| BackgroundSuperficial mycoses is a common disease and prevalent among patients in dermatological clinic. There are many obstacles, especially the etiological identification, high recurrence, met in epidemiological investigation and therapeutic studies. In this study of superficial mycoses, we investigated the etiological characteristics of clinical specimens, performed molecular identification of pathogenic fungi, and analyzed the relation between antifungal susceptibilities and genetic patterns of frequently seen pathogenic fungi.Material and methods1. Etiological analysis of clinical specimens: A total of 1948 clinical specimens including skin scrapings, nail scales and hairs, were examined directly under microscopy, and/or by culture(inoculated upon Sabouraud's glucose agar or other special medium). All isolated strains were identified by their characteristic micromorphology and morphologic forms of colonies .2. DNA extraction from filamentous fungi: Improved methods using benzyl chloride was compared with Yeast DNA extraction Kit(Lyticase+ Proteinase K) in extracting genomic DNA of Trichophyton rubrum and T. mentagrophytes.3. Molecular identification of etiological fungi: Minisatellites DNA primers and random primers were used in the polymerase chainreaction(PCR)to distinguish variations among strains of dermatophytes, nondermatophytes and yeast.4. Analysis of antifungal susceptibilities and genetic relatedness of pathogenic fungi: In this study, we examined the antifungal susceptibilities of of frequently seen dermatophytes strains to itraconzole, ketoconzole, fluconazole, terbinafine, natifine, 5-flucytosine and amphotericin B, using a modified microdilution methodology. Strain genetic relatedness of Trichophyton rubrum isolates was examined using random amplified polymorphic DNA (RAPD).Results1. Etiological analysis of clinical specimens: Approximately 18 genera , 36 species of fungi strains were isolatedfrom 783 culture-positive clinical specimens. These group of fungi included 473 stains of filamentous fungi (60.41%), 308 stains of yeast (39.34%). Trichophyton rubrum, T. mentagrophytes and Candida albicans among isolated strains were 24.52%, 16.48% and 12.64%, respectively. Clinical specimens received microscopy and culture, or both direct microscopic examination and culture gave positive results as 53.41%, 40. 28% and 66. 98%, respectively.2. DNA extraction from filamentous fungi:Extraction by improved benzyl chloride methods provided at lest 25 microgram of total DNA per gram mycelia. On agarose gels, the DNA appeared as good quality, with high molecular weight more than 21kb. It also gave satisfied results in molecular analysis even after been stored at -20℃ for 3 years. Whereas genomic fungal DNA yielded by Yeast DNA extraction Kit(Lyticase+ Proteinase K) was only 0.6 microgram per l.0x109 CPU.3. Molecular identification of etiological fungi: Different PCR-fingerprinting could be seen among strains of dermatophytes, nondermatophytes and yeast by using minisatellites DNA primers and random primers, especially amplification with primer (GACA)4 could give more distinct bands.4. Analysis of antifungal susceptibilities and genetic relatedness of pathogenic fungi:It was found, that MIC values of dermatophytes elevated within a certain period, but MIC endpoint determining should base on the results of 5-7 days after inoculation. For itraconzole, ketoconzole, fluconazole, terbinafine, natif ine and amphotericin B, MFCs were close to the MICs determined in sixth day after inoculation. MIC of 5-f lucytosine is the highest with a mean value above 128ng/ml. Different dermatophytes strains show different MICs to same antimycotic drug. Relative to the other agents tested, terbinafine and natifine possessed the highest antifungal activities against all of the dermatophytes.RAPD resulted in five DNA types of T. rubrum. DNA types of T. rubrum were not related with the sites strains isolated. But it was not clear if there was a relationship between genotype an...
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