We made a survey of the status of mycological laboratory techniques in Japan. Questionnaires were sent to clinical laboratories in various districts from June to July 1987. 182 institutions answered the questionnaire, with laboratories from university hospitals (40%), general hospitals (37%), commercial firms (4%) and other medical institutions (14%).
Fungus culture is performed by almost all laboratories, but some rely on commercial laboratories. The cultivation temperature and incubation time are remarkably different depending on the laboratory: 28% of laboratories use 35 to 37°C, and 13% terminate the culture in only 2 days; 64% perform identifications of both yeast-like fungi and molds, and 25% fo the laboratories only identify yeast-like fungi. Dermatophytes are not examined in 43%, and this is done by a dermatologist in a dermatological department, especially in university hospitals. As to the identification of major pathogenic fungi, almost all laboratories can identify Candida species, but 13% cannot identify Cryptococcus neoformans, 19% Trichosporon, 36% Geotrichum, 27% Aspergillus fumigatus, 38 to 47% dermatophytes, and 67% Fonsecaea. In spite of the large number of test requests from clinicians for the drug-susceptibility of fungi, this is performed by only a few laboratories.
The survey results indicate that the laboratory mycological test techniques in Japan are not yet sufficiently developed to cope with the requirements of clinicians. The improvement of methods, the dissemination of knowledge and the training of technicians are suggested to be most important.

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Methods of sampling and storage of specimens may influence results of the test. Tissue specimens should be collected not only from the infected portion but also from the neighboring area. Specimens should be kept in as appropriate a condition as possible. In our study, viable yeast cell counts in 4 urine samples were maintained at 4°C for 24 hours. Direct smear examination is important for rapid diagnosis. In 64 cases of deep-seated fungus infection excluding aspergillosis, positive rate of direct smear examination was over 80%. Because the amount of pathogen contained in a specimen is small, especially in deep-seated fungus infections, direct smear examination should be carefully carried out. Attention must be given to morphological difference in the invasive phase in man and in the cultivative phase. As differentiation of colonies on Sabouraud dextrose agar is difficult, it is desirable to apply several different media. When a small number of colonies is isolated, it is necessary to rule out the contaminant.

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During the past decade, much effort has been devoted to the development of automated microbiological diagnostic systems that are faster, more precise, more accurate, and labor saving. In fields related to clinical mycology, automated systems can be used for: (1) screening for urinary tract infection; and (2) identification of yeasts. For urine screening, various methods based on end-point assay, monitoring (rate assay) of growth curve, and quantification of fungal ATP are employed. The spectrum of mycotic identification is presently limited to the clinically important yeasts. The identification of automated systems is based on numerical identification using 20 to 30 biochemical reactions. The percent agreement of genus-species identification with conventional test procedures was reported to be 80 to 90%. It must be understood that the automated instruments are designed to produce accurate identification results unaffected by technical operator variables, rather than highly reproducible biotypes of organisms. Several anti-fungal reagents are now in clinical use. Thus, it will soon become necessary to utilize an automated susceptibility test in clinical mycological examinations.

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During the period from January to December 1987, we studied the identification and susceptibility of clinically isolated yeasts at Chiba University hospital. From outpatients, Candida albicans and Candida glabrata were mainly isolated from urine and vaginal discharge. From inpatients, yeasts were isolated from many kinds of specimens such as urine, sputum, faeces, vaginal discharge, and blood. C. albicans was frequently isolated from all specimens, and C. glabrata was isolated from faeces and vaginal discharge. Candida parapsilosis and Candida guilliermondii were only isolated from blood and Trichosporon beigelii was only isolated from urine.
We studied the background of the patients. For outpatients from whom yeasts were isolated from urine, many had an abnormality of the urinary tract, and for those from whom they were isolated from vaginal discharge, many patients had vaginitis. For inpatients, yeasts were largely isolated from those who had undergone surgical treatment, had a deficiency of immunity, and were medicated by wide spectrum antibiotics.
MICs were measured by microdilution method. There were few resistant strains among C. albicans and T. beigelii against flucytosine. T. beigelii showed lower susceptibility to amphotericin B than other species, but showed the highest susceptibility to miconazole.

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Incidence and species of fungi in fungemia in geriatric patients were determined. Of 1049 organisms isolated from blood cultures between 1972 and 1987, 75 (5.1%) were fungi, with Escherichia coli (24.4%), Staphylococcus aureus (10.6%), Klebsiella pneumoniae (8.9%), Pseudomonas aeruginosa (6.5%) and Enterococcus (6.0%). Incidences of fungi in 1972-1974, 1975-1977, 1978-1980, 1981-1983 and 1984-1986 were respectively 1.5%, 3.6%, 4.0%, 6.2% and 7.4%. The recent increase of incidence of fungi and S. aureus are noted. Candida albicans, C. tropicalis and C. glabrata were the most common fungi isolated from blood. Recent decrease of C. albicans and increase of C. parapsilosis and C. guilliermondii are noted. Most common portal of entry of fungemia was intravenous catheter infection. Endogenous fungemia associated with debilitating conditions such as hematological malignancy with antitumor chemotherapy was also observed. Wide use of intravenous catheters and broad-spectrum antibiotics and increase of compromised host contributes to the frequency of fungemia among the aged.

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Proteins and polypeptides extracted from guinea pig epidermis revealed candidacidal activity. In the present paper, we introduced the way of extraction and measurement of candidacidal assay. The substance was heat stable, and active in acidic pH against Candida.

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This report describes a patient with acute leukemia complicated with polymycotic septicemia caused by Candida albicans, C. krusei and Trichosporon beigelii (cutaneum). Oral flucytosine and intravenous miconazole were administered, but the patient died on the 59th hospital day. At autopsy, T. beigelii and C. albicans were isolated from the liver, and T. beigelii was isolated from the lung by cultural examination and pathological investigation by the peroxidase/anti-peroxidase solution method.
The patient was intubated with an intravenous catheter for hyperalimentation for a long time. The indwelling intravenous catheter appeared to be the major factor leading to the fungemia.
T. beigelii (cutaneum), a so-called “nonpathogenic yeast, ” must be considered a potential cause of fungemia.

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The efficacy of liposome-encapsulated amphotericin B in the treatment of Candida albicans infection in mice was investigated. Amphotericin B was entrapped in small unilamellar vesicles consisting of egg phosphatidylcholine, cholesterol and tocopherol succinate in a molar ratio of 5:2:1. Liposomes exhibited a good stability in serum with a small leakage of carboxy fluorescein. Amphotericin B encapsulated in liposomes resulted in protection of erythrocytes from lysis in isotonic saline. Based on organ distribution of [¹⁴C] labeled liposomes, the radioactivity was detected mainly in the liver, spleen, kidney and lung. The highest survival rate was observed at the dosage of liposomal-encapsulated amphotericin B which is lethal to these mice if administered as free amphotericin B.
These findings suggest that liposomal encapsulation would be advantageous for delivering amphotericin B in candidiasis.

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Malassezia folliculitis and Pityriasis versicolor are studied mycologically and histopathologically.
Positive rates of Pityrosporum spp., both by direct examination and culture study of follicular contents were very much higher in Malassezia folliculitis than in acne vulgaris or steroid acne. Pityrosporum was also increased in the normal looking skin of patients with Malassezia folliculitis compared to acne vulgaris, steroid acne and controls. Histopathologically, clusters of spores of the orbiculare type were seen in the dilated infundibula in Malassezia folliculitis.
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. In two cases, spores were seen continuously from the infrainfundibula to the orifices and transformed into hyphae only in the orifices.
It can be concluded that in Malassezia folliculitis, the increase of spores in the infundibula is important in its pathogenesis, whereas in Pityriasis versicolor, the yeast-mycelial transformation in the orifices plays an important role. In both diseases, normal looking skin also contained fungal elements and was considered to already be in the preclinical stages of the disease.

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An isolate of dematiaceous hyphomycete recovered from sea-water in Tsushima, Nagasaki, Japan was studied morphologically and physiologically. The colonies of the isolate (NHL 2975) were initially moist, greenish black, gradually becoming floccose, grayish olive, and attained 60mm in diameter after 60 days of growth at 27°C on Sabouraud's dextrose agar. Microscopically, round or scale-like bud scars were observed on the surface of thick projections (rachises) on hyphae. These bud scars were usually arranged on rachises in a sympodial manner, but sometimes overlapped each other to look like annellations. Conidia were one- or two-celled, ellipsoidal, short cylindrical or guitar-shaped, and often functioned as conidiogenous cells to produce secondary conidia in a sympodial or annellidic fashion. The isolate grew well on a medium containing 25% NaCl and hydrolyzed skim milk. At 37°C, growth of the isolate was restricted. These morphological and physiological characteristics permitted identification of the isolate as Hortaea werneckii (Horta) Nishimura et Miyaji.

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