Normal subjects included 110 healthy volunteers (60 males, 50 females); 0~80 years of age without any dermatoses of the examined regions. Sterile cotton swabs were moistened with wash fluid containing 0.1% Triton X-100 in 0.075 M phosphate buffer (pH 7.9), and rubbed gently, with rotation on the skin. Swabbing was performed for 10 sec on sites including: the scalp, forehead, cheek, chest, and thighs. The swabs were immediately placed in Leeming and Notman agar media. The investigations were conducted according to the Declaration of Helsinki Principles. Written informed consent was obtained from each subject before the procedure.

The yeast was cultured on agar plates with Leeming and Notman agar (LNA) - glycerol monoesterate (BDH, Poole, UK) 0.5 g, bacteriological peptone (Oxoid, Hampshire, UK) 20 g, glucose (Oxoid, Hampshire, UK) 5 g, yeast extract (Oxoid, Hampshire, UK) 0.1 g, ox bile (Merck, Darmstadt, Germany) 4 g, agar No.1 (Oxoid, Hampshire, UK) 12 g, Tween 60 (Yakuri, Osaka, Japan) 0.5 ml, glycerol (Tedia, Fairfield, USA) 1 ml, 10 ml of whole-fat cow's milk per liter that contained cycloheximide (Sigma, St Louis, MO, USA) 200 mg, and chloramphenicol (Sigma, St Louis, MO, USA) 50 mg. The plates were incubated at 34℃ for 14 days.

The yeasts grown in agar were harvested and resuspended in 0.4 ml of lysis buffer (100 mM Tris-HCl pH 8.0, 1.0% SDS, 2.0% Triton X-100, 10 mM EDTA, 100 mM NaCl). Equal volumes of phenol/chloroform/isoamyl alcohol (phenol: chloroform: isoamyl alcohol=25:24:1, v/v) and glass beads (0.5 mm) were added, and the mixture was vortexed for 10 min. The samples were centrifuged at 12,000×g for 15 min. Total DNA was precipitated from the aqueous fraction with isopropanol and centrifuged at 12,000×g for 20 min at 4℃. The DNA pellet was washed in 70% ethanol and resuspended in sterile water.

To amplify 26S rDNA from genomic DNA, the reaction mixture contained 25 mM of each dNTP, 10X PCR buffer, 5X Q buffer, 0.5 µM primer, 0.4 µm forward primer (5'-TAACAAGGATTCCCCTAGTA-3'), reverse primer (5'-ATTACGCCAGCATCCTAAG-3'), and 1.25 U Hot StarTaq polymerase in 50 µl reaction volume. Thirty five cycles with the following protocol were programmed: denaturation for 45 sec at 94℃; annealing for 45 sec 55℃; extension for 1 min at 72℃. After the confirmation of amplified 26S rDNA, the PCR products were purified using an Accu-Prep PCR purification kit (Bioneer, Daejeon, Korea). The absorbance at 260 nm and the 260/280 ratio (as a measure of DNA quality) were obtained for each purified PCR product sample using the NanoDrop spectrophotometer ND-1000 (NanoDrop Technologies) and the estimated concentration of DNA (ng/µl). The 26S rDNA was confirmed by running 50 ng of 26S rDNA on a 2% agarose gel. Two restriction enzymes, Hha1 (Takara Biomedicals, Otsu, Japan) and BstF51 (SibEnzyme, Novosibirsk, Russia) were used to perform the 26S rDNA-RFLP study of Malassezia. In this experiment, the restriction enzyme digestion was performed with 10 X PCR buffer, 10 U of the restriction enzyme, and PCR products 7.5 µl, of each sample was examined, from which the maximum expected concentration was 50 ng/µl (1 µg DNA in 20 µl recovery solution), which added up to 20 µl. After the reaction at 37℃ for 3 hours, the electrophoresis was performed with a 3.5% (w/v) NuSieve GTG agarose gel (FMC, Rockland, ME, USA) with 100 volts and stained with ethidium bromide. The restriction fragments were analyzed by the size and number of the DNA fragments under UV transillumination (Fig. 1).

To amplify rDNA from genomic DNA, a nested PCR was carried out using primers for Malassezia. The first amplification mix was carried out in a 20 µl reaction consisting of 2× pre mix (2 µl 10× buffer, 1.6 µl of 25 mM MgCl2, 0.25 µl of 10 mM dNTPs, nTaq-Hot DNA polymerase -Enzynomics-Korea), 1 µl of 2.5 µM primer and 10 ng of purified DNA. Primer pairs selecting for the Malassezia nana genes for ITS1, 5.8S rRNA, ITS2 are shown after an initial 5 min denaturation at 95℃, 20 cycles of 95℃ for 30 sec, 53.5℃ for 30 sec and 72℃ for 1 min were carried out, followed by a 7 min extension at 72℃ using a thermal cycler (Gene Amp PCR System 2400-Applied Biosystem, Monza, Italy). The reaction mixture for the second amplification round was the same as for the first, except for the "inner" primers [forward primer (5-GTCTCTGGCGCCTAACCCACTATA -3), reverse primer (5-TCCACGTACATACAACCATACGA-3)] were used instead of the "outer" primers [forward primer (5-GCCATACGGACGATAA-3), reverse primer (5-AGCAAATGACGTATCATGCCATGC-3)]. For the second amplification round 49 µl of amplification mix and 1 µl of the first amplification round PCR product were used. The thermal cycling was repeated as for the first amplification round using 30 cycles after the initial 2 min denaturation. Each amplification run contained a negative control, consisting of water and a positive control. Analysis of the PCR products was performed by 2% agarose gel electrophoresis followed by visualization with ethidium bromide (0.5 µg/ml) staining and UV illumination to confirm the expected products (Fig. 1).

Among the cultured samples of Malassezia, we first identified the species of the Malassezia yeasts using a 26S rDNA RFLP, and based on these findings, we conducted the nested PCR and compared the results of the 26S rDNA RFLP and nested PCR.

The overall positive culture rate of the Malassezia yeasts samples from different body sites of 110 persons was 63.6%, with 350 positive samples out of 550 samples.

Using 26S rDNA PCR-RFLP analysis, we successfully identified 11 species of Malassezia yeasts from the standard Malassezia species (Fig. 2). In addition, we identified six species of Malassezia yeasts from 350 positive samples. The results showed that M. globosa was identified most frequently in 116 samples (33.1%); M. restricta in 106 samples (30.3%); M. sympodialis in 55 samples (15.6%); M. furfur in 39 samples (11.1%); M. slooffiae in 5 samples (1.4%), and M. dermatitis in 6 samples (1.6%). As for the 23 samples with co-identification of more than two Malassezia species, they were excluded from the analysis for a more direct comparison between the RFLP and nested PCR methods (Table 1).

Using nested PCR analysis, we successfully identified 11 species of Malassezia yeasts from the standard Malassezia species (Fig. 3). In addition, 6 species of Malassezia yeasts were identified from 327 positive samples that were already confirmed by RFLP methods. These results showed that M. globosa was identified most frequently in 111 samples (31.7%); M. restricta in 105 samples (30.0%); M. sympodialis in 54 samples (15.4%); M. furfur in 39 samples (11.1%); M. slooffiae in 5 samples (1.4%), and M. dermatitis in 6 samples (1.7%) (Table 1).

The results of re-identifying and classifying Malassezia species using nested PCR among the 327 positive samples, where cases with two or more yeasts identified were excluded, showed that 320 out of 327 (98.8%) samples were identical with the results of the RFLP method. In cases with M. globosa, 111 out of 116 samples were identical, showing a 95.6% concordance rate, and among the 5 non-identical samples, 3 were later identified as M. obtusa (131 bp) and 2 as M. nana (114 bp) by the nested PCR. In addition, 105 out of 106 M. restricta samples identified by the RFLP method were identical, and the other 1 case was confirmed as M. obtusa (131 bp) by the nested PCR. As for M. sympodialis, 54 out of 55 samples were identical, and the other 1 case was confirmed to be M. nana (114 bp) by the nested PCR (Table 1).