In Candida albicans it is usually only the chlamydospores that accept intravital staining. However, it has been recognized in 33 out of 60 strains tested that staining of the suspensor cells from which the chlamydospores are epiphytically formed, and of other pseudohyphae as well, can take place in the same way as with the chlamydospores when an acidic solution of cotton blue (or methyl blue) is, employed. All the cells that became stainable in this way exhibited cell wall hypertrophy. When ultra-thin sections of the pseudohyphae that took up stain were examined under electron microscopy, marked hypertrophy of the inner layer of the cell wall and conspicuous vacuolation were found. Moreover, the ultrastructure was similar to that of chlamydospores, so that, without changing its external form, the pseudohyphae changed into chlamydospores. Observation over a period of the growth of the microcolonies revealed variations in speed between the transformation of the pseudohyphae into chlamydospores and the maturation of typical chlamydospores. The hypertrophy of the cells of this organism and the thickening of the cell walls were also seen to take place as, fundamentally, parts of the same process.
Observation of this transformation phenomenon of pseudohyphae into chlamydospores strongly suggested that typical chlamydospores are not specialized cells differentiated from pseudohyphae, but are the final-stage cells of pseudohyphae whose viability has deteriorated.

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A radioimmunoassay for the determination of Candida antigen in serum was described. A polyethylenglycol (PEG) method and a solid-phase method using Sepharose-coupled antibody were tested for this purpose. The latter was better in stability and susceptibility than the former. In both methods, pretreatment of test serum by heat was necessary to eliminate natural anti-Candida antibody present in the serum. Using established procedures and examining various factors implicated in these radioimmunoassay methods with ¹²⁵I-labeled glycoprotein toxin from C. albicans, a minute amount of glycoprotein toxin and C. albicans mannan as low as 10ng/ml could be determined.

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In order to investigate the difference between steroid hormone and cyclophosphamide treatment on the susceptibility of leukemic patients to deep-seated mycosis, experimental aspergillosis was studied in leukemic mice.
Mean survival time of the leukemic mice inoculated with Aspergillus fumigatus was 8 and a half days. When steroid hormone, which strongly impairs peripheral lymphocytes, was administered with inoculation, all mice died within 5 days and Aspergillus affected many organs with marked lymphocytopenia. On the other hand, cyclophosphamide induced mild lymphocytopenia, as compared with steroid hormone. When cyclophosphamide was administered with inoculation, fewer organs were affected by the fungus and some mice without marked lymphocytopenia survived for a relatively long time.
These results indicate that drugs such as steroid hormones, which induce more marked lymphocytopenia, have a profound effect on vulnerability to Aspergillus infection in the treatment of leukemia.

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The hemolytic active site of Asp-hemolysin from Aspergillus fumigatus was investigated using chemical modification techniques. As amino-terminal residues of Asp-hemolysin, arginine and threonine were detected by the dansyl method. The inhibition reaction with DTNB, 2-PDS, 4-PDS, NQS, TNBS or glyoxal suggested that sulfhydryl and amino group(s) might exist in the hemolytic active site. When one of the three sulfhydryl groups was rapidly modified with DTNB, its hemolytic activity was almost completely lost. The same results were obtained by modification with 2-PDS or 4-PDS, whereas the other two sulfhydryl groups reacted more slowly with DTNB in the presence of 1% SDS or 1N NaOH. The reaction with DTNB or 4-PDS suggested that two sulfhydryl groups were buried in the interior of the toxin molecule and one sulfhydryl group was exposed on the molecule. These findings also suggested that one sulfhydryl group and/or arginine group plays an important role in the hemolytic activity of Asp-hemolysin.

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We report the incidence of dermatophytoses in a colony of mentally and physically handicapped people. Among the 497 population in the colony, 237 patients (47.7%) had one or more dermatophytoses; more than half of them, both men and women, were in the thirties. There was no clear relationship between the incidence of infection and the degree of morbidity. The total number of dermatophytoses was 319, and they consisted of 202 tinea pedis, 2 tinea manuum, 100 tinea unguium, 10 tinea corporis, and 5 tinea cruris. In the tinea pedis cases, 71.3% were of the interdigital type. The species and numbers of isolated organisms were 85 strains of Trichophyton rubrum, 56 strains of T. mentagrophytes and 1 of Epidermophyton floccosum. The concurrence of two or three types of dermatophyte species was recognized in 22 cases of which tinea pedis associated with tinea unguium was most common; T. rubrum as a causative organism was cultured in the largest majority of these.

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Laboratory and clinical studies of ketoconazole were carried out and the results were as follows.
1. The drug concentrations in vaginal tissue, cervical tissue and vaginal fluid after a 300mg oral administration of ketoconazole were studied. Transfer of ketoconazole into these areas was proved.
2. The MICs of ketoconazole against Torulopsis glabrata were lower than those against Candida albicans. But clinical results were better in cases of C. albicans infection than in T. glabrata infection (initial cure rate: C. albicans-82.2%, T. glabrata-16.7%).
3. Ketoconazole was orally administered to 69 non-pregnant women with vulvovaginal mycosis. The patients were divided into 5 groups according to the dosage schedules. (Group 1: 100mg×1/day, Group 2: 200mg×1/day, Group 3: 100mg×2/day, Group 4: 300mg×1/day, Group 5: 100mg×3/day). All patients received the drug for 5 days. The overall initial cure rate was 73.9%. Among the 5 groups, Group 4 showed the best clinical and mycological results (initial cure rate: 84.2%).
4. No side effects were observed. And laboratory findings showed no significant changes.
Recurrences in vulvovaginal mycosis were mainly caused by the fungus which inhabit the rectum, urethra and the deep layer of the vaginal mucosal epithelium. There is a possibility that ketoconazole could be more useful in eradicating the fungus in above mentioned areas than conventional treatments.

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In vivo antifungal activities of cream and gel forms of 710674-S were evaluated by topical application on an experimental model of dermatophytosis (Arthroderma vanbreuseghemii) in guinea pigs. Evaluation was made in terms of (a) average lesion score and (b) rate of appearance of negative cultures from infected skin sections. 710674-S cream and gel forms (0.5, 1 and 2%) were effective in treating the dermatophyte model after 10 consecutive days of treatment initiated 5 days after infection began. The topical efficacy was found to excel in the order of 2%, 1% and 0.5% in both forms. 710674-S 1% cream was almost bioequivalent to the commercial clotrimazole 1% cream. The effect of 710674-S 1% gel seemed to be similar to those of 710674-S 1% cream and clotrimazole 1% cream.

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The therapeutic effects of ketoconazole on experimental cutaneous Trichophyton mentagrophytes infection in guinea pigs were investigated in comparison with clotrimazole. The lesion score at day 21 after infection was 3.60±0.92 in untreated animals, and 1.70±0.90, 1.70±0.78 and 2.90±1.14 in animals treated with 2% ketoconazole cream, 1% ketoconazole cream and 1% clotrimazole cream, respectively. Treatment with 2% and 1% ketoconazole cream produced significant lesion improvement (p<0.05), while 1% clotrimazole cream did not.
The therapeutic effects of ketoconazole on experimental cutaneous Candida albicans infection in guinea pigs were also investigated. The lesion score at day 21 after infection was 3.60±0.92 in untreated animals, and 0.00±0.00, 0.00±0.00 and 0.40±0.92 in animals treated with 2% ketoconazole cream, 1% ketoconazole cream and 1% clotrimazole cream, respectively. Treatment with antimycotics produced significant lesion improvement (p<0.05).

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Case 1: A 10 year-old girl first visited our clinic complaining of itchy scaly eruptions on her upper lip which she had noticed several days earlier. She had injured herself approximately 2 weeks prior to the consultation. Microsporum gypseum was identified as the causal agent. Using the hairbating technique, Trichophyton terrestre, Microsporum cookei, Trichophyton ajelloi, Chrysosporium anamorph of Arthroderma tuberculatum, Chrysosporium tropicum and Chrysosporium keratinophilum were isolated from soil in her home garden and a neighboring park as well as from the place of injury. M. gypseum, however was not found in these soils. Isolates of T. terrestre were identified by mating experiments and confirmed as anamorphs of Arthroderma quadrifidum and Arthroderma insingulare. To our knowledge, this is the first reported isolation of A. insingulare in Japan.
Case 2: A 30-year old postman visited our clinic with a one month old case of tinea pedis. Microsporum gypseum and Trichophyton mentagrophytes were isolated from the same lesion and the infection therefore considered to be of a dual cause.

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We report a 69 year-old male patient with Microsporum gypseum infection on his scrotum. The lesion was a form of annular erythema, not the diffuse erythema with scaling usually seen in the cases of tinea scroti due to anthropophilic dermatophytes. The fungus was seen as clusters of short hyphae in the scale from the lesion and was identified as M. gypseum. The patient had been confined to bed and the route of contagion was not detected. The lesion healed rapidly with topical application of isoconazole cream.

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Antifungal activities of structural analogs and metabolites of the synthetic antifungal agent ketoconazole (KCZ; KW-1414) were investigated. R-39519 (desacetyl deriv.) and R-44319 (trans isomer) had less antifungal activity against Candida sp. and dermatophytes than did ketoconazole. HLI-151 (oxidized deriv.) had no antifungal activity against any of the yeasts or fungi. Two known physiological metabolites of ketoconazole, R-43568 and T-1141 also showed no antifungal activity. Ketoconazole, R-39519, R-43568 and T-1141 showed no antibacterial activity against 12 strains of Lactobacillus sp. which are normal flora of the vagina. In blood of human volunteers administered p. o. 200mg of ketoconazole, no antifungal activity was detected except for ketoconazole by bioautography seeded with Kluyveromyces fragilis. In human urine of the same volunteers, no antifungal activity was detected by the bioautography.

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A case of subcutaneous cystic granuloma (phaeomycotic cyst) caused by Phialophora verrucosa, which affected the right lower leg of a 73 year-old female, is reported and several historic, diagnostic and therapeutic points of the disease are discussed. The patient injured her right leg at the seashore on Panay Island, the Philippines. After 5 weeks she noticed a subcutaneous abscess just below the wound. Dark walled septate hyphae with germ tubes were demonstrated in the wet mounts. The colony on Sabouraud agar was black with a surface covered by short mouse-gray aerial hyphae, and only phialides with collarettes were seen. On the biopsied specimen, an abscess with a central cavity was revealed between the deep dermis and the subcutis, which contained dark walled septate hyphae, germ tubes, chain formation and yeast-like cells as well as foreign bodies (sand). One month after curettage of the abscess, followed by gauze drainage, the lesion subsided without antifungal drugs and a year later there were no signs of recurrence.
This case is the 2nd report of a phaeomycotic cyst caused by P. verrucosa. The retention of foreign bodies in the tissue is probably an important causal factor of the disease, therefore curettage of an abscess which has foreign bodies may lead to a cure as shown in this report. Diagnosis of the phaeomycotic cyst rests on the demonstration of brown hyphae in wet mounts or in histological sections of biopsied tissue.

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C. albicans produced a keratinolytic proteinase (KPase) when cultivated in medium containing human stratum corneum (HSC) as the nitrogen source, but was unable to do so when cultivated in Sabouraud dextrose broth. The authors investigated the influence of culture medium pH and various kinds of protease inhibitors on the growth of C. albicans when cultivated in liquid medium containing HSC as the nitrogen source. Rapid growth of C. albicans was observed with weakly acidic media, particularly at pH4.0. From amongst the various kinds of protease inhibitors added to the media at pH4.0, pepstatin, a carboxyl protease inhibitor, most strongly inhibited the growth of C. albicans dependent upon its concentration. Pepstatin showed a strong antifungal effect indirectly, probably through KPase inhibition. This inhibitory effect of pepstatin was considerably stronger than that of the well-known antifungal agent, clotrimazole. Our results suggest that KPase may play an important role in the growth of C. albicans and that pepstatin has a possibility of being used as a new type of antifungal agent.

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Thirty-eight serum samples from patients with bronchopulmonary aspergillosis (24 patients with pulmonary aspergilloma, 13 with allergic bronchopulmonary aspergillosis and 1 with tracheal aspergillosis), and ninety-four sera for control (50 healthy adults, 20 with bronchial asthma, 10 with pulmonary tuberculosis, 2 with pulmonary nocardiosis and 12 with candidemia), were studied for antibody response against Aspergillus fumigatus antigens by means of agar gel double diffusion (DD), counterimmunoelectrophoresis (CIE) and indirect hemagglutination (IHA) tests. Metabolic antigens produced by a strain of A. fumigatus (ATCC 26430) were used for DD and CIE tests, while Aspergillus HA antigen (Roche) was used for the IHA test.
The sera from the patients with pulmonary aspergilloma showed high positive rates in the CIE (96%), DD (83%) and IHA (79%) tests, although sera from those with allergic bronchopulmonary aspergillosis had a higher positive rate in the IHA (85%) test than in the CIE (15%) and DD (8%) tests. Follow-up studies on patients with pulmonary aspergilloma showed that antibodies to the A. fumigatus antigens disappeared in two or three years after the operation or intracavitary infusion of antimycotics; IHA antibodies disappeared most rapidly.
The DD and CIE tests were simple, fast and highly sensitive methods for the diagnosis of pulmonary aspergilloma, but the IHA test was the most sensitive for bronchopulmonary aspergillosis. These serological methods are helpful not only in the diagnosis of bronchopulmonary aspergillosis, but also in the follow-up study after therapy.

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Ketoconazole was given to 12 mycosis patients: 2 suffered from candidemia, 4 from candiduria, 3 were thought to have mycotic pneumonia or pulmonary mycosis, 1 had aspergilloma, 1 a subcutaneous abscess by Candida, and 1 intracerebral cryptococcosis. The results of the clinical trials were markedly effective in 1 patient, effective in 5, slightly effective in 5 and ineffective in 1 patient. Ketoconazole was therefore considered effective in the treatment of 6 patients out of 12. Detected fungi were Candida sp. in 9 cases and Aspergillus sp. in 1 case. The causative agents disappeared in 7 cases, decreased in 1 case, were replaced from Candida albicans to Aerobacter cloacae in 1 and were unknown in 1 case. No particular side effects were observed except for an increase in GOT, GPT and Al-p in 1 case. Ketoconazole, in its formulation for oral use, is thought to be an effective antimycotic drug for deep mycosis at this time when there are only a few antimycotic drugs.

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