A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species Hossein Mirhendi a, * , Koichi Makimura b , Kamiar Zomorodian a , Tsuyoshi Yamada b , Takashi Sugita c , Hideyo Yamaguchi b a Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, PO Box 14155-6446, Tehran, Iran b Institute of Medical Mycology and Genome Research Center, Teikyo University, Tokyo, Japan c Department of Microbiology, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan Abstract A PCR-RFLP method targeted toward 26S rDNA and with 2 restriction enzymes, Cfo I and Bst F51, was developed to identify 11 Malassezia species. Not only type and standard strains but also 13 clinical isolates were identified successfully in this study. The results of identifications were confirmed by DNA sequencing. Keywords: Malassezia; Identification; PCR; RFLP; CfoI; Bst F51 Malassezia species are part of the resident skin flora of humans and other warm-blooded verte- brates (Leeming et al., 1989). These yeasts are associated with various superficial diseases, includ- ing pityriasis versicolor, seborrheic dermatitis, and folliculitis (Gupta et al., 2002; Tarazooie et al., 2004), as well as nosocomial bloodstream infec- tion in pediatric care units (Chryssanthou et al., 2001). The genus Malassezia has undergone several taxonomic revisions. In the reclassification by Gueho et al. (1996), seven distinct species were identified within this genus. Recently, on the basis of DNA relatedness, four new species have been included: Malassezia dermatis , Malassezia nana, Malassezia japonica , and Malassezia yamatoensis (Sugita et al., 2002, 2003, 2004; Hirai et al., 2004). Malassezia species can be identified through their morphological features and biochemical characte- rization (Guillot et al., 1996). However, these pheno- typic methods are usually time consuming, lack sufficient discriminatory power, and are unable to unambiguously differentiate newly identified species. * Corresponding author. Tel.: +98 21 89 51 583; fax: +98 21 646 22 67. E-mail addresses: mirhendi@yahoo.com, mirhendi@sphtums.com (H. Mirhendi).