[Recent advances in research on Malassezia microbiota in humans].

Malassezia species of lipophilic yeasts account for most fungal microbiota. Although they colonize healthy skin, they are also associated with several skin diseases, including pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and atopic dermatitis. To date, 14 members of the Malassezia genus have been identified. Of these, both M. globosa and M. restricta predominate, regardless of skin-disease type. Comprehensive analysis of fungal microbiota in the skin of patients with atopic dermatitis using an rRNA clone library method revealed that fungal microbiota cluster according to disease severity. The external ear canal and sole of the foot are colonized by specific Malassezia microbiota.

Sequence diversity of the intergenic spacer region of the rRNA gene of Cryptococcus albidus isolated from the skin of patients with atopic dermatitis and healthy individuals.

The yeast species Cryptococcus albidus var. albidus was found to more often colonize the skin surface of patients with atopic dermatitis (77.0%, 47/61) than that of  healthy subjects (37.5%, 15/40). The intergenic spacer 1 region of the rRNA gene of this species consists of four sequence types: I, II, III and IV. Types I and II were predominant among healthy subjects and atopic dermatitis patients, respectively.

Characterization of the skin fungal microbiota in patients with atopic dermatitis and in healthy subjects.

Patients with atopic dermatitis (AD) are highly susceptible to viral, bacterial, and fungal skin infections because their skin is dry and this compromises the barrier function of the skin. Therefore, the skin microbiota of patients with AD is believed to be different from that of healthy individuals. In the present study, the skin fungal microbiota of nine patients with mild, moderate, or severe AD and ten healthy subjects were compared using an rRNA clone library. Fungal D1/D2 large subunit analysis of 3647 clones identified 58 species and seven unknown phylotypes in face scale samples from patients with AD and healthy subjects. Malassezia species were predominant, accounting for 63%-86% of the clones identified from each subject. Overall, the non-Malassezia yeast microbiota of the patients was more diverse than that of the healthy individuals. In the AD samples 13.0 ± 3.0 species per case were detected, as compared to 8.0 ± 1.9 species per case in the samples taken from healthy individuals. Notably, Candida albicans, Cryptococcus diffluens, and Cryptococcus liquefaciens were detected in the samples from the patients with AD. Of the filamentous fungal microbiota, Cladosporium spp. and Toxicocladosporium irritans were the predominant species in these patients. Many pathogenic fungi, including Meyerozyma guilliermondii (anamorphic name, Candida guilliermondii), and Trichosporon asahii, and allergenic microorganisms such as Alternaria alternata and Aureobasidium pullulans were found on the skin of the healthy subjects. When the fungal microbiota of the samples from patients with mild/moderate to severe AD and healthy individuals were clustered together by principal coordinates analysis they were found to be clustered according to health status.

Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin.

BACKGROUND: Activation of caspase-14 occurs during terminal differentiation of keratinocytes and may play a role in filaggrin degradation. Therefore, down-regulation of caspase-14 may lead to impaired barrier function. OBJECTIVE: To compare the levels of active and total caspase-14 in healthy subjects in various age groups and in patients with atopic dermatitis (AD), using two enzyme-linked immunoassay (ELISA) systems. METHODS: We established four clones of monoclonal antibodies to caspase-14 and used clone 3 as the immobilizing antibody. A cleavage site-directed antibody, h14D146 [4] was used for specific quantification of active caspase-14 in extracts of tape-stripped corneocytes. Total caspase-14 was measured with a commercial antibody, H-99. RESULTS: The amount of caspase-14 remained constant (ca. 0.1% of extractable proteins) in healthy males from their twenties to their fifties. Caspase-14 was mostly in active form (71-94%) in these extracts. In contrast, caspase-14 level and active caspase-14 ratio were significantly decreased in females in their fifties and sixties. Contents of free amino acids were decreased in females in their sixties, and transepidermal water loss was increased in females in their forties and sixties. In patients with AD, active caspase-14 was markedly down-regulated compared to age-matched controls in both lesional (7.5%) and non-lesional skin (10.6%). Staining of active caspase-14 was considerably weaker in non-lesional skin and was hardly detectable in lesional skin with parakeratosis. CONCLUSION: Our new ELISA systems are effective tools to quantify activation of caspase-14. Our results indicate a role of caspase-14 in epidermal barrier function.

Anti-Malassezia-Specific IgE Antibodies Production in Japanese Patients with Head and Neck Atopic Dermatitis: Relationship between the Level of Specific IgE Antibody and the Colonization Frequency of Cutaneous Malassezia Species and Clinical Severity.

Atopic dermatitis of the head and neck (HNAD) is recognized as a separate condition. Malassezia, the predominant skin microbiota fungus, is considered to exacerbate atopic dermatitis (AD), especially HNAD. In the present study, we investigated the relationships between the levels of specific IgE antibodies, colonization frequency of eight predominant Malassezia species, and clinical severity in 61 patients with HNAD (26 mild, 24 moderate, and 11 severe cases). As clinical severity increased, the levels of specific IgE antibodies against eight Malassezia species also increased. Species diversity of the Malassezia microbiota in scale samples from patients was analyzed by nested PCR using species-specific primers. The clinical severity of HNAD was correlated with the total level of specific IgE antibodies against Malassezia species and the number of Malassezia species detected.

IgA pemphigus reacting exclusively to desmoglein 3.

IgA pemphigus is a rare disease which is classified into two major types: subcorneal pustular dermatosis (SPD) and intraepidermal neutrophilic IgA dermatosis (IEN). The autoantigen of the SPD type was identified as desmocollin1 (Dsc1), while the antigen of the IEN type is still unknown. We report a case of IgA pemphigus possessing antibodies exclusively against desmoglein3 (Dsg3). The patient also had ulcerative colitis, clinically severe oral mucosal lesions with pustule formation, and the "sunflower-like" pustular skin lesions. Histopathology showed intraepidermal bullae with numerous neutrophils. Direct immunofluorescence showed IgA deposits in the keratinocyte cell surfaces only in the lower epidermis. Indirect immunofluorescence using normal human skin as a substrate showed circulating IgA anti-cell surface autoantibodies at a titer of 1:160. cDNA transfection test for Dsc1-3 was negative. IgA antibodies to Dsg3, but not Dsg1, were detected with IgA-ELISA for Dsgs. Addition of diaphenylsulfone (DDS) to prednisolone and cyclosporine A significantly improved the oral and skin lesions. There have been rare cases showing IgA antibodies to either Dsg1 or Dsg3. These cases can be designated as IgA pemphigus foliaceus or IgA pemphigus vulgaris, respectively, according to the autoantigens. We report here a case of IgA pemphigus vulgaris complicated by ulcerative colitis.

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.

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.

Time-kill assay of itraconazole against Malassezia species.

The in vitro pharmacodynamic activity of itraconazole against Malassezia was determined by time-kill methods. Itraconazole showed fungistatic activity at concentrations greater than 2x minimum inhibitory concentration (MIC) for each of the isolates of all nine Malassezia species associated with humans. A concentration exceeding 4x MIC decreased the growth to less than the inoculum amount (1 x 10(5) cells/ml).

Quantitative analysis of cutaneous malassezia in atopic dermatitis patients using real-time PCR.

We quantified the cutaneous Malassezia in patients with atopic dermatitis using a real-time PCR assay. Seven to 12 times more Malassezia colonized the head and neck compared to the trunk or limbs, and the species M. globosa and M. restricta accounted for approximately 80% of all Malassezia colonization at any body site.

[Malassezia species in patients with seborrheic dermatitis and atopic dermatitis].

Malassezia species, organisms normally colonizing the skin surface, are thought to play a role as either the cause or an exacerbating factor in a number of skin conditions, including pityriasis versicolor, Malassezia folliculitis, seborrheic dermatitis (SD) and atopic dermatitis (AD). Using a non-cultural PCR method, we analyzed Malassezia spp. extracted from the skin surface of SD and AD patients. The species most commonly detected in both patient groups were M. globosa and M. restricta, and the number of Malassezia spp. In these patients was higher than in healthy subjects. After a topical application of 2% ketoconazole cream, changes in the population of Malassezia spp. in 20 intractable cases of AD were recorded. The addition of the 2% ketoconazole cream to the standard topical treatments was found to have reduced the Malassezia spp. population by 90%, and showed a clinical efficacy rate of 70%. Furthermore, a combination of azole agents and tacrolimus produced a synergistic anti-fungal effect against Malassezia spp. in vitro. A clinical trial using this drug combination conducted on the face and neck of patients with intractable AD showed a 66.6% efficacy rate in both the reduction of the flora and in clinical improvement. From these results it was evident that Malassezia is one of the factors exacerbating AD, and that removal of the organism results in an improvement in the clinical condition of the patient.

[Molecular analysis of Malassezia species isolated from three cases of Akatsuki disease (pomade crust)].

Malassezia spp. which normally colonize on the skin surface, are known as being either the cause or an exacerbating factor in a variety of skin conditions, including pityriasis versicolor, folliculitis, seborrheic dermatitis and atopic dermatitis. We report here three cases of Akatsuki disease (pomade crust). Scales and crusts were collected from the lesional skin and analyzed using a PCR-based non-culture method. Malassezia microflora in Akatsuki disease was compared to that of healthy subjects and atopic dermatitis patients. Samples were collected from upper and lower eyelids (Case 1), an operation scar (Case 2) and parietal scalp (Case 3). DNA was extracted from the scales and nested PCR was performed using specific primers for each species. Our analysis detected only M. obtusa and M. slooffiae in Cases 1 and 3 and only M. slooffiae in Case 2. Our previous data indicated that while M. globosa, M. restricta and M. sympodialis were common in healthy subjects, the two aforementioned species were rare, suggesting that the presence of M. obtusa and M. slooffiae in the subjects in the present study is correlated to the pathogenesis of Akatsuki disease.

Antifungal activities of tacrolimus and azole agents against the eleven currently accepted Malassezia species.

The lipophilic yeast Malassezia is an exacerbating factor in atopic dermatitis (AD) and colonizes the skin surface of patients with AD. With the goal of reducing the number of Malassezia cells, we investigated the antifungal activities of a therapeutic agent for AD, tacrolimus, and the azole agents itraconazole and ketoconazole against Malassezia species in vitro. We examined 125 strains of the 11 currently accepted Malassezia species by using the agar dilution method. All strains of the 11 Malassezia species were very susceptible to both azole agents, with MICs ranging from 0.016 to 0.25 mug/ml. Tacrolimus had antifungal activities against half of the strains, with MICs ranging from 16 to 32 mug/ml. Two of the major cutaneous floras, Malassezia globosa and Malassezia restricta, have several genotypes in the intergenic spacer region of the rRNA gene; the azole agents had slightly higher MICs for specific genotype strains of both microorganisms. A combination of azole agents and tacrolimus had a synergistic effect against Malassezia isolates, based on a fractional inhibitory index of 0.245 to 0.378. Our results provide the basis for testing these agents in future clinical trials to reduce the number of Malassezia cells colonizing the skin surface in patients with AD.

Genotype analysis of Malassezia restricta as the major cutaneous flora in patients with atopic dermatitis and healthy subjects.

Lipophilic yeasts of the genus Malassezia colonize the skin surface of humans and are an exacerbating factor in atopic dermatitis (AD). Two species, M. restricta and M. globosa are major cutaneous microflora in both AD patients and healthy subjects. We compared the DNA sequences of the intergenic spacer (IGS) region, located between the 26S and 5S rRNA genes of M. restricta colonizing the skin surfaces of 13 AD patients and 12 healthy subjects, and of three CBS stock strains as references. The IGS 1 sequences were divided into two major groups, corresponding to AD patients and healthy subjects. These findings suggest that a specific genotype of M. restricta plays a significant role in AD, although M. restricta commonly colonizes both AD patients and healthy subjects.

A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects.

Over the last few years, new Malassezia species have been found regularly in Japanese subjects. We isolated another new Malassezia species from a Japanese patient with seborrheic dermatitis (SD), and named it M. yamatoensis. In its physiological characteristics and the utilization of Tween by M. yamatoensis is similar to that of M. furfur and M. dermatis. It is distinguished by its growth temperature. To examine the distribution of the microorganism in the skin of patients with SD and atopic dermatitis (AD), and healthy subjects, we applied transparent dressings to the skin, and detected M. yamatoensis DNA using a non-culture-based method that consisted of nested PCR with specific primers. M. yamatoensis DNA was detected from 3 of 31 SD patients (9.7%), 5 of 36 AD patients (13.9%), and 1 of 22 healthy subjects (4.6%). Therefore, M. yamatoensis is a rare member of the cutaneous microflora.