Interlaboratory variability of caspofungin MICs for Candida spp. using CLSI and EUCAST methods: Should the clinical laboratory be testing this agent?

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1 AAC Accepts, published online ahead of print on 9 September 2013 Antimicrob. Agents Chemother. doi: /aac Copyright 2013, American Society for Microbiology. All Rights Reserved /28/13 (Revised version: AAC ) Interlaboratory variability of caspofungin MICs for Candida spp. using CLSI and EUCAST methods: Should the clinical laboratory be testing this agent? A. Espinel-Ingroff 1*, M.C. Arendrup 2, M.A. Pfaller 3, L.X. Bonfietti 4, B. Bustamante 5, E. Canton 6, E. Chryssanthou 7, M. Cuenca-Estrella 8, E. Dannaoui 9, A. Fothergill 10, J. Fuller 11, P. Gaustad 12, G. M. Gonzalez 13, J. Guarro 14, C. Lass-Flörl 15, S.R. Lockhart 16, J.F. Meis 17, C.B. Moore 18, L. Ostrosky-Zeichner 19, T. Pelaez 20, S.R.B.S. Pukinskas 21, G. St-Germain 22, M.W. Szeszs 23, and J. Turnidge 24 1 VCU Medical Center, Richmond, VA; 2 Unit of Mycology, Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen, Denmark; 3 JMI Laboratories and the University of Iowa, Iowa City, Iowa; 4 Adolfo Lutz Institute, Araçatuba City, Brazil; 5 Instituto de Medicina Tropical Alexander von Humboldt-Universidad Peruana Cayetano Heredia, Lima, Peru; 6 Unidad de Microbiologia Experimental, Centro de Investigacion, Hospital Universitario La Fe, Valencia, Spain; 7 Deptarment Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden; 8 Dept. Mycology, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain; 9 Institut Pasteur, Centre National de Reference Mycoses and Antifongiques, Unite de Mycologie Moleculaire, Paris, France; 10 University of Texas Health Science Center, San Antonio, TX; 11 The University of Alberta, Edmonton, Alberta, Canada; 12 Institute of Medical Microbiology, Rikshospitalet University Hospital, Oslo, Norway; 13 Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México; 14 Facultat de Medicina, IISPV, URV, Reus, Spain; 15 The Innsbruck Medical University, Division of Hygiene and Medical Microbiology, Innsbruck, Austria; 16 Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA; 17 Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital and Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands; 18 Mycology Reference Centre, Education and Research Centre, University Hospital of South Manchester, Manchester, UK; 19 University of Texas Health Science Center, Houston, TX; 20 Hospital General Universitario Gregorio Marañón, Facultad de Medicina-Universidad Complutense, Madrid, Spain; 21 Fungal Taxonomy Laboratories, Adolfo Lutz Institute, São Paulo City, Brazil; 22 Laboratoire de santé publique du Québec, Institut national de santé publique du Québec, Canada; 23 Mycology Department, Adolfo Lutz Institute, São Paulo City, Brazil; 24 University of Adelaide, Adelaide, Australia. 1

2 *Corresponding author address: 3804 Dover Rd, Richmond, VA 23221; Phone: (804) ; address: Abstract Although CLSI clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from: μg/ml for C. albicans and C. tropicalis; μg/ml for C. glabrata; μg/ml for C. krusei; variability was also similar among C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one twofold dilution of each other. These findings were consistent with available EUCAST data (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type [WT] (e.g., C. glabrata and C. krusei) as either non-wt or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead. Introduction The incidence and prevalence of invasive fungal infections is a major health problem, especially in the large population of immunocompromised patients and/or those with serious 2

3 underlying diseases (1,2). The most common fungal pathogens are Candida and Aspergillus species. The attributable mortality rate for candidemia is as high as 47% depending on the patient population and age (2,3). Three echinocandins (anidulafungin, caspofungin and micafungin) have been licensed for intravenous treatment and prevention of invasive Candida infections (including candidemia) (2). Standard conditions have been established by the Clinical and Laboratory Standards Institute (CLSI) for testing the susceptibilities of Candida spp. to the three echinocandins (4); in addition, the single susceptible clinical breakpoint (CBP) for echinocandin MIC interpreation has recently been adjusted to be species-specific (5,6). The main role of the new CBPs is to discriminate resistant mutants with amino acid substitutions in Fks1p or Fks2p proteins from susceptible wild type (WT) isolates as those have been associated with breakthrough or failure cases (5). Although the European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST) also has a broth microdilution method for echinocandin testing of Candida spp. as well as anidulafungin breakpoints (7,8), this organization has not established breakpoints for caspofungin. Significant interlaboratory variation in EUCAST caspofungin MIC ranges has precluded the establishment of interpretive cutoffs for this agent (9,10). Species-specific epidemiologic cutoff values (ECVs) have been defined for the three echinocandins and several species of Candida spp. using CLSI methodology performed by a single laboratory (11). To further validate these ECVs, echinocandin MIC data from multiple laboratories (17 centers) were gathered for Candida spp. The first step for ECV definition is the examination of each species/agent distribution from each laboratory, which is followed by the determination of each laboratory s individual modal MIC value (most frequent MIC for each species/agent combination) (12). ECVs for Candida spp. versus both anidulafungin and micafungin have been defined and are reported elsewhere (13). However, in collating caspofungin MIC distributions, we noted an excessive amount of MIC variability among participant laboratories (e.g., presumptive WT modes scattered across six-twofold dilutions), which precluded the calculation of caspofungin ECVs for Candida spp. Examination of EUCAST MIC distributions for the four common Candida spp. from seven laboratories revealed the same problem. This high degree of caspofungin MIC interlaboratory variation may potentially lead to incorrect categorization of susceptibility results. 3

4 The main objective of the present study is to report the CLSI caspofungin MIC variability for Candida spp. that was observed among 17 laboratories in Brazil, Canada, Europe, Mexico, Peru and the United States for 145 to 11,550 isolates (species-dependent) of C. albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis. We present corroborative EUCAST caspofungin MICs for Candida spp. gathered in 7 laboratories in Europe for 911 to 2,556 isolates (species-dependent) of C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis as well as comparative CLSI anidulafungin and caspofungin MIC distributions for C. albicans (anidulafungin MICs for 9,655 isolates and caspofungin MICs for 11,550 isolates from 11 and 14 laboratories, respectively). An attempt was made to elucidate the potential reason(s) for CLSI caspofungin MIC variability by examining the various testing conditions involved in MIC determination in each participant laboratory. Material and Methods Isolates. Echinocandin susceptibility testing of unique clinical isolates of Candida spp. was performed according to the CLSI broth microdilution method at the following medical centers: VCU Medical Center, Richmond, VA; JMI Laboratories and the University of Iowa, Iowa City, Iowa; Adolfo Lutz Institute, Araçatuba City, Brazil; Instituto de Medicina Tropical Alexander von Humboldt-Universidad Peruana Cayetano Heredia, Lima, Peru; Unidad de Microbiologia Experimental, Centro de Investigacion, Hospital Universitario La Fe, Valencia, Spain; University of Texas Health Science Center, San Antonio, TX; The University of Alberta, Edmonton, Alberta, Canada; Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México; Facultat de Medicina, IISPV, URV, Reus, Spain; The Innsbruck Medical University, Division of Hygiene and Medical Microbiology, Innsbruck, Austria; Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA; Canisius Wilhelmina Hospital, Nijmegen, The Netherlands; University of Texas Health Science Center, Houston, TX; Hospital General Universitario Gregorio Marañón, Facultad de Medicina-Universidad Complutense, Madrid, Spain; Fungal Taxonomy Laboratories, Adolfo Lutz Institute of São Paulo City, Brazil; 4

5 Laboratoire de santé publique du Québec, Canada and the Mycology Department, Adolfo Lutz Institute, São Paulo, Brazil. These laboratories were coded 1 to 20 (Tables 1 and 2). Similarly, EUCAST MICs were determined at the following medical centers: Statens Serum Institut, Copenhagen, Denmark; Karolinska University Hospital, Stockholm, Sweden; Instituto de Salud Carlos III, Madrid, Spain; Institut Pasteur, Centre National de Reference Mycoses and Antifongiques, Paris, France; Rikshospitalet University Hospital, Oslo, Norway; University Hospital of South Manchester, Manchester, UK and The Innsbruck Medical University, Innsbruck, Austria. These laboratories were coded 1 to 7 (Tables 3 and 4). The total of available CLSI caspofungin MICs for each species was: 11,550 for C. albicans (9,655 anidulafungin MICs), 145 for C. dubliniensis, 5,322 for C. glabrata, 262 for C. guilliermondii, 850 for C. krusei, 400 for C. lusitaniae, 4,880 for C. parapsilosis, and 2,958 for C. tropicalis. The total of available EUCAST caspofungin MICs for each species was: 2,556 for C. albicans, 948 for C. glabrata, 680 for C. parapsilosis, and 403 for C. tropicalis. Isolates were identified and stored at each medical center using standardized methodologies (14) and they were not evaluated for FKS mutations. In addition, CLSI and EUCAST MICs for one or both quality control (QC) isolates C. parapsilosis ATCC and C. krusei ATCC 6258 were obtained each time that testing was performed in each of the participant laboratories (4,5). Antifungal susceptibility testing. As defined in the CLSI and EUCAST broth microdilution standards documents (M27-A3 and EDef 7.2), inclusion criteria for CLSI-derived echinocandin MICs from the participant laboratories required the use of RPMI-1640 broth with 0.2% of dextrose, incubation temperatures at 35 C for 24 h and, a prominent inhibition of growth (>50% reduction relative to the growth control). For EUCAST-derived MICs, the required conditions included RPMI-1640 broth with 2% of dextrose, incubation temperatures at 35 C for 24 h and, a spectrophotometric prominent inhibition of growth (OD reduction of >50%) (4,7). In addition, CLSI and EUCAST MICs for QC isolates were obtained following the respective standard conditions. 5

6 Survey. To further investigate the possible causes of caspofungin MIC variability, a questionnaire was sent to the laboratories that provided the CLSI data, depicted in Tables 1 and 2. The questions were: (i) What was the source of the drug? (ii) At what temperature was the drug stored? (iii) Over what time period was the batch of powder used? (iv) What was the solvent used for preparing stock and subsequent dilutions? (v) Was the medium always the CLSI RPMI with 0.2% dextrose? (vi) Were the MICs always read at 24 h? (vii) Were MICs 50% or more growth inhibition? (viii) Were any of the data provided from YeastOne, Etest, or EUCAST methods? (ix) Were the microdilution trays read visually or was a spectrophotometer used? Data analysis. The MIC distributions of each of the species tested in each participating laboratory were first screened for evidence of grossly abnormal distributions and the presumptive WT modal (most frequent value) MICs were determined (12). In general, for ECV determination, grossly skewed distributions (distributions which had a modal MIC at the lowest or highest concentration tested and/or which were bimodal in the presumptive wild-type distribution) should be excluded (CLSI Establishing ECOFFs Workshop, Tampa, FL, January 22, 2013), but they were not for the present report. In addition, we only documented data for the less common species when those CLSI MICs originated from a minimum of four laboratories and the distribution from each laboratory comprised at least ten MICs for each species. Results and Discussion CLSI caspofungin MIC variability. In the present study, we observed significant interlaboratory variation in both (i) CLSI caspofungin MIC ranges and (ii) WT modes for most species evaluated. For example, we observed caspofungin MIC ranges of to 0.25 μg/ml and of 0.25 to 2 μg/ml for C. albicans (Table 1) and WT modes spanning five to six dilution steps among most of the species (0.016 to 0.5 μg/ml for C. albicans and C. tropicalis; to 0.5 μg/ml for C. glabrata; to 1 μg/ml for C. krusei). Wide modal ranges were also present among C. dubliniensis and C. lusitaniae distributions, but this was not the case for C. parapsilosis and C. guilliermondii (mostly 0.5 or 1 μg/ml and 0.25 or 0.5 μg/ml, respectively) 6

7 (Table 2). On the other hand, minimal interlaboratory variation was observed in the anidulafungin comparative distribution for C. albicans (modes, and 0.031µg/ml) (Table 1) suggesting that the variability is caspofungin-specific and not a general echinocandin problem. EUCAST caspofungin MIC variability corroborated CLSI data, where individual laboratories reported MIC ranges of to μg/ml and to 4 μg/ml for C. albicans (Table 3). EUCAST caspofungin modes were also variable for three of the four common species (0.016 to 0.5 μg/ml for C. albicans; to 0.5 μg/ml for C. glabrata; and to 0.5 μg/ml for C. tropicalis), while C. parapsilosis modes were constant between participant sites (either 1 or 2 μg/ml) (Table 4). Insufficient anidulafungin EUCAST MIC distributions precluded relevant comparisons between the two agents. It is currently not understood why variability is not a problem among caspofungin MIC distributions for C. parapsilosis or C. guilliermondii, since these data were gathered in the same laboratories using the same method as that for the other species. Perhaps, the stability of caspofungin MICs for these two species may be due to the higher concentrations of drug in the well and higher MICs. This could also explain why the CLSI modal MICs were in general lower at laboratories using DMSO as solvent (Tables 1 and 2) which improves solubility. More studies are needed in order to resolve these conflicting results. Caspofungin MIC interlaboratory variability was evident as early as 2002 with reported MIC ranges for C. albicans as low as to 1 μg/ml and as high as 0.25 to 4 μg/ml; similar discrepancies were observed for C. tropicalis (15). Earlier caspofungin range variability was attributed to the different panels of isolates that were evaluated in each study, but it does not seem plausible when considering the use of WT strains. Considerable interlaboratory variability of caspofungin MIC data was also reported in the original study that identified the optimum testing conditions for this agent and Candida spp. (overall agreement, 64.7 to 100% and agreement per species, 84 to 100% for C. albicans; 85.3% for C. glabrata, 41.4 to 85% for C. parapsilosis; and 41 to 85% for C. tropicalis; species data not included in that report) (15), Those authors concluded that interlaboratory agreement needed to be improved, which was corroborated in the present study. Caspofungin MIC variability has been previously reported by the EUCAST group as being dependent upon drug lot-to-lot inconsistency in potency and/or stability (10). Using different caspofungin lots, MICs were repeatedly determined for six ATCC strains resulting in wide caspofungin MIC ranges (e.g., to 0.25 μg/ml for the same strain of 7

8 C. albicans); results were similar for control isolates of the other species evaluated, with the exception of MICs for the C. krusei ATCC 6258 (0.25 to 1 μg/ml) and C. parapsilosis ATCC isolates (1 to 2 μg/ml). The agreement was acceptable (96 to 100%) in most laboratories regarding the CLSI recommended caspofungin MIC range (0.25 to 1 μg/ml) for the QC C. parapsilosis ATCC (5), but unacceptable in three laboratories (84 to 94%); most discrepancies occurred with MICs above the high end of the QC range, 2µg/ml. Modes ranged from 0.25 to 1 μg/ml in all laboratories, except one. There was more caspofungin modal variability for the QC isolate C. krusei ATCC 6258 (mode range, to 1 μg/ml), but the agreement was acceptable (95 to 100%) in most laboratories regarding the recommended range of to 1 μg/ml (5), the exceptions were MIC ranges in two laboratories (<0.125 to 2 μg/ml). The less pronounced overall variation for these two QC strains than for the clinical isolates suggests that these two control strains may not be sensitive enough to detect performance variations when testing this agent. Similar concerns have been raised by EUCAST (10). The CLSI plans to address this problem in the near future by collecting new data and using DMSO as the solvent for the selection of more appropriate control strains (16). Caspofungin powder. All 17 laboratories used caspofungin powder sourced from Merck in the US and other countries; 7 of the 17 laboratories (4,7,10,14,15,16, and 17) used a single lot. Discrepancies were noted in the powder storage temperature, where some laboratories stored their powder at either 4 o to 5 o C (laboratories 7,15,16, and 17) or -20 o C ( laboratories 6 and 14) and in the other 11 laboratories at -70 o C or below. This variation in storage conditions did not appear to explain the variations in MICs among the participating laboratories (Table 2). While most laboratories used their batch of powder for < 1 year, laboratories where data were gathered throughout >7 years used only two or three lots of caspofungin (laboratories 2,3,19,20); Merck s recommendation is two years. However, overall modes from those latter laboratories were among the lower values, e.g., the mode for C. albicans was and μg/ml (Tables 1 and 2). In addition, whilst we observed some bimodal distributions, especially for C. albicans, this type of abnormal distribution was only observed in data from laboratory 3, but not from laboratories 2, 19 or 20. Therefore, using the powder longer than two years, or any other related 8

9 powder issues, did not explain the problem. Unfortunately sufficient data on drug lot numbers were not available, so we were unable to determine that lot-to-lot variability caused the problem, as previously reported (10). Solvents for preparation of caspofungin stock solutions. Until 2012, the recommendation of the CLSI was the use of water as the solvent for the preparation of caspofungin stock solutions (17). Four (4, 17, 18, and 20) of the 17 laboratories used DMSO as the solvent, and laboratory 2 used both solvents. As shown in Tables 1 and 2, MICs derived using water vs. DMSO as the solvent could not account for all of the variability in the modes. It should be noted, however, that laboratories using DMSO as the solvent tended to have MIC distributions for each species with lower modal MIC values than those laboratories using water as the solvent suggesting that DMSO helps in avoiding loss of potency and higher MICs. Therefore, although the use of DMSO vs. water is not the sole explanation for the variability shown among the participating laboratories, it appears to play a role. Accordingly, the new and revised version of the document (M27-S4) lists DMSO as the solvent for echinocandins and fluconazole (5); EUCAST also strongly recommends the same for echinocandin susceptibility testing. Other testing parameters. Although each contributing laboratory was requested to provide only CLSI data and not a mixture of data from different methodologies (commercial methods included), our survey included specific questions (e.g., medium dextrose content, spectrophotometric vs. visual MICs, and 50% or more growth inhibition). We received capofungin MIC data from 19 laboratories; however, based on answers to the survey, data from 2 laboratories (8 and 13 used EUCAST medium) were not included in this report. Caspofungin MICs from the remaining 17 laboratories were determined according to the recommended CLSI testing conditions listed above (Antifungal susceptibility testing); MICs were read visually in 15 of the 17 laboratories, but variation was similar as that for the EUCAST data (all automated spectrophotometer reading) suggesting subjectivity in endpoint reading was not the source of variability. 9

10 Caspofungin new CBPs and MIC interpretation. Before 2004, caspofungin MICs for Candida spp. were obtained using a variety of testing conditions (18). Between 2008 and 2009, standard guidelines for caspofungin MIC determination (50% or more growth inhibition at 24 h) were described in the CLSI M27-A3 and M27-S3 documents (4,17). In 2008, the CLSI established a susceptible CBP (<2μg/ml) for echinocandins and all Candida spp. (17). Molecular studies identified the echinocandin antifungal activity target, the protein Fksp, glucan synthase, encoded by three FKS genes. In addition, elevated echinocandin MICs for clinical strains of Candida were associated with specific mutations in FKS genes and therapeutic failure or breakthrough infection (19-22). Using FKS 1 mutant strains, Garcia-Effron et al. (23,24) demonstrated that although caspofungin MICs >2 μg/ml captured almost 100% of FKS mutant strains, the MICs that differentiated these mutants from WT strains was lower (>0.5 μg/ml for C. albicans and >0.25 μg/ml for C. glabrata) for both anidulafungin and micafungin. By 2011, caspofungin species-specific ECVs were defined and CBPs were adjusted for six Candida spp.: C. glabrata (susceptible, μg/ml; resistant, 0.5 μg/ml), C. albicans, C. tropicalis, and C. krusei (susceptible, <0.25 μg/ml and resistant 1 μg/ml) and C. parapsilosis and C. guilliermondii (susceptible, <2 μg/ml; resistant, 8 μg/ml) (5,6,11). What is the impact in the clinical setting of new and lower species-specific CLSI caspofungin breakpoints? It has created a problem in some laboratories. As shown in Tables 1 and 2 (also according to some contributors comments), the variability in caspofungin MIC distributions will result in reporting too many WT strains as non-susceptible (or major errors) for most of the common species with the exception of C. parapsilosis. EUCAST has already recommended the use of anidulafungin MICs as markers for the echinocandins and to avoid the use of caspofungin MIC results for clinical decision making (8,10). It is expected that the CLSI will reach a similar conclusion and, in addition to the projected revision of MIC ranges for the QC isolates and the selection of more adequate strains, the issue of caspofungin variability will be addressed by this organization in the near future. CBPs are the same for all three echinocandins and each of the common species except for the lower endpoint for C. glabrata and micafungin. Therefore for the time being, either micafungin or anidulafungin MIC endpoints can be used as predictors of susceptibility or resistance of Candida spp. to caspofungin. 10

11 In summary, something unusual is going on with caspofungin testing. Although we have a great deal of caspofungin MICs from different geographical areas, it is evident that caspofungin ECVs cannot be defined using available MIC results. Routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended, especially in the clinical setting; in the meantime testing one of the other two echinocandins (anidulafungin or micafungin) could provide the desired susceptibility result. Available ECVs have been established based on data from a single laboratory and interpretation of MICs using the new CBPs or ECVs will result in some laboratories reporting too many isolates as resistant or non-wt isolates, mainly among C. grabrata, C. krusei and to certain extent amongst C. albicans and C. tropicalis, which could result in less effective therapy. Disclaimer: The findings and conclusions of this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. References 1. Arendrup MC, Fuursted K, Gahrn-Hansen B, Schonheyder HC, Knudsen JD, Jensen IM Seminational surveillance of fungemia in Denmark : increasing incidence of fungemia and numbers of isolates with reduced azole susceptibility. Clin. Microbiol. Infect. 14: Pappas PG, Kauffman CA, Andes D, Benjamin DK Jr, Calandra TF, Edwards JE, Filler SG, Fischer JF, Kullberg BJ, Ostrosky-Zeichner L, Reboli AC, Rex JH, Walsh TJ, Sobel JD Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin. Infect. Dis. 48: Barnes PD, Marr KA Risk, diagnosis and outcome of invasive fungal infections in haematopoietic stem cell transplant recipients. Br. J. Haematol. 139: Clinical and Laboratory Standards Institute Reference method for broth dilution antifungal susceptibility testing of yeasts, third ed. Approved standard M27-A3. Clinical and Laboratory Standards Institute, Wayne, PA. 5. Clinical and Laboratory Standards Institute Reference method for broth dilution antifungal susceptibility testing of yeasts; fourth informational supplement M27-S4. Clinical and Laboratory Standards Institute, Wayne, PA. 6. Pfaller MA, Diekema DJ, Andes D, Arendrup MC, Brown SD, Lockhart SR, Motyl M, Perlin DS, the CLSI Subcommittee for Antifungal Testing Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data 11

12 to arrive at species-specific interpretive criteria. Drug Resist. Update. 14: Arendrup MC, Cuenca-Estrella M, Lass-Flörl C, Hope WW, the EUCAST-AFST EUCAST Technical Note on the EUCAST Definitive Document EDef 7.2: Method for the determination of broth dilution minimum Inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST- AFST). Clin. Microbiol. Infect. 18: E246-E Arendrup MC, Rodriguez-Tudela J-L, Lass-Florl C,.the European committee on antimicrobial susceptibility testing- Subcommittee on antifungal susceptibility testing EUCAST technical note on anidulafungin. Clin. Microbiol. Infect.17: E Arendrup MC, Garcia-Effron G, Buzina W, Mortensen KL, Reiter N, Lundin C, Elvang Jensen H, Lass-Florl C, Perlin DS, Bruun B Breakthrough Aspergillus fumigatus and Candida albicans double infection during caspofungin treatment: laboratory characteristics and implications for susceptibility testing. Antimicrob. Agents Chemother. 53: Arendrup MC, Rodriguez-Tudela J-L, Park S, Garcia-Effron G, Delmas G, Cuenca-Estrella M, Gomez-Lopez A, Perlin DS Echinocandin susceptibility testing of Candida spp. using 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: Pfaller MA, Boyken L, Hollis RJ, Kroeger J, Messer SA, Tendolkar S, Jones RN, Turnidge J, Diekema DJ Wild-type MIC distributions and epidemiological cutoff values for the echinocandins and Candida spp. J. Clin. Microbiol. 48: Turnidge J, Kahmeter G, Kronvall G Statistical characterization of bacterial wild-type MIC value distributions and the determination of epidemiological cut-off values. Clin. Microbiol. Infect. 12: Pfaller MA, Espinel-Ingroff A, Bustamante B, Canton E, Fothergill A, Fuller J, Gonzales G, Guarro J, Lass-Flörl C, Lockhart SR, Martin-Mazuelos E, Meis JF, Ostrosky-Zeichner L, Pelaez T, St- Germain G, Turnidge J Wild-type MIC distributions and epidemiological cutoff values for anidulafungin and micafungin and Candida spp. In preparation. 14. Howell SA, Hazen KC Candida, Cryptococcus, and other yeasts of medical importance: mycology, p In Versalovic J, Carroll KC, Jorgensen JH, Funke G, Landry ML, Warnock DW(ed), Manual of clinical microbiology, 10th ed, vol 2. ASM Press, Washington, DC. 15. Odds FC, Motyl M, Andrade R, Bille J, Canton E, Cuenca-Estrella M, Davidson A, Durussel C, Ellis D, Foraker E, Fothergill AW, Ghannoum MA, Giacobbe RA, Gobernado M, Handke R, Laverdière M, Lee-Yang W, Merz WG, Ostrosky-Zeichner L, Peman J, Perea S, Perfect JR, Pfaller MA, Proia L, Rex JH, Rinaldi MG, Rodriguez-Tudela J-L, Schell WA, Shields C, Sutton DA, Verweij PE, Warnock DW Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species. J. Clin. Microbiol. 42: Clinical and Laboratory Standards Institute Minutes and agenda: Annual meeting of the Subcommittee for Antifungal Susceptibility Testing. Tampa, Florida, January 22,

13 Clinical and Laboratory Standards Institute Reference method for broth dilution antifungal susceptibility testing of yeasts; third informational supplement M27-S3. Clinical and Laboratory Standards Institute, Wayne, PA. 18. Espinel-Ingroff A, Cuenca-Estrella M, Canton E CLSI and EUCAST: working together towards a harmonized method for antifungal susceptibility testing. Curr. Fungal Infect. Rep. 7: Cleary JD, Garcia-Effron G, Chapman SW, Perlin DS Reduced Candida glabrata susceptibility secondary to an FKS1 mutation developed during candidemia treatment. Antimicrob. Agents Chemother.52: Desnos-Ollivier M, Bretagne S, Raoux D, Hoinard D, Dromer F, Dannaoui E Mutations in the FKS1 gene in Candida albicans, C. tropicalis and C. krusei correlate with elevated caspofungin MICs uncovered in AM3 medium using the method of the European Committee on Antibiotic Susceptibility Testing. Antimicrob. Agents Chemother. 52: Garcia-Effron G, Park S, and Perlin DS Caspofungin-resistant Candida tropicalis causing breakthrough fungemia in patients at high risk for hematologic malignancies. Antimicrob. Agents Chemother. 52: Thompson GR, Wiederhold NP, Vallor AC, Villareal NC, Lewis JS, Patterson TF Development of caspofungin resistance following prolonged therapy for invasive candidiasis secondary to Candida glabrata infection. Antimicrob. Agents Chemother. 52: Garcia-Effron G, Park S, Perlin DS Correlating echinocandin MIC and kinetic inhibition of FKS1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints. Antimicrob. Agents Chemother. 53: Garcia-Effron G, Lee S, Park S, Cleary JD, Perlin DS Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-ß-D-glucan synthase: implication for the existing susceptibility breakpoint. Antimicrob. Agents Chemother. 53:

14 Table 1. Caspofungin and andidulafungin MIC distributions of Candida albicans using the CLSI broth microdilution method in 11 to 14 laboratories a Laboratory No. Agent MIC 4* 20* 3 2* 18* Caspofungin b a MICs in μg/ml were determined following the M27-A3 standard conditions for testing echinocandins and Candida spp. (4). Bolded values, modes (most frequent MIC) of each distribution from each laboratory. *Laboratories that used DMSO to prepare caspofungin drug dilutions; the other laboratories used water as per Table 2 of M27-S3 document (16). b The total number of caspofungin MICs is 11,550 and of andifulafungin MICs 9,655 14

15 Table 2. Caspofungin modal MIC variability among independent laboratories using CLSI broth microdilution method for common and common Candida spp. a Species Mode (μg/ml) b No. of Isolates at Mode per Laboratory No. of Isolates c Laboratory No. 3 4* 20* 2* 18* C. albicans Laboratory No * 20* 1 2* * C. glabrata Laboratory No. 3 4* 2* 12 18* C. tropicalis Laboratory No * 2* 1 4* 9 10 C. krusei

16 Mode (μg/ml) b Species No. of Isolates c Laboratory No. 20* 18* 19 2* 4* 7 17* C. parapsilosis Laboratory No. 2* * 19 C. guelliermondii Laboratory No. 2* 18* 9 10 C. dubliniensis Laboratory No. 2* 18* C. lusitaniae a CLSI M27-A3 method (4). b Mode, most frequent MIC value of each species from each laboratory. c Total number of isolates at each mode. The total number of isolates was as follows: 11,550 C. albicans, 145 C. dubliniensis, 5,322 C. glabrata, 262 C. guilliermondii, 850 C. krusei, 400 C. lusitaniae, 4,880 C. parapsilosis, and 2,958 C. tropicalis. *Laboratories that used DMSO to prepare caspofungin drug dilutions; the other laboratories used water as per Table 2 of M27-S3 document (16). 16

17 Table 3. Caspofungin MIC distributions of Candida albicans using the EUCAST broth microdilution method in 7 laboratories a MIC Laboratory No. (μg/ml) > a MICs were determined following the EUCAST standard conditions for testing echinocandins and Candida spp. (7). Bolded values, modes (most frequent MIC) of each distribution from each laboratory. b The total number of EUCAST caspofungin MICs is 2,556 17

18 Table 4. Caspofungin modal MIC variability among independent laboratories using EUCAST broth microdilution method for common Candida spp. a Mode b Species (μg/ml) No. of Isolates at Mode per Laboratory No. of Isolates c Laboratory No C. albicans Laboratory No C. glabrata Laboratory No. C. tropicalis Laboratory No. C. parapsilosis a EUCAST method (7). b Mode, most frequent MIC value of each species from each laboratory. c Total number of isolates at each mode. The total number of isolates was as follows: 2,556 C. albicans, 948 C. glabrata, 680 C. parapsilosis, and 403 C. tropicalis. 18