Identification andDifferentiation of Trichophyton Mentagrophytes and T.rubrum by Polymerase Chain Reaction and Enzymatic Digestion

Introduction & Objective: Trichophyton rubrum and T. mentagrophytes are most common causative agents of dermatophytosis in the world. Differentiation of these species is important from the epidemiological and pathological point of view. Conventional methods including macroscopic and microscopic morphology and biochemical tests are time-consuming (in some cases it takes 3-4 weeks), laborious and still sometimes insufficient to identify these agents. The aim of this study was to use polymerase chain reaction followed by enzymatic digestion for differentiation of these 2 species. Materials & Methods: In this descriptive–experimental study one hundred strains were isolated from patients with dermatophytosis. Preliminary identification was done by morphological methods. DNA was isolated and purified by glass-bead methods and ITS1- 5.8SrDNA-ITS2 region was amplified by PCR and the amplicon was digested by the restriction enzyme MvaI. The products were visualized after agarose gel electrophoresis and staining. Differentiation of the species was based on sequence analysis and the electrophoretic patterns. Results: Morphological tests were not able to definitely differentiate the two tested species, especially for isolates with intermediate features. Using molecular methods, it was found that 45 isolates are T. rubrum and 54 are T. mentagrophytes. One isolate was Fusarium spp. Physiological tests were confirmed the results except for 4 isolates. It was also found that hair perforation test is more reliable than urease test for differentiation of these two species. Conclusion: We found that DNA-based method, although expensive, is a fast and reliable method for differentiation of T. rubrum and T. mentagrophytes. The frequency of mentioned species was almost similar in the tested isolates. The method is recommended for differentiation of other dermatophytes

Identification of Pathogenic Candida Species: PCR-Fragment Size Polymorphism (PCR-FSP) Method

"nBackground: The clinical importance of yeast infections has increased in recent decades. There are 10-15 pathogenic Candida species. The current morphological and physiological methods for identification of Candida species are generally not easy to interpret and may be expensive or time-consuming. In the present study, we introduce and use a new approach for the identification and differentiation of medically important yeast species of Candida. In this method, size polymorphism of the internal transcribed spacer regions, ITS1 and ITS2, of the ribosomal DNA in various Candida species is used as the basis of species recognition. "nMethods: The genomic DNA of 31 standard strains and 60 clinical isolates was extracted and PCR-amplified using two primer pairs (ITS1-ITS2 and ITS3-ITS4) separately. Both PCR products were mixed and analyzed after standard agarose gel electrophoresis. The species of the tested yeasts were identified by the electrophoretic patterns of the mixed PCR products of each sample, comparing the data obtained from the sequence analyses of ITS1 and ITS2 molecules. "nResults: By this method, with the exception of C. albicans and C. dubliniensis, we were able to clearly differentiate nearly all common pathogenic Candida species, including C. albicans, C. glabrata, C. gulliermondii, C. parapsilosis, C. tropicalis,      C. krusei, C. kefyr, C. lusinaniae and C. rugosa. All standard and clinical strains were identified correctly, without expensive methods such as sequencing and capillary electrophoresis. "nConclusion: It seems that the PCR-FSP method introduced in this study is the easiest molecular approach for the identification of a wide range of pathogenic Candida species and is applicable for diagnostic and epidemiological purposes in reference laboratories