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.

Three new combinations from the Cryptococcus laurentii complex: Cryptococcus aureus, Cryptococcus carnescens and Cryptococcus peneaus.

Fifteen strains of the Cryptococcus laurentii complex were reclassified based on sequence analyses of 18S rDNA, the D1/D2 region of the 26S rDNA and the internal transcribed spacer regions, as well as physiological and biochemical properties. The strains were divided into phylogenetic groups I and II. The type strain of C. laurentii (CBS 139T=ATCC 18803T=JCM 9066T=MUCL 30398T=NRRL Y-2536T) was in phylogenetic group I. Phylogenetic group II, which was phylogenetically distant from phylogenetic group I, clustered with Cryptococcus dimennae and Bullera globispora. In phylogenetic group I, the type strain of Torula aurea (CBS 318T=ATCC 32063T=IFO 0372T=NRRL Y-1582T) appeared to be a separate species from C. laurentii, and the designation Cryptococcus aureus comb. nov. is proposed for Torula aurea. Cryptococcus flavescens (formerly Torula flavescens, type strain CBS 942T=ATCC 10668T=DBVPG 6007T=MUCL 30414T) was treated as a synonym of C. laurentii; however, this is a distinct species. The type strains of Torulopsis carnescens (CBS 973T=ATCC 32064T=MUCL 30641T=NRRL Y-1503T), Rhodotorula peneaus (CBS 2409T=ATCC 13546T=MUCL 30643T=NRRL Y-2005T) and Cryptococcus victoriae belonged to phylogenetic group II. Two new combinations, Cryptococcus carnescens comb. nov. (type strain CBS 973T=ATCC 32064T=MUCL 30641T=NRRL Y-1503T) and Cryptococcus peneaus comb. nov. (type strain CBS 2409T=ATCC 13546T=MUCL 30643T=NRRL Y-2005T), are proposed from this group.

Effects of melanin upon susceptibility of Cryptococcus to antifungals.

Melanin is a recognized virulence factor in Cryptococcus neoformans; several pathogenetic mechanisms have been suggested. We studied melanin as an antifungal resistance factor. The growth of laccase-active strains of C. neoformans and C. albidus in L-DOPA resulted in the production of black pigment. The formal minimal inhibitory concentrations (MICs) of amphotericin B and fluconazole were not changed by melanization. However, when we examined those wells which contained inhibited cells, we found live cells only in wells containing melanized C. neoformans. In contrast, melanization did not protect C. albidus from killing by amphotericin B. In an amphotericin B time-kill study of C. neoformans, significantly more melanized cells than non-melanized survived for the first few hours. Fluorescence microscopy and flow cytometry analyses showed that fewer melanized cells were stained with the fluorescent dye MitoRed. Incubation of MitoRed (the model) or amphotericin B with melanin extracted from C. neoformans decreased the free concentrations of these substances. Fluconazole, in contrast, was not removed from solution by melanin. This suggests that neoformans cryptococcal melanin deposited amphotericin B in the cell wall binds, reducing its effective concentrations.

Sequence analysis of the ribosomal DNA intergenic spacer 1 regions of Trichosporon species.

We determined the sequence of the intergenic spacer (IGS) 1 region, which is located between the 26S and 5S rRNA genes, in 25 species of the genus TRICHOSPORON: IGS 1 sequences varied in length from 195 to 719 bp. Comparative sequence analysis suggested that the divergence of IGS 1 sequences has been greater than that of the internal transcribed spacer regions. We also identified five genotypes of T. asahii, which is a major causative agent of deep-seated trichosporonosis, based on the IGS 1 sequences of 43 strains. Most of the isolates that originated in Japan were of genotype 1, whereas the American isolates were of genotype 3 or 5. Our results suggest that analysis of IGS regions provides a powerful method to distinguish between phylogenetically closely related species and that a geographic substructure may exist among T. asahii clinical isolates.

Antifungal pharmacodynamic characteristics of amphotericin B against Trichosporon asahii, using time-kill methodology.

We determined the MIC of amphotericin B against 45 Trichosporon asahii isolates from various clinical and environmental sources, and used in vitro time-kill methods to characterize the relationship between amphotericin B concentrations and MIC for four representative T. asahii isolates. Amphotericin B had concentration-dependent antifungal activity. MICs ranged from 0.5 to 16 microg/ml, and most T. asahii isolates (76%, 34/45) were inhibited at safely achievable amphotericin B serum concentrations (< or = 2 microg/ml). However, 40% (18/45) of isolates were not killed at these concentrations (MFCs from 1.0 to 32 microg/ml). At concentrations > or = 2 x MIC, amphotericin B exhibited fungicidal activity (< 99.9% reduction in CFU) over a 12-hr time-period; the maximal effect was achieved at > or =4 x MIC. Susceptibility testing confirmed the resistance of T. asahii to amphotericin B, and in vitro pharmacodynamic results also suggest that amphotericin B is not suitable therapy for T. asahii infection.

Laccase and melanization in clinically important Cryptococcus species other than Cryptococcus neoformans.

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.

New yeast species, Malassezia dermatis, isolated from patients with atopic dermatitis.

Malassezia species are considered to be one of the exacerbating factors in atopic dermatitis (AD). During examination of the cutaneous colonization of Malassezia species in AD patients, we found a new species on the surface of the patients' skin. Analysis of ribosomal DNA sequences suggested that the isolates belonged to the genus MALASSEZIA: They did not grow in Sabouraud dextrose agar but utilized specific concentrations of Tween 20, 40, 60, and 80 as a lipid source. Thus, we concluded that our isolates were new members of the genus Malassezia and propose the name Malassezia dermatis sp. nov. for these isolates.