Inquiries were made to estimate the level of dermatological practice and to elucidate the mycological education of 147 recruits who applied to take the specialized examination offered by the Japanese Dermatological Association in August 2000. The potassium hydroxide (KOH) examination was performed on the following percentages of the 147 recruits: for tinea corporis, 79.6%; tinea manuum, 76.9%; pityriasis versicolor and tinea cruris, 74.1% and oral candidiasis, 70.1% respectively. Culture examination was applied by 30.6% of the recruits for sporotrichosis, 19.7% for dematiaceous fungal infection and 15.6% for kerion. Ninety-five percent of the recruits had been trained at university hospitals.
Another inquiry was made to directors of 380 dermatology clinics (88 university hospitals and 292 other clinics), in June 2000. KOH-examinations had been made at 94% of the university hospitals and 83% of the other clinics for all of suspected mycosis, whereas culture examinations were made at only 8% and 3% respectively. Further mycological examinations such as identification of the isolates to species level had been made at 73% of the university hospitals. It is postulated that 88% of the university hospitals have facilities to offer a medical mycology education to dermatologists. From these results it is concluded that the available mycological education is insufficient to satisfy the levels of dermatology specialists for clinical practice.

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To research the current status of mycological examinations in dermatology clinics in Japan, I sent out a questionnaire to 82 universities and 183 dermatologists in Miyagi and Gifu prefectures. A summary of the answers follows: a direct examination with potassium hydroxide solusion is usually performed, but in a few university clinics and half of the private clinics in both prefectures it is only used for a differential diagnosis in atypical cases.
Culture studies are performed sporadically, and at 3 universities no culture study of dermatophytoses is done at all. The ability to identify isolated fungi is reliable in half of the university clinics, but half of the professors of dermatology thought that these examinations should be left to a laboratory technician. The expertise needed to examine deep mycoses is generally quite poor. Based on these results the level of dermatomycology in Japan appeared to be gradually declining and may drop even lower in the near future. Therefore, I believe it is essential for the Japanese Society of Dermatology to establish an educational program of training courses of mycological examinations for young doctors and leaders. To raise the level of analyses of mycological examinations for rare pathogenic fungi, a system of consultation by specialists must be established.

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Blood and radiological tests are used to diagnose invasive aspergillosis (IA), but it remains unknown which is more useful. We recently made a prospective study to compare the usefulness of chest CT scan, latex agglutination test (LA) and determination of plasma (1→3)-beta-D-glucan (BDG) levels for IA diagnosis. Of 215 patients, 16 were diagnosed as definite IA. While sensitivities of LA and BDG were 44% and 63%, respectively, all the patients showed some abnormal signs on chest CT scans. On average, CT scans preceded LA by 7.1 days and BDG by 11.5 days. Interestingly, false-positive rate of the blood tests increased during neutropenia. Because lung is the usual portal of Aspergillus, chest CT scan may be more beneficial than the blood tests for early diagnosis of IA. Care is necessary in interpreting the results of these tests, especially when patients are neutropenic.
Blood tests must be sensitive and their results must be interpreted quantitatively. We recently developed a new quantitative diagnostic system for IA using real-time PCR. In vitro examination using 10² copies of Aspergillus DNA showed that linearity was obtained when there were more than 20 copies. We examined 323 samples taken from 122 patients with hematological malignancies. Blood samples were subjected to real-time PCR, enzyme-linked immunosorbent assay (EIA) and BDG. Sensitivity of real-time PCR, ETA and BDG was 79, 58 and 67%, respectively. Specificity of each assay was 92, 97 and 84%, respectively. Real-time PCR was highly sensitive for IA diagnosis, and quantification was accurate.

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Liver transplantation is one of the transplant fields where invasive mycosis is frequently encountered. The diagnosis of fungal infection in this population is often very difficult due to the fact that the liver itself is a key organ for immune defence for infection and due to immunosuppressive state of the patient. Although the majority of fungal infection is caused by Candida, Aspergillus infection is gradually increasing and mortality following invasive infection, often multifactorial, reaches to 70%. A characteristic feature of the infection in liver transplant recipients is the high incidence of preceding occult infection, often from the pretransplant period. Although the specificity is not satisfactory, peritransplant screening culture for fungi is a good prognostic parameter. Plasma β-D-glucan is also very useful in the decision for preemptive treatment, although its plasma level is potentially affected by the reticuloendothelial system of the grafted liver. Referring to these parameters, avoidance of excessive antibiotics and/or immunosuppressants, maintenance of graft function, and a high index of suspicion are always necessary.

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Invasive fungal infections remain one of the challenging complications after solid organ transplantation. Although, having a lower incidence than viral and bacterial infections, fungal infections carry the risk of higher mortality. Management of fungal infections involves several difficulties: the difficulty of early diagnosis including the lack of reliable diagnostic methods, limited effective therapy associated with side effects and drug interaction with immunosuppressive drugs.
Reduction of risk factors and preemptive antifungal therapy prevents and improves the outcome of invasive fungal infections in heart transplant recipients.

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Kidney transplantation therapy can be divided into two periods: the induction periods during which immunosuppressants are aggressively administered, and the maintenance periods during which treatment is continued at a maintenance dosage. During the induction periods, which usually lasts for several months after surgery, the host defense mechanism in the patient is so profoundly impaired that major infections, including deep-seated mycosis, occur in most cases.
The incidence of mycosis as a result of superinfection was quite high in the 1960's and 1970's, when steroids and antimetabolites were the mainstay immunosuppressants, and antibacterial agents were used without any specific purpose for fear of post-transplantation infections.
The new calcineurin inhibitor ciclosporin was developed around 1980 and has since become the primary immunosuppressant used to prevent graft dysfunction after transplantation. As a result of it's advent, use of steroids and antimetabolites has declined. Later studies have shown that ciclosporin selectively inhibits lymphocytes and provides more information on the pattern of bacterial infections after transplantation, making it possible to optimize the use of antibacterial agents. As a result, the incidence of bacterial infections and mycosis has dramatically declined.
Candida and Aspergillus are most frequently detected in post-transplantation mycosis. But Cryptococcus is also occasionally seen.
Antifungal agents such as flucytosine (5-FC) and ketoconazole are effective for deep-seated mycosis such as pulmonary infections which, occurring at a relatively early stage after transplantation, threaten a patient's life. However, the coadministration of ketoconazole must be carefully done because it increases blood concentrations of calcineurin inhibitors such as ciclosporin and tacrolimus by inhibiting the production of cytochrome P-450. Amphotericin B, which is effective but is associated with nephrotoxicity, is usually used in a mouthwash or inhalation therapy.
Dermatomycosis such as tinea versicolor due to fragility of the skin structure is not necessarily rare even in patients with a quasi-normal host defense mechanism and good renal function at a late stage in the maintenance phase.
This paper outlines change in post-transplantation mycosis in recent years and presents some case reports.

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Invasive deep mycoses following bone marrow and solid-organ transplantation remain a major cause of morbidity and mortality. Species of Candida and Aspergillus account for more than 80% of these mycoses. Because these infections are often difficult to diagnose and treat successfully, antifungal prophylaxis is recommended in high-risk patients. Fluconazole is useful in patients who are at risk of invasive candidiasis, including bone marrow transplants, liver and pancreatic transplants. Although invasive aspergillosis is frequent in patients with bone marrow, lung and heart transplantation, no established methods have been available for its prophylaxis. Recently, efforts to improve the efficiency of diagnostic tests have been directed toward the detection of fungal components or metabolites. The requirements for clinical use (monitoring) are as follows: capability of early diagnosis, quantitative measurement, and easy sampling and simple assay procedure. The detection of plasma (1→3)-β-D-glucan (BDG), a characteristic cell wall component of almost all fungi, is widely used in Japan. Twenty-seven episodes of fungemia were observed in our hematology ward and all were positive with BDG. Positive results were observed before the documentation of fungemia in 14 patients (51.9%). Although the positive rate of BDG also was 100% in 17 patients with invasive aspergillosis, it rose slightly at an early stage of the disease in 13 patients (76.5%). The determination of plasma BDG appears useful in the monitoring of deep fungal infection, but its usefulness for early diagnosis remains to be determined. The utility of detection of Aspergillus galactomannan by ELISA and fungal DNA by polymerase chain reaction are also discussed.

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Species identification of genus Malassezia is important in epidemiological and etiological studies, however, is difficult by the conventional system. A specific and rapid identification system based on sequences of internal transcribed spacer 1 of ribosomal DNA has therefore been developed. Using this system, we could identify two or more species mixed in the clinical samples.

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To elucidate the specificity of murine IgE antibody against Malassezia furfur, the immunoblot patterns of IgE in sera obtained from mice inoculated repeatedly in the nasal cavity with M. f. cells, or injected intraperitoneally with M. f. cells mixed with Al(OH)₃ gel were compared with those of IgE antibody detected in sera from patients with AD. Most of the murine IgE anti-Malassezia antibodies shared some antigenic bands with IgE in sera from patients with AD. The IgE antibody of murine also recognized Mal f4, one of the major allergens detected in patients with AD.

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We compared cutaneous colonization levels of Malassezia species in patients with AD and healthy subjects using nested PCR. Malassezia-specific DNA was detected in all 32 of the patients with AD. M. globosa and M. restricta were detected in approximately 90% of these patients, with M. furfur and M. sympodialis being detected in approximately 40% of the cases. In healthy subjects, Malassezia DNA was detected in 78% of the samples, M. globosa, M. restricta and M. sympodialis were detected at frequencies ranging from 44 to 61%, and M. furfur was found in 11% of healthy subjects.
Our results suggest that M. furfur, M. globosa, M. restricta and M. sympodialis are common inhabitants of the skin of both AD patients and healthy subjects, while the skin microflora of patients with AD shows more diversity than that of healthy subjects.

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We examined 52 patients with infantile seborrhoeic dermatitis (ISD) and 47 healthy 1-month-old infants. Yeast cells on the right side of the face were counted by direct microscopic examination, and isolates from the left side of the face were identified by Tween test. Yeast cells were more numerous patients with ISD than in the healthy infants. M. furfur and M. globosa were isolated from ISD patients at significantly higher rates than from healthy infants.

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We report a 63-year-old male with central venous catheter-related infection caused by Malassezia sympodialis after total gastrectomy for a gastric cancer. He had fever and his leukocyte counts and C-reactive protein were elevated 14 days after his operation. After his central venous hyperalimentation catheter was removed, the inflammatory signs immediately disappeared, suggesting an intravenous catheter-ralated infection. A yeast-like fungus was cultured in brain-heart infection semi-solid agar ten days later, and was diagnosed morphologically as Malassezia sp. This strain was identified as M. sympodialis by Tween assimilation test and was confirmed by whole-sequence of internal transcribed spacer 1 regions (ITS1). This is the first report of catheter-related infection caused by M. sympodialis. This strain grew and was subcultured on CHROMagar Candida^®, potato dextrose agar and Sabouraud agar. There have been no reports of such a lipid-independent Malassezia sp. except for M. pachydermatis. The mechanism of lipid independence of this strain is undetermined and future work is needed. Malassezia sp. is receiving increased attention as an etiologic pathogen of catheter-related fungemia in clinical microbiology laboratories and infectious disease sections.

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The possibility of one-week application of terbinafine cream for tinea pedis was studied in a doubleblind test at four institutes, comparing four-week application as a control. Of a total of forty-three patients studied, nineteen were randomized into a four-week application group, Group I, and twenty into a one-week application group, Group II. Group I was evaluated as moderate to extremely useful in twelve (63.2%) of the nineteen patients and Group II in twelve (60.0%) of the twenty patients. No statistical differences were observed between two groups.
These findings appeared to indicate that the short term, one-week application of terbinafine cream had results equivalent to the four-week application. This short-term treatment which aids in improving patient compliance and reducing the total amount of drug applied, thus lowering drug cost, is viewed as a useful way of treating tinea pedis.

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Histoplasmosis is distributed in tropical, subtropical and temperate zones of the world. The disease is one of the imported mycoses in Japan. To date, although more than 30 human and one canine case of histoplasmosis have been reported in Japan, some including that of the canine might have been infected domestically, since the patients have no history of going abroad. The pathogen of histoplasmosis is thus believed to be present in our country. We examined skin biopsies from two dogs in Tokyo and Kumamoto, and found fungal elements 1-2 or 2-4μm in diameter in the macrophages. In addition, the internal transcribed spacer region of the ribosomal RNA (ITS rRNA) gene was detected from DNA extracted from their paraffin-embedded tissues by polymerase chain reaction. The homology of DNA sequences for the ITS rRNA gene were correspondent to Ajellomyces capsulatus at a rate of more than 97.4%. Therefore, the two dogs were diagnosed as having been infected with Histoplasma capsulatum which is the anamorph of A. capsulatus. Since the dogs had no history of having been outside Japan and had not been brought from an endemic area, they might have been infected domestically. Further epidemiological surveys on canine histoplasmosis may be able to estimate autochthonous human cases in Japan.

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The measurement of serum (1→3)-β-D-glucan (β-glucan) in cases with deep seated mycosis is a useful diagnostic method. β-glucan has usually been measured using two different methods: by an alkali treatment, chromogenic automated kinetic assay (chromogenic assay), and by detergent dilution and heating methods, kinetic turbidimetric assay (turbidimetric assay). However, there are often large discrepancies in the β-glucan values measured by these two methods. In this study, we reexamined the values of β-glucan obtained by the two techniques, using 343 serum samples from 146 patients who had been treated in Kawasaki Medical School between January 1999 and May 1999, and then analyzed the reasons for the differences. Serum β-glucan results measured were evaluated by segregating them into three clinical categories: cases with proven deep mycosis, cases with probable deep mycosis and cases without deep mycosis. In addition, the β-glucan in the samples was suppressed by carboxy-methylated curdlan (CM-curdlan), and then was remeasured to find a non-specific reaction. Although a certain correlation was found between the serum β-glucan results measured by the two methods, the values measured by the chromogenic assay were, in general, higher than those measured by the turbidimetric assay. There were also many samples in the cases without deep mycosis that showed ositive values with the chromogenic assay, but not with the turbidimetric assay. With the turbidimetric assay, the addition of CM-curdlan suppressed the values of β-glucan in all samples; however, when measured by the chromogenic assay the values in many samples remained high. These results suggestthat a non-specific reaction which did not include β-glucan was detected by the chromogenic assay. Further studies are needed to evaluate the characteristics and comparable usefulness of the two assays.

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The dimorphic transition from yeast to pseudohyphae in Candida tropicalis occurs following the addition of ethanol to a synthetic medium containing glucose. We developed a method of subtractive gene cloning to isolate genes, of which the expression was apparently specific for pseudohyphal formation in this organism.
Subtraction was performed between sense-strand cDNAs instead of mRNAs from cells of the ethanol culture and anti-sense cDNAs linking to Dynabeads oligo(dT)₂₅ from those of the control culture. Dynabeads oligo(dT)₂₅ are paramagnetic beads with 25 nucleotide-long chains of deoxythymidines covalently linked to their surface and were expected to be easily collected using a magnet. This method using Dynabeads oligo(dT)₂₅ minimizes the degradation of mRNA and makes it easy to construct a cDNA library sufficient to analyze the genetic information on the yeast-to-hyphae transition.
Using this strategy, we identified several genes including a homologue of CPP1 coding tyrosine phosphatase and a homologue of nmt1⁺ encoding protein, which was reported to regulate thiamine biosynthesis.

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The infection rate, causative dermatophytes, and dermatophyte dissemination of tinea pedis in young healthy Japanese were studied by direct microscopic examination, slant cultures, and foot-press culture method. Questionnaires on subjective symptoms and treatments were also distributed.
Among fifty-eight medical students with a mean age of 23.9 years, thirteen (22.4%) showed positive by direct microscopic examination and T. mentagrophytes was more dominant than T. rubrum by slant cultures. In one hundred and sixteen student feet, twelve were infected and disseminating dermatophytes, four were infected but not disseminating, three were not infected but adhering dermatophytes. The infection rate of tinea pedis was thus 24.1%.
Among thirty-seven research workers (mean age: 34.8), twenty-one (56.8%) showed positive by direct microscopic examination. All the dermatophytes isolated by slant cultures were T. mentagrophytes. In seventy-four feet, twenty-nine were infected and disseminating, ten were infected but not disseminating, and three were adhering dermatophytes. The infection rate was 64.9%.
Among thirty-one females wearing boots (mean age: 21.0), seven (22.6%) were infected and T. mentagrophytes was more dominant. In sixty-two feet, eight were infected and disseminating, one was infected but not disseminating, and five were adhering dermatophytes.
The infection rate of tinea pedis was quite high and T. r/T. m rate was low in the three groups. Most of the patients had had no treatment and were disseminating dermatophytes.

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The study protocol was approved by the Ethical Committee of Sakai Municipal Hospital. A total of 40 outpatients with tinea unguium of the toenails, fingernails or both took part after giving voluntary written informed consent. Inclusion criteria were suggestive clinical appearance, a positive KOH preparation and an opaqueness of more than 50% of the nail length. The patients received 125mg terbinafine once a day. The medication was taken after the evening meal and treatment was continued for 12 weeks. Medication stopped in twenty patients who responded well and a follow-up study was continued for 64 weeks. In the other 20 patients, medication was continued for 24 weeks and the follow-up study continued for 64 weeks. At 4-week intervals, the patients were evaluated for their clinical and mycological statuses and adverse reactions.
Clipping of distal nail samples, including any attached subungual tissue, was done using nail clippers at 4-week intervals after cessation of therapy and the level of terbinafine was measured in the laboratory. No adverse reactions were detected.
Tinea unguium of fingernails (1) and the third toes (2) were cured easily in the short term in the 12 week therapy group. One case in the 24 week therapy group was excluded because systemic steroid therapy was started for bullous pemphigoid at 32 weeks. The results of treatment of big toe onychomycosis were compared between the 12 week (17) and the 24 week (19). In the former group, 9 (52.9%) showed complete cure and 8 (47.1%) showed relapse or worse after cessation of therapy was. In 24 week therapy group, complete cure was achieved in 14 (73.7%) and relapse or worse in 5 (26.3%). The cure rate between the 2 groups was statistically not significant. Terbinafine was detected in the target nails up to 24 weeks after cessation of medication in both groups. Onychomycosis or tinea pedis reappeared in a few cases 12 to 16 weeks after medication ceased in the 12 week group. Topical antimycological therapy is necessary after cessation of oral terbinafine.

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