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

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

Updates on the epidemiology of dermatophyte infections.

The spectrum of dermatophytes isolated from skin lesions had changed in last 70 years. Before the Second World War in Germany, Microsporum audouinii and Epidermophyton floccosum ranked the first, whereas Trichophyton rubrum is the most common dermatophyte since the fifties of last century, accounting for 80-90% of the strains, followed by T. mentagrophytes. This evolution is typical for Central and North Europe and it needs to be connected with the increase in the incidence of tinea pedis. In contrast, in Southern Europe and in Arabic countries, zoophilic dermatophytes, such as Microsporum canis or Trichophyton verrucosum, are the most frequently isolated. In Europe, especially in Mediterranean countries, the incidence of M. canis infection has strongly increased during the recent years and this dermatophyte is now the most prevalent in tinea capitis in children. An analysis of the frequency and distribution of tinea pedis in different occupations and leisure-time activities as well as the routes of infection are reported. The spreading of this disease in most developed countries of the world represents a considerable economic problem, since it was accompanied by a parallel increase in the frequency of onychomycosis which implies, as tinea pedis, large financial charges. In poor developing countries, mycoses appear endemically, primarily with children, and their treatment often fails because of the lack of efficient antifungals. The particular epidemiological situations of dermatophytoses and the pathogenic spectrum of dermatophytes are examined at the example of numerous countries