Malassezia species are associated with pityriasis (tinea) versicolor, seborrheic dermatitis, and atopic dermatitis. Presently, eleven species are accepted in the genus Malassezia. Of these, M. globosa and M. restricta colonize the skin surface in atopic dermatitis patients, suggesting that they play a significant role in exacerbating the condition. The two species have different genotypes in the intergenic spacer (IGS) region of the rRNA gene, which correspond to patients and healthy individuals. In addition, the antifungal susceptibilities of the genotypic strains to itraconazole and ketoconazole differ. We found a new analytically significant role of the rRNA gene in Malassezia species, in addition to its use for fungal taxonomy and identification.

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Mitochondrial cytochrome b genes of pathogenic yeasts and fungi were analyzed for identification and phylogenetic relationship. The species of genus Malassezia also were analyzed and each sequence was specific in the same domain of cytochrome b gene. Some species represented intraspecies variation. The structure and function of cytochrome b protein was retained and its substitution rates may be in proportion to the evolutionary period. The deduced amino acid sequence was encoded by each nucleotide sequence of cytochrome b gene, and the phylogenetic tree of eukaryote and basidiomycetous yeast was obtained using this sequence. The species of genus Malassezia formed one cluster in this tree, meaning that the concepts of this genus reflect its evolution. The mitochondrial cytochrome b gene analysis was valuable for the identification and phylogenetic analysis of the genus.

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Malassezia species have been recognized as members of the microbiological flora of human and animal skin; they are also considered to play an important role in the pathogenesis of folliculitis, atopic dermatitis and otitis externa. Therefore, the molecular characteristics were investigated to clarify the epidemiology and the pathogenesis of diseases associated with Malassezia species in human and animals. Molecular investigation was made of 105 clinical isolates of M. pachydermatis from dogs and cats by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD analysis and CHS2 gene analysis indicated that clinical isolates of M. pachydermatis were divided into four distinct genetic types (A, B, C and D). Type A was isolated from lesions of atopic dermatitis, flea allergic dermatitis, otitis externa, pyoderma and seborrheic (dermatitidis) in dogs and cats, and might be predominant on this.
The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of standard strains of 11 Malassezia species showed 11 distinct clusters of this species.

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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.

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Pityriasis versicolor (PV) is a superficial infection of the stratum corneum caused by Malassezia. Eleven species have been recognized within this genus, namely M. globosa, M. restricta, M. sympodiasis, M. furfur, M. obtusa, M. slooffiae, M. pachydermatis, M. dermatis, M. japonica, M. yamatoensis, M. nana. To examine the distribution of the microorganism in the skin of patients with PV, we detected Malassezia species without M. pachydermatis and M. nana using a non-culture-based method that consisted of nested PCR with specific primers. The most frequently isolated species were M. globosa and M. restricta (both 93.9%). M. globosa was detected in scales in which only the mycelial form (yeast cells<10/sample) were observed microscopically; M. restricta was not found. We suggest that M. globosa is the causative agent of PV.

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In terms of both diagnosis and treatment, onychomycosis remains a problem in Indonesia. To examine this situation, we performed a retrospective study of the incidence of onychomycosis involving 10 state university hospitals across the country.
We collected data from medical records of patients treated at these hospitals in 1997-1998 (study 1) and from a 1998-1999 multi-center Indonesian study on pulse-dose itraconazole therapy (study 2), both of which were part of onychomycosis campaigns with the objective of increasing the awareness of physicians of onychomycotic problems. Further, we analyzed data from year 2003 medical records of patients from 4 hospitals in Java (study 3). The average incidence of onychomycosis among fungal diseases increased to 4.7% in large cities that, from an average incidence within Indonesia of 3.5% in 1997-1998.
At only 0.5%, the incidence of onychomycosis among all skin diseases was low in both study 1 and 3. Female patients outnumbered male patients with a ratio of 1.5:1 to 2:1. In study 2, the fingernail was the site most frequently affected.
KOH examinations and fungal culture in study 1 (n=557) showed causative organisms to be Candida (50.1%), dermatophytes (26.2%), moulds (3.1%) and mixed infections (1.8%), with the remainder (18.7%) unidentified. Similar frequency of causative organisms was reported in study 2 (n=113) and in study 3 (n=183). People's low awareness of nail disease and consequent lack of concern as well as the limited diagnostic ability of KOH examination and fungal culture might influence the low incidence of the disease and the frequency of detection of the causative organisms.

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We report the efficacy of miconazole nitrate shampoo (Furfur Shampoo^®) to prevent T. tonsurans infection. Method: Experimental models were made from stratum corneum of healthy human heel or guinea pig skin. Before and/or after T. tonsurans was applied, samples were washed with miconazole nitrate shampoo, then inoculated on a Sabouraud culture plate. Result: Though miconazole nitrate shampoo did not eliminate T. tonsurans on the sample completely, the time required to develop the colony (9.9 days) was obviously extended (7.7 days by control). Discussion: We concluded that miconazole nitrate shampoo is a useful method of preventing T. tonsurans infection, and should be used every day (before/after the chance to infect).

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Alternaria is a saprophytic fungus that naturally subsists on decaying plant materials, but may be an opportunistic pathogen in immunocompromised hosts. We describe a case of dermal cutaneous alternariosis in a 70-year-old patient, who was on systemic steroid administration for autoimmune hemolytic anemia. The patient also had chronic heart failure, liver dysfunction, and diabetes mellitus. Infection was confirmed by histological examination, and multiple positive culture results. Treatment with oral terbinafine and itraconazole was ineffective. We review the literature of dermal cutaneous alternariosis reported in Japan; including our case, 15 cases have been reported. Recognition of Alternaria as a potential opportunistic pathogen is important for the differential diagnosis of dermatologic lesion, such as granulomatous lesion or ulcer, in immunocompromised hosts.

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A safe method is described for observing ultrastructure of highly infectious fungi by ultrathin sectioning electron microscopy. The fungal cells were first chemically fixed by glutaraldehyde to kill them. They were then rapidly frozen by propane slush in liquid nitrogen and freeze-substituted in acetone containing 2% osmium tetroxide. This method gave clear cell images with high resolution in a natural state, close to the image obtained by rapidly frozen freeze-substituted specimen of living cells. Although we have demonstrated the utility of this method using Exophiala dermatitidis and Cryptococcus neoformans, it could also be used for observing highly infectious fungi such as Coccidioides immitis.

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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.

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The aim of this study was to investigate the susceptibility of the different phenotypes of Candida albicans strains isolated from clinical specimens to three antifungal agents, fluconazole, itraconazole and voriconazole. Totally 215 specimens were collected from oropharyngeal, gastrointestinal and urogenital tracts of non-neutropenic patients who had received no previous prophylactic treatment. Each of the 215 C. albicans strains recovered was found to express one of the six phenotypes: smooth 73%, fuzzy 10.7%, irregular 2.3%, star 2.8%, ring 6% or stipple 5.1%.
The mean MICs for the six phenotypes of C. albicans strains ranged between 0.25μg/ml and 64μg/ml for fluconazole, 0.03μg/ml and 1μg/ml for itraconazole and 0.03μg/ml and 0.5μg/ml for voriconazole. The mean minimum inhibitory concentration (MIC) of fluconazole was consistently higher for C. albicans strains expressing the stipple phenotype. The antifungal susceptibility of the phenotypic switching requires attention, especially in patients who are clinically unresponsive to fluconazole chemotherapy or in cases of serious C. albicans infections of immunocompromised hosts.

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