Evaluation of multiplex real‐time PCR for identifying dermatophytes in clinical samples—A multicentre study

Abstract

The gold-standard method for dermatophyte identification involves direct microscopy and culture, which have inherent shortcomings. Only few molecular methods have been standardised for routine clinical work. This study aimed to develop and test a platform for identifying the most common dermatophytes in Israel using multiplex real-time polymerase chain reaction (RT-PCR). Specific primers were designed for the multiplex system (LightCycler 480) according to known cultures and validated by reference isolates. The dermatophyte detection rate was compared to smear and culture in 223 clinical samples obtained from a tertiary medical centre. Inconsistencies between methods were evaluated by sequencing. The RT-PCR was further evaluated in 200 community-based samples obtained from a health maintenance organisation and 103 military-personnel-based samples analysed at a central laboratory. In hospital-based clinical samples, complete concordance between methods was observed in 190 samples (85%; Kappa = 0.69). In most cases of non-concordance, sequencing was consistent with RT-PCR results. RT-PCR correctly identified all smear- and culture-positive cases in community and military-personnel samples. The results were available within 4 hours. The multiplex RT-PCR platform is a rapid and efficient method for identifying dermatophyte species in clinical samples and may serve as a first step in the diagnostic algorithm of superficial fungal infections.

Indian association of dermatologists, venereologists and leprologists (IADVL) task force against recalcitrant tinea (ITART) consensus on the management of glabrous tinea (INTACT)

“…RT-PCR is a rapid method that enables detection of dermatophytes from clinical specimens. [ 53 54 ] Research laboratories usually rely on sequencing of the internal transcribed spacer (ITS) region of the rDNA for sub-speciation and classification of dermatophytes. [ 55 ] Accurate subspecies identification helps understand the clinical and epidemiological implications of the genetic heterogeneity of dermatophytes.…”

Epidemiology and Diagnostic Perspectives of Dermatophytoses

“…In an attempt to increase the diagnostic response, real-time PCR can be performed as described by [67], who indicates multiplex RT-PCR as the fastest and most efficient method for the identification of dermatophyte species in clinical samples, or using pan-dermatophyte primers as described by [66] for the diagnosis of dermatophytes in nail samples. Commercial kits designed to facilitate the identification of dermatophytes by real-time PCR have also been developed.…”

New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions

“…An increasingly frequent method of species identification, also in mycology, is the quantification reverse transcription PCR technique (qRT-PCR) [23,26,35]. However, its need for adequate selection of reference genes is a limitation, which is associated with a strong dependence on the stability of the transcription level of these genes when in incubation conditions [26].…”