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We tested a global collection of Candida sp. strains against anidulafungin, caspofungin, and micafungin, using CLSI M27-A3 broth microdilution (BMD) methods, in order to define wild-type (WT) populations and epidemiological cutoff values (ECVs). From 2003 to 2007, 8,271 isolates of Candida spp. (4,283 C. albicans, 1,236 C. glabrata, 1,238 C. parapsilosis, 996 C. tropicalis, 270 C. krusei, 99 C. lusitaniae, 88 C. guilliermondii, and 61 C. kefyr isolates) were obtained from over 100 centers worldwide. The modal MICs (in μg/ml) for anidulafungin, caspofungin, and micafungin, respectively, for each species were as follows: C. albicans, 0.03, 0.03, 0.015; C. glabrata, 0.06, 0.03, 0.015; C. tropicalis, 0.03, 0.03, 0.015; C. kefyr, 0.06, 0.015, 0.06; C. krusei, 0.03, 0.06, 0.06; C. lusitaniae, 0.05, 0.25, 0.12; C. parapsilosis, 2, 0.25, 1; and C. guilliermondii, 2, 0.5. 05. The ECVs, expressed in μg/ml (percentage of isolates that had MICs that were less than or equal to the ECV is shown in parentheses) for anidulafungin, caspofungin, and micafungin, respectively, were as follows: 0.12 (99.7%), 0.12 (99.8%), and 0.03 (97.7%) for C. albicans; 0.25 (99.4%), 0.12 (98.5%), and 0.03 (98.2%) for C. glabrata; 0.12 (98.9%), 0.12 (99.4%), and 0.12 (99.1%) for C. tropicalis; 0.25(100%), 0.03 (100%), and 0.12 (100%) for C. kefyr; 0.12 (99.3%), 0.25 (96.3%), and 0.12 (97.8%) for C. krusei; 2 (100%), 0.5 (98.0%), and 0.5 (99.0%) for C. lusitaniae; 4 (100%), 1 (98.6%), and 4 (100%) for C. parapsilosis; 16 (100%), 4 (95.5%), and 4 (98.9%) for C. guilliermondii. These WT MIC distributions and ECVs will be useful in surveillance for emerging reduced echinocandin susceptibility among Candida spp. and for determining the importance of various FKS1 or other mutations.
The members of the echinocandin class of antifungal agents (anidulafungin, caspofungin, and micafungin) are now well recognized as the preferred, systemically active antifungal agents for the treatment of invasive candidiasis (IC), including candidemia (19). The in vitro activity of these agents against Candida spp. is also well-known (17, 24), and the Clinical and Laboratory Standards Institute (CLSI) Antifungal Subcommittee has established a clinical breakpoint (CBP) for susceptibility of ≤2 μg/ml for all three agents and all species of Candida (3, 4, 25). Recently, however, it has become evident that Candida infections involving strains with mutations in FKS1 (encodes the echinocandin target) do not necessarily have MICs above this CBP (2, 5-8, 14, 28). Likewise, kinetic studies of the glucan synthesis enzyme complex suggest that a lower MIC cutoff of 0.5 μg/ml may be more sensitive in detecting those strains with FKS1 mutations (7, 8). Given these considerations, we have conducted global surveillance of Candida spp. by using CLSI broth microdilution (BMD) methods to ascertain the wild-type (WT) MIC distribution for the three echinocandins and the eight most common species of Candida causing bloodstream infections (BSI). This information allows us to establish epidemiological cutoff values (ECVs) that may be used to assess the emergence of strains with FKS1 mutations and the decreased susceptibility to these agents (10, 27, 30).
A total of 8,271 clinical isolates obtained from more than 100 medical centers worldwide from 2003 through 2007 were tested. The collection included 4,283 strains of Candida albicans, 1,236 of Candida glabrata, 1,238 of Candida parapsilosis, 996 of Candida tropicalis, 270 of Candida krusei, 99 of Candida lusitaniae, 88 of Candida guilliermondii, and 61 of Candida kefyr. All isolates were obtained from blood or other normally sterile sites and represented the incident isolate from individual infectious episodes. The isolates were collected at individual study sites and were sent to the University of Iowa (Iowa City) for identification and susceptibility testing as described previously (20-23). The isolates were identified by standard methods (9) and stored as water suspensions until used in the study. Prior to testing, each isolate was passaged at least twice onto potato dextrose agar (Remel) and Chromagar Candida medium (Becton Dickinson and Company, Sparks, MD) to ensure purity and viability.
Reference powders of anidulafungin, caspofungin, and micafungin were obtained from their respective manufacturers. Stock solutions were prepared in water (caspofungin and micafungin) or dimethyl sulfoxide (anidulafungin), and serial 2-fold 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 M27-A3 (3) by using RPMI 1640 medium, an inoculum of 0.5 × 103 to 2.5 × 103 cells/ml, and incubation at 35°C. MICs were determined visually, after 24 h of incubation, as the lowest concentration of drug that caused a significant diminution (≥50% inhibition) of growth below control levels (16, 20, 25).
The definitions of WT and ECVs were those outlined previously (10, 26, 29, 30). A WT organism is defined as a strain which does not harbor any acquired resistance to the particular antimicrobial agent being examined (29, 30). The typical MIC distribution for WT organisms covers three to four 2-fold dilution steps surrounding the modal MIC (1, 11). Inclusion of WT strains in the present study was ensured by testing only the incident isolate for each infectious episode.
The ECV for each echinocandin and each species of Candida was obtained as described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (10), by considering the WT MIC distribution, the modal MIC for each distribution, and the inherent variability of the test (usually within 1 log2 dilution). In general, the ECV should encompass at least 95% of isolates in the WT distribution (29, 30). Statistical determination of ECVs for each species and antifungal agent was performed as described previously (29). Organisms with acquired resistance mechanisms may be included among those for which the MICs are higher than the ECV (1, 10, 11, 26).
The WT MIC distributions for the three echinocandins and each of the eight species of Candida are shown in Table Table1.1. These distributions clearly show the very low MICs typical of WT strains of C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr and the higher MICs typical of C. parapsilosis, C. guilliermondii, and C. lusitaniae for all three echinocandins.
The modal MICs (percentage of isolates with MICs equal to the mode is shown in parentheses) for anidulafungin, caspofungin, and micafungin, respectively, and each species are as follows (Table (Table2):2): C. albicans, 0.03 μg/ml (36.0%), 0.03 μg/ml (47.6%), 0.015 μg/ml (68.9%); C. glabrata, 0.06 μg/ml (57.8%), 0.03 μg/ml (59.1%), 0.015 μg/ml (75.6%); C. tropicalis, 0.03 μg/ml (49.5%), 0.03 μg/ml (48.4%), 0.015 μg/ml (40.2%); C. krusei, 0.03 μg/ml (58.9%), 0.06 μg/ml (51.9%), 0.06 μg/ml (78.1%); C. kefyr, 0.06 μg/ml (50.8%), 0.015 μg/ml (77.0%), 0.06 μg/ml (49.2%); C. lusitaniae, 0.5 μg/ml (43.4%), 0.25 μg/ml (46.5%), 0.12 μg/ml (52.5%); C. parapsilosis, 2 μg/ml (61.8%), 0.25 μg/ml (44.0%), 1 (54.6%); C. guilliermondii, 2 μg/ml (36.4%), 0.5 μg/ml (36.4%), 0.5 μg/ml (35.2%).
The ECVs (percentage of isolates with MICs that were less than or equal to the ECVs is shown in parentheses) were calculated as described by Turnidge et al. (29), taking into consideration the WT MIC distributions and the inherent variability of the BMD test method, and were as follows for each species and anidulafungin, caspofungin, and micafungin, respectively (Table (Table2):2): 0.12 μg/ml (99.7%), 0.12 μg/ml (99.8%), and 0.03 μg/ml (97.7%) for C. albicans; 0.25 μg/ml (99.4%), 0.12 μg/ml (98.5%), and 0.03 μg/ml (98.2%) for C. glabrata; 0.12 μg/ml (98.9%), 0.12 μg/ml (99.4%), and 0.12 μg/ml (99.1%) for C. tropicalis; 0.25 μg/ml (100%), 0.03 μg/ml (100%), and 0.12 μg/ml (100%) for C. kefyr; 0.12 μg/ml (99.3%), 0.25 μg/ml (96.3%), and 0.12 μg/ml (97.8%) for C. krusei; 2 μg/ml (100%), 0.5 μg/ml (98.0%), and 0.5 μg/ml (99.0%) for C. lusitaniae; 4 μg/ml (100%), 1 μg/ml (98.6%), and 4 μg/ml (100%) for C. parapsilosis; and 16 μg/ml (100%), 4 μg/ml (95.5%), and 4 μg/ml (98.9%) for C. guilliermondii.
Compared to the CBP value of ≤2 μg/ml, the ECVs are between 8- and 66-fold lower for the three echinocandins and C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr (Table (Table2).2). Whereas the CBP encompasses 99.9% to 100% of the isolates of these five species, the ECVs of each agent encompass 96% to 100% of the isolates, highlighting the small number of isolates of each species that fall outside of the WT distribution yet remain susceptible to each agent according to the CBP. In contrast, the ECVs for the three less susceptible species, C. lusitaniae, C. parapsilosis, and C. guilliermondii, are similar to the CBPs for all three of the echinocandins.
Generally speaking, CBPs are used to indicate those isolates that are likely to respond to treatment with a given antimicrobial agent administered at the approved dosing regimen for that agent, whereas the ECV can be used as the most sensitive measure of the emergence of strains with reduced susceptibility to a given agent (10, 11, 27). Although organisms whose MICs exceed the ECV show reduced susceptibility compared with the WT population and may exhibit one or more acquired resistance mechanisms, they may yet respond to clinical treatment, as their MIC may lie below the CBP (27).
Although the various clinical trials have shown that each of the three echinocandins can be used to treat candidemia and IC due to isolates of Candida spp. for which MICs are as high as 2 μg/ml (12, 13, 15, 18, 25), several recent reports of clinical resistance to caspofungin therapy (Table (Table3),3), as well as studies of glucan synthase (GS) enzyme kinetics (6-8), suggest that the CBP of ≤2 μg/ml may need to be adjusted to predict both clinical resistance as well as the emergence of strains with FKS1 mutations. In each of the cases shown in Table Table3,3, clinical failure of caspofungin therapy was associated with FKS1 mutations and MICs for all three echinocandins that were elevated compared to the WT but not necessarily higher than the CBP of ≤2 μg/ml. Application of the ECVs in Table Table22 would have recognized these strains as non-WT and thus likely to contain an acquired resistance mutation.
It is evident that only a small number (<4%) of isolates of C. albicans, C. glabrata, C. tropicalis, and C. krusei fall outside of the respective ECVs for each of the three echinocandins (Tables (Tables11 and and2).2). Almost all would be classified as susceptible using the CBP criteria despite the possibility that they may have an acquired FKS1 mutation. The questions that must be answered are (i) what proportion of these isolates do in fact contain a target enzyme mutation and (ii) is the presence of a mutation that does not result in an MIC that is greater than the CBP meaningful or necessary to detect?
Garcia-Effron et al. (7, 8) demonstrated that clinically resistant isolates of C. albicans and C. glabrata with mutations in FKS1 and/or FKS2 showed elevated MICs and altered GS enzyme kinetics for all three echinocandins. Importantly, an MIC of >0.5 μg/ml identified those strains with resistant GS for anidulafungin, caspofungin, and micafungin.
Likewise, Wiederhold et al. (31) examined 12 strains of C. albicans for which the MICs of anidulafungin (MIC range, 0.12 to 1 μg/ml), caspofungin (MIC range, 2 to 8 μg/ml), and micafungin (MIC range, 0.5 to 4 μg/ml) were elevated relative to the control (WT) MIC for each agent (0.03 μg/ml, 0.125 μg/ml, and 0.06 μg/ml, respectively). All 12 isolates were found to contain mutations in FKS1; however, the MICs exceeded the CBPs for 0 of 12 strains with anidulafungin, 9 of 12 with caspofungin, and 2 of 12 with micafungin. In contrast, all 12 would have been considered to have reduced susceptibility to caspofungin and micafungin, and nine would have been considered to have reduced susceptibility to anidulafungin, using the ECVs shown in Table Table2.2. Unfortunately, no clinical data concerning these strains were presented by the authors.
Thus, the ECVs determined for C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr will be important in detecting the emergence of decreased susceptibility to the echinocandins in ongoing surveillance efforts. The CBPs for these agents may serve the same purpose for C. parapsilosis and C. guilliermondii but appear to be too insensitive to be of epidemiological value in monitoring the more susceptible species. Future studies must include molecular analysis of FKS1 and FKS2 for the mutant strains with values that fall between the ECV and CBP to better understand the frequency and clinical importance of such mutations. The establishment of the WT MIC distributions and ECVs for each echinocandin and species of Candida will be useful in resistance surveillance and may prove to be an important step in the development of species-specific CBPs for this important class of antifungal agents.
Caitlin Howard provided excellent support in the preparation of the manuscript. The input of Gunnar Kahlmeter is gratefully acknowledged.
This work was supported in part by grants from Astellas and Pfizer.
Published ahead of print on 18 November 2009.