We performed Etest and broth microdilution (BMD) susceptibility testing of caspofungin, micafungin and anidulafungin against 67 clinical isolates of Aspergillus spp. and 10 Fusarium spp. Minimal effective concentrations (MECs) by BMD were read after 24 h of incubation at 35 degrees C and Etest MICs were read at 24 and 48 h. MECs < or =0.25 mg/l were obtained with caspofungin for all Aspergillus spp. tested but Etest MICs were < or =1 mg/l at 24 h. The agreement between caspofungin Etest and broth microdilution was good for all Aspergillus species tested (range 82.4-100%) except for A. niger and A. glaucus at 24 h of incubation. Micafungin and anidulafungin MEC and MIC results were lower than those of caspofungin (< or =0.015 mg/l) at 24 and 48 h for all Aspergillus tested. The agreement between the methods was excellent (100%) for micafungin and anidulafungin for all Aspergillus species tested. The three echinocandins were inactive against all isolates of Fusarium spp. showing MECs and MICs >8 mg/l. The Etest method could be a suitable procedure to test the susceptibility of most Aspergillus species to caspofungin, micafungin and anidulafungin; the best agreement was at 24 h.

Cryptococcal meningitis is one of the leading causes of death in HIV/AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA microsatellites) and analysis of two rDNA regions (IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C. deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C. neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty-nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C. neoformans strains were able to kill G. mellonella larvae more efficiently than C. deneoformans and hybrids between both species. Precisely molecular characterisation of C. neoformans species is important for an accurate patient's management.

The antifungal drug susceptibility of 70 Cryptococcus neoformans isolates obtained in Spain from 1994 to 1996 (23 isolates) and from 1997 to 2005 (47 isolates) was investigated. The MICs of fluconazole, itraconazole and voriconazole were determined by the modified Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) broth microdilution method. The MIC₅₀ and MIC₉₀ for itraconazole and voriconazole did not change significantly from 1994 to 2005. The MIC₅₀ of fluconazole remained stable and the MIC₉₀ decreased by 2 log₂ dilution in the isolates collected from 1997 to 2005. We conclude that the in vitro resistance to fluconazole decreased over an 11-year period. In addition, a tendency for the development of possible cross-resistance between the three triazoles was observed.

The ability of Sensititre YeastOne to discriminate isolates with reduced caspofungin susceptibility was determined against 36 Candida spp. (6 with a known FKS1 mutation). Results were compared with those of M27-A2. The MIC endpoint was 100% growth inhibition. Overall agreement (؎2 log 2 ) was 87.16%. Sensititre YeastOne detected strains with reduced caspofungin susceptibility.The NCCLS proposed reference standard methodology (M27-A2) (13) to test susceptibility of Candida spp. is laborintensive and requires 48 h of incubation. The marketed Sensititre YeastOne (SYO) microdilution colorimetric method has proved to be efficient for susceptibility testing of Candida and Aspergillus spp. (6,11,12,18,21).Caspofungin is a semisynthetic echinocandin with a broad spectrum of in vitro activity (5,15,19,22) that acts to inhibit (1,3)-␤-D-glucan synthesis. Methods for susceptibility testing of caspofungin have not been standardized. To facilitate the susceptibility testing for clinical laboratories and avoid the drawbacks of the NCCLS method, we have assayed the capacity of SYO to detect isolates with reduced caspofungin susceptibility.(This research was presented in part at the 42nd International Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 27 to 30 September 2002 [abstr. 1496.)Five Spanish laboratories collaborated in this study. Every participant tested 34 encoded strains, 25 blood culture isolates (Table 1), and 9 Candida albicans strains, 6 with reduced caspofungin susceptibility (2 heterozygous and 4 homozygous for FKS1 gene mutation) provided by Merck Research Laboratories (Rahway, N.J.) (9). Candida krusei ATCC 6258 and C. albicans ATCC 90028 were included as a quality control (QC) strain (13, 14) and reference strain, respectively.Stock solutions of fluconazole (Pfizer, Spain), flucytosine (Roche, Spain), itraconazole (Janssen Pharmaceutica, Belgium), amphotericin B (Sigma-Aldrich, Spain), and caspofungin (Merck Sharp & Dome, Spain; batch no. L-743,872-003M023) were frozen and sent to each participant the same day to prepare their own trays and perform the susceptibility tests (within 3 weeks) following the NCCLS microdilution methodology (13). The same batch of RPMI 1640 (RPMI) and RPMI with 2% dextrose (RPMI-G) (Oxoid, Spain) was used. The final concentrations were 64 to 0.12 g/ml for fluconazole and flucytosine and 16 to 0.03 g/ml for caspofungin, amphotericin B, and itraconazole. SYO panels with amphotericin B, itraconazole, and caspofungin at final concentrations of 16 to 0.016 g/ml and fluconazole and flucytosine at final concentrations of 64 to 0.12 g/ml were prepared by Trek Diagnostics Systems (United Kingdom).MICs were determined at 24 and 48 h and after 24 h in RPMI-G for the antifungal agents other than caspofungin. For each strain, 30 caspofungin MICs were recorded (5 for each test condition). The MIC endpoints were 100% growth inhibition (MIC 0 ) for caspofungin and amphotericin B and Ն50% growth inhibition (MIC 2 ) for azoles and flucytosine. SYO MIC endpoints were interpre...

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