Forty-eight-hour diagnosis of onychomycosis with subtyping of Trichophyton rubrum strains

Abstract

A novel strategy for the molecular identification of fungal agents of onychomycosis (including Trichophyton rubrum) has been designed based on the use of species-specific and universal primers in conjunction with a commercial kit that allows the extraction of DNA directly from the nail specimens. The microsatellite marker T1, which is based on a (GT)n repeat, was applied for the species-specific identification of Trichophyton rubrum. To evaluate how often Scopulariopsis spp. are detected in nail specimens, a second primer pair was designed to amplify specifically a 336-bp DNA fragment of the 28S region of the nuclear rRNA gene of S. brevicaulis and closely related species. Other fungal species were identified using amplification of the internal transcribed spacer (ITS) region of the rRNA gene, followed by restriction fragment length polymorphism analysis or sequencing. In addition, polyacrylamide gel separation of the T1-PCR product allowed subtyping of T. rubrum strains. We studied 195 nail specimens (the "nail sample") and 66 previously collected etiologic strains (the "strain sample") from 261 onychomycosis patients from Bulgaria and Greece. Of the etiologic agents obtained from both samples, T. rubrum was the most common organism, confirmed to be present in 76% of all cases and serving as the sole or (rarely) mixed etiologic agent in 199 of 218 cases (91%) where the identity of the causal organism(s) was confirmed. Other agents seen included molds (6% of cases with identified etiologic agents; mainly S. brevicaulis) and other dermatophyte species (4%; most frequently Trichophyton interdigitale). Simultaneous infections with two fungal species were confirmed in a small percentage of cases (below 1%). The proportion of morphologically identified cultures revealed by molecular study to have been misidentified was 6%. Subtyping revealed that all but five T. rubrum isolates were of the common type B that is prevalent in Europe. In comparison to microscopy and culture, the molecular approach was superior. The PCR was more sensitive (84%) than culture (22%) in the nail sample and was more frequently correct in specifically identifying etiologic agents (100%) than microscopy plus routine culture in either the nail or the strain samples (correct culture identifications in 96% and 94% of cases, respectively). Using the molecular approach, the time for diagnosing the identity of fungi causing onychomycosis could be reduced to 48 h, whereas culture techniques generally require 2 to 4 weeks. The early detection and identification of the infecting species in nails will facilitate prompt and appropriate treatment and may be an aid for the development of new antifungal agents.

FIG. 1.

PCR-RFLP of the ITS (ca. 1,000 bp) with MvaI. Lanes 1 and 14, molecular size markers in base pairs; lanes 2, 3, and 16, T. interdigitale CBS 558.66, CBS 165.66, and CBS 435.73; lanes 4 and 17, clinical isolates of C. albicans; lane 5, Epidermophyton floccosum CBS 358.93; lane 6, T. rubrum CBS 392.58 (B type); lane 7, T. rubrum CBS 289.86 (A type); lane 8, T. yaoundei CBS 730.88 (D type); lane 9, Microsporum vanbreuseghemii CBS 243.66; lanes 10 to 13, clinical isolates 1052Gr, 7112Gr, 3702Gr, and 346Gr; lane 15, T. mentagrophytes CBS 501.48; lane 18, T. rubrum CBS 100084 (A type); lanes 19 and 20, clinical isolates 817P and 5162Gr; lanes 21 to 25, DNA extracted from nails 684H2, 154Gr, 431Gr, 7962Gr, and 1024Gr.

FIG. 2.

Microsatellite polymorphisms in different clinical isolates. Lanes 1 and 14, molecular size markers in base pairs; lanes 2 and 20, CBS 289.86 (A type); lanes 3 and 19, CBS 392.58 (B type); lane 4, CBS 374.92 (C type); lane 5, CBS 305.60 (D type); lanes 6 to 13, clinical isolates 13673Gr, 1045Gr, 1034Gr, 12882Gr, 7312Gr, 641Gr, 11073Gr, 11352Gr, and 1373Gr; lanes 15 to 18, clinical isolates 1218Gr, 1159Gr, 1812Gr, and 10702Gr; lanes 21 to 25, clinical isolates 696Gr, 785Gr, 3598Gr, 247Gr, and 3571Gr.