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Journal of Clinical Microbiology, April 2006, p. 1419-1427, Vol. 44, No. 4
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.4.1419-1427.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Forty-Eight-Hour Diagnosis of Onychomycosis with Subtyping of Trichophyton rubrum Strains

National Center of Infectious and Parasitic Diseases, Department of Microbiology, Sofia, Bulgaria,¹ Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands,² Mycological Laboratory, State Hospital,³ Department of Dermatology, Aristotelian University of Thessaloniki, Thessaloniki, Greece,⁴ Institute of Microbiology and Hygiene, Department of Parasitology (Charité), Humboldt University, Berlin, Germany⁵

Received 26 July 2005/ Returned for modification 2 September 2005/ Accepted 18 January 2006

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.

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