Identification of Malassezia species from patient skin scales by PCR-RFLP

Abstract

This study was aimed at the development of a DNA-based procedure directly applicable to pathological skin scales and at the assessment of its value in rapid laboratory confirmation and identification of each of the seven Malassezia species. These lipophilic basidiomycetous yeasts in predisposed individuals are involved in pityriasis versicolor, seborrheic dermatitis, blepharitis, folliculitis, atopic dermatitis and fungemia. Standard identification procedures to species level are available, but so far no system for direct detection and characterization of Malassezia species in clinical specimens is available. Malassezia DNA was extracted from pathological skin scales by a modified hexadecyltrimethylammonium bromide (CTAB) method and amplified by single and nested polymerase chain reaction (PCR), assays using the general fungal ITS 1/4 and 3/4 primers for amplification of sequences from the Malassezia major ribosomal DNA complex. Restriction fragment length polymorphism (RFLP) analysis of PCR products was used in subsequent species identification. DNA extracted from culture-positive skin scales was also tested by PCR and the RFLP patterns obtained were analyzed. A total of 36 isolates were tested. Distinct pure culture and skin-scale ITS 3/4 HinfI and AluI restriction patterns differentially identified M. furfur, M. globosa, M. restricta, M. sympodialis, M. pachydermatis, M. obtusa and M. slooffiae. Malassezia DNA was extracted from pathological skin scales and RFLP identified solitary and multiple Malassezia species in the same specimen. Molecular identification was confirmed by cultures and biochemical tests. Concurrent detection and identification of Candida and Yarrowia species was also feasible from skin scales. The proposed method, described for the first time, could provide a sensitive and rapid detection and identification system for Malassezia species, which may be applied to epidemiological surveys and routine practice.