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We determined the in vitro activities of anidulafungin, caspofungin, and micafungin against 526 isolates of Aspergillus spp. (64 A. flavus, 391 A. fumigatus, 46 A. niger, and 25 A. terreus isolates) collected from over 60 centers worldwide from 2001 through 2007. Susceptibility testing was performed according to the CLSI M38-A2 method. All three echinocandins—anidulafungin (50% minimum effective concentration [MEC50], 0.007 μg/ml; MEC90, 0.015 μg/ml), caspofungin (MEC50, 0.015 μg/ml; MEC90, 0.03 μg/ml), and micafungin (MEC50, 0.007 μg/ml; MEC90, 0.015 μg/ml)—were very active against Aspergillus spp. More than 99% of all isolates were inhibited by ≤0.06 μg/ml of all three agents.
The echinocandins anidulafungin, caspofungin, and micafungin are a group of recently introduced systemically active antifungal agents that inhibit the (1,3)-β-d-glucan synthase activity of Candida and Aspergillus spp. (3, 7, 12). Although each of these agents has been studied clinically for treatment of invasive aspergillosis (IA) (5, 10) (Pfizer, Inc., unpublished data [http://clinicaltrials.gov/ct2/show/NCT00531479]), only caspofungin has been approved for treatment of IA in patients refractory to or intolerant of other licensed antifungal agents (10, 17).
All three echinocandins are considered to have broad in vitro activity against most species of Aspergillus (1, 6, 16). As patient exposure to echinocandins broadens, however, the number of infecting strains with reduced susceptibility may increase (2, 13). Indeed, sporadic treatment failures or breakthrough infections consistent with clinical resistance have been documented in association with so-called high-minimum-effective-concentration (high-MEC) isolates (i.e., isolates for which the MECs range from 0.25 to 8 μg/ml) (2, 9, 15). These observations underscore the importance of antifungal susceptibility testing of echinocandins for activity against Aspergillus spp. in order to detect unusual resistance profiles as these agents are used more often worldwide.
Whereas the relative activities of these agents against Candida spp. have been well studied (14), there is a lack of head-to-head comparisons of the in vitro activities of all three of these agents against Aspergillus spp. (1).
We provide a unique head-to-head comparison of all three clinically available echinocandins by using Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) methods for a global collection of 526 clinical isolates of Aspergillus spp.
Between January 2001 and December 2007, 526 unique patient isolates of Aspergillus spp. (64 A. flavus species complex, 391 A. fumigatus species complex, 46 A. niger species complex, and 25 A. terreus species complex isolates) were obtained from more than 60 different medical centers worldwide for testing against the three echinocandins. The isolates were obtained from a variety of sources, including sputum, bronchoscopy, and tissue biopsy specimens. All isolates were identified using standard microscopic morphology determination (8) and were stored as spore suspensions in sterile distilled water at room temperature until they were used in the study. Before being tested, each isolate was subcultured on potato dextrose agar (Remel, Lenexa, KS) to ensure viability and purity.
Reference powders of anidulafungin (Pfizer), caspofungin (Merck), and micafungin (Astellas) were obtained from their respective manufacturers. Stock solutions were prepared in water (caspofungin and micafungin) or dimethyl sulfoxide (anidulafungin), and serial twofold dilutions in RPMI 1640 medium (Sigma, St. Louis, MO) buffered to pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid) buffer (Sigma) were made.
BMD testing was performed in accordance with the guidelines in CLSI document M38-A2 (4) by using RPMI 1640 medium, an inoculum of 0.4 × 104 to 5 × 104 CFU/ml, and incubation at 35°C. The MEC was determined, after 48 h of incubation, as the lowest concentration of drug at which short, stubby, and highly branched hyphae were observed (4, 11). Quality control was ensured by testing the following strains, as recommended in M38-A2 (4): Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, and A. flavus ATCC 204304.
The MEC distributions for each of the three echinocandins and the four species of Aspergillus are shown in Table Table1.1. First of all, it should be noted that all three echinocandins demonstrate excellent potency and spectra, with more than 99% of all isolates inhibited by ≤0.06 μg/ml of all three agents. The MEC50 and MEC90 values for all isolates combined were 0.007 μg/ml and 0.015 μg/ml, respectively, for anidulafungin and micafungin and 0.015 μg/ml and 0.03 μg/ml, respectively, for caspofungin. The results by species complex (expressed as percentages of isolates inhibited by ≤0.06 μg/ml of anidulafungin, caspofungin, and micafungin, respectively) were as follows: for the A. flavus species complex, 100%, 100%, and 100%; for the A. fumigatus species complex, 100%, 99%, and 100%; for the A. niger species complex, 100%, 100%, and 100%; and for the A. terreus species complex, 100%, 100%, and 100%.
The results of this study constitute the largest head-to-head comparison of the in vitro activities of anidulafungin, caspofungin, and micafungin against Aspergillus spp. that has been reported to date. Antachopoulos et al. (1) have compared the MECs and inhibitions of metabolic activity for the three echinocandins for a much smaller collection of isolates of Aspergillus spp. (27 isolates), using both germinated and nongerminated conidia. They found that anidulafungin exhibited the lowest MECs and that caspofungin exhibited the highest MECs for nongerminated conidia. This difference was minimized when germinated conidia were tested. There was a significant correlation between the degrees of maximal metabolic inhibition caused by the different echinocandins at both the species level (greater inhibition for A. flavus) and the strain level. Furthermore, for each drug and species, the maximal metabolic inhibition values obtained for germinated and nongerminated conidia did not differ significantly, suggesting that the degree of metabolic inhibition induced by the echinocandins was not significantly altered in the presence of germinated conidia in comparison to that in the presence of nongerminated conidia (1).
The CLSI M38-A2 BMD uses a nongerminated conidial inoculum and as such supports the findings of Antachopoulos et al. (1), showing excellent and broad-spectrum activity of all three echinocandins against a large collection of Aspergillus isolates. Both anidulafungin and micafungin were slightly more active than caspofungin; however, 99% to 100% of all isolates were inhibited at the low MEC of ≤0.06 μg/ml by all three agents. The MECs of all agents tended to be slightly higher (1 log2 dilution) for the A. fumigatus species complex than for the other three species.
In summary, we have performed a head-to-head challenge of anidulafungin, caspofungin, and micafungin against a large, globally diverse collection of Aspergillus species isolates by using the CLSI M38-A2 BMD method. The results of the study demonstrate the comparable and excellent levels of inhibitory activity of the three agents and the distinct lack of isolates with significantly decreased susceptibility to one or more of the echinocandins. These data provide a baseline level of in vitro activity of these agents against Aspergillus spp. that may be used to add perspective to other studies of clinical and in vitro echinocandin activity. For example, Madureira et al. (9) reported four cases of breakthrough IA in patients undergoing empirical or prophylactic therapy with caspofungin for which MECs for all three echinocandins were obtained. The MECs for caspofungin ranged from 0.25 μg/ml to 8 μg/ml, those for anidulafungin were 0.125 μg/ml, and those for micafungin ranged from 0.25 μg/ml to 4 μg/ml. In each case, the MECs for each of the echinocandins were outside the MEC distributions shown in Table Table1,1, with the greatest deviations seen with caspofungin. Continued surveillance using the CLSI BMD method is warranted to monitor the activities of these agents against Aspergillus spp. and to detect those unusual isolates with reduced susceptibility for further study.
We thank Caitlin Howard for excellent secretarial support.
This study was supported in part by research and educational grants from Astellas, Merck, and Pfizer.
Published ahead of print on 26 August 2009.