Caspofungin Etest Susceptibility Testing of Candida Species: Risk of ...

Caspofungin Etest Susceptibility Testing of Candida Species: Risk of Misclassification of Susceptible Isolates of C. glabrata and C. krusei when Adopting the Revised CLSI Caspofungin Breakpoints Maiken Cavling Arendrup,a Michael A. Pfaller,b and the Danish Fungaemia Study Group Unit of Mycology, Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen, Denmark,a and JMI Laboratories and the University of Iowa, Iowa City, Iowa, USAb

The purpose of this study was to evaluate the performance of caspofungin Etest and the recently revised CLSI breakpoints. A total of 497 blood isolates, of which 496 were wild-type isolates, were included. A total of 65/496 susceptible isolates (13.1%) were misclassified as intermediate (I) or resistant (R). Such misclassifications were most commonly observed for Candida krusei (73.1%) and Candida glabrata (33.1%). The revised breakpoints cannot be safely adopted for these two species.

T

he CLSI breakpoints for the three echinocandins have been revised (16). The motivation behind the change was emerging data suggesting that the initial breakpoint defining susceptibility (S) for all Candida species and echinocandins (ⱕ2 mg/liter) failed to identify isolates with FKS hot spot gene mutations as resistant (1, 2, 4, 6, 9–11, 14, 16). Such isolates have recently been associated with a decreased response rate in animal models (1, 19, 20, 22) and with a higher risk of failure in clinical practice (1, 8, 12, 17). EUCAST has established breakpoints for anidulafungin and recommends anidulafungin MIC testing as a marker for the echinocandin class of drugs (3). EUCAST has not set caspofungin breakpoints and does not currently recommend caspofungin MIC testing for clinical decision making. This is due to unacceptably high variation among caspofungin MIC values from different centers which, at least in part, has been linked to high variability in the performance of different lots of pure caspofungin substance (1, 3, 4). Whereas the CLSI and EUCAST reference methodologies, including the associated breakpoints, have been carefully validated using wild-type and mutant isolates, it is less well understood how these breakpoints perform when applied on commercial susceptibility tests. We have recently shown that by using caspofungin Etest and the revised CLSI breakpoints, an FKS1 mutant isolate

was successfully identified in a Swedish collection of blood isolates (5). However, it was noted that the caspofungin MICs for Candida glabrata and Candida krusei, as detected by Etest, were very close to the susceptibility breakpoint, suggesting a risk of misclassification of susceptible isolates among these two species (5). We therefore investigated the performance of caspofungin Etest using a collection of Danish blood isolates, including a higher number of C. glabrata and C. krusei isolates. The isolates were, with one exception, all classified as echinocandin susceptible using the EUCAST method and anidulafungin testing as a marker for echinocandin susceptibility (3, 18). A total of 497 blood isolates were included (278 Candida albicans, 9 Candida dubliniensis, 136 C. glabrata, 26 C. krusei, 24 Can-

Received 15 February 2012 Returned for modification 26 March 2012 Accepted 27 April 2012 Published ahead of print 7 May 2012 Address correspondence to Maiken Cavling Arendrup, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.00355-12

TABLE 1 Susceptibility of 497 Candida isolates to caspofungin as determined by Etest compared to susceptibility to anidulafungin as determined by the EUCAST reference microdilution methodology EDef7.1

* The dotted line symbolizes the EUCAST breakpoint separating isolates categorized as anidulafungin (and, hence, echinocandin) S and R (3). The gray shading indicates the intermediate category defined by CLSI (caspofungin Etest endpoints) (16). CNT, concentration not tested. ** For anidulafungin, the EUCAST breakpoints are listed (3). For caspofungin, the upper limit of the wild-type Etest endpoint distribution (WT-UL) obtained in this study is shown. *** Species-specific breakpoints have not been established for C. dubliniensis; however, as this species is very similar to C. albicans and has not been differentiated from this species in the data set underlying the selection of breakpoints, we have applied the C. albicans breakpoints for C. dubliniensis in this study.

July 2012 Volume 56 Number 7

Antimicrobial Agents and Chemotherapy

p. 3965–3968

aac.asm.org

3965

Arendrup et al.

FIG 1 Caspofungin MIC distributions obtained by Etest (above the x axis) compared to caspofungin MICs obtained by CLSI M27-A3 (below the x axis) for each of the five common Candida species that were used for the CLSI breakpoint selection process (14).

dida parapsilosis, and 24 Candida tropicalis). The isolates were obtained from unique candidemia episodes (January 2010 to March 2011) and were categorized as echinocandin susceptible or resistant using anidulafungin EUCAST EDef 7.1 reference microdilution MIC determination as recommended (3, 18). Anidulafungin breakpoints were as follows: for C. albicans, S ⱕ 0.03, resistance (R) ⬎ 0.03 mg/liter; for C. glabrata, C. krusei, and C. tropicalis, S ⱕ 0.06, R ⬎ 0.06 mg/liter (with no intermediate category) (3). Caspofungin Etest was performed as recommended by the manufacturer using RPMI 1640-2% glucose agar, and endpoints were read after 24 h of incubation. Endpoints in between the 2-fold dilution scale were adjusted to the nearest upper 2-log dilution for comparison. Isolates were classified as susceptible (S), intermediate (I), or resistant (R) using the following CLSI caspofungin breakpoints: for C. albicans, C. krusei, and C. tropicalis, S ⱕ 0.25, I ⫽ 0.5, R ⬎ 0.5 mg/liter; for C. glabrata, S ⱕ 0.125; I ⫽ 0.25, R ⬎ 0.25 mg/liter; and for C. parapsilosis, S ⱕ 2, I ⫽ 4, R ⬎ 4 mg/liter (16). The FKS gene was sequenced for isolates classified as anidulafungin resistant as previously described (5). Using the EUCAST anidulafungin susceptibility testing as the gold standard, 496/497 isolates were classified as echinocandin

3966

aac.asm.org

susceptible (Table 1) (3). By Etest, the caspofungin MIC50 and the upper limit of the wild-type Etest endpoint distribution (WT-UL) were, respectively, 0.06 and 0.25 mg/liter for C. albicans, C. dubliniensis, and C. tropicalis, 0.125 and 0.5 mg/liter for C. glabrata, 0.5 and 1 mg/liter for C. krusei, and 0.5 and 2 mg/liter for C. parapsilosis (Table 1). Application of the revised CLSI breakpoints for caspofungin to the Etest results found that 65/496 echinocandin-susceptible isolates (13.1%) were misclassified as I or R. Such misclassifications were most commonly observed for C. krusei (73.1% misclassified as I) and for C. glabrata (31.6% misclassified as I and 1.5% as R), whereas only a single C. albicans isolate (0.4%) was misclassified as I and no isolates belonging to the other species were misclassified. One C. tropicalis isolates was classified by EUCAST anidulafungin microdilution as echinocandin resistant (MIC of 0.25 mg/liter) and found to harbor a heterozygous S80S/P alteration. This isolate was correctly classified as caspofungin R with an Etest endpoint of ⬎32 mg/liter. The revised CLSI breakpoints were carefully selected in order to provide optimal separation between wild-type isolates and isolates with resistance mutations and were established either at the epidemiological cutoff value (ECV) (C. glabrata) or a single step


Caspofungin Etest and Candida Species

higher (16). CLSI caspofungin MIC50s and ECVs were 0.03 and 0.125 mg/liter, respectively, for C. albicans, C. glabrata, C. krusei, and C. tropicalis (14). The values are, however, one to three dilution steps lower than those found in our study for Etest endpoints (Fig. 1). A comparison of these distributions by species reveals that the revised CLSI susceptibility breakpoint bisects the caspofungin Etest endpoint distributions for C. glabrata and C. krusei, thus leading to random classification of wild-type isolates as either S or I/R (Fig. 1). One of the strengths for the reference microdilution methods is that growth inhibition is evaluated relative to the growth control for the specific isolates, thereby reducing variation associated with differences in inoculum concentration and growth rate. The automated reading applied in the EUCAST method additionally avoids potential subjectivity in the endpoint reading, thereby minimizing variability even further compared to that of the endpoint reading for agar diffusion methods. However, the Etest MIC50 values reported in this study are in agreement with those reported previously (0.125 to 0.25 mg/liter for C. glabrata and 0.25 to 1 mg/liter for C. krusei) (2, 5, 7, 13, 15, 21), and thus, our findings of a high risk of misclassification will apply to all routine laboratories using caspofungin Etest and CLSI breakpoints. Moreover, in this study, all Etest endpoints were read after 24 h of incubation. For C. glabrata, a second reading after 48 h is recommended by the manufacturer, particularly in cases of weak growth. Previous studies have shown that the Etest MICs are typically similar or 1 dilution step higher after 48 h, and thus, a second reading would further increase the risk of misclassifications (5). In conclusion, this study illustrates the caveats associated with the adoption of reference breakpoints for commercial methods when MIC distributions do not exactly mirror one another. In the case of caspofungin Etest, the revised CLSI breakpoints can be safely adopted for C. albicans, C. dubliniensis, C. parapsilosis, and C. tropicalis but not for C. glabrata and C. krusei. According to this and our previous studies, an Etest susceptibility breakpoint of ⱕ0.5 for C. glabrata and C. krusei would lead to a better classification of the isolates (2). ACKNOWLEDGMENTS We thank Birgit Brandt for excellent technical assistance. We thank Pfizer for providing anidulafungin pure substance. The study was financially supported in part by an unrestricted research grant from the investigator-initiated study programs of Gilead and Pfizer. M.A.P. has been a consultant for Astellas, Pfizer, Merck, Schering, Esai, MethylGene, and Becton Dickinsen, has been an invited speaker for Astellas, Pfizer, Merck, and Schering, and has received research funding (but not for this particular study) from Astellas, AB Biodisk, bioMérieux, Pfizer, Schering, Merck, Esai, and MethylGene. M.C.A. has been a consultant for Astellas, Merck, Pfizer, and SpePharm, has been an invited speaker for Astellas, Cephalon, Merck Sharp & Dohme, Pfizer, ScheringPlough, and Swedish Orphan, and has received research funding (but not for this particular study) from Astellas, Gilead, Merck, and Pfizer. The members of the study group have no conflicts to declare with regard to this study. The participants in the Danish Fungaemia Study Group in Denmark include the following: M. C. Arendrup (coordinator), Unit of Mycology, Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen; E. Dzajic, Sydvestjysk Sygehus, Esbjerg, and Vejle Sygehus, Vejle; H. K. Johansen, Rigshospitalet, Copenhagen University Hospital, Copenhagen; P Kjældgaard, Sygehus Sønderjylland, Sønderborg; J. D. Knudsen, Hvidovre University Hospital, Hvidovre; L. Kristensen, Herning Hospital, Herning; C. Leitz, Sydvestjysk Sygehus, Viborg;

July 2012 Volume 56 Number 7

L. E. Lemming, Skejby Hospital, Aarhus University Hospital, Aarhus; L. Nielsen, Herlev University Hospital, Herlev; B. Olesen, Hillerød Hospital, Hillerød; F. S. Rosenvinge, Odense University Hospital, Odense; B. L. Røder, Slagelse Sygehus, Slagelse; and H. C. Schønheyder, Aalborg Hospital, Aarhus University Hospital, Aalborg.

REFERENCES 1. Arendrup MC, et al. 2009. Breakthrough Aspergillus fumigatus and Candida albicans double infection during caspofungin treatment: laboratory characteristics and implication for susceptibility testing. Antimicrob. Agents Chemother. 53:1185–1193. 2. Arendrup MC, et al. 2010. Echinocandin susceptibility testing of Candida species: comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest, disk diffusion, and agar dilution methods with RPMI and IsoSensitest media. Antimicrob. Agents Chemother. 54:426 – 439. 3. Arendrup MC, et al. 2011. EUCAST technical note on anidulafungin. Clin. Microbiol. Infect. 17:E18 –E20. 4. Arendrup MC, et al. 2011. Echinocandin susceptibility testing of Candida spp. using the EUCAST EDef 7.1 and CLSI M27-A3 standard procedures: analysis of the influence of bovine serum albumin supplementation, storage time, and drug lots. Antimicrob. Agents Chemother. 55:1580 –1587. 5. Axner-Elings M, Botero-Kleiven S, Jensen RH, Arendrup MC. 2011. Echinocandin susceptibility testing of Candida isolates collected during a 1-year period in Sweden. J. Clin. Microbiol. 49:2516 –2521. 6. Baixench MT, et al. 2007. Acquired resistance to echinocandins in Candida albicans: case report and review. J. Antimicrob. Chemother. 59:1076 – 1083. 7. Chryssanthou E, Cuenca-Estrella M. 2002. Comparison of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing proposed standard and the E-test with the NCCLS broth microdilution method for voriconazole and caspofungin susceptibility testing of yeast species. J. Clin. Microbiol. 40:3841–3844. 8. Garcia-Effron G, Kontoyiannis DP, Lewis RE, Perlin DS. 2008. Caspofungin-resistant Candida tropicalis strains causing breakthrough fungemia in patients at high risk for hematologic malignancies. Antimicrob. Agents Chemother. 52:4181– 4183. 9. Garcia-Effron G, Lee S, Park S, Cleary JD, Perlin DS. 2009. Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-␤-D-glucan synthase: implication for the existing susceptibility breakpoint. Antimicrob. Agents Chemother. 53:3690 –3699. 10. Garcia-Effron G, Park S, Perlin DS. 2009. Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints. Antimicrob. Agents Chemother. 53:112–122. 11. Garcia-Effron G, Park S, Perlin DS. 2011. Improved detection of Candida sp. fks hot spot mutants by using the method of the CLSI M27-A3 document with the addition of bovine serum albumin. Antimicrob. Agents Chemother. 55:2245–2255. 12. Hakki M, Staab JF, Marr KA. 2006. Emergence of a Candida krusei isolate with reduced susceptibility to caspofungin during therapy. Antimicrob. Agents Chemother. 50:2522–2524. 13. Laverdiere M, Restieri C, Habel F. 2002. Evaluation of the in vitro activity of caspofungin against bloodstream isolates of Candida species from cancer patients: comparison of Etest and NCCLS reference methods. Int. J. Antimicrob. Agents 20:468 – 471. 14. Pfaller MA, et al. 2010. Wild-type MIC distributions and epidemiological cutoff values for the echinocandins and Candida spp. J. Clin. Microbiol. 48:52–56. 15. Pfaller MA, et al. 2010. Comparison of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Etest methods with the CLSI broth microdilution method for echinocandin susceptibility testing of Candida species. J. Clin. Microbiol. 48:1592–1599. 16. Pfaller MA, et al. 2011. Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria. Drug Resist. Updat. 14:164 –176. 17. Pfeiffer CD, et al. 2010. Breakthrough invasive candidiasis in patients on micafungin. J. Clin. Microbiol. 48:2373–2380. 18. Rodriguez-Tudela JL, et al. 2008. EUCAST definitive document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts. Clin. Microbiol. Infect. 14:398 – 405. 19. Slater JL, et al. 2011. Disseminated candidiasis caused by Candida albi-

aac.asm.org 3967

Arendrup et al.

cans with amino acid substitutions in Fks1 at position Ser645 cannot be successfully treated with micafungin. Antimicrob. Agents Chemother. 55: 3075–3083. 20. Spreghini E, et al. 2012. 2011. Comparative analysis of micafungin, caspofungin, and anidulafungin against a difficult-to treat fungal opportunistic pathogen: Candida glabrata. Antimicrob. Agents Chemother. 56: 1215–1222. 21. Vale-Silva L, Pinto P, Lopes V, Ramos H, Pinto E. 9 September 2011,

3968

aac.asm.org

posting date. Comparison of the Etest and a rapid flow cytometry-based method with the reference CLSI broth microdilution protocol M27-A3 for the echinocandin susceptibility testing of Candida spp. Eur. J. Clin. Microbiol. Infect. Dis. doi:10.1007/s10096-011-1390-z. 22. Wiederhold NP, Najvar LK, Bocanegra RA, Kirkpatrick WR, Patterson TF. 2011. Caspofungin dose escalation for invasive candidiasis due to resistant Candida albicans. Antimicrob. Agents Chemother. 55:3254 – 3260.
