[Mycological examination of domestic unpolished rice and mycotoxin production by isolated Penicillium islandicum].

Fungi growing on domestic rice were examined from April to June, 2003. One hundred samples of rice, which had been harvested in the autumn of 2002, were collected from the local market, and 15 samples of stored rice, which had been harvested in 2001 and stored in warehouses under government control, were used as samples. From each sample, 50 grains (100 grains in total) were plated on potato-dextrose agar (PDA) and malt yeast 40% sucrose agar (M40YA) containing chloramphenicol after being washed with sterile distilled water to remove any microorganisms on the surface, and incubated at 25 degrees C for a week. For most of the rice samples harvested in the preceding year, the proportion of grains infected with fungi was less than 20% of the total grains tested. In about half the samples of rice stored for one and half years, more than 80% of the grains were infected with fungi that grew on M40YA. The major genera of fungi isolated from the rice harvested in the preceding year were Penicillium and Alternaria, and those from the rice stored for one and a half years were Aspergillus, Penicillium and Eurotium. P. islandicum, A. versicolor, A. ochraceus and others were isolated as possible mycotoxin-producers in the mycoflora of domestic rice. P. islandicum was isolated from 3 samples, and 82% of the grains were infected with this fungus in one sample. All three isolates from these samples appeared to produce luteoskyrin on Czapek yeast extract agar, based on TLC and HPLC analysis.

Multicenter evaluation of commercial frozen plates for microdilution broth antifungal susceptibility testing of yeasts and comparison of MIC limits recommended in NCCLS M27-A2.

A commercial kit, Frozen Plate for Antifungal Susceptibility Testing of Yeasts, Eiken (Eiken Chemical Co., Ltd., Tokyo), was tested in a multi-institute study to evaluate the agreement between interinstitute MICs and National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 recommendation limits of MIC value. The kit was reported as a method equivalent to the standardized guidelines for antifungal susceptibility testing by the Committee for Clinical Laboratory Standards-1994, the Japanese Society for Medical Mycology, and which is widely used in Japan for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole. The degrees of inter-institute and NCCLS agreements were good to excellent especially with 48-hr incubation for all antifungal agents. However, the percent agreements to NCCLS recommendations against itraconazole were poor. Overall, MIC values obtained using the frozen plate antifungal susceptibility testing kit, with 48-hr incubation, were thought to be reliable and convenient alternatives to the data obtained by the NCCLS M27-A2 reference macrodilution and microdilution method. This kit will allow matching of results between international laboratories. However, the MIC value for itraconazole requires careful interpretation.

Comparative evaluation of standard dilution method and commercial kit for frozen plate antifungal susceptibility testing of yeasts using 200 clinical isolates.

A comparative evaluation of standard microdilution methods and a commercial kit for frozen plate antifungal susceptibility testing of yeasts was performed using amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole on 200 yeast isolates. The isolates included 100 strains of Candida albicans, eight of C. tropicalis, twelve of C. parapsilosis, eight of C. glabrata, five of Cryptococcus neoformans, thirteen of Trichosporon asahii, and 54 other strains of seven other species of ascomycotic yeasts. Microdilution testing was performed according to the standard method for antifungal susceptibility testing published by the Japanese Society for Medical Mycology (JSMM), which are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) M27-P. The commercial kit was prepared according to the manufacturer's instructions. The degree of agreement within +/-1 dilution for 200 clinical isolates against five antifungal agents was excellent with values for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole of 100%, 99.0%, 97.5%, 97.0%, and 97.0%, respectively. Overall, the frozen plate antifungal susceptibility testing kit provided convenient and reproducible results comparable to those obtained with the JSMM standard method.

In-situ detection of Aspergillus fumigatus.

An in-situ hybridisation (ISH) technique to detect Aspergillus fumigatus in infected tissues was developed in which 568-bp, 333-bp and 154-bp PCR products of the alkaline proteinase gene were employed. Dot-blot hybridisation with the 568-bp probe on a membrane containing genomic DNA from several different fungi including A. flavus, A. niger, Penicillium spp., Mucor racemosus or Pseudallescheria boydii gave negative results. ISH was done on formalin-fixed, paraffin-embedded pulmonary tissues from rats infected with A. fumigatus and renal tissues from mice infected with A. fumigatus, A. flavus or A. niger. The 568-bp probe reacted strongly in ISH with both A. fumigatus and A. flavus, and weakly with A. niger. The 333-bp probe also reacted in ISH with A. fumigatus and A. flavus, although the intensity was weaker. However, in ISH with the 154-bp probe, there was no positive signal with any Aspergillus spp. These results demonstrate that A. fumigatus and A. flavus can be specifically detected in infected tissues by ISH with the 568-bp probe. This technique could be applicable to clinical specimens for molecular diagnosis of aspergillus infections.