(2) Echinocandin Susceptibility Testing. MATERIALS AND METHODS Organisms. We tested a total of 3,764 clinical isolates of Candida spp. obtained from ⬎100 medical centers worldwide (8, 9, 34). The collection included 2,010 isolates of C. albicans, 566 isolates of C. glabrata, 539 isolates of Candida parapsilosis, 426 isolates of C. tropicalis, 105 isolates of C. krusei, 52 isolates of Candida guilliermondii, 42 isolates of C. lusitaniae, and 24 isolates of Candida kefyr. All were incident isolates from individual patients and were obtained from blood or other normally sterile body fluids. Among the included isolates of C. albicans, C. glabrata, C. tropicalis, and C. krusei were 73 isolates (11 C. albicans, 4 C. tropicalis, 3 C. krusei, and 55 C. glabrata) with documented fks resistance mutations. The isolates were identified by use of the Vitek and API yeast identification systems (bioMérieux, Inc., Hazelwood, MO, USA) supplemented with conventional methods as needed (35). The isolates were stored as water suspensions until use. Prior to testing, each isolate was passaged at least twice on potato dextrose agar (Remel, Lenexa, KS, USA) and CHROMagar Candida (Becton, Dickinson, Sparks, MD, USA) to ensure purity and viability. The presence or absence of a mutation in the hot spot (HS) regions of fks1 and fks2 (C. glabrata only) was determined as described previously (36, 37). Antifungal susceptibility testing. All isolates were tested for in vitro susceptibility to caspofungin and micafungin using CLSI BMD methods (38, 39). The MIC results for the agents were read following 24 h of incubation. In all instances, the MIC values were determined visually as the lowest concentration of the drug that caused significant growth diminution levels (38, 39). We used the recently revised CBPs to identify the strains of the 6 most common species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. guilliermondii) that were susceptible (S), intermediate (I), or resistant (R) to caspofungin and micafungin (40, 41): caspofungin and micafungin MIC values of ⱕ0.25 g/ml, 0.5 g/ml, and ⱖ1 g/ml were considered to be susceptible, intermediate, and resistant, respectively, for C. albicans, C. tropicalis, and C. krusei, and MIC results of ⱕ2 g/ml, 4 g/ml, and ⱖ8 g/ml were categorized as susceptible, intermediate, and resistant, respectively, for C. parapsilosis and C. guilliermondii; caspofungin MIC values of ⱕ0.12 g/ml, 0.25 g/ml, and ⱖ0.5 g/ml and micafungin MIC values of ⱕ0.06 g/ml, 0.12 g/ml, and ⱖ0.25 g/ml were considered to be susceptible, intermediate, and resistant, respectively, for C. glabrata. In addition to the CBPs for these species, epidemiological cutoff values (ECVs) were established in order to provide a sensitive means of separating WT from non-WT strains (those that possess an intrinsic or acquired resistance mutation). The ECVs for caspofungin and micafungin for each species are 0.12 g/ml and 0.03 g/ml for both C. albicans and C. glabrata, 1 g/ml and 4 g/ml for C. parapsilosis,. January 2014 Volume 52 Number 1. 0.12 g/ml and 0.12 g/ml for C. tropicalis, 0.25 g/ml and 0.12 g/ml for C. krusei, and 2 g/ml and 2 g/ml for C. guilliermondii, respectively (41). CBPs have not been established for caspofungin and micafungin and less common species, such as C. lusitaniae and C. kefyr. The caspofungin and micafungin ECVs for these two species are 1 g/ml and 0.5 g/ml for C. lusitaniae and 0.03 and 0.12 g/ml for C. kefyr, respectively (41). Candida spp. isolates for which caspofungin or micafungin MICs exceed the ECVs are considered to be non-WT and may harbor acquired mutations in the fks genes (17). Quality control was performed as recommended in CLSI documents M27-A3 (39) and M27-S4 (38) using C. krusei strain ATCC 6258 and C. parapsilosis strain ATCC 22019. Analysis of results. All MIC results (in g/ml) for micafungin were directly compared with those for caspofungin by regression statistics and by scattergram (data not shown). The error rate bounding method to minimize intermethod interpretive error was also applied using the interpretive criteria described above. The acceptable error rate limits used in this comparison were those cited in CLSI document M23-A3 (40, 42) and in other studies (43, 44). The definitions of the errors used in this analysis are as follows: a very major error (VME), or a false-susceptible error, was a susceptible result for the surrogate marker micafungin and a resistant result for caspofungin; a major error (ME), or a false-resistant error, was a resistant result for micafungin and a susceptible result for caspofungin; and minor errors occurred when the result for one of the agents was susceptible or resistant and that for the other agent was intermediate. In general, for an agent to be considered a reliable surrogate marker, the VME rate should be ⱕ1.5% of all results, and the absolute categorical agreement (CA) between methods should be ⱖ90% (33, 40, 42). In addition to the above analysis, we will also examine the detectability of fks mutants of C. albicans and C. glabrata using the CBPs and ECVs for each echinocandin.. RESULTS AND DISCUSSION. Table 1 depicts the MIC distribution profiles for micafungin and caspofungin determined for 3,764 strains of Candida spp. using BMD methods validated by the CLSI (39). Overall, 3,694 isolates (98.2%) were susceptible, 20 isolates (0.5%) were intermediate, and 50 isolates (1.3%) were categorized as resistant to micafungin. Similarly, 3,682 isolates (97.8%) were susceptible, 20 isolates (0.5%) were intermediate, and 62 isolates (1.7%) were resistant to caspofungin. The modal MIC for micafungin was 0.015 g/ml (1,323 results [35.1%]) compared to 0.03 g/ml (1,362 results [36.2%]) for caspofungin. There was a strong positive correlation (r ⫽ 0.84) between the micafungin and caspofungin MIC values (data not shown). Overall, the essential agreement (MIC ⫾2 dilutions) was 97.2%. Decreased potencies of both micafungin and caspofungin were observed among C. parapsilosis (modal MICs, 1 g/ml and 0.5 g/ml, respectively) and C. guilliermondii (modal MICs, 0.25 g/ml and 0.5 g/ml, respectively). The highest rates of resistance to both agents were observed with C. glabrata: 5.8% were resistant to micafungin and 7.9% were resistant to caspofungin. Among the 33 isolates of C. glabrata that were resistant to micafungin, 30 isolates (90.9%) possessed a mutation in fks1 or fks2, and among 45 isolates that were resistant to caspofungin, 40 (88.9%) possessed a mutation in the fks gene (Table 1). The extent of cross-resistance between micafungin and caspofungin can be seen more clearly in Table 2. For the 3,694 isolates that were susceptible to micafungin, 3,672 (99.4%) were also susceptible to caspofungin. There were seven isolates that were susceptible to micafungin and resistant to caspofungin; of those, four isolates were C. glabrata, two isolates were C. guilliermondii, and one isolate was C. krusei, and two of the four C. glabrata isolates contained an fks mutation. Among the 50 isolates that were resis-. jcm.asm.org 109. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. were established by the CLSI (9), the extreme intra- and interlaboratory variations in caspofungin MIC results are of great concern (4, 11, 13). The fact that micafungin MIC values appear to be much less variable from laboratory to laboratory (M. A. Pfaller et al., unpublished data) suggests that this echinocandin may be the preferred reagent for the in vitro testing of echinocandins against Candida. Previously, we have shown that fluconazole may serve as a surrogate marker for evaluating the susceptibility of Candida to ravuconazole (32), voriconazole (33), and posaconazole (34). This same approach should be applicable to caspofungin, while the more reliable test reagent, micafungin, may be applied for the prediction of susceptibility and resistance to these echinocandins. In the present study, we utilized a large database of susceptibility test results, all determined by CLSI BMD methods and that included results for 73 fks mutant strains, to provide a robust analysis of cross-resistance between the two agents and additionally to examine the usefulness of micafungin as a surrogate marker for evaluating caspofungin susceptibility and resistance among WT Candida spp..
(3) Pfaller et al.. TABLE 1 MIC distributions of caspofungin and micafungin versus Candida spp., including strains with fks mutations using CLSI methods Candida sp. (no. of isolates tested). No. of isolates with an MIC (g/ml) (no. of isolates with fks mutation) of: 4. ⱖ8. 8 (8) 3 (3). 2 (2) 5 (5). 14 (13) 2 (2). 1. Echinocandins. 0.007. 0.015. 0.03. 0.06. 0.12. 0.25. 0.5. 1. 2. C. albicans (2,010). Caspofungin Micafungin. 33 177. 539 1,357. 892 375. 502 80 (1). 22 5 (1). 12 (2) 1. 3 (2) 4 (4). 4 (4) 8 (2). 3 (3) 3 (3). C. glabrata (566). Caspofungin Micafungin. 29. 34 437 (3). 273 (1) 39 (7). 189 (6) 14 (5). 16 (3) 14 (10). 9 (5) 11 (9). 14 (11) 10 (10). 7 (6) 2 (1). Caspofungin Micafungin. 1. 1 1. 1. 17. 35 2. 208 25. 219 93. 49 285. 7 133. C. tropicalis (426). Caspofungin Micafungin. 4 7. 137 126. 191 169. 82 101. 3 (1) 15 (1). 4 2. 2 (1) 5 (3). 2 (2). 1. Caspofungin Micafungin. 1 3. 44 75 (1). 32 15. 19 (1) 1. 6 1 (1). 2 (1). 9. Caspofungin Micafungin. 1. 1 2. 2 3. 4 4. 13 23. 22 18. 7 1. 1. 1 2. 20 25. 18 13. 2 2. C. krusei (105). C. guilliermondii (52). C. lusitaniae (42). Caspofungin Micafungin. C. kefyr (24). Caspofungin Micafungin. 3. 20. 1 9. tant to micafungin, 43 isolates (86.0%) were also resistant and seven isolates (14.0%) were susceptible to caspofungin. Similarly, 17 of the 20 (85.0%) isolates categorized as intermediate to micafungin were either intermediate or resistant to caspofungin. Thus, 99.4% of the micafungin-susceptible and 85.7% of the micafungin-nonsusceptible (NS) (I plus R) isolates were susceptible and nonsusceptible, respectively, to caspofungin. When the micafungin test result category (S, I, or R) was used to predict the caspofungin category, the absolute categorical agreement (CA) between the test results was 98.8%, with only a 0.2% VME (false-susceptible error) and ME (false-resistant error) rate and a 0.8% minor error rate (Table 3). Among the eight species of Candida tested, the CA was ⱖ92% (range, 92.4% to 100.0%) for all species. Generally, the discrepancies in the categorical results were minor errors. VMEs were seen more often with C. glabrata, C. krusei, and C. guilliermondii; however, only the VME rate involving C. guilliermondii (3.8%) exceeded the allowable VME rate of ⱕ1.5% (Table 3) (40, 42, 43). Clearly, it is important to detect those isolates of Candida that possess an acquired mutation in the fks gene (9, 17), and in that regard, micafungin performs quite well as a surrogate marker. Among all 73 fks mutant strains, 14 (19.2%) were susceptible to both caspofungin and micafungin, and 54 (74.0%) were intermediate or resistant to both, for an overall concordance of 93.2%. Furthermore, for the 11 isolates of C. albicans with a mutation in fks1, nine isolates (82.0%) were either intermediate or resistant to both micafungin and caspofungin and two isolates were susceptible to both agents (Tables 1 and 4). Using the micafungin ECV for C. albicans (0.03 g/ml), all 11 isolates would be classified as nonWT, indicating that they were likely to contain an acquired resistance mutation. There were a total of 55 isolates of C. glabrata that contained a mutation in fks1 or fks2 (Tables 1 and 4). Of these, 10 isolates (18.2%) were susceptible to both micafungin and caspo-. 110 jcm.asm.org. 1. 1 (1) 1 (1) 1. 2. 15. TABLE 2 Use of micafungin to predict susceptibility patterns of caspofungin employing 3,764 clinical isolates of Candida spp. from a global surveillance program. Candida sp. (no. of isolates tested). Micafungin susceptibility categorya. No. (%) of isolates in caspofungin categoryb: S. I. S I R. 1,994 (99.2). 1 (0.1) 2 (0.1). C. glabrata (566). S I R. 509 (90.0) 3 (0.5). 6 (1.1) 3 (0.5). C. parapsilosis (539). S I R. 538 (99.8). 1 (0.2). C. tropicalis (426). S I R. 420 (98.6). C. krusei (105). S I R. 96 (91.4). 6 (5.6). 1 (1.0) 1 (1.0) 1 (1.0). C. guilliermondii (52). S I R. 49 (94.2). 1 (2.0). 2 (3.8). C. lusitaniae (42). WT Non-WT. 42 (100.0). C. kefyr (24). WT Non-WT. 24 (100.0). C. albicans (2,010). a b. 6 (0.3). R. 2 (0.1) 5 (0.2) 4 (0.7) 8 (1.4) 33 (5.8). 1 (0.2) 4 (1.0). 1 (0.2). S, susceptible; I, intermediate; R, resistant; WT, wild type. MIC interpretive criteria for each species as shown in reference 41.. Journal of Clinical Microbiology. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. C. parapsilosis (539).
(4) Echinocandin Susceptibility Testing. TABLE 3 Absolute categorical agreement and error rates when the micafungin results were used to predict the caspofungin susceptibility of Candida spp. % ofa: CA. VME. ME. C. albicans (2,010) C. glabrata (566) C. parapsilosis (539) C. tropicalis (426) C. krusei (105) C. guilliermondii (52) C. lusitaniae (42) C. kefyr (24). 99.6 96.3 99.8 99.6 92.4 94.2 100.0 100.0. 0.0 0.7 0.0 0.0 1.0 3.8 0.0 0.0. 0.2 0.0 0.0 0.2 0.0 0.0 0.0 0.0. 0.2 3.0 0.2 0.2 6.6 2.0 0.0 0.0. All Candida spp. (3,764). 98.8. 0.2. 0.2. 0.8. a. CA, categorical agreement; VME, very major errors; ME, major errors.. fungin, 5 isolates (9.1%) were susceptible to micafungin and either intermediate or resistant to caspofungin, and 40 (72.7%) were intermediate or resistant to both agents (Table 4). The overall concordance between the two methods (testing of micafungin versus caspofungin) using the CLSI CBPs to classify fks mutant strains of C. glabrata as susceptible or nonsusceptible was 90.9%. Using the ECVs of 0.03 g/ml for micafungin and 0.12 g/ml for caspofungin to differentiate WT from non-WT strains of C. glabrata, nine strains (16.4%) were WT and 44 strains (80.0%) were non-WT for both agents (overall concordance, 96.4%). There were four strains of C. tropicalis and three of C. krusei that harbored an fks mutation. Of these, 3 strains of C. tropicalis (75.0%) and 2 strains of C. krusei (66.7%) were intermediate or resistant to both agents, whereas one strain of each species was susceptible to both agents. All of these would be considered non-WT using the micafungin ECV of 0.12 g/ml for each species. The most frequently encountered mutations in this collection occurred at position S663 (18 isolates), followed by F659 (nine isolates), S645 (seven isolates), and S629 (six isolates), and four isolates each contained mutations at positions F625, F641, and I634 (Table 4). Previous reports indicate that isolates of C. glabrata with the S663F mutation respond in vivo to high doses of either micafungin or caspofungin, whereas isolates with the S629P mutation fail to respond to even the highest dose of either agent (21). Notably, the pharmacodynamic target for the isolate with the S663F mutation was below the human area under the concentration curve (AUC) using standard doses of micafungin but not caspofungin, suggesting that differences in the echinocandin MIC values observed with individual fks mutations may be associated with differential antifungal activity in vivo (21). These findings are supported by those of Spreghini et al. (19), who found in a comparison between micafungin, caspofungin, and anidulafungin that micafungin showed the best in vitro and in vivo activities against two resistant mutants of C. glabrata bearing specific mutations in the fks2 HS region. Mutations at positions S663 and F659 in C. glabrata have been associated with breakthrough infections in patients receiving echinocandin therapy (22, 23, 27), whereas patients infected with C. glabrata strains containing the I1379V and I634V mutations (i.e., susceptible to both micafungin and caspofungin) tended to respond to therapy with either agent. January 2014 Volume 52 Number 1. MIC (g/ml) for: Candida sp.. fks mutation. Micafungin. Caspofungin. C. albicans. S629P S629P F641Y F641S S645P S645P S645P S645F S645Y D648Y P649H. 1 2 0.5 0.5 1 0.5 2 0.5 2 0.06 0.12. 2 2 1 0.5 1 1 2 0.5 1 0.25 0.25. C. glabrata. F625Y F625S F625S F625S S629P S629P S629P S629P, R631S L630I R631G D632Y D632E D632E I634V I634V I634V I634V F659S F659S F659S F659V F659V F659V F659V F659Y F659Y L662W L662W S663F S663F S663P S663P S663P S663P S663P S663P S663P S663P S663P S663P S663P S663P S663P S663P S663P S663Y. 0.03 0.12 0.5 0.5 1 0.5 4 16 0.015 0.06 0.015 0.25 0.25 0.03 0.03 0.015 0.03 0.06 0.06 0.06 0.25 0.12 0.25 0.25 0.12 0.25 0.5 0.25 0.12 0.12 0.25 4 2 2 0.5 16 0.5 0.12 4 0.5 0.12 4 0.5 0.5 4 0.5. 0.12 0.5 2 2 16 16 16 2 0.03 0.12 0.12 0.5 1 0.06 0.06 0.06 0.06 0.5 0.25 0.5 8 2 4 2 1 2 1 0.5 0.25 0.25 1 16 2 4 2 16 8 0.5 16 8 0.5 16 8 1 16 0.5 (Continued on following page). jcm.asm.org 111. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. Candida sp. (no. of isolates tested). Minor errors. TABLE 4 Isolates of C. albicans, C. glabrata, C. tropicalis, and C. krusei harboring fks mutations.
(5) Pfaller et al.. TABLE 4 (Continued) MIC (g/ml) for: Candida sp.. Micafungin. Caspofungin. L664R R665G R665S D666Y P667F delF658 I1379V I1379V P1371S. 0.25 0.12 0.12 0.06 0.12 2 0.03 0.03 0.03. 0.5 0.5 0.5 0.25 1 16 0.06 0.06 0.25. C. tropicalis. F641S F641S S645P S645P. 1 0.12 1 1. 1 0.12 2 2. C. krusei. F655C R1361G L701M. 0.5 2 0.06. 1 16 0.25. (22). Regarding the C. albicans mutants in this study, three of the 11 fks mutants contained the S645P mutation, a genotype that cannot be treated with conventional doses of micafungin or caspofungin (45). Taken together, these results suggest a linkage between the increased echinocandin MIC results, specific fks mutations, and the potential for a successful clinical outcome (19, 21, 22, 27). Previously, we conducted similar analyses using fluconazole results to predict the susceptibilities of Candida spp. to the newer triazoles as a proof of concept regarding the use of surrogate markers or class representatives for antifungal susceptibility testing (32–34). This strategy has been used for decades in antibacterial susceptibility testing to develop practical alternatives for the microbiology laboratory when specific diagnostic susceptibility reagents produce unreliable results or are limited or unavailable (40, 44). Given the issue of unacceptable variability in caspofungin MIC results, we concur with the recommendations of the EUCAST that the caspofungin MIC results for Candida spp. should not be used for clinical decision making at this time (10). As shown herein, MIC testing with micafungin is highly predictive of caspofungin categorical results, and it reliably detects clinically important fks mutations. In addition to providing a strategy for predicting caspofungin susceptibility and resistance among Candida spp., these results provide strong support for concerns regarding the issue of crossresistance between micafungin and caspofungin (5, 15, 16, 18, 22, 26). By using an extensive global collection of clinically important isolates, including fks mutant strains, we validate concerns originating from single-center case series and rightly focus attention on C. glabrata as the species most likely to demonstrate cross-resistance between these two studied echinocandins. Micafungin functioned well as a surrogate marker for caspofungin susceptibility when applied to this large collection of clinically significant isolates of Candida spp. The absolute CA of 98.8%, with only 0.2% VMEs among the 3,764 isolates tested, easily meets the recognized criteria for a reliable surrogate marker in antibacterial susceptibility testing (40, 44). The excellent con-. 112. jcm.asm.org. ACKNOWLEDGMENTS The global antifungal surveillance programs that served as the source of data used in the development of the manuscript were supported in part by Pfizer, Inc., and Astellas. We acknowledge the excellent technical assistance of S. Benning in the preparation of the manuscript. JMI Laboratories, Inc., received research and educational grants in 2011 to 2013 from AIReS, the American Proficiency Institute (API), Anacor, Astellas, AstraZeneca, Bayer, bioMérieux, Cempra, Cerexa, ContraFect, Cubist, Dipexium, Furiex, GlaxoSmithKline, Johnson & Johnson (J&J), LegoChem Biosciences, Inc., Meiji Seika Kaisha, Merck, Nabriva, Novartis, Pfizer, PPD Therapeutics, Premier Research Group, Rempex, Rib-X Pharmaceuticals, Seachaid, Shionogi, The Medicines Company, Theravance, and Thermo Fisher Scientific. Some JMI employees are advisors and/or consultants for Astellas, Cubist, Pfizer, Cempra, CerexaForest, J&J, and Theravance. These authors have no conflicts of interest with regard to speakers’ bureaus and stock options. D. Diekema has received research funding from Cerexa, bioMérieux, Pfizer, T2Biosystems, and PurThread, Inc. D. Diekema has no conflicts of interest with regard to speakers’ bureaus or consultant and/or advisor work.. REFERENCES 1. Cornely OA, Bassetti M, Calandra T, Garbino J, Kullberg BJ, Lortholary O, Meersseman W, Akova M, Arendrup MC, Arikan-Akdagli S, Bille J, Castagnola E, Cuenca-Estrella M, Donnelly JP, Groll AH, Herbrecht R, Hope WW, Jensen HE, Lass-Flörl C, Petrikkos G, Richardson MD, Roilides E, Verweij PE, Viscoli C, Ullmann AJ, ESCMID Fungal Infection Study Group. 2012. ESCMID guideline for the diagnosis and management of Candida diseases 2012: non-neutropenic adult patients. Clin. Microbiol. Infect. 18(Suppl 7):19 –37. http://dx.doi.org/10 .1111/1469-0691.12039. 2. Pappas PG, Kauffman CA, Andes D, Benjamin DK, Jr, Calandra TF, Edwards JE, Jr, Filler SG, Fisher JF, Kullberg BJ, Ostrosky-Zeichner L, Reboli AC, Rex JH, Walsh TJ, Sobel JD, Infectious Diseases Society of America. 2009. Clinical practice guidelines for the management of candi-. Journal of Clinical Microbiology. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. fks mutation. cordance between the micafungin and caspofungin results in categorizing the fks mutants also provides further validation of this approach. The major limitation of this study, given the interlaboratory variability of caspofungin MICs, is the fact that the data were obtained from only two laboratories. This may lead to concerns regarding the generalizability of the findings to other laboratories. These concerns are somewhat mitigated by the inclusion of a large number of fks mutants in the study. In conclusion, we have demonstrated the existence of crossresistance between micafungin and caspofungin, with the greatest emphasis on C. glabrata. Furthermore, we have shown that in the face of the unreliable caspofungin MIC results documented elsewhere, a prediction of caspofungin susceptibility in a medical center currently performing antifungal susceptibility testing of micafungin can be accomplished by using the micafungin result as a surrogate marker for caspofungin susceptibility and resistance. Arguably, the most important role of in vitro susceptibility testing is to predict the resistance of the infecting organism to the agent under consideration for use in a patient (46). The occurrence of false-resistant and false-susceptible errors with this application of the class representative concept to the echinocandin antifungal was low and was considered very acceptable for use as a surrogate marker. 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