تعیین گونه مالاسزیاهای جدا شده از بیماران مبتلا به پیتیر یازیس ورسیکالر و درماتیت سبوروئیک، با روش PCR-RFLP

Background and Purpose: Lipophilic yeast of the genus Malassezia are members of normal human cutaneous micro flora which are also associated with several skin diseases. It is strongly suspected that Malassezia species are responsible for pityriasis versicolor (PV), and seborrhoeic dermatitis (SD). Considering various sensitivities among Malassezia species to antifungal, accurate species identification will facilitate the treatment of relevant diseases. Malassezia species can be identified through their morphological features and bio-chemical characteristics. However, these phenotypic methods are usually time consuming, and lack sufficient discriminatory power. Development of DNA- based methods for detection and identification of Malassezia species provide helpful alternatives for solving problems. The aim of this study was to examine the distribution of Malassezia species in patients with pityriasis versicolor and seborrhoeic dermatitis, using molecular methods. Materials and methods: A total of 63 clinical isolates of Malassezia spp, 30 strains isolated from patients with PV and 33 strains isolated from patients with SD, were studied. To investigate the strains at molecular level, genomic DNA of Malassezia isolates were extracted and amplified within the ITS1 region (located between 18S and 5.8S rDNA) by polymerase chain reaction (PCR) assay. DNA sequencing of ITS1 of rDNA in Malassezia spp was performed to type the species. Restriction fragment length polymorphism (RFLP) analysis of ITS1 PCR product with two restrictive enzymes CFOI and BSTF5I, was used in subsequent species identification. Results: In this study, 37 patients with PV (20 females, 17 males 2 to 64 years old), 81.1% (30 case) yielded growth of Malassezia in culture, while the frequency of isolation of M. globosa was 53.3% (16 case), M .furfur 40% (12 case) and M. sympodialis 6.7% (2 case). Of the 41 patients with SD (22 females, 19 males 1 to 52 years old), 80.5% (33 case) yielded growth of Malassezia in culture, while the frequency of M. furfur was 42.4% (14 case), M.globosa 39. 4% (13 case), M. restrict 15.2% (5 case) and M. sympodialis 3% (1 case). This PCR-RFLP Profile allows us to clearly identify important Malassezia species. The results of the PCR-RFLP analyses of clinical isolates were in complete agreement with those from DNA sequencing, morphological features and bio-chemical characterization. Conclusion: In patients with PV, the most frequently isolated species were M. globosa, followed by M .furfur. However, in patients with SD, the most frequently isolated species were M. furfur, followed by M. globosa. The PCR-RFLP system applied for the ITS1 fragment of the rDNA is a reliable, simple and rapid method for identification of the most important Malassezia species, but further work will be necessary in applying these techniques to additional patients.