This is a reptrospective and comprehensive review of studies on the action mechanism of antifungal agents which have been done in my laboratory in collaboration with a number of my colleagues. Since elucidation of the action mechanism by which an antifungal agent inhibits fungal growth is essential for evaluating its clinical usefulness for the treatment of systemic and/or superficial mycoses, we have been exploring with a varying types of both existing and newly developed antifungal agents to specify the exact targets of their action on cellular and molecular levels using biochemical, biophysical and electron microscopical techniques. The antifungal agents thus far studied include: several minor topical drugs such as variotin, haloprogin and ciclopiroxolamine; flucytosine; azoles; thiocarbamates; allylamines; wall-active agents aculeacin A and HM-1 killer toxin; and an amino acid analog RI-331. The most intensive studies have been done with imidazoles because of their clinical importance and the complexity of the mechanism of their antifungal action. We found that yeast-form cells of a dimorphic fungus Sporothrix schenckii may work as a useful experimental cellular system for biochemical studies of those antifungal agents which have no significant activity against Candida or any other yeast.
The results of these studies will contribute not only to better understanding the biological characteristics of existing antifungal drugs but to development of some novel types of candidate-drugs.

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In recent years candida species have emerged as a major human fungal pathogen, causing both superficial mucocutaneous and life-threatening systemic disease in alarming proportions around the globe. Fungal infections of skin generally reflect some breakdown in the host's defense system, which, as we know, is quite complex. In the infectious process, however, both host and parasite factors must determine the outcome of any mutual encounter. This review highlights the multitude of host and fungal factors which potentiate skin infections with candida species (1). In addition, I will consider natural protective factors which might be amplified to treat or prevent such infections in susceptible individuals. The bibliography is limited to more inclusive reviews and specific points for emphasis.

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A black yeast-like fungus was isolated from house dust in Hamamatsu, Shizuoka, Japan. It was compared with a culture derived from the neotype of Hortaea werneckii and 15 cultures isolated from patients with tinea nigra in Okinawa and identified as Hortaea werneckii. The colonies of the fungus were blcak, yaest-like and glossy on various kinds of agar media. Conidia were dark, elliptical, fusiform, or peanut-shaped, and one or two-celled. The septum of two-celled conidia was darker than their cell wall. Hyphae were light to dark brown, 3.5-5μm wide, and with many septa. Apical and lateral walls of hypha, which had small pegs with irregular frills, produced conidia. Both ends of two-celled conidia also produced conidia secondarily. The isolate hydrolyzed skim milk and showed a tolerance to salt and high osmotic tension. There have been several reports of tinea nigra in Okinawa and Kyusyu, southern parts of Japan. The present isolation of H. werneckii, however, shows the possibility of occurrence of the disease in Honsyu, the country's main island.

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In disseminated fungal infections, the most frequent site of the entrance of causative fungi is the lung. However, the process of the dissemination of such infections from the lung remains unclear. The present study was done to produce disseminated candidiasis by inoculating Candida albicans into the lung of compromised mice and to describe the process of its hematogenous dissemination.
ICR female mice were administered prednisolone sodium (60mg/kg), cyclophosphamide (60mg/kg) and cefazolin sodium (900mg/kg) intraperitoneally to develop leukopenia of less than 4, 000/mm³. A spore suspension of 2×10⁶ live cells of Candida albicans MCLS-2 was inoculated intratracheally into control and drug-treated animals.
The drug-treated animals developed disseminated candidiasis from the lungs soon after inoculation. The mechanical destruction of vessel walls by fungi was important for the fungal invasion into the vessel lumens. The dissemination was greater when the fungal cells invaded vessel lumens which were more than 50μm in diameter. On the other hand, no dissemination occurred when the fungal cells only entered capillary lumens which were less than 50μm in diameter because fibrin thrombi enclosed the cells.
It is concluded that the dissemination of Candida from the lung may occur in the compromised state, especially in leukopenia and when the vessel lumens with diameters wider than the capillaries are invaded by fungus.

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Kerion celsi caused by Trichophyton violaceum in a 10-year-old boy with tinea corporis and tinea manum is reported. He developed several nodules on the head, and erythematous plaques with fine scales on the ears and right hand. Direct examination of infected hairs using the KOH method revealed large-pored endothrix. Isolated organisms from all lesions showed a slight red color on oatmeal dextrose agar, and many microconidia were observed on brain heart infusion agar enriched with thiamine. The organisms were therefore identified as Trichophyton violaceum. Histopathological findings showed peri- and intra-follicular abscesses and organisms were seen in the hairs. When the patient was treated with griseofulvin, all lesions apparently disappeared; but the erythematous plaques on the ears recurred after about one year. These results indicate that oatmeal dextrose agar medium is useful in the differentiation between T. violaceum and T. glabrum, and that T. violaceum was carried subclinically by the patient for about one year.

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Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis and is known as a temperature-dependent dimorphic fungus.
Even though several routes of transformation from a mycelial to yeast forms have been reported, the route via chlamydospore is the most important.
At this time, conditions of temperature, nutrients, population of yeast cells and concentration of agar which influence chlamydospore formation are examined.
P. brasiliensis strain Pb-18 was used in this experiment. Its yeast cells were mixed with agar media, and were cultured at various temperatures. The results were as follows:
1. At 25°C, more chlamydospores were formed in poor media than in rich ones.
2. At over 25°C, the number of chlamydospores increased in proportion to the increase in temperature.
3. Chlamydospores were most frequently formed when 10⁶ yeast cell units were mixed with 25ml of medium.
4. One and 2.0% agars were the most adequate concentrations for chlamydospore formation.

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In order to identify patients with pulmonary cryptococcosis, we performed screening by the latex agglutination test for detection of serum cryptococcus antigen in patients with respiratory disease between 1981 and June 1986. We found 49 positive cases (1.9%) among 2, 623 tested patients. Of those 49 positive cases, only 3 cases were confirmed to be true positive (0.1%) and the other 46 cases were false positive, including 20 cases due to rheumatoid factor. In addition, there were 3 false negative cases, as determined by histological examination and fungal culture.
Using a new latex agglutination test involving pretreatment of patient's serum with a protease solution to remove the rheumatoid factor, we found 8 cases of pulmonary Cryptococcosis among 1, 231 patients with respiratory disease between July 1986 and 1988. All 8 cases were true positives, and there were no false positive cases. This new latex agglutination test was not interfered by rheumatoid factor. However, two cases showed a false negative result.
On the other hand, we also assayed the serum antibody titers by an agglutination test using a formalin killed yeast cell suspension in 861 patients with respiratory disease. Among 36 positive cases (4.2%), 3 cases were diagnosed as pulmonary cryptococcosis.
The latex agglutination test, especially when combined with pretreatment of serum with protease, is thus a highly useful diagnostic screening tool for detection of patients with pulmonary cryptococcosis. It is difficult to diagnose pulmonary cryptococcosis by radiological techniques because the X-ray findings are variable and usually not specific. We also stress that latent infection with Cryptococcus neoformans is not as rare a disease as had been thought.

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Since it was recently proposed that Nocardia asteroides (N. asteroides group) should be divided into three species, i. e., N. asteroides sensu stricto, N. farcinica and N. nova, the N. asteroides group (26 strains) which was isolated from clinical cases and identified in this laboratory were re-identified on the basis of their physiological and biochemical characteristics. They were re-identified into 3 species: N. asteroides, N. farcinica and N. nova containing 11, 8 and 7 strains, respectively. To our knowledge, this is the first paper in which prevalence of infection due to N. nova in Japan has been recognized.

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A-62-year old female, under treatment with systemic corticosteroids, developed widespread ulcerative lesions on her left leg. Histological examination revealed granulomatous inflammatory changes containing few microorganisms, typical of Prototheca species. Culture of the specimen using several different media, however, yielded no microorganisms. This is the second case of protothecosis reported in Japan. Possible reasons for the failure to isolate pathogens were discussed.

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[in Japanese]

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