Direct molecular analysis of Malassezia species from the clinical samples of patients with pityriasis versicolor.

Abstract

Species identification of Malassezia using culture-dependent methods is time-consuming due to their fastidious growth requirements. This study aimed to evaluate a rapid and accurate molecular method in order to diagnose the pityriasis versicolor (PV) and identify Malassezia species from direct clinical samples. Skin scraping or tape samples from patients with PV and healthy volunteers as the control group were collected. Diagnosis of PV was confirmed by direct microscopic examination. The DNA extraction was performed according to the steel-bullet beating method. Polymerase chain reaction-restriction fragment length polymorphism assay using HhaI restriction enzyme was applied for the identification and differentiation of Malassezia species. The PCR method was able to detect Malassezia in 92.1% of specimens which were also confirmed with microscopic examination. Statistically, a significant association was observed between the results of the two assays (P < 0.001). Moderate agreement was identified between the two methods to diagnose the PV in both populations (Kappa: 0.55). Considering microscopic examination as the gold standard method for confirmation of PV, the sensitivity, specificity, positive predictive value, and negative predictive value values of the PCR assay for recognition of PV were 85%, 75%, 92%, and 60%, respectively. M. globosa and M. restricta were the most prevalent species isolated from patients. In this study, the two-step molecular method based on the amplification of the D1/D2 domain and digestion of the PCR product by one restriction enzyme was able to diagnose and identify Malassezia directly from clinical samples. Consequently, it can be said that the molecular-based method provides more facilities to identify fastidious species, such as M. restricta.