Molecular analysis of Malassezia microflora in the lesional skin of psoriasis patients

Systemic and focal infections by microorganisms have been known to induce or exacerbate psoriasis. To investigate the role of Malassezia species in the development of psoriasis, we analyzed the Malassezia microflora in psoriasis patients using a nested polymerase chain reaction (PCR) assay, and compared it with those in atopic dermatitis (AD) patients and healthy subjects. Fungal DNA was directly collected from the lesional and non-lesional skin of the trunk of 22 psoriasis patients by applying a transparent dressing. The extracted DNA was amplified by using specific primers designed for the PCR in the intergenic spacer or internal transcribed spacer area of the ribosomal RNA. All nine of the Malassezia species were detected at different rates from the 22 psoriasis patients. The overall detection rates in lesional and non-lesional skin of M. restricta, M. globosa and M. sympodialis were high (96%, 82% and 64%, respectively), whereas the detection rates of the other species were relatively low. However, there was no difference in the rates between lesional and non-lesional skin areas. The average number of Malassezia species detected in overall sites of the psoriasis patients was 3.7 +/- 1.6 species, although this fact showed no correlation with the severity of the symptoms. The number of Malassezia species detected was 4.1 +/- 1.9 in the AD patients, and 2.8 +/- 0.8 in the healthy subjects, suggesting that the skin microflora of psoriasis patients and AD patients show greater diversity than that of healthy subjects.     

Quantitative analysis of Malassezia in the scale of patients with psoriasis using a real-time polymerase chain reaction assay

BACKGROUND: It has been suggested that Malassezia is associated with the development of skin lesions in psoriasis because of the response of the scalp lesions in psoriasis to antifungal agents. Malassezia restricta and M. globosa are the two major members of the cutaneous Malassezia flora in patients with psoriasis, although they have not been analysed quantitatively. OBJECTIVES: This study quantified the two major cutaneous Malassezia species in psoriatic scale from different body sites using a real-time polymerase chain reaction (PCR) assay. METHODS: Scale samples were collected from lesional and nonlesional skin of 20 Japanese patients with psoriasis and fungal DNA was extracted from the samples directly. All the Malassezia species, including the two major species M. globosa and M. restricta, were quantified with high accuracy, using a real-time PCR assay. RESULTS: Colonization by M. restricta was approximately five times higher at all body sites than colonization by M. globosa. Malassezia colonization was significantly lower in patients with hyperlipidaemia than in patients with normolipidaemia. CONCLUSIONS: Malassezia restricta is the predominant species in psoriatic scale.     

Cutaneous Malassezia flora in atopic dermatitis differs between adults and children

BACKGROUND: Malassezia species are suspected to be involved in the development of skin lesions in atopic dermatitis (AD) when the response of adult AD to anti-inflammatory treatments is poor. However, a comparative analysis of Malassezia flora between adults and children with AD has not been performed. OBJECTIVES: To compare the cutaneous Malassezia flora between adults and children with AD. METHODS: Scale samples were collected from skin lesions of 58 patients with AD in the head and neck regions (28 males and 30 females; 31 children and 27 adults), and fungal DNA was extracted from the samples directly. The number and identities of the Malassezia species were analysed with high accuracy using a polymerase chain reaction-based culture-independent method. The in vivo level of anti-Malassezia IgE antibody was also assayed. RESULTS: Malassezia restricta was the predominant species in the children with AD, while both M. restricta and M. globosa predominated in the adults. The adults showed increased sensitization in terms of anti-Malassezia-specific IgE responses in the sera to both M. globosa and M. restricta in comparison with the children. CONCLUSIONS: The cutaneous Malassezia flora differs significantly between the two age groups.     

从花斑癣患者的皮损区及非皮损区分离和鉴定马拉色菌

目的 研究花斑癣患者皮损区及非皮损区马拉色菌菌种构成;不同解剖部位、皮损颜色及各菌种的分布情况;患者病情和年龄与菌种构成的关系。方法 用无菌胶带粘取113例花斑癣患者皮损区及非皮损区共629个部位的皮屑,分别接种于含菜子油培养基中分离马拉色菌,用生理生化及形态学方法鉴定菌种。结果 皮损区与相对应的非皮损区马拉色菌分离阳性率无差别,非皮损区额部和胸背部分离阳性率高于上、下肢。共分离到565株马拉色菌,鉴定出合轴马拉色菌(44.78%)、糠秕马拉色菌(32.94%)、球形马拉色菌(11.68%)、钝形马拉色菌(5.84%)及限制马拉色菌(4.76%)共5个种,有27处(5.01%)同时分离到两种菌。皮损区与非皮损区菌种构成无明显差别,限制马拉色菌主要从额部分离出。菌种构成与皮损面积无关,但与皮损颜色和患者年龄有关。皮损颜色与病程无关。结论 花斑癣患者皮损区与非皮损区马拉色菌分离阳性率和菌种构成基本一致,与病情无关,而不同解剖部位、皮损类型及年龄患者的菌种构成有一定差异。     

Antigenic components of Malassezia species for immunoglobulin E antibodies in sera of patients with atopic dermatitis

Antigenic components of Malassezia furfur, M. globosa, M. restricta, M. slooffiae, and M. sympodialis were studied for immunoglobulin E antibodies in sera of patients with atopic dermatitis (AD). Antigenic components were extracted from Malassezia cells by treatment with beta-mercaptoethanol, referred to as 2-ME extract. CBB staining and lectin blots using Con A, LCA, PHA-E4, PNA or RCA120 showed that the 2-ME extracts contained several species-dependent components that differed quantitatively and qualitatively among the Malassezia species at the protein level. In the Western blot with the 2-ME extracts, of 54 sera of the patients with AD (54 patients), the patients' IgE antibodies most frequently recognized components showing molecular weights of 43-46 kDa for M. slooffiae, 12-22 kDa for M. sympodialis, 35-40 kDa for M. restricta, 45-50 kDa for M. globosa, and of 67-72 kDa for M. furfur, respectively. In the correlative study, in which the total band intensities generated for each extract in Western blot were compared among the Malassezia species, the intensity for M. globosa was well correlated with that for M. sympodialis (r=0.756). In the Western blot inhibition test, the 2-ME extract of M. globosa partially inhibited the reaction of the antigenic components of other Malassezia species with the patient's IgE antibodies. These results indicated that Malassezia species contained both species-specific and common antigenic components at the IgE antibody level.     

Malassezia globosa and restricta: breakthrough understanding of the etiology and treatment of dandruff and seborrheic dermatitis through whole-genome analysis

Dandruff and seborrheic dermatitis (D/SD) share an etiology dependent upon three factors: sebum, microbial metabolism (specifically, Malassezia yeasts), and individual susceptibility. Advances in microbiological and analytical techniques permit a more detailed understanding of these etiologic factors, especially the role of Malassezia. Malassezia are lipid-dependent and demonstrate adaptation allowing them to exploit a narrow niche on sebum-rich skin. Work in our and our collaborators' laboratories has focused on understanding these adaptations by detailed analysis of biochemistry and gene expression. We have shown that Malassezia globosa and M. restricta predominate on dandruff scalp, that oleic acid alone can initiate dandruff-like desquamation, that M. globosa is the most likely initiating organism by virtue of its high lipase activity, and that an M. globosa lipase is expressed on human scalp. Considering the importance of M. globosa in D/SD (and the overall importance of commensal fungi), we have sequenced the M. globosa and M. restricta genomes. Genomic analysis indicates key adaptations to the skin environment, several of which yield important clues to the role Malassezia play in human disease. This work offers the promise of defining new treatments to D/SD that are targeted at changing the level or activities of Malassezia genes.     

Distribution of Malassezia species in pityriasis versicolor and seborrhoeic dermatitis in Greece. Typing of the major pityriasis versicolor isolate M. globosa

BACKGROUND: The expansion of the genus Malassezia has generated interest in the epidemiological investigation of the distribution of new species in a range of dermatoses, on which variable results have been reported from different geographical regions. No data are thus far available from South-east Europe (Greece). OBJECTIVES: To study the distribution of Malassezia species in pityriasis versicolor (PV) and seborrhoeic dermatitis (SD) and to investigate whether polymorphisms in the internal transcribed spacer (ITS) 1 region facilitate detection of M. globosa and M. sympodialis subtypes. METHODS: In total, 109 patients with PV and SD and positive Malassezia cultures were included in the study. Age, gender, primary/recurrent episode, disease extent and clinical form of PV were recorded. ITS 1 polymorphisms of M. globosa and M. sympodialis type and clinical strains were investigated by polymerase chain reaction (PCR)-single-strand conformational polymorphism (SSCP) analysis. RESULTS: Malassezia globosa was the prevalent species isolated from PV and SD either alone (77% and 39%, respectively) or in combination (13% and 18%, respectively) with other Malassezia species. The pigmented form of PV was strongly correlated with the female gender. PCR-SSCP differentiated five subgroups of M. globosa with one being associated with extensive clinical disease. All M. sympodialis isolates displayed a homogeneous ITS 1 PCR-SSCP profile. CONCLUSIONS: Malassezia species isolation rates were in agreement with those reported from South-west Europe. PCR-SSCP of the ITS 1 is useful for highlighting prospective clinical implications of M. globosa subtypes.     

The prevalence of Malassezia yeasts in patients with atopic dermatitis, seborrhoeic dermatitis and healthy controls

Cultures for Malassezia yeasts were taken from both normal-looking skin and lesional skin in 124 patients with atopic dermatitis, 16 patients with seborrhoeic dermatitis and from normal skin of 31 healthy controls. Positive Malassezia growth was found in fewer patients with atopic dermatitis (56%) than in patients with seborrhoeic dermatitis (88%) or in healthy controls (84%, p<0.01). In the patients with atopic dermatitis, fewer positive cultures were found in lesional (28%) than in non-lesional skin (44%, p<0.05), while positive cultures were found in 75% of both lesional and non-lesional skin of patients with seborrhoeic dermatitis (not significant). M. sympodialis dominated in patients with atopic dermatitis (46%) and in healthy controls (69%). In patients with seborrhoeic dermatitis both M. sympodialis and M. obtusa were cultured in 43%. A Malassezia species extract mixture would increase the possibility of detecting IgE sensitization to Malassezia in patients with atopic dermatitis.     

An update on the microbiology,immunology and genetics of seborrheic dermatitis

The underlying mechanism of seborrheic dermatitis (SD) is poorly understood but major scientific progress has been made in recent years related to microbiology, immunology and genetics. In light of this, the major goal of this article was to summarize the most recent articles on SD, specifically related to underlying pathophysiology. SD results from Malassezia hydrolysation of free fatty acids with activation of the immune system by the way of pattern recognition receptors, inflammasome, IL-1β and NF-kB. M. restricta and M. globosa are likely the most virulent subspecies, producing large quantities of irritating oleic acids, leading to IL-8 and IL-17 activation. IL-17 and IL-4 might play a big role in pathogenesis, but this needs to be further studied using novel biologics. No clear genetic predisposition has been established; however, recent studies implicated certain increased-risk human leucocyte antigen (HLA) alleles, such as A*32, DQB1*05 and DRB1*01 as well as possible associations with psoriasis and atopic dermatitis (AD) through the LCE3 gene cluster while SD, and SD-like syndromes, shares genetic mutations that appear to impair the ability of the immune system to restrict Malassezia growth, partially due to complement system dysfunction. A paucity of studies exists looking at the relationship between SD and systemic disease. In HIV, SD is thought to be secondary to a combination of immune dysregulation and disruption in skin microbiota with unhindered Malassezia proliferation. In Parkinson's disease, SD is most likely secondary to parasympathetic hyperactivity with increased sebum production as well as facial immobility which leads to sebum accumulation.     

Identification of Malassezia yeast species isolated from patients with pityriasis versicolor

Pityriasis versicolor (PV) is a disease with worldwide distribution. Twelve different species of Malassezia yeast have been described. The objective of this study was to determine which species of Malassezia are more prevalent in patients with pityriasis versicolor. Samples were collected by scraping the lesions of 87 patients with a clinical suspicion of pityriasis versicolor. The samples were then submitted to fungal microscopy and culture to identify the species. The species found were: Malassezia sympodialis (30%), Malassezia furfur (25.7%), Malassezia globosa (22.7%), Malassezia restricta (12.1%), Malassezia obtusa (7.6%) and Malassezia slooffiae (1.5%).     

Cutaneous Malassezia microbiota of healthy subjects differ by sex, body part and season

Malassezia is a component of normal cutaneous resident microbiota. The aim of this study was to quantitatively clarify the differences in cutaneous Malassezia microbiota in healthy subjects by sex, body part and season. Samples were collected from the forehead, cheek, upper chest and upper back of 20 healthy men and 20 healthy women (average age 32 years) in summer and winter by the swab method. Malassezia DNA was analyzed using a real-time PCR system. As a result, in sex, body parts and season, men, the upper trunk and summer showed the highest total numbers of cutaneous Malassezia species on average. There were also differences depending on the analytical method. The predominant species were M. restricta on the face of men, M. globosa and M. dermatis on the upper trunk of men, and M. globosa and M. sympodialis on the upper trunk of women. This study clarified that the cutaneous Malassezia microbiota of healthy subjects differed by sex, body part and season.     

Epidemiology of pityriasis versicolor in Adana, Turkey

Pityriasis versicolor is a common superficial mycoses of the skin. It is now recognized that the causative organisms of this infection are different species of Malassezia . The aim of this study was to determine the distribution of Malassezia species in patients with pityriasis versicolor in Adana, Turkey. In total, 97 patients positive for Malassezia elements, namely, yeast cells and short hyphae in microscopic examination, were included in the study. All samples were inoculated in plates containing modified Dixon's medium. However, only 44 of the patients (45.4%) showed Malassezia spp. in culture. Malassezia globosa (47.7%) was the most commonly isolated species followed by Malassezia furfur (36.4%) and Malassezia slooffiae (15.9%). Mixed Malassezia species were not isolated. In conclusion, M. globosa was found to be the predominant PV isolate in Adana, Turkey.     

Identification of Malassezia species isolated from scalp skin of patients with psoriasis and healthy subjects

The role of Malassezia species in psoriasis is still undetermined, but several reports have associated these lipophilic yeasts with the development of skin lesions in psoriasis. The aim of our study was to analyze the prevalence of Malassezia species in the scalp lesions of patients with psoriasis and assess the distribution of the species according to patient sex, age, and duration of the disease. Forty psoriatic patients with scalp involvement and the same number of clinically healthy individuals were included in the study. The samples were obtained by scraping the skin surface of the scalp of all subjects and then incubated on modified Dixon agar. The yeasts isolated were identified by their morphological and physiological properties according to Guillot et al method. M.globosa in its yeast phase was a predominant species (55%), followed by M.slooffiae (18%) and M.restricta (10%), the latter being the most common species isolated from healthy scalp skin. We found significant difference in the distribution of Malassezia species between psoriatic and healthy scalp skin and in the distribution of Malassezia species according to the severity of the scalp involvement.     

Atopy patch test reactions to Malassezia allergens differentiate subgroups of atopic dermatitis patients

BACKGROUND: The yeast Malassezia is considered to be one of the factors that can contribute to atopic dermatitis (AD). OBJECTIVES: To investigate the reactivity to Malassezia allergens, measured as specific serum IgE, positive skin prick test and positive atopy patch test (APT), in adult patients with AD. METHODS: In total, 132 adult patients with AD, 14 with seborrhoeic dermatitis (SD) and 33 healthy controls were investigated for their reactions to M. sympodialis extract and three recombinant Malassezia allergens (rMal s 1, rMal s 5 and rMal s 6). RESULTS: Sixty-seven per cent of the AD patients, but only one of the SD patients and none of the healthy controls, showed a positive reaction to at least one of the Malassezia allergens (extract and/or recombinant allergens) in at least one of the tests. The levels of M. sympodialis-specific IgE in serum correlated with the total serum IgE levels. Elevated serum levels of M. sympodialis-specific IgE were found in 55% and positive APT reactions in 41% of the AD patients with head and neck dermatitis. A relatively high proportion of patients without head and neck dermatitis and patients with low total serum IgE levels had a positive APT for M. sympodialis, despite lower proportions of individuals with M. sympodialis-specific IgE among these groups of patients. CONCLUSIONS: These results support that Malassezia can play a role in eliciting and maintaining eczema in patients with AD. The addition of an APT to the test battery used in this study reveals a previously overlooked impact of Malassezia hypersensitivity in certain subgroups of AD patients.     

特应性皮炎患者面部、上肢和背部皮损真菌群落多样性分析

【摘要】 目的 探讨特应性皮炎（AD）患者面部、上肢和背部皮损真菌群落多样性和结构特征。方法 对2015年9 - 10月来自成都医学院第一附属医院皮肤科门诊的10例AD患者面部、上肢和背部皮损区和10例健康对照相应部位皮肤采样，提取样品DNA行MiSeq高通量测序，进行多样性指数分析、物种组成分析及主成分分析。两组间比较采用独立样本t检验，多组间均数比较采用单因素方差分析，两两组间多重比较采用LSD-t检验。结果 多样性指数分析显示，AD患者组面部、上肢、背部皮损区样本Shannon指数均显著高于健康对照组相应区域（t值分别为2.67、2.37、3.34，均P < 0.05）。物种组成分析显示，马拉色菌属在AD患者组面部、上肢、背部皮损区和健康对照组相应区域样本中均占主要地位，其中球形马拉色菌和限制马拉色菌丰度合计约为80%。AD患者组总样本念珠菌属、曲霉属丰度显著高于健康对照组（t值分别为3.515、2.137，均P < 0.05）。AD患者组与健康对照组面部各主要真菌属丰度差异均无统计学意义（均P > 0.05）；AD患者组上肢念珠菌属丰度显著高于健康对照组（t = 3.186，P < 0.05），背部曲霉属丰度显著高于健康对照组（t = 2.736，P < 0.05）。AD患者组、健康对照组面部、上肢、背部样本间主要真菌属丰度差异均无统计学意义（P > 0.05）。轻中重度AD患者组样本间各主要真菌属丰度比较，差异均无统计学意义（均P > 0.05）。主成分分析显示，AD患者组面部、上肢、背部皮损样本真菌群落未按病情严重程度聚类。结论 AD患者面部、上肢、背部皮损区真菌群落多样性明显高于健康对照相应区域，马拉色菌为AD患者上述部位皮损和健康对照相应区域的优势菌种，AD患者皮损区样本中真菌群落组成与疾病严重程度可能无相关性。     

重度特应性皮炎患者急性期和缓解期皮肤微生态研究

目的:探索重度特应性皮炎（AD）成人患者急性期及缓解期皮肤微生态结构和功能的变化特点。方法:采集2019年10月至2020年11月于广州市皮肤病防治所门诊就诊的4例成人重度AD患者急性期和缓解期5个部位（面颊、肘窝、手背、腹部、下肢）皮屑标本,利用第二代高通量测序技术进行宏基因组测序,构建皮肤微生物样本基因集,获得各个...     

花斑糠疹和马拉色菌毛囊炎菌种分布特点分析

目的探讨花斑糠疹和马拉色菌毛囊炎的菌种分布特点。方法从临床诊断为花斑糠疹和马拉色菌毛囊炎的患者分离培养菌种,通过形态学和生理生化学方法鉴定菌种,并比较两组患者菌种分布情况。结果共收集花斑糠疹病例161例,培养阳性121株,其中合轴马拉色菌46株,糠秕马拉色菌13株,球形马拉色菌29株,钝性马拉色菌33株;马拉色菌毛囊炎135例,培养阳性114株,其中合轴马拉色菌52株,糠秕马拉色菌43株,球形马拉色菌13株,钝性马拉色菌6株。两种疾病菌种分布差异有显著性(P<0.005)。结论花斑糠疹和马拉色菌毛囊炎菌种分布存在差异,可能为两种疾病不同临床表现的原因之一,但结果尚需慎重解释,需要分子水平的进一步研究。     

Malassezia globosa as the causative agent of pityriasis versicolor

BACKGROUND: The taxonomic revision carried out in 1996 on the genus Malassezia, which now comprises seven different species, made necessary a re-evaluation of the data concerning the ecology and pathogenicity of these lipophilic yeasts. Very little has been published since then. OBJECTIVES: The aim of this work was to contribute to the knowledge of the aetiology of pityriasis versicolor (PV) with a mycological study made according to the new species. METHODS: Ninety-six patients with PV completed the study. In all of them, samples were taken from the lesions for direct microscopy with KOH+ Parker ink and culture. Samples were also taken from normal skin of the trunk and the forehead of the same patients for culture. Cultures were made in modified Dixon medium in Petri dishes, incubated at 31 degrees C and the isolates were identified by morphological and physiological characteristics. RESULTS: In the PV lesions, direct microscopy always showed the typical mixture of globose blastoconidia and pseudomycelium. Only one patient, having received previous topical antimycotic treatment, was negative. The spherical yeasts observed in vivo were morphologically identical to the globose yeasts characteristic of M. globosa. In culture, M. globosa was found in 97% of cases, alone in 60% of them and associated with M. sympodialis in 29% and M. slooffiae in 7%. These two species were also found in similar percentages on the clinically uninvolved skin of the trunk, whereas M. globosa was not isolated at these sites. However, on the forehead, a small number of colonies of M. globosa was recovered in 12% of cases. CONCLUSIONS: These results support the data previously reported and strongly suggest that M. globosa in its mycelial phase is the causative agent of PV.     

In vitro activity of R126638 and ketoconazole against Malassezia species

The in vitro activity of a new triazole R126638 against Malassezia yeasts was compared with that of ketoconazole. With the agar dilution technique, minimal inhibitory concentrations were lower for R126638 compared with ketoconazole against Malassezia globosa, M. obtusa, M. slooffiae, M. restricta and two strains of M. sympodialis. On human stratum corneum in vitro, both R126638 and ketoconazole were very effective in reducing the production of hyphae from 15% to 1% with R126638 and to 2% with ketoconazole. Scanning electron microscopy did not reveal obvious surface differences between untreated cultures and cultures exposed to ketoconazole or R126638 in the concentration range 0.01-1 microg/ml. However, transmission electron microscopy showed partial to complete necrosis of the cytoplasmic organelles of Malassezia yeasts. The combined scanning electron microscopy and transmission electron microscopy findings confirm earlier observations of the "mummifying" effect of azoles against Malassezia spp. In conclusion, R126638 is an interesting new triazole with high activity against the Malassezia yeasts, which are involved in pityriasis versicolor and seborrhoeic dermatitis.