About 620-bp genomic DNA fragments of CHS1 genes were amplified from 13 species of dermatophytes by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of CHS1 gene fragments of these dermatophyte species revealed that 3 genera of Epidermophyton, Microsporum and Trichophyton were genetically different from each other. The molecular analysis of CHS1 genes will provide useful information for the identification of dermatophytes. The species-specific primers were designed from the nucleotide sequences of CHS1 gene in 3 teleomorphs of T. mentagrophytes. Using these primers the PCR analysis identified the clinical isolates of T. mentagrophytes from rabbit as A. benhamiae.
By PCR analysis with the dermatophyte specific primer pair, dermatophyte DNA could be diagnosed directly and rapidly in clinical skin samples.
The full length of CHS1 and CHS2 genes of Arthroderma benhamiae (one of the teleomorphs of T. mentagrophytes) was sequenced by 5'-RACE and 3'-RACE methods using cDNA as a template. The full length cDNA sequences of CHS1 gene (3158bp) and CHS2 gene (3392bp) were proved to encode 890 and 419 amino acids, respectively.
The amino acid sequences of A. benhamiae CHS1 and CHS2 in the conserved regions shared, respectively, about 70% and 80% sequence similarity with those of the other filamentous ascomycetes registered in the data base of the GeneBank. RT-PCR analysis suggested that chitin synthase inhibitors (nikkomycin Z and polyoxin D) might stimulate the expression of CHS1 mRNA in A. benhamiae, and not the expression of CHS2 mRNA.

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Although approximately 80, 000 fungi are known, less than 1% are associated with human infection. However, their taxonomy has long been insufficient. During the last decade, DNA sequence analysis was introduced to the taxonomy of pathogenic fungi. Taxonomic advances in the field of medical mycology are helping to identify the causative agents of infectious diseases accurately, facilitating diagnosis and treatment. For example, Malassezia furfur was long considered the major microflora in atopic dermatitis, yet recent studies have indicated that this is not the case, as M. furfur is taxonomically heterogeneous and consists of five species. DNA sequence analysis resolved its taxonomic heterogeneity. Similar examples can be seen in “Candida albicans and C. dubliniensis” and “Trichosporon cutaneum and T. asahii”. DNA sequence analysis also enables accurate identification of fungi. At present, almost all pathogenic fungi can be identified by determining the D1/D2 26S rDNA and ITS region of rRNA gene. This paper describes the practical taxonomy and identification of pathogenic fungi based on DNA sequence analysis.

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The current status of molecular biological methods to control mycoses was described. New tools for detecting of specific fungal DNA by reaction at a constant temperature with Loop-mediated isothermal amplification of DNA (©LAMP, Eiken Chemical Co., Ltd.), and for strain typing by multilocus sequence typing (MLST) will greatly aid in managing complex fungal infection.

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Clinically important resistance of fungal pathogens to azole antifungal drugs is most frequently caused by the over-expression of energy-dependent drug efflux pumps. These pumps usually belong to either the ATP-binding cassette (ABC) family or the major facilitator superfamily (MFS) class of membrane transporter. Little is known about how these pumps work and there is urgent need to develop pump antagonists that circumvent resistance. The expression system is based on an S. cerevisiae AD1-8u^- strain deleted in seven major ABC transporters which has reduced background and endogenous efflux activity. Plasmid pABC3 was engineered to allow functional hyper-expression of foreign proteins in this host. The main advantages of our system are its cloning efficiency: the use of homologous recombination to stably integrate single copy constructs into the host genome under the control of a highly active transcriptional regulator. The expression system has been used to clone and express genes encoding drug efflux pumps from several pathogenic fungi. Furthermore, functional over-expression of human P-glycoprotein was also demonstrated. The protein hyperexpression system will be useful for the screening of pump inhibitors and the study of membrane protein pumping mechanisms. This system has been used to screen chemicals for pump inhibitors. It was found that FK506 and milbemycins chemosensitized pump-expressing and fluconazole-resistant strains and inhibited pump ATPase activity.

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A large number of fungi are associated with allergic disorders. There are many problems in using non-standardized fungal extracts for diagnosis of fungal allergy. These problems can be solved by using genetically engineered recombinant allergens. At present, more than 70 fungal allergens have been cloned and sequenced, and the recombinant forms of several of these are commercially available.
Measurement of IgE antibodies to these commercially available recombinant allergens could provide the tools useful for characterizing the differential sensitization pattern in relation to a paticular disease and the allergenic cross-reactivity among fungi.
Only a limited number of recombinant allergens are available at present, thus further studies on the molecular biology of fungal allergens are needed so that more recombinant allergens can be used in the clinical field.

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The major antifungal agents currently used in clinics fall into classes of either antibiotics or azoles. Recent introduction of a candin-antibiotic, micafungin, into clinical practice is expected to greatly improve the outcome of therapy in deep mycoses. However, there still exist many mycoses which are hard to treat even with application of a variety of antifungal agents.
With this situation of chemotherapy in mycoses, development of novel antifungal agents with good profiles in efficacy and safety and superior to those currently available are anticipated to be discovered by exploratory research. The major target worldwide in the research and development of novel antifungal agents is azole-class compounds. However, among the antifungal antibiotics now being developed, several compounds are being subjected to clinical evaluation based on their novel mechanisms of action and on their non-susceptible feature of cross-resistance to existing antifungal agents.

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Preclinical evaluation of the efficacy of an antifungal agent is basically conducted by measuring both the in vitro and the in vivo antifungal activity of the drug using appropriate infection models.
Although the first requisite for a method measuring in vitro activity is to obtain results with good reproducibility, an additional requirement is that there be good correlation with the in vivo activity, as described later. For the first condition, in recent years the National Committee for Clinical Laboratory Standards in the United States and the Standardization Committee of the Japanese Society for Medical Mycology have proposed reference techniques with the objective of standardizing drug susceptibility testing; these have been used extensively in measuring antifungal activities of novel agents. However, there are several issues involved when these methods are applied to newly developed drugs. First, standard methods are for particular currently available antifungal agents, but MIC determining standards have not been established for other agents. Reproducibility is therefore not guaranteed. Second, there is a question of whether reliable results can be obtained to test an antifungal spectrum with a limited number of fungal species. On the other hand, in vivo evaluation of novel antifungal agents is extremely important to predict the clinical outcome at the preclinical stage. The important requirements for this in vivo experimental system are: use of an animal model of mycosis that resembles the pathophysiology in humans; use of an administration schedule corresponding to that used in clinical studies, and evaluation of the therapeutic effect considering the dose and administration period.
This review presents the present status of preclinical evaluation test methods and discusses the issues.

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[in Japanese]

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The incidence of deep-seated mycosis has recently been increasing, while the number of clinically available antifungal agents is very limited and each agent has some drawbacks. We focused on the triazole class that is known to have good profiles as antifungal agents. After researching them, we found that CS-758 had excellent profiles in terms of antifungal spectrum, activity, and safety. Until this candidate was obtained, we experienced the following difficulties: (1) In vivo activity did not always reflect in vitro activity. (2) The relationship between in vivo activity and pharmacokinetic profile was important in selecting a candidate as an antifungal agent. (3) Suitable infection-models had to be established to predict clinical efficacy. (4) It was necessary to demonstrate superiority over marketed drugs to develop a novel agent. These experiences give us good ideas for future development of novel antifungal agents.

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Hyalohyphomycoses caused by Pseudallescheria boydii and Scedosporium apiospermum have recently been on the increase. To find the appropriate treatment for this emerging disease, we examined the antifungal susceptibility of 10 isolates of P. boydii and 17 isolates of S. apiospermum, most of which were isolated from clinical specimens. When the NCCLS M38-P microdilution method was used, itraconazole showed strong antifungal activities, while amphotericin B had little efficacy. A new triazole agent, voriconazole showed a strong effect against isolates of P. boydii and S. apiospermum (MIC₅₀ 0.06μg/ml), whereas micafungin, a newly developed echinocandin, had little effect (MIC₅₀>16μg/ml). There was no significant difference in the susceptibilities between P. boydii and S. apiospermum isolates against any antifungal agents. Our study suggests that voriconazole is a promising new drug in these infections, and that the same antifungal strategy can be employed in the infections by P. boydii and S. apiospermum.

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The restriction fragment length polymorphism (RFLP) in the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene (rDNA) was analyzed on Fonsecaea pedrosoi isolates kept in the Department of Dermatology, Kanazawa Medical University, Japan.
On the bases of the RFLP patterns with Dde I and Msp I, 131 isolates were classified into 5 types (D1-D5) and 4 types (M1-M4), respectively.
Combining the RFLP patterns with Dde I and Msp I, the isolates were further classified into 6 rDNA-types which corresponded to the 6 mtDNA-types reported by Kawasaki et al. based on the mtDNA-RFLP patterns, except for a single strain of mtDNA-type 7, which was indistinguishable from mtDNA-type 2.
The strains of each rDNA-type formed a Glade on the phylogenetic tree constructed from sequences of the ITS regions. ITS-RFLP analysis discriminated F. pedrosoi from 11 other species of pathogenic phaeoid fungi except F. compacta. These results strongly suggest that the typing based on ITS-RFLP is reliable and that F. pedrosoi and F. compacta are conspecific.
Compared with mtDNA-RFLP analysis, ITS-RFLP analysis is less tedious, permits simultaneous analysis of many samples and gives equivalent results rapidly. This analysis is therefore useful for typing or epidemiologically investigating F. pedrosoi and for differentiating it from other dematiaceous fungi.

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Candida albicans is a pathogenic dimorphic fungus. When yeast cells were pre-incubated in YPD medium at 25°C and released into HFM7 medium containing 4% serum at 37°C, germ tubes emerged within 0.5h. To determine whether mRNA or protein synthesis was necessary for germ tube formation, we examined the effects of mRNA and protein syntheses inhibitors on this formation. In the presence of cycloheximide, cells were unbudded and no germ tube was observed. However, in the presence of actinomycin D, germ tube formation was observed while budding growth and true hyphae elongation were blocked. Next, we measured mRNA or protein accumulation during induction of germ tube formation in the presence of the inhibitors. In the presence of cycloheximide, protein was not synthesized, while in the presence of actinomycin D, mRNA synthesis decreased to 6.3% and protein synthesis to 37.7%. The condition we found which allows only germination but not budding or filamentation might be convenient to use in screening genes involved in the initial stage of morphological change in C. albicans.

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