Relationship Between Phenotypic and Genotypic Characteristics of Trichophyton mentagrophytes Strains Isolated from Patients with Dermatophytosis.

According to epidemiological, clinical and mycological criteria, it has long been admitted that the Trichophyton mentagrophytes species includes two varieties: a zoophilic variety (var. mentagrophytes) and an anthropophilic variety (var. interdigitale) that involve the upper and the lower part of the body, respectively. The further application of molecular techniques to the characterization of dermatophyte strains showed that this classification is unreliable. The aim of our study was to assess the usefulness of PCR-RFLP (restriction fragment length polymorphism) and sequencing in the characterization of T. mentagrophytes strains taken from Tunisian patients. The study was carried out in 2008 in the laboratory of Parasitology-Mycology of Farhat Hached University Hospital, Sousse, Tunisia. A total of 133 strains were isolated from 133 patients addressed to the laboratory for dermatological lesions very evocative of dermatomycosis. Eighty strains were isolated from lesions located on the lower part of the body (onychomycosis, tinea pedis) and 53 strains from the upper part of the body (tinea capitis, tinea corporis). All strains were submitted to mycological examination (direct microscopic examination and culture on Sabouraud medium) and further investigated by using RFLP analysis of the PCR-amplified ITS1-5.8 s-ITS2 region of the ribosomal DNA and the MvaI restriction enzyme. In addition, 62 strains were further submitted to a sequencing of the ITS1-5.8 s-ITS2 region. On the basis of mycological criteria, all strains were diagnosed as T. mentagrophytes. All strains produced the same RFLP pattern and were identified as T. mentagrophytes interdigitale regardless of the location of lesions. Out of the 62 sequenced strains, 16 were found anthropophilic and 46 were zoophilic. In conclusion, all strains provisionally diagnosed as T. mentagrophytes on the basis of mycological criteria were shown to belong to T. interdigitale by using PCR-RFLP and sequencing irrespective of the site of lesions. The predominance of zoophilic strains needs further investigation.

Multiplex PCR assay for the detection of common dermatophyte nail infections.

Onychomycosis is one of the most prevalent dermatophytic diseases. Mycological methods used in the conventional diagnosis may not be optimal. Multiplex (MX) PCR was reported as a reliable alternative. Dermatophyte gene sequence records were used to design a MX PCR for detection and identification of dermatophytes in nail specimens. A MX PCR method based on the amplification of the chitin synthase 1 and internal transcribed spacer genes was developed. The study included 93 strains of dermatophytes and non-dermatophytic fungi, six dermatophytic reference strains and 201 nail specimens from patients with dermatophytic onyxis. DNA extraction directly from nail samples was carried out by using the QIAamp DNA extraction kit (Quiagen). A set of primers was designed and their specificity was assessed. MX PCR detected the causal agent in specimens from which Trichophyton rubrum and T. interdigitale grew in culture and also identified a dermatophyte species in an additional 32 specimens that were negative in microscopy and culture. None of the investigated non-dermatophytic strains was positive. Sensitivity of MX PCR was higher as compared to mycological examination (97% vs. 81.1%). MX PCR for direct detection of dermatophytes from nail samples yielded mixed flora in 32.8% of samples. MX PCR proved sensitive and adequate for the diagnosis of dermatophytic onychomycosis. It is much adapted to cases where culture is negative or contaminated by overgrowing moulds, which makes the identification of the causal agent problematic.

Clinical and mycological features of onychomycosis in central Tunisia: a 22 years retrospective study (1986-2007).

Onychomycosis is the most frequently encountered nail disease and may be difficult to diagnose and treat. The objective of this study was to determine the prevalence, the clinical and mycological characteristics of onychomycosis in central Tunisia. It is a retrospective study performed over a 22-year period (1986-2007). It included 7151 patients (4709 women and 2442 men) with suspected fingernails and/or toenails onychomycosis. The patients were referred to the Mycology-Parasitology Laboratory of Farhat Hached hospital in Sousse for mycological examination. Both direct microscopy and culture of the nail material were performed to diagnose and identify the causative fungal species. Onychomycosis was confirmed in 78.6% of investigated patients (5624/7151). The positivity rate was higher in women as compared with men. In both men and women, fingernails were most frequently involved than toenails. No significant relation was found between gender and toenails onychomycosis, whereas fingernails were frequently involved in women. As far as aetiological agents are considered, dermatophytes, yeast and moulds were responsible for 49.9%, 47.4% and 2.7% of onyxis cases respectively. In fingernail infections, yeast were the most frequent fungi (83.6%), Candida albicans being the leading species (51.6%). In contrast, in toenail infections, dermatophytes were more frequent (74.1%). Trichophyton rubrum was by far the dominant species (88.1%). Yeast were observed more frequently in women whereas dermatophytes were more common in men. Moulds were involved in 4.2% of cases. The most frequent species were Aspergillus sp. and Chrysosporium sp. Onychomycosis is a frequent disease in central Tunisia. T. rubrum is the predominant agent in toenails infection and yeast, mainly C. albicans, in fingernails onychomycosis.

Evaluation of Chitine synthase (CHS1) polymerase chain reaction assay in diagnosis of dermatophyte onychomycosis.

BACKGROUND: Onychomycosis is one of the most prevalent dermatophytic diseases. Mycological methods used in the conventional diagnosis may not be optimal. PCR was reported as a reliable alternative in the diagnosis of dermatophytosis. MATERIALS AND METHODS: A PCR method based on the amplification of the chitin synthase 1 gene was developed. The study included 119 strains of dermatophytes and non dermatophytic fungi, eight dermatophytic reference strains and 201 nail specimens from patients with dermatophytic onyxis. DNA extraction was carried out by using the QIAamp DNA extraction kit (Quiagen). RESULTS: PCR positivity was based on the production of a specific 432 bp fragment. None of the investigated non dermatophytic strains was positive. Sensitivity of PCR was higher as compared to mycological examination (90.5% vs. 81.1%). PCR was positive in 31 onyxis cases with positive direct examination but negative or contaminated culture. In contrast, PCR was negative in 10 cases where both direct examination and culture were found positive. CONCLUSION: PCR is an adequate tool for the diagnosis of dermatophytic onychomycosis. It is much adapted to cases where culture is negative or contaminated by overgrowing molds, which makes the identification of the causal agent problematic.