There were 60 healthy adults and 60 patients with SD (30 males, 30 females) who participated in this study. The patients were chosen among those who had visited the dermatology outpatient clinic at Konkuk University Medical Center, from July 2005 to June 2008. The healthy adults were grouped depending on their age (20 patients in each age group: 31~40 years, 41~50 and 51~60). Excluded patients included those who had been administered adrenocortical hormones, those who had undergone light therapy or antimycotic treatment within 2 months of the examination date, as well as those who had been treated with topical antifungals within 1 month and/or with topical corticosteroids within 1 week. All participants were told not to wash their face or use moisturizer (not to apply any products) on the examination day. According to the Declaration of Helsinki, the participants were informed of the physical and mental risks that could happen during the study, and written consents were obtained before their participation.

Specimens were gathered from forehead, cheek and chest, and were sampled by scrub-wash technique, based on the method that Williamson and Kligman suggested5,6. A stainless tube, which has an interior area of 4.909 cm², was set on the selected skin: forehead, cheek and chest, and then 1 ml of detergent7 (0.01% NaH₂PO₄H₂O₂, 1.01% Na₂HPO₄, 0.1% Triton X-100 [pH 7.9]) was put into the tube. After rubbing the skin by a glass rod for 1 minute, the sample was taken out by a pipette and stored in a different container. Then, 1 ml of the detergent was put into the stainless tube, and the specimen was repetitively sampled and added to the first sample. 100 µl of the sampled specimen was then mixed with 900 µl of the detergent with 50% concentration, and 100 µl was taken from the mixture, evenly applied on the Leeming-Notman medium, and cultured at 34℃ for 14 days8.

For DNA extraction and PCR analysis of skin isolates, this study adopted colony PCR analysis which was developed to extract DNA directly from a colony of a PCR tube and to amplify 26S rDNA at the same time instead of direct genomic DNA extraction methods. A colony of Malassezia yeast was taken and transferred to a PCR tube and warmed up 3 times a day for 1 minute, in a double boiler by using a microwave, and then the tube was moved into ice water. The PCR reaction mixture (0.25 mM deoxynucleoside triphosphate, 10X PCR buffer, 5X Q buffer, 0.5 µM primers, 1.25 U Hot StarTaq polymerase, 20 mM MgSO₄) was added and vortex mixing was done. Then, PCR, using Mastercycler 5333 (Eppendorf, Hamburg, Germany), was performed immediately. To amplify 26S rDNA, a primer that can amplify all 11 standard strains at once was chosen. The sequence was: forward, 5'-TAACAAGGATTCCCCTAGTA-3' and reverse, 5'-ATTACGCCAGCATCCTAAG-3'9. The conditions in the early stage of the reaction were at 95℃ for 14 minutes for predenaturation, at 94℃ for 45 seconds for denaturation, at 55℃ for 45 seconds for annealing, at 72℃ for 1 minute for extension of 40 cycles, and at 72℃ for 7 minutes for the last extension. Amplified DNA was visualized by electrophoresis on a 1.5% (w/v) agarose gel with Ethidium bromide (0.5 µg/ml) by using 1 X TAE migrating buffer (pH 8.0, 40 mM Tri-acetate 1 mM EDTA).

After checking the amplified 26S rDNA, the PCR product was purified using the Accu-Prep PCR purification kit (Bioneer, Daejeon, Korea). For the 26S rDNA RFLP analysis of Malassezia yeasts, 2 restriction enzymes were used: Hha I (Takara Biomedicals, Otsu, Japan) and BtsC I (SibEnzyme, Novosibirsk, Russia). The restriction enzyme reaction was conditioned with 10X PCR buffer, 10 U restriction enzyme, and reaction mixture including 7.5 µl of the PCR product to become 20 µl. After 3 hours of reaction at 37℃, electrophoresis was performed in the TAE buffer with 3.5% (w/v) NuSieve GTG agarose gel (FMC, Rockland, ME, USA) by 100 volts. Then, it was stained with ethidium bromide, and the size and number of DNA fragment were checked by a UV transilluminator to analyze RFLP patterns10.

Chi-squared tests (SPSS) were used to find the difference in the distribution of the Malassezia species between patients with SD and healthy adults from equivalent age groups. The results are considered to be statistically significant when the p-value is less than 0.05.

Specimens were collected from 3 body sites: forehead, cheek, and chest, in 60 SD patients and 60 healthy adults. Malassezia yeasts of the patient group were cultured in 93 out of 180 specimens, and thus the identification rate was 51.7%. Five Malassezia species (M. sympodialis, M. restricta, M. furfur, M. globosa, M. dermatis) were identified in the group of SD patients (Fig. 1, 2). The healthy control group, on the other hand, showed 7 Malassezia species (M. globosa, M. restricta, M. sympodialis, M. furfur, M. dermatis, M. slooffiae, M. obtusa), and the identification rate was 63.9% (115/180) (Table 1, 2). Within age groups, a high identification rate was shown in of the 30s (31~40 years) in both patient and healthy adult groups: 66.7% (40/60) in the patient group, 71.7% (43/60) in the healthy adult group. Within body parts, in both patient and healthy adult groups, a high identification rate was shown in the forehead: 56.7% (34/60) in the patient group and 66.7% (40/60) in the healthy adult group.

Identifying and classifying the Malassezia yeasts cultured on each body part according to ages, in the patients in their 30s and 40s, M. restricta (30s: 31.6%, 40s: 16.6%) was identified as the major species, and in their 50s, M. sympodialis (20.0%) was identified as the major species. In the healthy adult groups, M. sympodialis (25.0%), M. restricta (21.6%) and M. globosa (21.7%) were identified to be predominant in the 30s, 40s, and 50s, respectively (Table 1). Comparing the difference in the distribution of the Malassezia species between the patient and control groups by equivalent ages, there was no statistically significant difference (Fig. 3).

Identifying the Malassezia yeasts cultured from patients' body parts, in whole parts M. restricta was identified more than other species (19.5%, 35 cases). Both in the experiment and control groups by different body parts, M. restricta was identified frequently in the forehead and cheek. In the chest, on the other hand, M. sympodialis (16.7%) and M. globosa (26.7%) were identified as common species in both the patient and healthy adult groups, respectively (Table 2). The results showed no significant difference in the distribution of the Malassezia species in different body parts in either the patient or healthy control groups (Fig. 4).