Optimal growth conditions for the determination of the antifungal susceptibility of three species of dermatophytes with the use of a microdilution method.

As a prerequisite to standardization of dermatophyte susceptibility testing, conditions that support optimal growth of different dermatophyte species must be established. Eighteen isolates of Trichophyton spp. (T rubrum, T mentagrophytes, T tonsurans) were grown in 4 different media: RPMI 1640 with L-glutamine, without sodium bicarbonate and buffered at pH = 7.0; antibiotic medium #3 (Penassay); yeast nitrogen base with 0.5% dextrose buffered at pH = 7.0; and Sabouraud dextrose broth. Incubation for 6 days at 35 degrees C produced the following results: RPMI and Sabouraud dextrose supported equally sufficient growth for all strains tested; Penassay supported growth of only 33% of the isolates tested, and buffered yeast nitrogen base did not support growth of any isolates. RPMI was selected as the optimal medium, and organisms were tested at both 30 degrees C and 35 degrees C with a standardized inoculum density of 10(3) conidia/mL. No temperature differences were noted in the amount of growth of the dermatophytes tested. With RPMI at an incubation temperature of 35 degrees C, 3 inoculum sizes (10(3), 10(4), and 10(5) conidia/mL) were tested against 4 antifungal agents: griseofulvin, itraconazole, terbinafine, and fluconazole. Inoculum size did not affect minimum inhibitory concentration (MIC) results for itraconazole or terbinafine, but a larger inoculum produced a slightly higher MIC for griseofulvin and a noticeably higher MIC for fluconazole. Our data support the use of RPMI 1640, 35 degrees C, and 4 days as an incubation temperature and time, respectively, and an inoculum of 10(3) conidia/mL as optimal conditions for the determination of the antifungal susceptibility of dermatophytes.

Multicenter comparison of the sensititre YeastOne Colorimetric Antifungal Panel with the National Committee for Clinical Laboratory standards M27-A reference method for testing clinical isolates of common and emerging Candida spp., Cryptococcus spp., and other yeasts and yeast-like organisms.

National Committee for Clinical Laboratory Standards (NCCLS) standard guidelines are available for the antifungal susceptibility testing of common Candida spp. and Cryptococcus neoformans, but NCCLS methods may not be the most efficient and convenient procedures for use in the clinical laboratory. MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined by the commercially prepared Sensititre YeastOne Colorimetric Antifungal Panel and by the NCCLS M27-A broth microdilution method for 1,176 clinical isolates of yeasts and yeast-like organisms, including Blastoschizomyces capitatus, Cryptococcus spp., 14 common and emerging species of Candida, Hansenula anomala, Rhodotorula spp., Saccharomyces cerevisiae, Sporobolomyces salmonicolor, and Trichosporon beigelii. Colorimetric MICs of amphotericin B corresponded to the first blue well (no growth), and MICs of the other agents corresponded to the first purple or blue well. Three comparisons of MIC pairs by the two methods were evaluated to obtain percentages of agreement: 24- and 48-h MICs and 24-h colorimetric versus 48-h reference MICs. The best performance of the YeastOne panel was with 24-h MICs (92 to 100%) with the azoles and flucytosine for all the species tested, with the exception of C. albicans (87 to 90%). For amphotericin B, the best agreement between the methods was with 48-h MIC pairs (92 to 99%) for most of the species tested. The exception was for isolates of C. neoformans (76%). These data suggest the potential value of the YeastOne panel for use in the clinical laboratory.