Comparison of Nested PCR and RFLP for Identification and Classification of Malassezia Yeasts from Healthy Human Skin

Abstract

Background: Malassezia yeasts are normal flora of the skin found in 75∼98% of healthy subjects. The accurate identification of the Malassezia species is important for determining the pathogenesis of the Malassezia yeasts with regard to various skin diseases such as Malassezia folliculitis, seborrheic dermatitis, and atopic dermatitis. Objective: This research was conducted to determine a more accurate and rapid molecular test for the identification and classification of Malassezia yeasts. Methods: We compared the accuracy and efficacy of restriction fragment length polymorphism (RFLP) and the nested polymerase chain reaction (PCR) for the identification of Malassezia yeasts. Results: Although both methods demonstrated rapid and reliable results with regard to identification, the nested PCR method was faster. However, 7 different Malassezia species (1.2%) were identified by the nested PCR compared to the RFLP method. Conclusion: Our results show that RFLP method was relatively more accurate and reliable for the detection of various Malassezia species compared to the nested PCR. But, in the aspect of simplicity and time saving, the latter method has its own advantages. In addition, the 26S rDNA, which was targeted in this study, contains highly conserved base sequences and enough sequence variation for inter-species identification of Malassezia yeasts. (Ann Dermatol 21(4) 352

A study on malassezia micro flora in the skin of healthy individuals in North Kerala, India

“…The highest detection rates of M. globosa is in agreement with studies from Iran Korea and Spain and stands in contrast with the other studies from India Canada and Russia in which they reported M. sympodialis as the commonest species, Bosnia and Herzegovina as M. restricta and Iran as M. furfur from normal healthy skin. 15,14,25,19,12,16,18 The differences between this study and others may not only be due to the result of variation in species on the skin of individuals in different countries, but also by the difference in sampling methods i.e. ; swabbing, a relatively insensitive method, scraping or adhesive transparent tape method, difference in culture media used (SDA with olive oil, mDA, LNA) and difference in incubation temperature of inoculated culture media.…”

“…This is in agreement with the studies done in normal healthy Korean subjects at 62.4% and 63.6%. 13,14 An Iranian study showed similar rates at 62%. 15 Higher rate of Malassezia colonization compared with the present study was also noticed in studies done on healthy human skin in Bosnia and Herzegovina (65%) and Sweden (84%).…”

“…20 Mixed cultures were also observed in a couple of studies from normal healthy Korean subjects based on molecular based studies. 14,13 However, diverse sites yielded different species.…”

“…A total of 120 apparently healthy men and 120 women without any skin diseases or who were not treated for Malassezia associated skin diseases were selected for the study. They were grouped according to their age as (0-10), (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), (21)(22)(23)(24)(25)(26)(27)(28)(29)(30), (31-40), (41-50), (> 51) where each group consisted of 40 subjects (20 men and 20 women). The specimens consisted of scrapings collected from chest, back and neck from each subject totaling 720 sites.…”

Detection of Malassezia Species Isolated From Patients With Pityriasis Versicolor and Seborrheic Dermatitis Using Nested-PCR

“…Therefore, for successful treatment, identifying the disease-causing agent is necessary. Methods for identifying these species include culture-dependent methods, such as physiological methods, and molecular methods (2,(10)(11)(12).…”