This review provides abstracts of our research for which the year 2000 prize of The Japanese Society for Medical Mycology was awarded. The study consists of 4 fields: 1) Ultrastructure and biochemistry of the cell walls of dermatophytes. 2) Freeze-fracture electron microscopic study on the membrane systems of pathogenic fungi. 3) Action mechanisms of antifungal agents in terms of membrane structure and functions. 4) Dimorphism and virulence of pathogenic fungi in terms of molecular biology of membrane lipids. Since the detailed contents of these studies were reported in my previous review article (Jpn J Med Mycol 41: 211-217, 2000), I would like to mention these studies only briefly here, together with a detailed review of the septal cell wall architecture of dermatophytes, which I did not cover in my earlier articles.

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This describes a new and reliable species identification and classification system for dermatophytes based on the cluster analysis of nuclear ribosomal internal transcribed spacer 1 (ITS1) DNA sequences. In this system, some phenotypically similar species construct a compact monophyleic cluster which seems to be a “species”. This ITS1 sequence based species is called an “ITS1-genospecies”. The classification of genospecies is a practical concept for DNA sequence based species identification. It is possible to perform species identification and/or strain typing of 25 major dermatophytes (anamorphic genera Trichophyton, Microsporum, Epidermophyton, and the teleomorphic genus Arthroderma), some of which are hard to identify from their morphological features, by demonstrating their dendrogram using this system.

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Cryptococcus neoformans consists of two varieties and is divided into five serotypes: serotypes A, D and AD (C. neoformans var. neoformans) and serotypes B and C (C. neoformans var. gattii). This article deals with the investigation on the serotype of C. neoformans by molecular analysis technique in place of the immunological method with antisera against the capsule component of the yeast. For easier and more precise epidemiological surveillance, twenty-seven isolates of C. neoformans were molecularly analyzed by a RAPD method. This method differentiated these isolates of C. neoformans into 4 groups corresponding to the serotypes A, D, AD and complex of serotypes B and C. These results indicated that serotype A, D and AD could be differentiated by the molecular analysis technique described here. Furthermore, nucleotide sequences of CAP59 genes from five serotypes of C. neoformans were analyzed for their phylogenetic relationship. Approximately 600-hp genomic DNA fragments of the CAP59 gene were amplified from each isolate by PCR and sequenced. The CAP59 nucleotide sequences of C. neoformans showed more than 90% similarity among the five serotypes. The phylogenetic analysis of their sequences was divided into three clusters: serotype A and AD, serotype B and C, and serotype D. These results also indicated that serotype B and C isolates belonging to var. gattii were genetically homogeneous and closely related.

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The Extramural Mycology Program of the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID) has organized and implemented a five workshop series in medical mycology during a critical period in the evolution of contemporary medical mycology (1992 to 2000; http://www.niaid.nih.gov/research/dmid.htm). The goals of the workshop series were to: initiate interactions; build collaborations; identify research needs; turn needs into opportunities; stimulate molecular research in medical mycology; and summarize recommendations emerging from the workshop proceedings. A recurring recommendation in the series was to foster communications within and beyond the field of medical mycology. US-Japan interactions were noted as one specific example of potential information exchange for mutual benefit. The first formal action directed at this recommendation was the workshop “Emergence and Recognition of Fungal Diseases” convened under the auspices of the US-Japan Cooperative Medical Science Program (USJCMSP; http://www.niaid.nih.gov/dmid/us%5Fjapan/default.htm) in Bethesda, Maryland USA on 30 June 1999 (D. M. Dixon & T. Matsumoto, co-chairs). A major goal of the workshop was to present contemporary medical mycology to the Joint Committee of the USJCMSP through representative research presentations in order to make the Committee aware of current status in the field, and the potential for scientific interactions. The second formal action is the workshop, under the auspices of the Japanese Society for Medical Mycology “Medical Perspectives of Fungal Genome Studies” scheduled for 28 November 2000 in Tokyo, Japan (T. Matsumoto & D. M. Dixon, co-chairs). The NIAID Mycology Workshop series recommended interactions between the following groups: academic and pharmaceutical; medical and molecular (model systems); medical and plant pathogens; basic and clinical; mycologists and immunologists. The first two US-Japan workshops can be viewed as consistent with these recommendations, and serve as a Western/Eastern gateway for exchange. The focus of the second US-Japan workshop on genome projects for the medically important fungi provides an excellent model for international communications. Given the tsunami of information that is flowing from genomics and bioinformatics, it is clear that global interactions will be essential in managing and interpreting the data.

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During the 44th meeting of The Japanese Society for Medical Mycology in Nagasaki, 2000, a forum was held entitled “Advances in Molecular Biology of Dermatophytes”. Based on the subject, target molecules and kind of approach, we selected seven presentations from over 100 of the poster abstracts. Six of them concerned identification and one concerned viability.
Summaries of the 7 presentations are given in this article. Of presentations on the identification methods, 5 demonstrated their usefulness: 1) A sequence analysis of ITS 1 region in ribosomal DNA of several Microsporum species showed “ITS 1 genospecies Arthroderma otae” to be composed of A. otae, M. canis, M. equinum and M. audouinii. 2) RAPD may be useful for identifying isolates which are not clearly identifiable by conventional biological techniques. 3) Sequence analysis of CHS 1 was shown to be a rapid tool for species level identification of M. gypseum. 4) PCR-SSCP analysis was also useful for discrimination of dermatophytes with high reproducibility and sensitivity. 5) Strain identification of A. benhamiae isolates may be possible using RFLP analysis of NTS regions in ribosomal DNA. The other presentation concerning identification pointed out some important problems: RFLP of mitochondrial DNA and ITS sequencing of A. benhamiae showed that the results are sometimes in conflict with those obtained from biological techniques, or in some cases, between other molecular techniques. This implies that our concept of fungal species needs to be re-examined and perhaps amended.
The presentation on viability introduced quantitative analysis of mRNA of ACT gene, a new application of a molecular technique. Since the mRNA expresses only in living cells, the method is highly useful as an indicator of fungal viability.

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A 71-year-old man was referred to our department on January 30, 1998 with hard red papules that had developed on the philtrum in mid-January. On January 2, the patient had received high-dose steroid therapy (pulse therapy) for cluster asthma attacks and antibiotics at the Department of Internal Medicine of our hospital. Infiltrative, protruding reddish plaques were observed on the philtrum, which contained a number of small pustules at sites corresponding to hair follicles. There was partial opacity and slight irregularity of the nail plates on the first and second toes of the right foot. Fungal elements were detected from a lesion on the mustache and the nail. Histological examination of the lesion on the philtrum revealed infiltration of inflammatory cells comprising neutrophils, lymphocytes, and macrophages around the hair follicles. Beard hair and nail cultures revealed Candida albicans A, indicating that the patient had candidal sycosis and candidal onychia. He was treated with oral fluconazole (100mg/day). The lesion was clinically improved within 50-days. Recently, extensive use of steroids and antibiotics has produced an increase in reports of patients with Folliculitis barbae Candidomycetica. We believe that the present case was also induced by high-dose steroid therapy and antibiotics.

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A non-pigmented and asporogenic fungal strain without taxonomically useful characteristics was isolated from sputum of a patient with chronic bronchitis. Although the taxonomic position could not be determined based on traditional morphological criteria, the fungus was identified as an Aspergillus fumigatus mutant strain by molecular techniques including ITS sequence analysis and PCR identification system using a PCR primer pair specific for A. fumigatus. Usefulness of these molecular techniques for the identification of non-classifiable fungal strains in a clinical laboratory is discussed.

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Candida dubliniensis, a recently described Candida species, is frequently isolated from oral candidiasis in human immunodeficiency virus infected individuals. To detect the organism rapidly, we have developed specific oligonucleotide primers based on the sequences of mitochondrial cytochrome b gene. These primers selectively amplified DNA only from C. dubliniensis; the DNAs of all pathogenic Candida species tested, as well as those of medically relevant yeasts such as Cryptococcus neoformans, and Trichosporon cutaneum, were not amplified. This is the first report describing the effectiveness of cytochrome b gene in PCR based detection of an organism, and we hope the system will be useful as a microbiological tool for rapid detection of C. dubliniensis.

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