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。     

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.     

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%).     

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.     

Molecular analysis of Malassezia microflora in seborrheic dermatitis patients: comparison with other diseases and healthy subjects

Malassezia species colonize the skin of normal and various pathological conditions including pityriasis versicolor (PV), seborrhoeic dermatitis (SD) and atopic dermatitis (AD). To elucidate the pathogenic role of Malassezia species in SD, Malassezia microflora of 31 Japanese SD patients was analyzed using a PCR-based, culture-independent method. Nested PCR assay using the primers in the rRNA gene indicated that the major Malassezia species in SD were M. globosa and M. restricta, found in 93 and 74% of the patients, respectively. The detection rate and number of each species varied similarly in SD, PV and healthy subjects (HSs), whereas AD showed higher values. Real-time PCR assay showed that the lesional skin harbored approximately three times the population of genus Malassezia found in nonlesional skin (P<0.05), and that M. restricta is a significantly more common species than M. globosa in SD (P<0.005). Genotypic analysis of the rRNA gene showed that the M. globosa and M. restricta from SD patients fell into specific clusters, and could be distinguished from those collected from HSs, but not from those colleted from AD patients. Our results indicate that certain strains of M. restricta occur in the lesional skin of SD patients.     

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.     

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.     

Molecular analysis of Malassezia species isolated from Israeli patients with pityriasis versicolor

Background Pityriasis versicolor (PV) is a common superficial fungal infection of the skin caused by Malassezia species. The clinical significance of each of these species is not fully understood. M. furfur has long been identified as the causative agent of PV. Several recent studies suggest that M. globosa is most frequently associated with PV. Objectives The aim of this study was to examine the prevalence of Malassezia species in affected and unaffected skin in Israeli patients with PV using a polymerase chain reaction (PCR)‐based culture‐independent method. Methods Samples were taken from affected skin of 75 patients with PV (35 females and 40 males, age range 18–65 years) who visited our outpatient clinic and from unaffected skin in 26 patients for direct microscopy and detection of Malassezia species using a PCR‐based method. Results The major Malassezia species in PV was M. globosa, found in 97.3% (73 of 75) of samples from affected skin and 80.8% (21 of 26) samples of unaffected skin. M. restricta was associated with M. globosa in 1.3% (one of 75) of patients. No cases of M. furfur were detected in this study. There was no difference in the distribution of Malassezia species between affected and unaffected skin. Conclusion Based on the PCR test, we have shown that M. globosa is probably most frequently associated with PV.     

Skin colonization by Malassezia species in neonates: a prospective study and relationship with neonatal cephalic pustulosis

OBJECTIVES: To assess skin colonization by Malassezia species in full-term healthy newborns, to investigate factors associated with colonization, and to look at acnelike cephalic pustulosis associated with this carriage. DESIGN: Samples were obtained from neonates and their mothers 0 to 5 days after birth and again 3 weeks later. Clinical patterns of common acnelike pustulosis were reported as mild (<10 papulopustules), moderate (> or =10 papulopustules), or absent. Direct examination and culture of sample. Identification of yeasts was based on microscopic and physiologic criteria. SETTING: A maternity hospital and the pediatric dermatology unit of a university hospital. PARTICIPANTS: Consecutive series of 102 neonates and their mothers. MAIN OUTCOME MEASURES: Incidence of skin colonization and type of Malassezia species found in neonates and correlation with neonatal cephalic pustulosis (neonatal acne). RESULTS: At the first visit, 11 neonates and 36 mothers had cultures positive for Malassezia. Malassezia sympodialis and Malassezia globosa were preferentially cultured. At 3 weeks, 29 (52%) of 56 neonates and 18 (32%) of 56 mothers had cultures positive for only M sympodialis and M globosa. Breastfeeding was not associated with a higher prevalence of Malassezia carriage in neonates. Malassezia colonization was higher when pustulosis was more severe and M sympodialis was found in pustules. CONCLUSIONS: Malassezia colonization begins at birth and increases in the first weeks of life. A high prevalence of M sympodialis in neonates is noted from birth. Its association with neonatal acne is confirmed. Further investigation is needed to study the role of sebum secretion rate and quality in the neonatal period.     

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。     

Molecular analysis of Malassezia species isolated from hospitalized neonates

Abstract:  Malassezia species are a part of the skin microflora of neonates. Under certain circumstances, they can cause diseases ranging from simple pustulosis to lifethreatening fungemia in newborn infants. Little information is available about the epidemiology of Malassezia species in neonates. In the present study, we successfully isolated Malassezia yeasts from 68.7% of hospitalized neonates. Using the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFPL), M. furfur (88.06%) was identified as the most isolated species, followed in frequency by M. globosa (10.48%), M. obtusa (0.73%), and M. slooffiae (0.73%). Among the variables studied, only a longer stay in the ward resulted in a higher colonization rate. Using multiple logistic regression, only the type of hospital and ward had some effects on the colonization rate. Our results supported the hypothesis that neonates acquire Malassezia flora through direct contact with their mothers or hospital personnel.     

球形马拉色菌过增殖特应性皮炎小鼠模型的建立

 目的:探讨球形马拉色菌过增殖小鼠特应性皮炎(AD)模型的建立方法。方法：将18只BALB/c小鼠随机分为6组：对照组、AD组、NS/M组、AD+NS/M组、Oil/M组和AD+Oil/M组，每组3只。通过耳皮肤涂布MC903溶液诱导小鼠特应性皮炎模型后，再分别使用橄榄油和生理盐水作为基质配置球形马拉色菌混悬液涂抹小鼠耳皮肤，以构建真菌过增殖模型，并通过观察其临床表现、皮损评分、真菌学和组织病理学变化，扫描/透射电镜观察毛发结果等评估。结果：与AD组相比：AD+Oil/M组小鼠耳部湿疹样皮炎加重，耳周毛发有明显脱落，皮损评分升高（q=5.95, P<0.01）；组织病理学结果显示AD+Oil/M组表皮明显增厚，真皮浅层致密的炎症细胞浸润，伴部分毛囊受损，毛干退化；PAS染色和真菌荧光染色可见角质层内大量球形马拉色菌；扫描/透射电镜下见毛干表面及毛干内均有大量球形马拉色菌。NS/M组和AD+NS/M组小鼠耳组织PAS染色和真菌荧光染色仅见散在数个马拉色菌。结论：应用橄榄油混悬球形马拉色菌液涂布皮肤可成功建立马拉色菌过增殖小鼠特应性皮炎模型。     

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.     

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.     

从包皮龟头炎患者皮损处分离鉴定马拉色菌

目的：了解马拉色菌属各菌种在包皮龟头炎皮损处的构成及其在发病中的作用。方法：从患处取材直接镜检查见马拉色菌孢子和(或)菌丝的包皮龟头炎患者作为研究对象。用胶带法取材后分别接种在含菜子油的培养基及无放线菌酮的沙堡培养基分离菌种。依据生理生化和形念学特点及转种到科玛嘉显色培养基和米粉吐温80琼脂培养结果鉴定出马拉色菌和(或)念珠菌。结果：81例患者中有57例(70．37％)培养并鉴定出马拉色菌(共60株)，其中糠秕马拉色菌20株(33．33％），合轴马拉色菌18株(30．00％)，钝形马拉色菌17株(28．33％)，球形马拉色菌5株(8．33％)。有37例同时分离到念珠菌(其中72．97％为白念珠菌）。44例仅检出马拉色菌的患者中有23例接受抗真菌治疗。结论：糠秕马拉色菌、合轴马拉色菌、钝形马拉色菌是包皮龟头炎患者皮损处的主要菌种；马拉色菌可能单独或与念珠菌协同引起包皮龟头炎。     

球形马拉色菌致难辨认花斑糠疹2例

报告2例球形马拉色菌引起的难辨认花斑糠疹。均为青年男性,临床见背部或躯干边界较清的红斑,周边鲜红,上覆少许鳞屑,表现为体癣样,但经皮损取材真菌镜检,真菌培养和电镜检查,确诊为球形马拉色菌引起的花斑糠疹。     

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.     

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.     

马拉色菌与人角质形成细胞株共同培养液对培养人黑素细胞生长及酪氨酸酶mRNA表达的影响

目的:观察不同马拉色菌与人角质形成细胞株共同培养液对培养人黑素细胞生长及酪氨酸酶mRNA表达的影响,探讨花斑癣的色素改变是否与马拉色菌的不同菌种有关。方法:将球形、钝形、糠秕、合轴马拉色菌分别与人角质形成细胞株共同培养,24 h后收集上清液,按一定比例加入黑素细胞培养体系中继续培养24 h:①用四甲基偶氮唑兰比色法观察对黑素细胞生长的影响;②用实时荧光定量PCR法测定黑素细胞酪氨酸酶mRNA的表达。结果:各共同培养液对黑素细胞生长无明显影响,但除球形马拉色菌与人角质形成细胞共同培养液可上调黑素细胞酪氨酸酶mRNA的表达(P<0.05)外,未见其它种马拉色菌与人角质形成细胞株共同培养液对黑素细胞酪氨酸酶mRNA的表达有影响。结论:球形马拉色菌与人角质形成细胞株共同培养液刺激黑素细胞后,能提高酪氨酸酶mR-NA的表达,该菌可能与花斑癣的色素沉着有关。