To elucidate the role of Malassezia species in atopic dermatitis (AD) requires investigation of the Malassezia microflora on the skin of AD patients. Previously, M. furfur was considered the dominant species in the microflora, however, this microorganism has been reclassified into five species and reanalysis of the microflora based on the current Malassezia taxonomy is therefore needed. Malassezia is more difficult to isolate and culture than other pathogenic yeasts such as Candida and Cryptococcus, making it difficult to elucidate the microflora of AD patients accurately. We developed a PCR-based non-culture method that does not require the use of isolation or culture techniques. Of the members of the genus Malassezia, M. globosa colonized the skin of both AD patients and healthy subjects more frequently than other Malassezia species. In addition, we found polymorphisms in the intergenic spacer 1 region of the M. globosa rRNA gene. The genotypes of the microorganisms obtained from AD patients were significantly different from those obtained from healthy subjects. We believe that a specific genotype of M. globosa is responsible for exacerbation of AD.

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Although many exacerbating factors for atopic dermatitis (AD) have been discussed, we are focusing on fungus antigen as a pathogenesis for this condition. About half of the patients were sensitized by Candida albicans and/or Malassezia furfur (MF) using IgE. Patients with severe eruption tended to have a higher concentration of specific IgE. IgE to purified antigens such as manganese superoxide dismutase (MnSOD), cyclophilin, and Malf2 from MF was also detected, while the pattern of positive IgE was varied among the patients so that the major allergen could not be determined. Skin testing gave a positive reaction to MF after 24 hours as well as an immediate type reaction; this delayed type reaction was AD specific since a small number of patients with bronchial asthma showed a positive response to MF. Peripheral mononuclear cells co-cultured with crude MF antigen in vitro produced IL-5 in some AD patients. This response was correlated with the severity of facial eruption, indicating that Th2 type response to MF might make these eruptions worse. MF was easily detected from various skin regions, but we were not able to explain why fewer colonies were obtained from a region with dermatitis than from a non-dermatitis region. From these results, we speculate there are patients who have IgE and Th2 cells which respond to MF. The exact mechanism, however, is still obscure as to how normal flora such as MF can react and exacerbate AD. Further investigations should be done to learn more about the relationship between AD and MF.

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Antigenic components extracted by treatment with β-mercaptoethanol from M. furfur, M. globosa, M. restricta, M. slooffiae, and M. sympodialis were studied for immunoglobulin E antibodies in sera of patients with atopic dermatitis. CBB staining and lectin blots of the extracts showed that each Malassezia species contained species-dependent components at the protein level. In a Western blot with the 2-ME extracts, IgE antibodies against the Malassezia species were found in sera of 83% (for M. globosa), 74% (for M. sympodialis), 65% (for M. furfur), 56% (for M. restricta) and 50% (for M. slooffiae) of the AD patients. 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. A major component of M. globosa was isolated from the 2-ME extract of this fungus by ion-exchange column chromatography and was referred to as Malg46b. Dot blot with the Malg46b containing fraction immunologically reacted with 69% of the sera of the patients, and with 83% of the sera of those who were positive for IgE antibodies to the 2-ME extract of M. globosa in Western blot. The intensities generated for each dot correlated well with the total intensities generated for the 2-ME extract of M. globosa in Western blot (r=0.763). The polyclonal antibody to Malg46b reacted strongly only with the 2-ME extract of M. globosa and reacted slightly with M. restricta. These results indicate that a glycoprotein, Malg46b of M. globosa, is dominantly expressed in this fungus and is a possible major antigen for IgE antibodies in patients with AD.

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Seborrheic dermatitis (SD) is a disease characterized by erythema and accompanied by greasy scale in the seborrheic region. The mechanism by which the disease occurs is still unknown. The genus Malassezia is involved in aggravating SD. Objective diagnosis of SD has yet been established. Atopic dermatitis and psoriasis or contact dermatitis are often confused with SD. One method to differentiate SD from other skin diseases is direct microscopic examination. Mild corticosteroids are effective in treatment of this condition, although, many cases recur within a few days. Antifungal agents are also effective in the treatment of SD by reducing the number of spores, which results in prolongation of the time to recurrence. It is my recommendation that antifungal agents be the first choice of therapy.

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With the recent advances in DNA sequencing technology, a succession of entire genome sequences have been published. A number of genome projects are underway in pathogenic fungi. From these, we present the history and current status of the genomic analysis of Candida albicans. The sequencing project for this organism has been undertaken at Stanford University, and is now nearing the end.

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The emergence of azole-resistant Candida spp. is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis in HIV-infected patients. Several mechanisms can cause this resistance.
An important mechanism of azole resistance is reduced intracellular accumulation of the drug. Among the multidrug efflux transporters, ABC transporters and the major facilitator superfamily are reported to cause the resistance.
Erg11p, sterol C14α-demethylase, is a target of azole derivatives. It was reported that ERG11 over-expression had only a modest effect on the development of azole resistance. However, mutations in the ERG11 gene can cause the resistance, probably by reducing binding of azole to the target enzyme. We sequenced the ERG11 gene in a high-level azole resistant C. albicans strain, Darlington, and found that two amino acid substitutions, Y132H and I471T, had been encoded in the Darlington ERG11 gene. To assess the significance of these substitutions, we replaced one of the two copies of ERG11 gene in an azole-susceptible strain of C. albicans with a copy of the Darlington ERG11 and this resulted in a modest increase in azole resistance. Furthermore, to estimate the effect of Y132H and I471T individually, ERG11 genes with either or both mutations were expressed in S. cerevisiae. The I471T substitution, not previously described, conferred azole resistance when overexpressed alone and increased this resistance when added to the Y132H substitution.
Alterations in the sterol biosynthetic pathway are another resistance mechanism. Inhibition of 14α-demethylase by azole results not only in ergosterol depletion but also in accumulation of methylated sterol 14α-methylergosta-8, 22 (28)-dien-3β, 6α-diol. We deleted the ERG3 gene, which encodes a sterol 5, 6-desaturase, in C. albicans, and the deletion resulted in reduced susceptibility of the mutant to azoles. Sterol analysis revealed that erg3 mutant lost both ergosterol and diol when cultured with fluconazole.

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The dimorphic transition from yeast to pseudohyphae in the petroleum-assimilating yeast Candida tropicalis occurs following the addition of ethanol to glucose semi-defined medium. Subtractive gene cloning was performed on the cDNA from the yeast-growing control culture and on that from the ethanol-supplemented one (the ethanol culture). A homologue of Schizosaccharomyces pombe nmt1⁺ or Saccharomyces cerevisiae THI5 was isolated from the cDNA fraction as a preferentially expressed gene for the ethanol culture. This homologue was tentatively called Ctnmt1⁺, since exogenous thiamine repressed its expression in C. tropicalis growth media. The ethanol culture showed a biphasic pattern of growth phases and the expression of Ctnmt1⁺ occurred at the first growth phase. The supplementation of thiamine to the ethanol culture at the first phase was followed by repression of Ctnmt1⁺ expression and also delay of pseudohyphal growth: filamentous growth was inhibited and chains of yeast cells were formed. A Ctnmt1⁺ disruptant of this organism did not show thiamine auxotrophy and produced pseudohyphal filaments even in the control culture. The supplementation of oxythiamine, an analog of thiamine, to the control culture was followed by the appearance of pseudohyphal filaments, indicating the participation of thiamine during the process of pseudohyphal growth in this organism.

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The PHO system is an ingenious mechanism by which the yeast Saccharomyces cerevisiae regulates the expression of a set of genes involved in phosphate metabolism in response to the change of phosphate concentrations in the environment. A key factor in this mechanism is the Pho85 kinase, which has been discovered as a negative regulator of the PHO system. One of the genes isolated in our laboratory in screening the protein kinase genes from Candida albicans was identified as a homologue (CaPHO85) of the PHO85 of S. cerevisiae, based on the following results. a) Pho85 is the polypeptide with the highest homology to CaPho85 (62% identity) among the S. cerevisiae genome sequence. b) The position of insertion of the intron is quite similar between CaPHO85 (in the 7th codon of the N-terminal MTGSSSQ) and S. cerevisiae PHO85 (in the 6th codon of the N-terminal MSSSSQ). c) The nucleotide sequences in the intron possess the consensus sequences for yeast intron: the 5'-splice-site, internal, and the 3'-splice-site sequence. d) CaPHO85 complemented the S. cerevisiae pho85 mutation. e) CaPho85 contains all of the consensus sequences for the ATP-binding domain and for the kinase domain found in S. cerevisiae Pho85.

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A system to regulate gene expression is a convenient tool to explore gene function (s) not only in prokaryote but also in eukaryote. Such manipulation tools are scarce in the medical mycology field due to its complexity and diploidity. Although systems to regulate gene expression have been constructed, most of them are restricted in their application to particular culture conditions due to the nature of the promoter used. This motivated us to establish a new regulatable expression system that can function regardless of culture conditions, including in a host. In this review, a new system using tetracycline or its derivative as a molecular switch is introduced, which can function in several culture conditions, and in a host. We also show that the system can be applied to the selection of antifungal drug targets, which is the first step in a target-based strategy for drug discovery.

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The Japanese Society for Medical Mycology (JSMM) decided in 2002 to establish guidelines for the clinical evaluation of antifungal agents. The JSMM committee presents here guidelines for the clinical evaluation of topical antifungal agents in the dermatology field. “Guidelines for the Clinical Evaluation of Antibiotic Agents” established by the Japanese Society of Chemotherapy were referred to, and the diseases subjected to clinical evaluation include tinea (tinea pedis and tinea glabrosa), cutaneous candidiasis, and pityriasis versicolor.
Among superficial mycoses, tinea pedis is viewed as the pivotal disease because it is intractable and is the most common. Therefore, the clinical efficacy of antifungal agents for external use in this condition should be established, and tinea pedis is subjected to phase III clinical studies. If efficacy of the antifungal agents is confirmed in the treatment of tinea pedis, a comparative study need not necessarily be performed for tinea glabrosa. If the number of patients is adequate for statistical analysis, a comparative study should be considered for both cutaneous candidiasis and pityriasis versicolor. However, if the number of patients is low, the efficacy of the agents should be evaluated based on their antifungal activity on pathogens and the results of open trials, and a comparative study is not necessarily performed for such diseases. The safety should be strictly evaluated.

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We studied the effectiveness of short-term treatment of tinea corporis and tinea cruris with oral terbinafine at 250mg/day for 2-3 days. The treatment on an open study basis consisted of two groups: the first group (n=17) was given 250mg/day for two consecutive days, and the second group (n=24) was given the same dose for three consecutive days. No patient was treated topically. Effectiveness was evaluated at the end of the second week both clinically and mycologicaliy (KOH examination and culture). In the two-day group, five cases showed an excellent response, three had a good response and nine had a fairly good response. Patients with good response or better comprised 47.1% of the total, while those with excellent response stood at 29.4%. The negative mycological examination ratio was 47.1%. In the three-day group, 12 cases showed an excellent response, four a good response while eight had a fairly good response. Patients with a good response or better comprised 66.7% of the group, while cases with an excellent response comprised 50.0%. The negative mycological examination ratio stood at 66.7%. The overall effectiveness evaluation showed no statistically significant difference between the two treatment groups in the Wilcoxon's rank sum test. No side effect was observed in either group. These findings showed that terbinafine therapy of tinea cruris is effective even with a short-term treatment of 2-3 days at a small dose.

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The prophylactic effects of a Basidiomycetes preparation, AHCC, against lethal Candida albicans infection were investigated in nontreated or immunosuppressed mice. In the cyclophosphamide-or 5-fluorouracil (5-FU)-treated leukopenic mice and nontreated mice, the intraperitoneal administration of AHCC prior to C. albicans infection clearly prolonged the survival periods of the infected mice. In doxorubicin-treated mice, AHCC was less but significantly effective. On the other hand, in prednisolone-treated mice, AHCC was not effective. Oral administration of AHCC also protected the 5-FU-treated mice from lethal Candida infection, as indicated by prolongation of the survival periods and inhibition of Candida growth in the kidneys of these mice and by the increase in a number of neutrophils in their peripheral blood. These results suggested that AHCC may display a protective role against opportunistic fungal infection in leukopenic hosts.

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The role of chlamydospores in the conversion process from a mycelial-to-yeast form using the slide culture method was studied. Three clinical isolates and two other isolates from armadillo, belonging to the fungal species Paracoccidioides brasiliensis, were cultured on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA) and brain heart infusion dextrose agar (BHIDA). Initially, the mycelial forms of each isolate were grown at 25°C for 7, 14, 30 or 60 days on slide cultures and then the temperature was shifted to 35°C. Interestingly, the slide cultures of all the isolates at 25°C formed chlamydospores on either SDA or BHIDA, whereas, on PDA medium, aleurioconidia were formed. If the slide cultures on BHIDA were incubated at 35°C for 7 to 14 days, multiple budding forms could be observed. This phenomenon was not evident in the slide cultures of SDA or PDA. The results of this morphological study indicate that in P. brasiliensis, chlamydospores may play an important role in the conversion process from a mycelial-to-yeast form.

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Immunomagnetic separation (IMS) was utilized for the selective isolation of Cryptococcus neoformans from environmental sources, such as soils and pigeon droppings. Magnetic beads coated with anti-cryptococcal IgG (serotypes A and B) were used to isolate the fungus. In a modeled spiking experiment using C. neoformans serotype A strain and anti-serotype A antibody, the recovery rate of the cells was more than 47%. Specificity experiments using C. neoformans and Candida albicans showed that the beads, when coated with specific antibody for C. neoformans, were highly effective for the separation of C. neoformans strains from C. albicans (more than 97%). The IMS of serotype B cells with purified anti-serotype B antibody indicated a high specificity. When this IMS technique was applied to soils and pigeon droppings, C. neoformans cells were selectively isolated from 3 out of 8 samples, and C. neoformans DNAs were identified by PCR. Therefore C. neoformans cells were thus selectively isolated and the efficiency of the technique further confirmed.

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