%) samples. Twenty-eight of the isolates grown in culture were identified as T.rubrum, and two as T.mentagrophytes with T.rubrum/T.mentagrophytes-specific nPCR protocol. In direct application, 67 (55%) of the clinical samples were found positive with pan-dermatophyte nPCR and 65 (53%) were positive with T.rubrum/T. mentagrophytes-specific nPCR. Samples which were negative in direct microscopic examination were also negative in culture. Nine of them were found positive with pan-dermatophyte nPCR and eight were positive with T.rubrum/T.mentagrophytes-specific nPCR. Two of the 30 samples which were positive in culture were negative in direct pan-dermatophyte nPCR, and one of them was negative in T.rubrum/T. mentagrophytes-specific nPCR. Three samples which were positive by pan-dermatophyte nPCR, gave negative result with T.rubrum/T.mentagrophytes-specific nPCR. Sequence analysis was performed for these three samples and all were identified as T.rubrum. In evaluation of concordance between the methods, the agreement of direct microscopy and culture was moderate (kappa value; kappa= 0.48), the agreement of direct microscopy and both protocols of nPCR was high (kappa= 0.78) and the agreement of both nPCR protocols with each other was excellent (kappa= 0.93). Our data indicated that two different nPCR methods used for the laboratory diagnosis of dermatophytosis yielded higher positivity in less time than the culture method. In conclusion, nPCR was considered to be useful in identification of dermatophytosis from either direct clinical samples or culture-isolated strains.