Biological typing of the lipophilic yeast Malassezia furfur was attempted in taxonomical and epidemiological studies of this unique and medically important species. Forty strains of M. furfur comprising 12 cultures stocked in our laboratory and 28 fresh isolates from patients with pityriasis versicolor were used in this study. These strains were identified as M. furfur on the basis of lipid requirement, chromaticity with Diazonium Blue B test, positive urea hydrolysis and specific characters of the ubiquinone system. Based on the morphological characteristic of colonies grown on Dixon medium at 27°C for 10 days, the strains were grouped into 5 types, Types I, II, III, IV and V. Slide-agglutination test with the cells of each strain and antiserum generated by immunizing a rabbit with each representative strain of the 5 colonial types revealed that all the strains have a common antigen and a type-specific antigen. Types I-IV were clearly distinguished from Type V by theier ability to assimilate Tween 20-85, ammonium sulfate and sodium glutamate and theier susceptibility to two antifungal antibiotics, nikkomycin Z and blasticidin S. Additionally, electrophoretic analyses of the activities of 10 kinds of somatic enzymes extracted from growing M. furfur cells suggested the existence of several different groupsof strains in terms of alloenzymic pattern.
From these results, we are led to the possibility that M. furfur can be grouped into 5 morphological and serological types, with which some physiological characteristics were associated. The DNA typing of these different biological types is in progress.

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The genome of Malassezia pachydermatis isolates from dogs was resolved into six chromosomes using pulsed-field gel electrophoresis, and their molecular sizes were caluculated as 820, 1, 100, 1, 400, 1, 470, 1, 660 and 1, 820 Kb, respectively. Comparison of electrophoretic patterns suggests that the chromosomes of M. pachydermatis were homozygous.

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Malassezia furfur, which is frequently isolated from human skin, has also been detected on animals including cattle, sheep and pelicans.
M. pachydermatis is present on various kinds of animals but very rarely causes them infection. However, a great many cells of M. pachydermatis are sometimes found growing on the surface of lesions such as external otitis and dermatitis in animals. Further studies are required to disclose the role of this fungus in the pathogenesis of the lesions.

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Lipase activity of Malassezia furfur is associated with insoluble fractions of the organisms. The pH optima of the lipase was acidic at 5.0, which is very similar to skin surface pH. The enzyme activity was strongly activated by a lipase activator, sodium taurocholate (STC). Addition of STC to the culture medium activated cell growth and induced hyphae. Results suggested that M. furfur lipase plays an important role in cell growth.

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Pityriasis versicolor and Malassezia intertrigo were studied mycologically and clinically. The main results were as follows: 1) Positive rates of Malassezia furfur by culture were much higher in the lesions and the normal looking skin of patients with Pityriasis versicolor than those of controls. 2) Malassezia furfur was frequently isolated from the scalp, but was not isolated from the tongue. 3) The hypopigmented lesions increased after sunburn and treatment, and in autumn and winter. 4) Positive rates of Malassezia furfur after treatment were very low by direct examination but relatively high by culture. 5) Patients who were negative by culture on completion of treatment seldom had recurrence within 2 months. 6) In the hair follicles of Pityriasis versicolor, hyphae were seen only in the orifices and acroinfundibula; however, spores of the orbiculare type were also seen in the infrainfundibula. 7) In direct examination and culture of the scales in our case of Malassezia intertrigo, large numbers of Pityrosporum ovale and a few Pityrosporum orbiculare cells were seen.

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Malassezia folliculitis is a common skin disease with distinct clinical entity, first described in 1968 by Graham et al.. Lesions appear on the upper back, chest and/or upper arms. They exhibit 2-6mm dome-shaped erythematous papules and pustules. Histopathological examination reveals dilated hair follicles and perifollicular cellular infiltration. Abundant yeast-like morphology of Malassezia furfur is observed mainly in the hair follicles. Etiologic fungus is recognized by potassium hydroxide preparation. Most cases are successfully treated with topical antimycotics.

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Contact sensitivity to extracts from Malassezia furfur (M. furfur) were studied in patients with atopic dermatitis (AD). In a patch test, 75 (64) % of 118 patients with AD responded positively to M. furfur extract, compared with 1 (3%) of 40 healthy volunteers. However, no significant statistical correlations were found between contact sensitivity to the Yeast and any of the following factors: age, sex, distribution of skin lesions, presence of pruriginous papules, history of infantile seborrheic dermatitis, or concomitance of other atopic diseases. In addition, 25 (54%) of 46 patients with AD showed a immediate response to M. furfur in a prick test, whereas none of 11 healthy volunteers showed any response. There was no clear correlation between the delayed and immediate hypersensitivity reactions. Lymphocyte transformation test with M. furfur antigen confirmed that those with positive patch test reactions showed significantly higher stimulation index. Based on these results, we believe that M. furfur plays a role as an antigen derived from the host environment in the exacerbation of skin lesions of AD.

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Seventy-three strains of Candida species isolated from human lesions at the 2nd University Hospital of Norman Bethune University of Medical Sciences were identified by Candida check. Sixty-four strains of C. albicans were isolated from 26 of 28 cases with cutaneous candidosis, 15 of 16 cases with oral candidosis, 16 of 20 cases with vaginal candidosis, 6 of 8 cases with respiratory candidosis, and one case with candida septicemia. Sixty out of all 64 strains of C. albicans revealed serotype A. Seven strains of C. tropicalis were isolated from 2 cases with cutaneous candidosis, one case with oral candidosis, 2 cases with vaginal candidosis, and 2 cases with respiratory candidosis. Two strains of C. parapsilosis were isolated from 2 cases with vaginal candidosis.
With the exception of vaginal candidosis, these results are similar to the incidence of Candida species reported in Japan.

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We have used an Aspergillus fumigatus culture filtrate antigen for serodiagnostic methods, however, this antigen does not dissolve completely in distilled water. To improve this and other problems, we prepared 10 different antigens and carried out comparative studies on them. The antigens were prepared from filtrates of culture of A. fumigatus that had been incubated at 25°C for 1-10 weeks in Sabouraud liquid medium.
Based on the results of double diffusion methods, the antigen obtained after 6 weeks of culture was most effective for double diffusion tests against the sera of aspergilloma patients. Immunoelectrophoresis showed more precipitin lines with antigens obtained after 9 and 10 weeks of culture than with other antigens. Some cross precipitin lines have appeared in double diffusions, however, so we surmise that several changes occurred in the cultures.
In conclusion, we found that the antigen obtained from 6-week culture filtrates at 25°C is more useful than the antigen obtained from 3-week culture filtrates at 37°C for demonstrating precipitin reactions against the sera of aspergilloma patients.

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A 24-year-old housewife came to our department on Oct. 1st, 1986 with a complaint of nail changes in the nail of her right thumb. Ten months previously she had injured the thumb with a sewing machine needle. Three weeks later she noticed pustule formation under the nail in the same location.
The thumb was punctured for drainage and onychectomy was performed three times but in vain. Light microscopic features in a 20% KOH sample of the scales of the nail revealed oddly shaped elements. In all of three mycologic culture examinations of the scales, colonies of Aspergillus niger was diagnosed detected, so the disease as onychomycosis due to Aspergillus niger.

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A case of fungemia due to Hansenula anomala is reported. The infection developed in a 35-year-old woman with acute myelocytic leukemia who was receiving immunosuppression therapy and intravenous hyperalimentation (IVH). From cultures of both peripheral blood specimens and IVH-catheter tips taken during a febrile episode, a species of yeast was isolated repeatedly as the sole organism. After treatment with intravenous fluconazole (400mg/day), the fever was reduced and the blood became culture-negative. The patient later died of disseminated intravascular coagulation (DIC) associated with underlying disease. The morphology and biochemical characteristics of the isolate were compatible with the features of Candida pelliculosa (perfect state: H. anomala). The isolate was identified by the shape of ascospores produced which is peculiar to H. anomala. Susceptibility of the isolate to currently available antifungal agents was as follows (MIC): amphotericin B, 1.25μg/ml; flucytosine, <0.04μg/ml; miconazole, 1.25μg/ml; itraconazole, 5.00μg/ml and fluconazole, 1.25μg/ml. Human infections due to H. anomala are rare and this is only the third case in Japan. From now on this organism should be recognized as one of the pathogens of opportunistic infections in patients undergoing IVH-treatment.

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A series of experiments was carried out to elucidate the pathogenesis of congenital cutaneous candidiasis.
1) In in vitro study on the proliferation rate and morphological changes of C. albicans cultivated in human amniotic fluid, human serum and Sabouraud's broth, respectively, the proliferation rate decreased significantly and the development of germ tubes and pseudohyphae increased in the former two more than in Sabouraud's broth.
2) In experimental infection of fetal rat skin by intrauterine injection of a C. albicans suspension (10⁶ cells/ml) only histological changes of adhesion and superficial invasion of Candida spores with germ tubes to the horny layer at 24 hrs were observed, suggesting the initial stage of infection. Forty-eight hours after injection there were clinical signs of scattered pustules and a unique histological finding of hyphal invasion through the epidermis to the dermis, where many polymorphonuclear leukocytes had infiltrated.
3) The phagocytic activity and intracellular killing activity of fetal rat neutrophils against C. albicans cells were compared with those of adult rat neutrophils. The fetal rat neutrophils phagocytized at a rate of approximately 30% in one hour, and this was significantly lower than that by adult rat neutrophils. The two neutrophil groups showed almost the same intracellular killing activity: less than 1% within one hour.
These results indicate that rat skin 48 hrs before parturition can easily be infected by C. albicans.

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The in vivo efficacies of pradimicin derivatives, BMS-181184 and BMY-28864, and benanomicin A were evaluated comparatively in experimental systemic candidiasis and aspergillosis in cyclophosphamide-treated neutropenic mice. Compounds were given intravenously once daily for 5 consecutive days beginning immediately after the infection of 10LD₅₀ of either Candida albicans A9540 or Aspergillus fumigatus IAM 2034. BMS-181184 was most effective in reducing the mortality among mice infected with C. albicans A9540, giving a PD₅₀ value of 33mg/kg/day, while BMY-28864 and benanomicin A gave PD₅₀ values of 50 and 71mg/kg/day, respectively. Against A. fumigatus IAM 2034, BMS-181184 and BMY-28864 were equally effective, giving PD₅₀ values of 41 and 43mg/kg/day, respectively, while benanomicin A was less effective, giving a PD₅₀ value of 81mg/kg/day.

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Orally administered amphotericin B (AmB) induced protective activity against infections due to vaccinia virus and Candida albicans in mice. This activity was also confirmed in immunocompromised mice. Analyses of the kinetics of peripheral blood cells treated with oral AmB indicated an increased ratio of polymorphonuclear leukocytes (PMN). Enhancement of γ-interferon (IFN) production and of NK-cell activity was also observed. The data were considered to indicate that γ-IFN produced by oral AmB has an important part as one of the factors protecting mice against both candidiasis and vaccinia virus infection.

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Therapeutic efficacy of orally administered terbinafine (TBF) was evaluated in an experimental trichophytosis model, a guinea pig infected with Trichophyton mentagrophytes on the dorsal skin. Assessment was made on the basis of degree of improvement in local symptoms and the negative culture rate of the infected skin tissue.
The therapeutic efficacy of TBF was shown at a dose of 25 mg/kg/day or more. TBF exhibited high efficacy, significantly greater than that of either of the two reference drugs, ketoconazole or griseofulvin, used at the same dose.

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