Hideaki Taguchi

Fungi in bathwater and sludge of bathroom drainpipes

Samples of bathwater from 14 homes and 22 public bathhouses and sludge in drainpipes from 19 house-hold bathrooms were plated out onto potato dextrose agar supplemented with chloramphenicol. Several media were used to study colony morphology of the isolates and the thermotolerance and alkaline tolerance of each isolate were examined.Eleven sludge samples produced 12 isolates of Exophiala jeanselmei, 2 of E. dermatitidis and 1 of E. moniliae. Five household bathwater samples produced 2 isolates of E. jeanselmei, 4 of E. dermatitidis and 1 of E. alcalophila. One isolate of E. jeanselmei, 2 of E. dermatitidis, 3 of E. moniliae and 2 of unidentified Exophiala species were recovered from 6 samples of the bathwater dissolving ‘Chinese medicine’ in the bathtubs of public bathhouses. One isolate of E. jeanselmei was recovered from the 15 samples of bathwater from public bathhouses. Bathwater and sludge in bathroom drainpipes may be an important habitat of Exophiala species.

Determination of ploidy in Cryptococcus neoformans by flow cytometry

We determined the ploidy of Cryptococcus neoformans (28 strains) isolated from patients and nature. The cellular DNA content of these strains, which stained with propidium iodide in comparison to that of two authentic haploid strains, was determined by flow cytometry. All the strains exhibited diphasic histograms. In case of the authentic haploid strains, the first peak was centred around channel 9, and the second peak around channel 18. Most strains exhibited this type of histogram. Some strains exhibited another type of histogram: the first peak was centred around channel 18, and the second one around channel 35. In flow cytometry, the channel number is correlated with the intensity of fluorescence, namely, in proportion to the channel number the DNA content in the cells increases. The cellular DNA content of the second type of histograms showed twice that of the authentic haploid strains, and thus, five of 28 isolates were concluded to be diploid, and the others haploid.

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 micro g/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.

The isolation of Cryptococcus neoformans from pigeon droppings and serotyping of naturally and clinically sourced isolates in China.

This is the first report on the isolation ofCryptococcus neoformans from pigeon droppings in China and their serotypes.C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified asC. neoformans by a commercially available kit for carbon source assimilation test and Christensens urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that onlyC. neoformans var.neoformans strains could be isolated from pigeon droppings, although the varietygattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells ofC. neoformans strains in same samples of pigeon droppings were observed.

Survival rates of some terrestrial microorganisms under simulated space conditions

In connection with planetary quarantine, we have been studying the survival rates of nine species of terrestrial microorganisms (viruses, bacteria, yeasts, fungi, etc.) under simulated interstellar conditions. If common terrestrial microorganisms cannot survive in space even for short periods, we can greatly reduce expenditure for sterilizing space probes. The interstellar environment in the solar system has been simulated by low temperature, high vacuum (77 k, 4 x 10(-6) torr), and protons irradiation from a Van de Graaff generator. After exposure to a barrage of protons corresponding to about 250 years of irradiation in solar space, Tobacco mosaic virus, Bacillus subtilis spores, Aspergillus niger spores and Clostridiun mangenoti spores showed survival rates of 82%, 45%, 28%, and 25%, respectively. Furthermore. pathogenic Candida albicans showed 7% survival after irradiation corresponding to about 60 years in space.

Human cryptococcosis: relationship of environmental and clinical strains of Cryptococcus neoformans var. neoformans from urban and rural areas.

Forty-five clinical and 55 environmental strains of Cryptococcus neoformans var. neoformans from São Paulo, Brazil, were tested for their susceptibilities to amphotericin B, fluconazole, itraconazole, and flucytosine by the broth microdilution method according to the National Committee of Clinical Laboratory Standards guidelines. Electrophoretic karyotypes analysis by counter-clamped homogeneous electrophoresis was used to compare their genetic relatedness. Molecular typing revealed three clinical profiles very similar to two environmental profiles and an identical environmental and clinical profile. The results showed that human cryptococcosis can be acquired from environmental strains, which had similar minimum inhibitory concentration values to clinical strains, for antifungal agents.

Evaluation of skin test for chromoblastomycosis using antigens prepared from culture filtrates ofFonsecaea pedrosoi, Phialophora verrucosa, Wangiella dermatitidis andExophiala jeanselmei

Antigenic substances were prepared from culture filtrates ofFonsecaea pedrosoi, Phialophora verrucosa, Wangiella dermatitidis andExophiala jeanselmei. These antigenic substances were evaluated for detecting cutaneous delayed hypersensitivity in rats experimentally-infected withF. pedrosoi, P. verrucosa, W. dermatitidis, E. jeanselmei, Cladosporium carrionii andFonsecaea compactum and in patients with chromoblastomycosis caused byF. pedrosoi. TheF. pedrosoi antigen elicited positive reactions in all of the animals infected withF. pedrosoi and in 5 of 6 patients. TheP. verrucosa, W. dermatitidis andE. jeanselmei antigens elicited positive reactions in all of the animals infected with the homologous species. These antigens displayed cross-reactivity in some of the animals and patients, whereas more than half of them exhibited positive reactions only to the antigens prepared from the homologous species. These results suggest that a delayed-type skin test using the antigens prepared by the authors may be useful not only for the diagnosis of chromoblastomycosis but also for the identification of species of the causative agent.

Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC)

In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusarium solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusarium genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1α gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both assays were shown to be extremely sensitive even when fungal cells were mixed with human cells, detecting 3 germinated conidia spiked in 3mL of human blood. To apply our new real-time PCR system to the molecular diagnosis of fusariosis, we evaluated its efficacy using a mouse model of invasive F. solani infection. Plasma and whole blood samples of infected mice were tested using the real-time PCR system. The sensitivity of the real-time PCR system was found to be 100% (n=4) in plasma samples. In contrast, no amplification signal was detected in whole blood samples. This system could provide a rapid and precise diagnostic tool for early diagnosis, which is necessary for appropriate treatment and improvement of prognosis of disseminated fusariosis.

In Vitro Antifungal Activity of Epigallocatechin 3-O-Gallate against Clinical Isolates of Dermatophytes

Previously, we reported that epigallocatechin 3-O-gallate (EGCg) has growth-inhibitory effect on clinical isolates of Candida species. In this study, we investigated the antifungal activity of EGCg and antifungal agents against thirty-five of dermatophytes clinically isolated by the international guidelines (M38-A2). All isolates exhibited good susceptibility to EGCg (MIC50, 2-4 µg/mL, MIC90, 4-8 µg/mL, and geometric mean (GM) MICs, 3.36-4 µg/mL) than those of fluconazole (MIC50, 2-16 µg/mL, MIC90, 4-32 µg/mL, and GM MICs, 3.45-25.8 µg/mL) and flucytosin (MIC50, MIC90, and GM MICs, >64 µg/mL), although they were less susceptible to other antifungal agents, such as amphotericin B, itraconazole, and miconazole. These activities of EGCg were approximately 4-fold higher than those of fluconazole, and were 4 to 16-fold higher than flucytosin. This result indicates that EGCg can inhibit pathogenic dermatophyte species. Therefore, we suggest that EGCg may be effectively used solely as a possible agent or combined with other antifungal agents for antifungal therapy in dermatophytosis.

Antifungal susceptibility of Aspergillus fumigatus clinical isolates collected from various areas in Japan

Azole resistance among clinical isolates of Aspergillus fumigatus is becoming a serious problem in Europe, but the status in Japan is not yet known in detail. The aim of this study was to determine the present status of azole resistance in A. fumigatus in Japan. We employed 171 clinical isolates of A. fumigatus sensu stricto collected from 1987 to 2008 at the Medical Mycology Research Center, Chiba University, Japan for azole resistance determination. Identification of all isolates were re-examined both from the aspect of morphology and molecular phylogeny. The antifungal susceptibility of these isolates was tested based on the CLSI M38-A2 broth microdilution method. In our collection, only 1 (0.6%) and 2 isolates (1.2%) showed elevated MIC to voriconazole and itraconazole, respectively. Our study disclosed that the frequency of azole resistance in A. fumigatus still remains low in this collection.