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.
Background Patients with a previous medical history of nonmelanoma skin cancers (NMSCs) often develop multiple or recurrent malignant lesions around the site of the primary tumour. This finding led to the field cancerization theory, which suggests that the entire epithelial surface of the regional skin has an increased risk for the development of malignant lesions. Management of field change is challenging, taking into account the high impact of NMSCs on public health and healthcare costs. Objectives We sought to investigate whether field-photodynamic therapy (PDT) of extreme photodamaged skin would prevent new NMSCs, in comparison with a control area receiving placebo-PDT, in patients with clinical and histological signs of field cancerization. Methods Forty-five patients, previously diagnosed as having NMSCs of the face or scalp, with actinic keratoses symmetrically distributed over the same regions, were randomized for field treatment with 20% aminolaevulinic acid (ALA)-PDT on one side and placebo-PDT on the other. During the next 12-month period of follow up, patients were clinically evaluated for new NMSCs. Results A significant delay in the mean time of appearance and a reduction in the total number of new lesions were observed in the field-PDT protocol, when compared with the control. Conclusions The results obtained showed that field therapy with ALA-PDT confers a significant preventive potential against the formation of new NMSCs in patients with field changes.
In the 5-year period 1994-1998, 13957 patients were examined in the Mycological Laboratory of the State Hospital for Skin and Venereal Diseases, in Thessaloniki, Greece. Of the 2766 patients presenting with onychomycoses (20%), 67% were women and 33% were men. In the toenail infections, dermatophytes were most often isolated (72.3%), especially in women, followed by moulds (9.6%) and yeasts (2%); 16.1% of the infections were mixed. In the fingernail infections mostly yeasts were isolated (72%), especially in women, followed by dermatophytes (10%) and moulds (5.6%); 12.4% of the infections were mixed.
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