Measurement of (1→3)-β-D-glucan for invasive fungal infection is used practically in Japan. The problem of false positive results due to the frequent occurrence of non-specific reaction in alkaline treatment, chromogenic automated kinetic assay to measure (1→3)-β-D-glucan had been recognized in Japan. But this important problem was resolved in July 2005 by improvement made in the pretreatment reagent in a (1→3)-β-D-glucan measurement kit. In this manuscript, we describe the process of improvement of this kit and its clinical usefulness.

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A 72 year-old man was referred to our department with white curd-like material on the surface of his tongue as well as the mucosal surface of the lower lip, after unsuccessful treatment with itraconazole for 3 weeks. He also had a history of depression and had received topical steroid and/or antibiotics treatment for persistent oral aphtha and irritation of his upper lip for 4 years. A diagnosis of oral candidiasis was made through positive KOH direct microscopic examination and he was instructed to rinse his oral mucosal lesion with amphotericin B syrup. Although no significant eruption was observed on his upper lip at his first visit, he applied the steroid ointment for 4 weeks and came back to our clinic with his upper lip red and swollen. It was also covered with yellow crusty material mixed with a pustule. Histological examination of the lips revealed non-specific chronic inflammation in the mid to lower dermis. Hyphae in the cornea detected by PAS and Grocott staining. KOH direct microscopic examination from the pustule and crust showed positive pseudohyphae although no sign of parasitism to the hair was seen. Candida albicans and Candida parapsilosis were detected by culture from the crust and a biopsy sample. He was successfully treated with 2 courses of pulse therapy of oral itraconazole for sycosis candidiasis, accompanied by 2% miconazole gel for oral candidiasis.

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The biological properties of elastase inhibitor from Aspergillus flavus (AFLEI) were investigated. AFLEI was produced at the highest rate when casamino acid was used as the nitrogen source. When a mixture of AFLEI (approx. molecular weight, 7,500) and elastase from A. flavus (approx. molecular weight, 40,000) was detected using anti-AFLEI antibody, molecular weight of the detected mixture was approximately 48,000, indicating that AFLEI and elastase bound at a proportion of 1 : 1. When immunocompromised mice administrered of immunosuppressive (cyclophosphamide) were infected by inhalation of A. flavus and administered amphotericin B (AMB) alone or in combination with AFLEI, survival rate tended to be higher with combination treatment than with AMB alone.
Moreover, although extensive bleeding was seen in pathology sections taken from rat lung resected 24 hr after purified elastase was administered to the lung via the bronchus, this bleeding was inhibited by AFLEI. These findings indicate that for the treatment of aspergillosis, combination of an existing antifungal agent with AFLEI can be expected to provide greater therapeutic benefits than administration of an antifungal agent alone.

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An experimental study was conducted between January 2002 and April 2003 for the detection of delayed hypersensitivity to Fonsecaea pedrosoi metabolic antigen (chromomycin) in skin tests. A total of 194 subjects were attended by spontaneous demand at the Infectious and Parasitic Diseases outpatient clinic of the Federal University of Maranhão-UFMA and at the Department of Microbiology, Federal University of Minas Gerais-UFMG and classified into three groups: patients with chromoblastomycosis caused by F. pedrosoi (n=20), healthy subjects (n=86) and patients with other diseases (n=88). For the skin test, 0.1 ml of the antigen was applied to the anterior side of the right forearm and 0.1 ml Smith medium was applied to the anterior side of the left forearm as control. The results were analyzed 48 h after inoculation of the antigen and an induration ≥ 5 mm was considered to indicate a positive test. A cellular immune response to chromomycin was detected in 18 (90.0%) of the 20 patients with chromoblastomycosis caused by F. pedrosoi, and one of the patients with a negative test had reactional leprosy. Eighty-five (98.8%) of the 86 healthy subjects presented a negative reaction and only one reacted positively to the antigen. The skin test was negative in all 88 (100%) patients with other diseases, such as dermatophytosis, paracoccidioidomycosis, pulmonary aspergilloma, candidiasis, pityriasis versicolor, tuberculosis, leprosy, tegumentary leishmaniasis and syphilis, and one case of chromoblastomycosis caused by Rhinocladiella aquaspersa. Chromomycin was effective in detecting delayed hypersensitivity in patients with chromoblastomycosis caused by F. pedrosoi, with a sensitivity and specificity of 90.0% and 98.8%, respectively. These results suggest that this antigen can be used in the auxiliary diagnosis of the disease and also in epidemiological studies for determination of the prevalence of chromoblastomycosis infection in endemic areas.

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The genotypes and phenotypes of 77 isolates derived from ascospores produced from two genetically different Arthroderma benhamiae were studied. Specifically, mitochondrial DNA (mtDNA) type, nuclear DNA (nDNA) type, mating type, colony texture, growth rate, urease activity, red pigmentation and hair perforation were examined.
The nDNA types based on the restriction fragment length polymorphism (RFLP) in the internal transcribed spacer (ITS) region of ribosomal RNA (rRNA) genes and mating types were inherited from parents independently. mtDNA type was inherited from only one parent.
All the phenotypes, except hair perforation and mating type, showed great variations. Those seemed not to be a conclusive factor for species identification. Additionally, these characteristics appeared in variable combinations suggesting that they are not interrelated. The intensity of red pigmentation varied even within a colony, implying that it is not a strain-specific characteristic. Hair perforation was observed in isolates of all but one atypical strain, and therefore could be one characteristic of this species.

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As there is not yet a standardized in vitro susceptibility test of micafungin (MCFG), we evaluated the methods of such testing, focusing on the judgment method of MIC, based on the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2, M38-A, the proposed standards of The Japanese Society for Medical Mycology (JSMM) for yeast (JSMM-Y) and for filamentous fungi (JSMM-F) against Candida spp. and Aspergillus spp.
The judgment of MIC value was performed spectrophotometrically and visually in both (NCCLS and JSMM) assays. Only the spectrophotometric MIC judgment against Aspergillus spp. in the NCCLS assay used two end points: 80% inhibitory concentration (IC80) of the growth control and 50% inhibitory concentration (IC50). The end point for the visual judgment against Aspergillus spp. in the NCCLS assay was determined to be no growth from the small clumps of altered hyphae in the microtiter plate. The other MIC judgments used an IC80 end point.
The MICs of MCFG for Candida spp. were ≤ 0.0039-1μg/ml in both judgments by the two assays. In contrast, there was a large difference of the MIC values against Aspergillus spp. between the NCCLS assay results using the IC80 end point and the visual judgment (> 4μg/ml and 0.0078-0.0313μg/ml). However, the MICs using the IC50 end point and those by JSMM assay agreed with the result of the visual assessment. Therefore, we recommend the JSMM assay, the NCCLS assay using the IC50 end point or the novel visual judgment for the susceptibility testing of MCFG against Aspergillus spp.

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Pseudallescheria boydii is a ubiquitous filamentous fungus. We report a case of cutaneous P. boydii infection of the left knee in a 79-year-old Japanese man who was receiving oral predonisolone (25 mg/day) for radiation pneumonitis after radiation therapy on left breast cancer. He presented with a 2-week-history of a lesion on the left knee. A biopsy specimen from the skin lesion revealed granulomatous inflammation with hyphae. Culture of the pus from the skin specimen confirmed the diagnosis of cutaneous P. boydii infection. rDNA ITS sequence was analyzed to confirm the mycological diagnosis. The patient was treated orally with 200 mg/day of itraconazole. The lesion was gradually cured and left a hypertrophic scar. Cutaneous injury may be responsible for an incidence of localized infection. Such rare fungus infection in immunocompromised patients who have a persistent traumatic skin ulcer needs to be ruled out. An opportunistic infection in immunocompromised patients can be life-threatening and prompt treatment based on accurate diagnosis is important.

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Dimethyl sulfoxide (DMSO) is commonly used as a solvent for antifungal drugs. Earlier the author has reported the inhibitory effect of DMSO on the growth of many strains of dermatophytes' colonies in dermasel agar and proposed that this could cause the variations between results of different studies for the evaluation of the activities of antifungal drugs. In studies regarding the determination of the effect of antifungal drugs on the germination of arthrospores of dermatophytes it was observed that relatively higher concentrations of DMSO were being used as a solvent for the antifungal drugs, the final concentration in the media being 5%. Therefore, the present study was aimed at determining the effect of different concentrations of DMSO (1.25 to 10%) on the growth of germ tubes of arthrospores of Trichophyton mentagrophytes and Candida albicans, in glucose peptone broth. With DMSO 10% there was a negligible growth of germ tubes of both the arthrospores and yeast; between 2.5 and 7.5% there was a rather linear dose-related inhibitory effect; whereas 1.25% had insignificant effect from controls. The present study shows that besides other factors, variations in the results of the susceptibility tests of antifungal drugs might occur due to the effect of DMSO on the growth of fungi and differences in the final concentration of DMSO in the medium.

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