Five-hour diagnosis of dermatophyte nail infections with specific detection of Trichophyton rubrum

Abstract

A rapid two-step DNA extraction method and a multiplex PCR for the detection of dermatophytes in general and Trichophyton rubrum specifically were developed and evaluated with DNA extracted from pure cultures and from clinically diseased nails. DNA from the following dermatophytes was used: Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum gypseum, Microsporum nanum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton soudanense, Trichophyton terrestre, Trichophyton tonsurans, Trichophyton verrucosum, and Trichophyton violaceum. Human DNA and DNA from the following nondermatophyte fungi were included as controls: Alternaria, Aspergillus niger, Candida albicans, Candida glabrata, Candida krusei, Malassezia furfur, Saccharomyces cerevisiae, and Scopulariopsis brevicaulis. A total of 118 nail samples received for routine microscopy and culture for dermatophytes were subsequently tested by the two PCRs separately and in a multiplex format. Using DNA extracted from pure cultures and the pan-dermatophyte PCR, the T. rubrum-specific PCR sequentially and in a multiplex format correctly detected all dermatophytes and additionally correctly identified T. rubrum. Comparison of the traditional diagnostic evaluation (microscopy and culture) of nail samples with PCR on DNA directly extracted from the nails showed excellent agreement between PCR and microscopy, but the number of samples with dermatophyte species identification was increased considerably from 22.9% to 41.5%, mainly due to the identification of T. rubrum by PCR in microscopy-positive but culture-negative samples. In conclusion, this 5-hour diagnostic test was shown to increase not only the speed but also the sensitivity of investigation for nail dermatophytosis.

FIG. 1.

Example of Trichophyton rubrum-specific and pan-dermatophyte PCR product analysis. Lanes: 1 and 12, molecular size marker (fragment sizes, 501, 489, 404, 331, 242, 190, 147, 111, and 110 bp); 2 and 3, results of T. rubrum-specific PCR performed for T. mentagrophytes DNA (lane 2) and T. rubrum DNA (lane 3); 4 to 11, results of pan-dermatophyte PCR performed for Microsporum audouinii (lane 4), T. mentagrophytes var. mentagrophytes (lane 5), Trichophyton schoenleninii (lane 6), Trichophyton terrestre (lane 7), T. rubrum (lane 8), T. tonsurans (lane 9), Trichophyton soudanense (lane 10), and Epidermophyton floccosum (lane 11).

FIG. 2.

Example of Trichophyton rubrum-specific and/or pan-dermatophyte multiplex PCR product analysis. Lanes: 1, molecular size marker (100-bp DNA ladder); 2 to 6, results of multiplex PCR performed for DNA extracted directly from nail specimens diagnosed by conventional methods as negative (lane 2), M. audouinii (lane 3), T. rubrum (lane 4), T. mentagrophytes (lane 5), and Aspergillus sp. (lane 6).