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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Distribution of Malassezia species in pityriasis versicolor and seborrhoeic dermatitis in Greece. Typing of the major pityriasis versicolor isolate M. globosa.

BACKGROUND: The expansion of the genus Malassezia has generated interest in the epidemiological investigation of the distribution of new species in a range of dermatoses, on which variable results have been reported from different geographical regions. No data are thus far available from South-east Europe (Greece). OBJECTIVES: To study the distribution of Malassezia species in pityriasis versicolor (PV) and seborrhoeic dermatitis (SD) and to investigate whether polymorphisms in the internal transcribed spacer (ITS) 1 region facilitate detection of M. globosa and M. sympodialis subtypes. METHODS: In total, 109 patients with PV and SD and positive Malassezia cultures were included in the study. Age, gender, primary/recurrent episode, disease extent and clinical form of PV were recorded. ITS 1 polymorphisms of M. globosa and M. sympodialis type and clinical strains were investigated by polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) analysis. RESULTS: Malassezia globosa was the prevalent species isolated from PV and SD either alone (77% and 39%, respectively) or in combination (13% and 18%, respectively) with other Malassezia species. The pigmented form of PV was strongly correlated with the female gender. PCR-SSCP differentiated five subgroups of M. globosa with one being associated with extensive clinical disease. All M. sympodialis isolates displayed a homogeneous ITS 1 PCR-SSCP profile. CONCLUSIONS: Malassezia species isolation rates were in agreement with those reported from South-west Europe. PCR-SSCP of the ITS 1 is useful for highlighting prospective clinical implications of M. globosa subtypes.