Sensititre YeastOne Caspofungin Susceptibility Testing of Candida Clinical Isolates: Correlation with Results of NCCLS M27-A2 Multicenter Study

Abstract

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...

Comparison of Three Statistical Methods for Establishing Tentative Wild-Type Population and Epidemiological Cutoff Values for Echinocandins, Amphotericin B, Flucytosine, and Six Candida Species as Determined by the Colorimetric Sensititre YeastOne Method

“…Table 3 shows the ECVs obtained for the SYO method, determined by the different above-mentioned methods (three statistical methods, those using two dilutions above the MIC 50 and modal MIC, and the median ECV of the five methods) and those published by Pfaller et al for the CLSI. In general, the median ECVs for the SYO method were very similar to those for the CLSI method: 82.1% of ECVs were equal to or within one 2-fold dilution of those reported for the CLSI method, in agreement with the MIC values for contrasting these two methods (3,21). The greatest difference was for C. tropicalis, for which the ECVs of AND were 3 dilutions higher for the SYO method.…”

“…Table 1 shows the MIC values of echinocandins for C. albicans strains with fks mutations. The in vitro activities of the antifungal agents tested, which have been reported elsewhere (16), were similar to those observed by other authors using the SYO method (1,3,7,21). Table 2 depicts the WT MIC distributions of the five antifungal agents tested.…”

“…methods in each center, stored in a water suspension, and sent to the reference center (Hospital Universitario La Fe, Valencia, Spain) for posterior studies. Four C. albicans strains with reduced CAS susceptibility (2 heterozygous and 2 homozygous for FKS1 gene mutation) were included to assess the ability of the methods to identify non-WT isolates (3,5,13).…”

Paracoccidioides brasiliensis: chemical and molecular tools for research on cell walls, antifungals, diagnosis, taxonomy

“…MICs for caspofungin in the pathogenic yeastlike phases of several dimorphic fungi, fluctuate according to species and strains within species, e.g., B. dermatitidis (0.5-8 lg/ ml) or H. capsulatum (<0.125 lg/ml; 0.5-4 lg/ml) [65]. On the other hand, values for Candida, a socalled caspofungin-sensitive genus, also shows ranges of action, e.g., C. albicans (0.03->16 lg/ml), C. krusei (0.03-2 lg/ml), C. glabrata (0.03-2 lg/ ml), C. tropicalis (0.06-1 lg/ml), or C. parapsilosis (0.5-16 lg/ml) [66]. As deduced by a large-scale study carried out simultaneously in 17 American and European laboratories, under strictly controlled identical conditions [67], considerable interlaboratory variability of MIC data on caspofungin are found when testing Candida and Aspergillus isolates, results that suggest the need for careful interpretation of the antifungal effect of this drug.…”

The Resistant-Population Cutoff (RCOFF): a New Concept for Improved Characterization of Antimicrobial Susceptibility Patterns of Non-Wild-Type Bacterial Populations

“…Epidemiological cutoffs (ECOFFs), as defined by the European Committee for Antimicrobial Susceptibility Testing (EUCAST), separate wild-type from non-wild-type populations, reviving the concept of microbiological breakpoints (4)(5)(6)(7)(8)(9). EUCAST uses the ECOFF as one of several tools in the process of CBP setting.…”