Molecular Biological Identification of Malassezia Yeasts Using Pyrosequencing

BACKGROUND: A Pyrosequencing assay has been used in identification of fungal species such as Candida or Aspergillus and diagnosis of pathogenic bacteria such as Helicobacter pylori but there has been no report on successful isolation and identification of Malassezia yeasts using the pyrosequencing method. OBJECTIVE: Examine the applicability and plausibility of the pyrosequencing method in identification of the Malassezia species. METHODS: At internal transcribed spacer (ITS) sites 1 and 2, three primers were developed using Pyrosequencing Assay Design Software (Biotage AB). Pyrosequencing was performed on 11 standard strains and 83 genomic DNA samples obtained from 66 healthy controls aged from 1 to 80. RESULTS: The eleven Malassezia standard species and 83 genomic DNA samples were successfully identified using the pyrosequencing assay. CONCLUSION: The pyrosequencing method is a new tool for analysis of Malassezia yeasts, and its precision and rapidity suggests its clinical applicability.

Molecular Biological Identification of Malassezia Yeasts Using Pyrosequencing

BACKGROUND: A Pyrosequencing assay has been used in identification of fungal species such as Candida or Aspergillus and diagnosis of pathogenic bacteria such as Helicobacter pylori but there has been no report on successful isolation and identification of Malassezia yeasts using the pyrosequencing method. OBJECTIVE: Examine the applicability and plausibility of the pyrosequencing method in identification of the Malassezia species. METHODS: At internal transcribed spacer (ITS) sites 1 and 2, three primers were developed using Pyrosequencing Assay Design Software (Biotage AB). Pyrosequencing was performed on 11 standard strains and 83 genomic DNA samples obtained from 66 healthy controls aged from 1 to 80. RESULTS: The eleven Malassezia standard species and 83 genomic DNA samples were successfully identified using the pyrosequencing assay. CONCLUSION: The pyrosequencing method is a new tool for analysis of Malassezia yeasts, and its precision and rapidity suggests its clinical applicability.

A Comparison Study between Culture Based Technique and Op-site Non-Culture Based Technique for Identifying Malassezia Yeasts on Normal Skin / 대한의진균학회지

BACKGROUND: Culture based technique, a traditional method for extraction of DNA from a cultured colony, was complex in culture conditions and was associated with a lower chance of successful culture. Recently, non-culture based technique, which skipped the culture process and directly extracted fungal DNA and differentiated Malassezia species, has been introduced. OBJECTIVE: Using 26S rDNA PCR-RFLP, the authors identified Malassezia yeasts and compared the yield of Malassezia DNA by the traditional culture based technique and the non-culture based technique via Op-site adhesive tape. METHODS: DNA of Malassezia yeasts were extracted using the culture based technique and the non-culture based technique from normal adults. Comparison was performed in order to clarify the differences between these two techniques. RESULTS: Use of the culture based technique resulted in a culture rate of 57.8% (78 out of 135 samples). On the other hand, using the non-culture based technique, fungal species were identified from all 135 samples. Using both techniques, M. globosa was the most identified species. The identification rate of the non-culture based technique was 100%; however, 7 repeats of PCR were required to reach 100% identification. Among samples from five body sites, those from the thigh required 5.5 repeats of PCR. CONCLUSION: The non-culture based technique was better than the culture based technique. However, due to the low amount of DNA extracts from the body sites with low habitation of Malassezia yeasts, repeated PCR was required for differentiation of Malassezia species.

Skin Characteristics in Patients with Pityriasis Versicolor Using Non-Invasive Method, MPA5

BACKGROUND: Skin pigmentary changes of pityriasis versicolor may occur as either hyperpigmented or hypopigmented lesions, depending on the outcome of interactions between Malassezia yeasts and the skin, such as lipoperoxidation process, stimulus of inflammatory cell to melanocytes, and increased thickness of keratin layer. OBJECTIVE: To investigate skin characteristic factors that enhance the susceptibility to Malassezia yeasts and provoke different color changes of pityriasis versicolor patients. METHODS: To clarify these factors, we investigated the skin characteristics of pityriasis versicolor patients, using a non-invasive method known as MPA 5(R) (Courage and Khazaka, Germany). A total of 90 normal healthy subjects and 30 pityriasis versicolor patients were included in this study. RESULTS: Both hyperpigmented and hypopigmented pityriasis versicolor skin lesions showed higher humidity, increased sebum excretion rate and increased transepidermal water loss (TEWL) values than normal healthy subjects. But no significant difference of specific Malassezia yeasts species between hyperpigmented and hypopigmented skin lesions was evident. CONCLUSION: These results indicate that higher humidity and increased sebum level provide a better growing environment of Malassezia yeasts in the skin, leading to the assumption that interaction between Malassezia yeasts and skin barrier materials makes disruption of skin barrier causing increased TEWL.

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.

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.

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.

Therapeutic Effect of Itraconazole in Seborrheic Dermatitis / 대한의진균학회지

BACKGROUND: Seborrheic dermatitis is a very common chronic inflammatory disease. The causal factor of the disease is still unknown, but early investigators focused on the role of Malassezia yeasts. These yeasts are also normal skin commensals, thus their importance as pathogens in this disorder came to be doubted. However, it was subsequently found that treatment of seborrheic dermatitis with an antifungal agent not only resulted in clinical improvement but also reduced the number of Malassezia yeasts on the skin. OBJECT: The purpose of this study is to confirm relationship between seborrheric dermatits and Malassezia yeast, and to evaluate the therapeutic efficacy of oral itraconazole in the seborrheic dermatitis. METHODS: Using the scrub-wash technique in the glabella and swabbing technique in the scalp, the number of cultured Malassezia yeasts were counted in 30 patients with seborrheic dermatitis and 20 control persons. The patients took itraconazole, 100mg/day, during 4weeks. The clinical and mycologic score were measured at the initial evaluation, followed after 2weeks and 4weeks RESULT: The number of Malassezia yeasts in patient with seborrheic dermatits were significantly higher than in normal control group. There was statistically significant decrease in the clinical and mycological score after a 4 week trial of oral itraconazole in the seborrheic dermatitis group. CONCLUSION: This study indicates that Malassezia yeast may be one of the important causative factor of seborrheic dermatitis and itraconazole plays an important role in the treatment of seborrheic dermatitis

The Distribution of Malassezia Yeasts on Normal Human Skin by Culture Study Using the Scrub-wash Techique / 대한피부과학회지

BACKGROUND: Cutaneous vasculitis associated with viral hepatitis seems to occur as a hypersensitivity reaction against the circulating viral antigens. Hepatitis B virus(HBV)-encoded X antigen(HBxAg) is known to participate in the carcinogenesis of hepatocellular carcinoma(HCC) by the inactivation of p53. However, HBxAg has been found in chronic infiammatory lesions without the overexpression of p53. Accordingly, not only EBsAg and HBcAg but also HBxAg may be involved in HCC-associated cutaneous vasculitis, regardless of the alteration of p53. OBJECTIVE: This study was conducted to investigate the expression of HBV-encoded antigens in cutaneous vasculitis accompanied by HBV hepatopathy. Additionally, we have compared the expression of 3 HBV antigens and p53 between vasculitic patients with HCC and in others showing HCC-non-associated vasculitis. METHODS: Immunohistochemically, we examined the expression of HBsAg, HBcAg, and HBxAg in the tissue specimens taken from the vasculitic lesions of the 33 HBsAg-positive enrolled patients with cutaneous vasculitis proven by skin biopsy. RESULTS: 1. The immunohistochemical positivity rate to HBsAg in vasculitic patients with HBV hepatopathy was 66.7% overall. It was 90% in HCC-associated vasculitic subjects and 56.5% in the vasculitic subjects without HCC, respectively. 2. We found the expression of HBxAg in 80% of the vasculitic subjects showing HCC. The vasculitic patients without HCC showed 17,3% of the positivity rate to HBxAg. 3. We could not find the overexpression of p53 in the vasculitic tissue specimens of the HCC patients without the cutaneous metastasis from primary HCC. CONCLUSION: HBsAg, HBcAg and HBxAg may participate in the pathogenesis of cutaneous vasculitis with HBV hepatopathy, regardless of tumorigenesis.

The Distibution of Malassezia Yeasts on Normal Human Skin by Culture study Using the Swabbing Technique / 대한피부과학회지

BACKGROUND: The distribution of Malassezia yeasts on normal human skin was varied according to the age and race of the volunteers and the methodologies used. In Korea, most reports of Malassezia yeast distribution have relied on direct skin smears rather than culture methods. OBJECTIVE: The aim of this work was to perform a comprehensive survey of the distribution of Malassezia yeasts on normal human skin to provide a base line for a companion study of Malassezia yeasts in patients with various dermatoses. METHODS: Malassezia yeasts were cultured using the swabbing technique from the scalp, forehead, chest, upper back, upper arm and upper thigh in 137 subjects, infancy to 80 years of age. RESULTS: Malassezia yeasts were present in the lowest incidence(0-30%) on six sites of infants and present in 60.0-66.7% on the sebum-rich sites (scalp, forehead, chest, upper back) of children aged 1-9. Malassezia yeasts were present in 80.0-86.7% on the sebum-rich sites of the elderly group(over 60 years of age), about the same frequency as in the middle-aged groups. The population density of these organisms was significantly higher on the upper back than on the forehead, chest, upper arm and upper thigh in all age groups except the infant group and the group aged 1 to 9 (p<0.05). There were no regular quantitative variations in the distribution of Malassezia yeasts on a given site between age groups. On Leeming and Notman media, besides three morphotypes of Malassezia yeasts reported by Cunningham et al(1990), one additional type was identified. CONCLUSION: The results showed regional variations in the distribution of Malassezia yeasts in all ages except infancy and no regular age variations on a given site. Additionally, four colony types of Malassezia yeasts were found. The findings of our study coold help to investigate the role of Malassezia yeasts in related disorders.

The Isolation of Malassezia Yeasts in the Comedones of Acne Vulgaris / 대한의진균학회지

BACKGROUND: Both Malassezia yeast and Propionibacterium acnes form part of the normal flora of the human skin and hair follicles. The former is the etiological agent of Malassezia(Pityrosporum) folliculitis and the latter is one of the major factors in the pathogenesis of acne vulgaris. These two follicular diseases can coexist on a certain area of the skin, but there have been few reports about their coexistence in the literature. OBJECTIVE: The aim of this work was to investigate the distribution of Malassezia yeasts in the comedones of patients clinically diagnosed as acne vulgaris for elucidation of the coexistence of the two diseases, and for information on the predominance of the colonized Malassezia species and on relationship between certain species and Malassezia folliculitis. METHODS: The spore load in the comedonal plugs of 32 patients with acne vulgaris was graded using direct microscopy of KOH/Parker ink mounts. The comedonal specimens were cultured on Looming & Notman's media and the isolated Malassezia yeasts were identified to species level by their colony morphologies, microscopic morphologies and physiological characteristics. RESULTS: On direct microscopy, 8 of 32 patients (25%) showed a 4+ spore load, which is considered as a diagnostic grading index of Malassezia folliculitis. The predominant Malassezia(M.) species from 32 patients with acne vulgaris were M. restricta, M. globosa, M. furfur in descending order. Three strains of M. restricta, 4 strains of M, globosa, 2 strains of M. furfur and 1 strain of M. obtusa were isolated from the comedones of the 8 patients with 4+ spore load. CONCLUSION: This study shows that Malassezia folliculitis might coexist with acne vulgaris on the face, but there was no relationship between certain species and Malassezia folliculitis. The results suggest that antibiotic resistant acne vulgaris should be examined by direct microscopy of KOH/Parker ink mounts to confirm the coexistence of Malassezia folliculitis and acne vulgaris.

The Distribution of Malassezia Yeasts on the Skin of Neonates and Infants / 대한의진균학회지

BACKGROUND: Malassezia yeasts are normal skin flora of humans. But skin colonization appear to be controversial during neonate. OBJECTIVE: We prospectively studied the distribution of Malassezia yeasts on clinically normal skin of neonates and infants for providing the basic data for proving the relationship of Malassezia yeasts and pathogenesis of the diseases of neonates and infants. METHODS: A total of 200 subjects were studied using the direct smear test with 20% Parker ink/KOH solution. The numbers of the Malassezia yeasts per high power field were counted according to a bacterial index of lepra bacilli in patients of leprosy. In order to identify risk factors for the distribution of Malassezia yeasts, we compared sex, mode of delivery, gestational age, birthweight during the first week of life by statistical method of the logistic regression. RESULTS: Of the 200 neonates and infants under 12 weeks, 121 (60.5%) revealed Malassezia yeasts in at least a part of five examined sites. The prevalence of Malassezia yeasts was increased according to the age. No association was found between the incidence of Malassezia yeasts and sex, mode of delivery, gestational age, birth weight. CONCLUSION: We conclude that Malassezia yeasts colonize on the skin of neonates and infants.