Nested PCR for Detection of Malassezia Species from Patient Skin Scales and Clinical Strains

Lim, Se Woong; Shin, Myung Geun; Lim, Ju Young; Yun, Sook Jung; Kim, Seong Jin; Lee, Seung Chul; Won, Young Ho; Lee, Jee Bum

Abstract

BACKGROUND: Due to recent identification of new Malassezia (M.) species, M. dermatis, and M. equi, the genus Malassezia was revised into eleven species that have been isolated from human and animal skin. This has further substantiated the need for molecular techniques to distinguish the various Malassezia species.
OBJECTIVE
We aimed to make the nested polymerase chain reaction (PCR) using species-specific primers with specificity and sensitivity as a diagnostic tool for differentiating the various Malassezia species from skin scales and fungal cells rapidly and accurately. In addition, we evaluated the common causative Malassezia species in the patients with seborreheic dermatitis, pityriasis versicolor or pityrisporum folliculitis.
METHODS
Malassezia species-specific primers were designed based on DNA sequencing of the ribosomal RNA gene. The standard strains of eight members of the genus Malassezia such M. pachydermatis, M. furfur, M. sympodialis, M. globosa, M. obtuse, M. restricta, M. slooffiae, and M. dermatis were used for positive control. Each Malassezia species was cultured separately and two or three standard species were cultured together on Modified Leeming and Notman agar (MLNA) media plates. In addition, twenty-five clinical strains of Malassezia species isolated from the skin of patients with dermatological conditions and twenty-three samples of skin scale were used as well.
RESULTS
The nested PCR assay with primers for all eight Malassezia species were species-specific since it amplified DNA only from the target Malassezia species, and could differentiate mixed, that is, the two or three Malassezia species of all standard strains grown on MLNA medium precisely. Detection of Malassezia species from clinical strains and patient skin scales using the nested PCR assay was 96% (24/25) and 87% (20/23), respectively. M. globosa, M. sympodialis, M. restricta were the most common causative Malassezia species in patients with pityriasis versicolor, pityrosporum folliculitis, seborrheic dermatitis, respectively.
CONCLUSION
Nested PCR using species-specific primers is useful and reliable in the detection of various Malassezia species from patient skin scales as well as cultured fungal cells.