Five-Hour Diagnosis of Dermatophyte Nail Infections with Specific Detection ofTrichophyton 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.

Identification of Infectious Agents in Onychomycoses by PCR-Terminal Restriction Fragment Length Polymorphism

“…In the present study, a similar approach was adopted, and TRFLP analysis was used to identify infectious fungi based on differences in their 28S rDNA amplicons. Other DNA sequences, such as that of the chitin synthase 1 gene or small ribosomal subunit 18S rRNA, were successfully used for fungal species delineation and identification (7,27,28). The polymorphism of the internal transcribed spacers (ITS) of ribosomal DNA regions (ITS1 and ITS2) flanking the DNA sequence composing the 5.8S rDNA is the most discriminating tool for distinguishing different fungi (1).…”

Rapid Detection of FKS -Associated Echinocandin Resistance in Candida glabrata

“…C. glabrata DNA was prepared by incubating a single colony of a strain in 100 l of extraction buffer (60 mM NaHCO 3 , 250 mM KCl, 50 mM Tris, pH 9.5) at 95°C for 10 min followed by an addition of 100 l of 2% bovine serum albumin (16).…”

The Use of Polymerase Chain Reaction in Laboratory Diagnosis of Dermatophytosis

“…Klinik örneklerden DNA ekstraksiyonu, Brillowska-Dabrowska ve arkadaşlarının 8 bildirdiği yöntemle yapıldı. Buna göre 60 mM sodyum bikarbonat (NaHCO 3 ), 250 mM potasyum klorür (KCl) ve 50 mM Tris karışımından oluşan ekstraksiyon tamponu hazır-landı (pH: 9.5).…”

“…mentagrophytes'e özgül nPCR arasında iyi düzeyde (κ= 0.78), pan-dermatofi t nPCR ile T.rubrum/T.mentagrophytes'e özgül nPCR arasında mükemmel düzeyde uyum bulunmuştur (κ= 0.93). Hat 4,5,[7][8][9][10]12,16,19,21: Negatif örnekler; Hat 6,11,[13][14][15]17,18,20, …”