Epidemiologic Study of Malassezia Yeasts in Acne Patients by Analysis of 26S rDNA PCR-RFLP

BACKGROUND: Although acne is a common follicular inflammatory dermatosis, studies of the relationship between Malassezia yeasts and acne have rarely been conducted. OBJECTIVE: We sought to identify Malassezia yeasts from acne patients and establish a relationship between specific types of species of Malassezia and acne. METHODS: Sixty acne patients were enrolled. Each strain obtained was identified as one of eleven species by 26S rDNA PCR-RFLP. We then compared these data with those of age- and sex-matched healthy subjects. RESULTS: Growth of Malassezia was evident in fewer patients with acne (50%) in comparison to controls (70.6%). M. restricta was dominant in patients with acne (23.9%), whereas M. globosa was most common (26.7%) in healthy controls. In the patients group, the rate was the highest (71.7%) in the twenties and, in terms of body site, the rate was the highest (60%) in the chest. In the control group, the rate was the highest (75.0%) in the thirties and in the forehead (85.0%). CONCLUSION: The detection rate of Malassezia yeasts was conspicuously low in the acne patients group. Statistically significant differences were observed between the patient and the control groups in the twenties and thirties, and in terms of body site, in the forehead and chest.

Distribution of Malassezia Species on the Scalp in Korean Seborrheic Dermatitis Patients

BACKGROUND: Malassezia species play an important role in the pathogenesis of seborrheic dermatitis. In particular, M. restricta and M. globosa are considered to be the predominant organisms in seborrheic dermatitis of Western countries. However, species distribution of Malassezia in seborrheic dermatitis has not been clearly determined yet in Asia. OBJECTIVE: To identify the distribution of Malassezia species on the scalp of seborrheic dermatitis patients in Korea using 26S rDNA PCR-RFLP analysis. METHODS: A total of 40 seborrheic dermatitis patients and 100 normal healthy volunteers were included in this study. For the identification of Malassezia species, the scalp scales of the subjects were analyzed by 26S rDNA PCR-RFLP analysis. RESULTS: The most commonly identified Malassezia species were M. restricta in the seborrheic dermatitis patients, and M. globosa in the normal controls. In the seborrheic dermatitis group, M. restricta was identified in 47.5%, M. globosa in 27.5%, M. furfur in 7.5%, and M. sympodialis in 2.5% of patients. In the healthy control group, M. globosa was identified in 32.0%, M. restricta in 25.0%, M. furfur in 8.0%, M. obtusa in 6.0%, M. slooffiae in 6.0%, and M. sympodialis in 4.0% of subjects. CONCLUSION: M. restricta is considered to be the most important Malassezia species in Korean seborrheic dermatitis patients.

Epidemiologic Study of Malassezia Yeasts in Patients with Malassezia Folliculitis by 26S rDNA PCR-RFLP Analysis

BACKGROUND: So far, studies on the inter-relationship between Malassezia and Malassezia folliculitis have been rather scarce. OBJECTIVE: We sought to analyze the differences in body sites, gender and age groups, and to determine whether there is a relationship between certain types of Malassezia species and Malassezia folliculitis. METHODS: Specimens were taken from the forehead, cheek and chest of 60 patients with Malassezia folliculitis and from the normal skin of 60 age- and gender-matched healthy controls by 26S rDNA PCR-RFLP. RESULTS: M. restricta was dominant in the patients with Malassezia folliculitis (20.6%), while M. globosa was the most common species (26.7%) in the controls. The rate of identification was the highest in the teens for the patient group, whereas it was the highest in the thirties for the control group. M. globosa was the most predominant species on the chest with 13 cases (21.7%), and M. restricta was the most commonly identified species, with 17 (28.3%) and 12 (20%) cases on the forehead and cheek, respectively, for the patient group. CONCLUSION: Statistically significant differences were observed between the patient and control groups for the people in their teens and twenties, and in terms of the body site, on the forehead only.

Molecular Analysis of Malassezia Microflora on the Skin of the Patients with Atopic Dermatitis

BACKGROUND: The yeasts of the genus Malassezia are members of the normal flora on human skin and they are found in 75~80% of healthy adults. Since its association with various skin disorders have been known, there have been a growing number of reports that have implicated Malassezia yeast in atopic dermatitis (AD). OBJECTIVE: The aim of the present study is to isolate the various Malassezia species from AD patients by using 26S rDNA (ribosomal Deoxyribonucleic acid) PCR-RFLP and to investigate the relationship between a positive Malassezia culture and the severity of AD. METHODS: Cultures for Malassezia yeasts were taken from the scalp, cheek, chest, arm and thigh of 60 patients with atopic dermatitis. We used a rapid and accurate molecular biological method 26S rDNA PCR-RFLP, and this method can overcome the limits of the morphological and biochemical methods. RESULTS: Positive Malassezia growth was noted on 51.7% of the patients with atopic dermatitis by 26S rDNA PCR-RFLP analysis. The overall dominant species was M. sympodialis (16.3%). M. restricta was the most common species on the scalp (30.0%) and cheek (16.7%). M. sympodialis (28.3%) was the most common species on the chest. The positive culture rate was the highest for the 11~20 age group (59.0%) and the scalp showed the highest rate at 66.7%. There was no significant relationship between the Malassezia species and SCORing for Atopic Dermatitis (SCORAD). CONCLUSION: The fact that the cultured species was different for the atopic dermatitis lesion skin from that of the normal skin may be due to the disrupted skin barrier function and sensitization of the organism induced by scratching in the AD lesion-skin. But there was no relationship between the Malassezia type and the severity score. The severity score is thought to depend not on the type, but also on the quantity of the yeast.

Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP

BACKGROUND: This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age. OBJECTIVE: The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls. METHODS: 26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods. RESULTS: The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients. CONCLUSION: According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups.

The Investigation on the Distribution of Malassezia Yeasts on the Normal Korean Skin by 26S rDNA PCR-RFLP

BACKGROUND: Malassezia yeasts are normal flora of the skin that are discovered in 75~98% of health subjects, but since its association with various skin disorders have been known, many studies have been conducted in the distribution of the yeasts. OBJECTIVE: To isolate, identify, and classify Malassezia yeasts from the normal human skin of Koreans by using the rapid and accurate molecular biology method (26S rDNA PCR-RFLP) which overcome the limits of morphological and biochemical methods, and to gather a basic database that will show its relation to various skin diseases. METHODS: Malassezia yeasts were cultured from clinically healthy human skin using scrub-wash technique at five sites (forehead, cheek, chest, upper arm, and thigh) and swabbing technique at scalp in 160 participants comprised of 80 males and 80 females aged from 0 to 80. Identification of obtained strains were placed into the one of the 11 species by 26S rDNA PCR-RFLP. RESULTS: An overall positive culture rate was 62.4% (599/960). As shown in the experiment groups by their age, the positive culture rate was the highest (74.2%) in the age 21~30 and 31~40 (89/120). In the experiment groups by different body areas, the scalp showed the highest positive culture rate of 90% (144/160). On analysis of 26S rDNA PCR-RFLP, M. globosa was the most predominant species in the age 0~10 (32.8%), 11~20 (28.9%), 21~30 (32.3%). M. restricta was identified as predominant species in the age 41~50 (27.9%), 61~70 (31.5%) and 71~80 (24.0%). In the age 31~40 years, M. sympodialis was found to be the most common species (24.6%). According to body site, M. restricta was more frequently recovered in the scalp (56.8%), forehead (39.8%) and cheek (24.0%) and while M. globosa was more frequently recovered in the chest (36.8%). Higher positive culture rates of Malassezia yeasts were shown in male subjects than female counterparts in all body areas except scalp (p<0.05). Especially in this study, M. dermatis, newly isolated Malassezia species from atopic dermatitis patient in Japan, was isolated and identified in 19 cases (1.9%) in healthy subjects. CONCLUSION: The key is to recognize the existence of a difference in the type of Malassezia species in different ages as well as body areas, which reflects differing skin lipid levels in various ages and different body areas. Moreover, 26S rDNA PCR-RFLP analysis which was opted in this study could provide a sensitive and rapid identification system for Malassezia species, which may be applied to epidemiological surveys and clinical practice.

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

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.

A Mycological and Molecular Biological Study of Malassezia dermatis Isolated from Korean / 대한피부과학회지

BACKGROUND: Malassezia (M.) yeasts are lipophilic fungi which are regarded as normal flora of the skin, and are recovered in 75~98% of healthy adults. Gueho et al reclassified the Malassezia yeasts into 7 species (M. furfur, M. obtusa, M. globosa, M. slooffiae, M. sympodialis, M. pachydermatis, M. restricta) on the basis of molecular biology and by employing an interdisciplinary approach of morphology, microstructurology and physiology. Recently novel species of the genus Malassezia have been discovered as a result of molecular analysis. But there are no additional reports in Korea regarding newly reported Malassezia species because most identification and classification of Malassezia in Korea depend on classical methods and research on molecular biologic application is insufficient. OBJECTIVE: Five clinical isolates of M. dermatis were isolated from the skin of healthy subjects without skin disease or seborrheic dermatitis patients using molecular biology techniques for the first time in Korea. Hence the present study describes mycological and molecular biological characteristics of these five isolates as a novel species of M. dermatis. METHODS: Morphological and biochemical analyses, such as colony morphologies, microscopic morphologies and physiological characteristic were done targeting 5 clinical isolates of M. dermatis. Molecular techniques, namely, 26S rDNA PCR-RFLP, 26S rDNA and internal transcribed spacer region 1 (ITS1) sequencing, were done for identification and phylogenetic systematic analysis. RESULTS: Five clinical isolates of M. dermatis showed positive in the catalase test. No growth is obtained on Sabouraud's dextrose agar (SDA) without lipid supplementation but all grew in 0.5% Tween 60 and 0.1% Tween 80 added 2% glucose/1% peptone culture medium. Round and ellipsoidal yeast cells and budding of the yeast cells were observed under microscope, resembling M. sympodialis, M. furfur, and M. nana. The 26S rDNA PCR-RFLP pattern showed the same pattern as M. dermatis (JCM 11348), the standard strain. 26S rDNA and ITS1 sequencing were performed for exact identification, showing 99% accordance with M. dermatis (AB070361), and M. dermatis (AB070356), confirming the species to be new, the first to be reported in Korea. Phylogenetic trees based on the D1/D2 domains of the 26S rDNA sequences and nucleotide sequences of the ITS 1 region showed that the isolates were conspecific and belonged to the genus Malassezia and crusted with M. sympodialis. CONCLUSION: Taking a molecular biological classification approach, we have successfully isolated 5 cases of M. dermatis-the first in Korea. Although it is not known whether M. dermatis plays a role in Malassezia-related skin disease, this species was part of the microflora in both patients with seborrheic dermatitis and healthy subjects.

The Application of 26S rDNA PCR-RFLP in the Identification and Classification of Malassezia Yeast / 대한의진균학회지

BACKGROUND: Malassezia yeast are lipophilic fungi that are found in 75~80% of healthy adults. The yeast are known to be associated with pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and recently its pathogenicity is being expanded to other various skin disorders, such as atopic dermatitis and acne vulgaris. Up to present, mycological studies on Malassezia yeast have been carried out mostly through morphological analysis and biochemical analysis. Recently however, various molecular biological techniques are being preferred over morphological analysis, which is not a suitable method for establishing taxonomic relationship between species, and more or less time-consuming. OBJECTIVE: We sought to implement novel molecular biology technique, namely 26S rDNA PCRRFLP method in identifying and classifying Malassezia yeast, and assess its clinical applicability. METHODS: Eleven standard strains and eight clinical isolates were thoroughly examined with special attention to the shape of the colonies, size and change in media. Subsequently, the colonies were classified according to Gueho classification. For molecular analysis, RFLP analysis was carried out after DNA was isolated from each organism and 26S rDNA was amplified through PCR. The results of identification were confirmed by 26S rDNA sequencing. RESULTS: In PCR analysis to amplify the 26S rDNA, a 580bp PCR band was seen in all of eleven standard colonies. On analysis of PCR-RFLP of 26S rDNA using restriction enzymes Hha1 and BstF51, all of the database in the restriction pattern of each species was attained. On analyzing eight clinical isolates, a restriction pattern which was interspecifically distinguishable, was identified, and the result was in accord with the pattern obtained from 26S rDNA PCR-RFLP of standard colonies. Out of eight, seven clinical isolates colonies was in accord with the result of 26S rDNA PCR-RFLP. In order to assess the precision of 26S rDNA PCR-RFLP, 26S rDNA sequencing was performed, whose result was in accord with 26S rDNA PCR-RFLP analysis. CONCLUSION: As evidenced above, 26S rDNA PCR-RFLP analysis could provide a sensitive and rapid identification system for Malassezia species, which may be applied to epidemiological surveys and clinical practice