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Comparison of micafungin MICs as determined by the Clinical and Laboratory Standards Institute broth microdilution method (M27-A3 document) and Etest for Candida spp. isolates
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Diagnostic Microbiology and Infectious Disease 70 (2011) 54 – 59 www.elsevier.com/locate/diagmicrobio
Mycology
Comparison of micafungin MICs as determined by the Clinical and Laboratory Standards Institute broth microdilution method (M27-A3 document) and Etest for Candida spp. isolates Ana Espinel-Ingroff a,⁎, Emilia Cantónb , Teresa Pelaezc , Javier Pemánd b
a VCU Medical Center, Richmond, VA 23298, USA Unidad de Microbiología, Centro de Investigación, Hospital Universitario La Fe, Valencia 46009, Spain c Hospital General Universitario Gregorio Marañon, Madrid 28007, Spain d Servicio Microbiología, Hospital Universitario La Fe, Valencia 46009, Spain Received 8 November 2010; accepted 10 December 2010
Abstract Micafungin Etest and Clinical and Laboratory Standards Institute (CLSI) MICs were compared for 337 Candida spp. isolates. The performance of Etest for testing the susceptibilities of Candida spp. to micafungin was evaluated by the assessment of both categorical (CA) and essential (EA) agreements. The CA was evaluated 2 ways: (i) by the ability of Etest to separate resistant (nontreatable) from susceptible (treatable) isolates by using the newly adjusted species-specific micafungin clinical breakpoints (CBPs) that are available for most of the common species tested and (ii) by the ability to separate wild type (WT) from non-WT isolates or those harboring FKS mutations (with reduced echinocandin susceptibility) by using micafungin epidemiologic cutoff values (ECVs). Etest and CLSI MICs were in EA when the MICs were within 2 log2 dilutions. Based on agreement percentages, our data indicated that Etest is suitable to test micafungin for most of the Candida species evaluated (overall EA 94.7%; overall CA according to CBPs 97.2% and according to ECVs 97.3%). However, the number of resistant isolates was small, so further evaluations are needed with a higher number of such isolates including more resistant or those with known mechanisms of resistance (non-WT). © 2011 Elsevier Inc. All rights reserved. Keywords: Etest micafungin; Micafungin MICs; Micafungin MICs for Candida; Etest versus reference MICs
1. Introduction Echinocandins (anidulafungin, caspofungin, and micafungin) are available for intravenous treatment and prevention of Candida and Aspergillus infections, especially for patients with recent azole exposure (Espinel-Ingroff et al., 2009; Pappas et al., 2009). In 2008, the Clinical and Laboratory Standards Institute (CLSI) established susceptibility testing guidelines and clinical breakpoints (CBPs) for the 3 echinocandins and the most common Candida spp. (susceptible MIC ≤2 μg/mL and nonsusceptible MIC N2 μg/ mL) (CLSI, 2008b). However, kinetic characterization of Candida albicans (Garcia-Effron et al., 2009a) and Can-
⁎ Corresponding author. Tel.: +804-358-5895. E-mail addresses: [email protected], [email protected] (A. Espinel-Ingroff). 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.12.010
dida glabrata (Garcia-Effron et al., 2009b) indicated that isolates harboring mutations in FKS1 and FKS2 regions had MICs below the susceptible CBP of ≤2 μg/mL. These results suggested that CBPs needed to be adjusted. More recently, epidemiologic cutoffs (ECVs) were proposed for several Candida spp. and the 3 echinocandins (Pfaller et al., 2010a). Because of this newer information, the CLSI is proposing a susceptible CBP (MIC ≤0.25 μg/mL) that would capture at least 95% of the clinical trials isolates of C. albicans, Candida tropicalis, and Candida krusei; however, the susceptible and resistant CBPs for C. glabrata and micafungin are ≤0.06 and ≥0.25μg/mL, respectively. The resistant endpoint for C. albicans, C. tropicalis, and C. krusei is ≥1 μg/ mL because it has been documented that these isolates are usually clinically resistant and harbor FKS mutations (CLSI, 2010; Pfaller et al., 2011). The susceptible CBP for Candida parapsilosis and Candida guilliermondii continues to be ≤2 μg/mL and the resistant cutoff is ≥8 μg/mL (Table 1).
A. Espinel-Ingroff et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 54–59 Table 1 CLSI proposed species-specific clinical breakpoints (CBPs) and ECVs for micafungin and selected Candida speciesa Species
C. albicans C. glabrata C. krusei C. tropicalis C. parapsilosis C. guilliermondii
MIC (μg/mL) S
I
R
ECVs
≤0.25 ≤0.06 ≤0.25 ≤0.25 ≤2 ≤2
0.5 0.12 0.5 0.5 4 4
≥1 ≥0.25 ≥1 ≥1 ≥8 ≥8
0.03 0.03 0.12 0.12 4 4
S = susceptible; I = intermediate; R = resistant. a Adopted from CLSI (2010), Pfaller et al., (2010a), and Pfaller et al., (2011).
We have evaluated the suitability (essential [EA] and categorical [CA] agreements) of micafungin Etest MICs for Candida spp. bloodstream isolates from 3 medical centers (VCU Medical Center, Richmond, VA; the Hospital La Fe, Valencia, Spain; and Hospital Gregorio Marañon, Madrid, Spain). The CA was evaluated according to both ECVs and newly proposed CBPs (available for 6 of the common species tested) (CLSI, 2010; Pfaller et al., 2010a, 2011). By using the CBPs for the comparison, we evaluated the ability of Etest to detect the resistant or nontreatable isolates and by using the ECVs the ability to separate wild type (WT or isolates with no acquired resistance mechanisms) from those harboring mechanisms of resistance with reduced susceptibility to micafungin (Turnidge et al., 2006). 1.1. Isolates The total set of isolates from the 3 laboratories included 125 C. albicans, 52 C. glabrata, 11 C. guilliermondii, 39 C. krusei, 39 C. tropicalis, 14 less common Candida spp., and 57 C. parapsilosis group (10 Candida metapsilosis, 16 Candida orthopsilosis, and 31 Candida parapsilosis; Table 2). The C. parapsilosis group was identified by using specific primers based on the intron of the RPSO gene (Canton et al., 2010). Among the 6 C. albicans echinocandin-resistant (CLSI MIC, N1 μg/mL) and 16 non-WT FKS mutant isolates (CLSI micafungin MICs N0.03 μg/mL), there were 3 well-documented heterozygous and one homozygous strains (Douglas et al., 1997; Jacobsen et al., 2007); one caspofungin-resistant C. krusei isolate was also included (Canton et al., 2009). C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 isolates were tested as QC; micafungin MICs were within the established QC limits (CLSI 2008a, 2008b).
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and Etest strips (0.002–32 μg/mL). MICs were the lowest drug concentration at which the border of the elliptical inhibition intercepted the strip scale, ignoring trailing growth or microcolonies inside the ellipse. CLSI MICs were the lowest micafungin concentration that showed ≥50% inhibition. 1.3. Definitions The WT is the population of organisms/MICs in a species/ antifungal agent combination with no acquired resistance mechanisms (Turnidge et al., 2006). The high WT MIC has been defined as the WT cutoff value or the ECV (Pfaller et al., 2010a; Turnidge et al., 2006) in contrast to the non-WT microorganisms with acquired or mutational resistance mechanisms (Dalhoff et al., 2009). On the other hand, the drug concentration that classifies the organisms as treatable (susceptible) or nontreatable (resistant) or the predictor of clinical outcome is now defined as the CBP (Dalhoff et al., 2009, Turnidge and Patterson, 2007). CBPs are established based on clinical trial data, global susceptibility surveillance, resistance mechanisms, and PK/PD parameters from model systems. 1.4. Data analysis For the comparison between the 2 methods, MICs from the 3 laboratories were pooled. Micafungin MICs were in EA when the discrepancies between the 2 methods were within 2 log2 dilutions. CA between the 2 methods was evaluated as follows: errors were calculated according to each available species-specific ECV and CBP (Table 1); they were considered very major errors when the reference MIC indicated resistance (according to the species CBP) or nonWT (an isolate with changed susceptibility phenotype or with an MIC above the corresponding ECV) and the Etest susceptibility or WT. Errors were considered major errors when the Etest categorized the isolate as resistant or non-WT and the reference method as susceptible or WT. Since intermediate CBPs are also available for some of the species, errors were considered minor when there was a single shift between the 2 results (e.g., susceptible to intermediate). A linear regression analysis using the least-square method (Pearson correlation coefficient; MS Excel software) was performed by plotting Etest versus reference MICs for correlation between the methods.
1.2. Antifungal susceptibility testing
2. Results and discussion
Each of the 3 participant laboratories tested their set of isolates; 2 of the 3 laboratories tested the C. albicans mutants. Micafungin (Astellas Pharma, Tokyo, Japan) MICs were determined by both CLSI M27-A3 and Etest methods after 24 h at 35 °C (CLSI, 2008a). Etest MICs were determined according to the manufacturer's instructions (AB BIODISK, Solna, Sweden) using RPMI agar (2% glucose)
A new or commercial method such as the Etest should identify resistant strains to the drug being evaluated in the same manner as the reference method or yield similar MICs as those by this method. Therefore, both CA and EA between Etest and CLSI reference methods were evaluated by using available ECVs as well as CBPs as listed in Table 1. To evaluate such performance, our set of isolates included
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echinocandin-resistant (micafungin MICs N1 μg/mL) and non-WT isolates (micafungin MICs above the CLSI microdilution species-specific ECV) among the C. albicans, C. glabrata, and C. krusei isolates; however, only 4 of our isolates (among the C. albicans) have known fks gene mutations. Echinocandin resistance for C. albicans, C. glabrata, C. krusei, and other species has been associated with high MICs (N1 μg/mL), mutations in the FKS gene, and therapeutic failure (Baixench et al., 2007; Desnos-Ollivier et al., 2008; Garcia-Effron et al., 2009a, 2009b; Kahn et al., 2007). To our knowledge this is the first evaluation of the suitability of Etest micafungin MICs for Candida spp. by using the newly species-specific proposed CBPs. Our CLSI MIC data for most species were the same or one dilution higher than those previously published (Pfaller et al., 2010a) as demonstrated by our MIC90s (encompassing 90% of isolates, Table 2). The susceptibilities of C. metapsilosis, C. orthopsilosis, and C. parapsilosis were variable by both methods as previously described (MIC ranges 0.03–0.5, 0.25–2, and 0.06-4 μg/mL, respectively (Canton et al., 2010). The overall EA was 94.7% (R = 0.814) and it was N90% for most species, except for C. tropicalis (89.7%), C. parapsilosis (87.1%), and the less common species listed as miscellaneous in Table 2. The EA in the present study was similar to a prior comparison of the 2 methods (EA, N90%) (Pfaller et al., 2010b), but superior to that reported for anidulafungin Etest and CLSI MICs (Espinel-Ingroff et al., 2010). In the latter study, EA agreement was b90% for most species, but 100% for C. tropicalis. The acceptable EA is
N90% (CLSI, 2007). Our micafungin Etest results usually were the same or one dilution lower than reference results, while anidulafungin Etest MICs have been reported as mostly higher (Espinel-Ingroff et al., 2010). These differences could be attributed to anidulafungin (non–watersoluble agent) diffusion problems. Table 3 depicts the CA between CLSI and Etest for the species for which both susceptible and resistant speciesspecific microdilution CBPs are available (C. albicans, C. guilliermondii, C. krusei, C. parapsilosis, C. tropicalis and more recently C. glabrata [not listed in Table 3]) (CLSI, 2010; Pfaller et al., 2011). The CLSI subcommittee has not yet defined the susceptible CBP for the less common species listed in Table 2 as miscellaneous. There were no very major errors for any of 2 species that had micafungin-resistant isolates (C. albicans and C. krusei). That means that both methods were able to identify 6 of the 7 micafungin-resistant isolates (MICs N1 μg/mL). The Etest MIC for one of the C. albicans laboratory mutants was 1 μg/mL, while the CLSI MIC was 0.25 μg/mL (major error). This strain was moderately resistant to caspofungin in the mouse model of disseminated candidiasis (Douglas et al., 1997) and was categorized as non-WT. The overall number of minor and major errors was small (2% and 0.8%, respectively), and most of them were found among the isolates of C. albicans (2 major or 1.6%, and 3 minor or 2.4%). There was only one minor error each among C. krusei, C. grabrata and C. parapsilosis isolates, where the resistant C. krusei isolate (MIC 1 μg/mL), the susceptible C. glabrata (MIC
Table 2 Susceptibilities of 337 isolates of Candida spp. to micafungin as determined by the CLSI broth microdilution (M27-A3) and Etest methods Species (no. tested)
C. albicans (125) C. glabrata (52) C. guilliermondii (11) C. krusei (39) C. metapsilosis (10) C. orthopsilosis (16) C. parapsilosis (31) C. tropicalis (39) Miscellaneous speciesd (14) Overall (337) a
Methoda
BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest
MIC (μg/mL)b
EA (%)c
MIC range
MIC50
MIC90
≤0.016–2 ≤0.016–2 ≤0.016–0.062 ≤0.016–0.125 0.032–0.5 ≤0.016–1 0.032–1 ≤0.016–0.5 0.032–0.5 0.12–0.25 0.25–2 0.25–1 0.06–4 0.03–1 ≤0.016–0.125 ≤0.016–0.062 ≤0.016–0.125 ≤0.016–4 ≤0.016–4 ≤0.016–2
0.016 0.016 ≤0.016 ≤0.016 0.25 0.25 0.062 0.062 0.25 0.25 1 0.25 1 0.5 0.016 0.016 0.016 0.06 0.032 0.03
0.25 0.25 0.032 0.032 0.5 1 0.25 0.062 0.5 0.25 2 1 2 1 0.125 0.032 0.062 0.5 1 0.5
96.8 100 90.9 94.9 90 100 87.1 89.7 78.6 94.7
BMD = CLSI M27-A3 broth microdilution MICs (50% inhibition); MICs by both tests were determined after 24 h of incubation. MIC50 and MIC90 were the MICs encompassing 50% and 90% of the isolates tested, respectively. c Agreement between BMD and Etest MICs. d Including C. famata (5 isolates), C. dubliniensis, C. kefyr (2 isolates), C. lusitaniae, C. lipoltyca, C. pelliculosa, C. rugosa, and Saccharomyces cerevisae (1 isolate each). b
A. Espinel-Ingroff et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 54–59
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Table 3 CA agreement between micafungin CLSI broth microdilution and Etest MIC pairs of Candida spp. using species-specific CBPs Species (no. values)
C. albicans (125) C. krusei (39) C. guilliermondii (11) C. parapsilosis (31) C. tropicalis (39) Total (245)
Methoda
BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest
% of MIC by categoryb,c
%CAd
% of errors
S
I
R
92.8 92 94.8 94.9 100 100 96.8 100 100 100 95.1 95.1
2.4 1.6 2.6 5.1 0 0 3.2 0 0 0 2 1.6
4.8 6.4 2.6 0 0 0 0 0 0 0 2.9 3.3
Major
Very Major
Minor
1.6
0
2.4
96
0
0
2.6
97.4
0
0
0
0
0
3.2
0
0
0
0.8
0
2
100 96.8 100 97.2
a
BMD = CLSI M27-A3 broth microdilution MICs (50% inhibition); MICs were determined by both tests after 24 h of incubation (CLSI, 2008a; 2008b). CBP or category by microdilution method: (i) S = susceptible MICs ≤0.25 μg/mL for C. albicans, C. krusei, and C. tropicalis and MICs ≤2 μg/mL for C. guilliermondii and C. parapsilosis; R = resistant MICs of ≥1 μg/mL for the former 3 species and MICs of ≥8 μg/mL for the latter 2 species; I = intermediate (CLSI, 2010; Pfaller et al., 2011). c Percentages of BMD and Etest MICs that were within each CBP. d Percentages of BMD and Etest MIC pairs that were in agreement regarding each CBP. b
0.03 μg/mL; not listed in Table 3) and the intermediate C. parapsilosis isolate (MIC 4 μg/mL) were categorized by Etest as either intermediate or susceptible, respectively (MICs 0.5, 0.12 and 0.06 μg/mL). In contrast to a prior anidulafungin Etest evaluation (Espinel-Ingroff et al., 2010), the CA was excellent for C. parapsilosis (96.8%) in the present study. Our highest MICs for this species were mostly 1 μg/mL by both methods, while the susceptible CBP is ≤2 μg/mL and the resistant ≥8 μg/mL for this species; Etest anidulafungin MICs were unusually higher than reference values in that earlier evaluation. The CA agreement was 100% for C. tropicalis and C. guilliermondii, but we did not have echinocandin-resistant or echinocandin-intermediate
isolates among these species. The Food and Drug Administration target for major errors is b3% and b1.5% for very major errors (FDA, 2003). Therefore, according to the available CBPs for the 6 Candida spp., Etest could be considered suitable for testing the antifungal susceptibility of these species to micafungin, but a future evaluation with more resistant isolates of the most common Candida species is necessary. By using micafungin ECVs, we were able to evaluate the performance of Etest for testing C. glabrata since ECVs are available for this species and the other 5 species listed in Table 4 (Pfaller et al., 2010a). However, ECVs are not available for C. metapsilosis, C. orthopsilosis, and the less
Table 4 CA agreement between micafungin CLSI broth microdilution and Etest MIC pairs of Candida spp. using ECVs Species (no. of values)
Methoda (ECV)b
C. albicans (125)
BMD (0.03) Etest BMD (0.03) Etest BMD (4) Etest BMD (0.12) Etest BMD (4) Etest BMD (0.12) Etest BMD (NA) Etest
C. glabrata (52) C. guilliermondii (11) C. krusei (39) C. parapsilosis (31) C. tropicalis (39) Total (297) a b c d
% of MIC by categoryc
% of errors
%CA agreementd
≤ECV
N ECV
Major
Very major
87.2 86.4 96.2 98.1 100 100 89.7 94.9 100 100 100 100 92.6 93.3
12.8 13.6 3.8 1.9 0 0 10.3 5.1 0 0 0 0 7.4 6.7
1.6
0.8
97.6
1.9
3.8
94.3
0
0
0
5.1
0
0
100
0
0
100
1
1.7
BMD = CLSI M27-A3 broth microdilution MICs (50% inhibition); MICs were determined by both tests after 24 h of incubation. ECV by microdilution method (species-specific ECVs in μg/mL by the CLSI BMD method as defined by Pfaller et al., 2010a). Percentages of BMD and Etest MICs that were within each ECV. Percentages of BMD and Etest MIC pairs that were in agreement regarding each ECV.
100 94.9
97.3
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common species listed as miscellaneous under Table 1; with the exception of C. orthopsilosis, MICs for those species were ≤0.5 μg/mL. The CA according to Etest's ability to separate non-WT (or isolates with MICs above the ECV) from WT (or isolates with MICs equal or below the ECV) isolates was also excellent (94.3–100%, species dependent). There were 16 C. albicans with MICs above the ECV (CLSI MICs N0.03 μg/mL); one of these isolates was categorized as WT by the Etest (0.8% very major error) and 2 WT as nonWT (1.6 % major errors). Similar discrepant results were obtained among isolates with MICs above the ECV of C. glabrata (2 isolates, 1.9% and 3.8% major and very major errors, respectively) and of C. krusei (4 isolates, 5.1% very major errors), however, the CA agreement according to the recently developed CBPs was 98.1% (results not shown in Table 3). None of our isolates of C. parapsilosis and C. guilliermondii were outside the ECV of 4 μg/mL; these results are in agreement with the WT distribution of Pfaller et al., (2010a), where the highest micafungin MIC for the 1238 C. parapsilosis isolates was 2 μg/mL and there was only one isolate of C. guilliermondii for which the micafungin MIC was N4 μg/mL among their 88 isolates. It is interesting that in a prior comparison of echinocandin Etest and CLSI MICs using ECVs, the CA was excellent with the 3 echinocandins for most of the species; the exception was for C. krusei and caspofungin (81.8%) (Pfaller et al., 2010b). Seven isolates harboring fks1 and fks2 mutations were included among their isolates of C. albicans, C. glabrata, and C. tropicalis; both Etest and CLSI were able to differentiate WT strains from the 7 isolates harboring gene mutations, especially when using both micafungin and caspofungin as the antifungal agent. Although we had only 4 C. albicans mutants, our results corroborate those previous results. While the FDA's target for very major errors applies mostly to CBPs, percentages of these errors were unsuitably high regarding the identification of non-WT isolates among C. glabrata and C. krusei. Therefore, caution should be used doing interpretations of Etest data for these 2 species since Etest could miss some of the non-WT isolates. In conclusion, our data indicated suitable percentages of both EA and CA (the latter by using both CBPs and ECVs for the analysis) for most of the species evaluated. In addition, Etest has not been evaluated in multicenter studies to assess its reliability and ability to identify either echinocandin-resistant or non-WT isolates. Such studies are essential with large numbers of isolates, including welldocumented resistant isolates. References Baixench MT, Aoun N, Desnos-Ollivier M, Garcia-Hermoso D, Bretagne S, Ramires S, Piketty C, Dannaoui E (2007) Acquired resistance to echinocandins in Candida albicans: case report and review. J Antimicrob Chemother 59:1076–1083. Cantón E, Espinel-Ingroff A, Pemán J, del Castillo L (2010) In vitro fungicidal activities of echinocandins against Candida metapsilois,
C. orthopsilosis and C. parapsilosis evaluated by time-kill studies. Antimicrob Agents Chemother 54:2194–2197. Cantón E, Pemán J, Valentín A, Espinel-Ingroff A, Gobernado M (2009) In vitro activities of echinocandins against Candida krusei determined by three methods: MIC and minimal fungicidal concentration measurements and time-kill studies. Antimicrob Agents Chemother 53:3108–3111. Clinical and Laboratory Standards Institute (CLSI) (2007) Development of in vitro susceptibility testing criteria and quality control parameters; approved standard, third edition, M23-A3. Wayne, PA: CLSI. Clinical and Laboratory Standards Institute (CLSI) (2008a) Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard, third edition, M27-A3. Wayne, PA: CLSI. Clinical and Laboratory Standards Institute (CLSI) (2008b) Reference method for broth dilution antifungal susceptibility testing of yeasts; informational supplement, third edition, M27-S3. Wayne, PA: CLSI. Clinical and Laboratory Standards Institute (CLSI) (2010) Minutes of the Subcommittee on Antifungal Susceptibility Testing meeting, June 2010 and January 2011, Atlanta, GA. Dalhoff A, Ambrose PG, Mouton JW (2009) A long journey from minimum inhibitory concentration testing to clinically predictive breakpoints: deterministic and probabilistic approaches in deriving breakpoints. Infection 37:296–305. Desnos-Ollivier M, Bretagne S, Raoux D, Hoinard D, Dromer F, Dannaoui E (2008) 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: 3092–3098. Douglas CM, D'Ippolito JA, Shei GJ, Meinz M, Onishi J, Marrinan JA, Li W, Abruzzo GK, Flattery A, Bartizal K, Mitchell A, Kurtz MB (1997) Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-β-D-glucan synthase inhibitors. Antimicrob Agents Chemother 41:2471–2479. Espinel-Ingroff A, Canton E, Martin-Mazuelos E, Pemán J (2009) Pharmacotherapy of Candida infections with echinocandins. Clin Med Therapeutics 1:889–897. Espinel-Ingroff A, Canton E, Martin-Mazuelos E, Pemán J (2010) Comparison of anidulafungin MICs determined by the Clinical and Laboratory Standards Institute broth microdilution method (M27-A3 document) and Etest for Candida species isolates. Antimicrob Agents Chemother 54:1347–1350. Food and Drug Administration (2003) Class II special controls guidance document:antimicrobial susceptibility test systems; guidance for industry. Washington, DC: Food and Drug Administration. Garcia-Effron G, Park S, Perlin DS (2009a) Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints. Antimicrob Agents Chemother 53:112–122. Garcia-Effron G, Lee S, Park S, Cleary JD, Perlin DS (2009b) Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-β-D-glucan synthase: implications for the existing breakpoint. Antimicrob Agents Chemother 53:3690–3699. Jacobsen MD, Whyte JA, Odds FC (2007) Candida albicans and Candida dubliniensis respond differently to echinocandin antifungal agents in vitro. Antimicrob Agents Chemother 51:1882–1884. Kahn JN, Garcia-Effron G, Hsu MJ, Park S, Marr KA, Perlin DS (2007) Acquired echinocandin resistance in a Candida krusei isolate due to modification of glucan synthase. Antimicrob Agents Chemother 51:1876–1878. Pappas PG, Kauffman CA, Andes D, Benjamin Jr DK, Calandra TF, Edwards Jr JE, Filler SG, Fisher JF, Kullberg BJ, Ostrosky-Zeichner L, Reboli AC, Rex JH, Walsh TJ, Sobel JD (2009) Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 48: 503–535.
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Diagnostic Microbiology and Infectious Disease 70 (2011) 54 – 59 www.elsevier.com/locate/diagmicrobio
Mycology
Comparison of micafungin MICs as determined by the Clinical and Laboratory Standards Institute broth microdilution method (M27-A3 document) and Etest for Candida spp. isolates Ana Espinel-Ingroff a,⁎, Emilia Cantónb , Teresa Pelaezc , Javier Pemánd b
a VCU Medical Center, Richmond, VA 23298, USA Unidad de Microbiología, Centro de Investigación, Hospital Universitario La Fe, Valencia 46009, Spain c Hospital General Universitario Gregorio Marañon, Madrid 28007, Spain d Servicio Microbiología, Hospital Universitario La Fe, Valencia 46009, Spain Received 8 November 2010; accepted 10 December 2010
Abstract Micafungin Etest and Clinical and Laboratory Standards Institute (CLSI) MICs were compared for 337 Candida spp. isolates. The performance of Etest for testing the susceptibilities of Candida spp. to micafungin was evaluated by the assessment of both categorical (CA) and essential (EA) agreements. The CA was evaluated 2 ways: (i) by the ability of Etest to separate resistant (nontreatable) from susceptible (treatable) isolates by using the newly adjusted species-specific micafungin clinical breakpoints (CBPs) that are available for most of the common species tested and (ii) by the ability to separate wild type (WT) from non-WT isolates or those harboring FKS mutations (with reduced echinocandin susceptibility) by using micafungin epidemiologic cutoff values (ECVs). Etest and CLSI MICs were in EA when the MICs were within 2 log2 dilutions. Based on agreement percentages, our data indicated that Etest is suitable to test micafungin for most of the Candida species evaluated (overall EA 94.7%; overall CA according to CBPs 97.2% and according to ECVs 97.3%). However, the number of resistant isolates was small, so further evaluations are needed with a higher number of such isolates including more resistant or those with known mechanisms of resistance (non-WT). © 2011 Elsevier Inc. All rights reserved. Keywords: Etest micafungin; Micafungin MICs; Micafungin MICs for Candida; Etest versus reference MICs
1. Introduction Echinocandins (anidulafungin, caspofungin, and micafungin) are available for intravenous treatment and prevention of Candida and Aspergillus infections, especially for patients with recent azole exposure (Espinel-Ingroff et al., 2009; Pappas et al., 2009). In 2008, the Clinical and Laboratory Standards Institute (CLSI) established susceptibility testing guidelines and clinical breakpoints (CBPs) for the 3 echinocandins and the most common Candida spp. (susceptible MIC ≤2 μg/mL and nonsusceptible MIC N2 μg/ mL) (CLSI, 2008b). However, kinetic characterization of Candida albicans (Garcia-Effron et al., 2009a) and Can-
⁎ Corresponding author. Tel.: +804-358-5895. E-mail addresses: [email protected], [email protected] (A. Espinel-Ingroff). 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.12.010
dida glabrata (Garcia-Effron et al., 2009b) indicated that isolates harboring mutations in FKS1 and FKS2 regions had MICs below the susceptible CBP of ≤2 μg/mL. These results suggested that CBPs needed to be adjusted. More recently, epidemiologic cutoffs (ECVs) were proposed for several Candida spp. and the 3 echinocandins (Pfaller et al., 2010a). Because of this newer information, the CLSI is proposing a susceptible CBP (MIC ≤0.25 μg/mL) that would capture at least 95% of the clinical trials isolates of C. albicans, Candida tropicalis, and Candida krusei; however, the susceptible and resistant CBPs for C. glabrata and micafungin are ≤0.06 and ≥0.25μg/mL, respectively. The resistant endpoint for C. albicans, C. tropicalis, and C. krusei is ≥1 μg/ mL because it has been documented that these isolates are usually clinically resistant and harbor FKS mutations (CLSI, 2010; Pfaller et al., 2011). The susceptible CBP for Candida parapsilosis and Candida guilliermondii continues to be ≤2 μg/mL and the resistant cutoff is ≥8 μg/mL (Table 1).
A. Espinel-Ingroff et al. / Diagnostic Microbiology and Infectious Disease 70 (2011) 54–59 Table 1 CLSI proposed species-specific clinical breakpoints (CBPs) and ECVs for micafungin and selected Candida speciesa Species
C. albicans C. glabrata C. krusei C. tropicalis C. parapsilosis C. guilliermondii
MIC (μg/mL) S
I
R
ECVs
≤0.25 ≤0.06 ≤0.25 ≤0.25 ≤2 ≤2
0.5 0.12 0.5 0.5 4 4
≥1 ≥0.25 ≥1 ≥1 ≥8 ≥8
0.03 0.03 0.12 0.12 4 4
S = susceptible; I = intermediate; R = resistant. a Adopted from CLSI (2010), Pfaller et al., (2010a), and Pfaller et al., (2011).
We have evaluated the suitability (essential [EA] and categorical [CA] agreements) of micafungin Etest MICs for Candida spp. bloodstream isolates from 3 medical centers (VCU Medical Center, Richmond, VA; the Hospital La Fe, Valencia, Spain; and Hospital Gregorio Marañon, Madrid, Spain). The CA was evaluated according to both ECVs and newly proposed CBPs (available for 6 of the common species tested) (CLSI, 2010; Pfaller et al., 2010a, 2011). By using the CBPs for the comparison, we evaluated the ability of Etest to detect the resistant or nontreatable isolates and by using the ECVs the ability to separate wild type (WT or isolates with no acquired resistance mechanisms) from those harboring mechanisms of resistance with reduced susceptibility to micafungin (Turnidge et al., 2006). 1.1. Isolates The total set of isolates from the 3 laboratories included 125 C. albicans, 52 C. glabrata, 11 C. guilliermondii, 39 C. krusei, 39 C. tropicalis, 14 less common Candida spp., and 57 C. parapsilosis group (10 Candida metapsilosis, 16 Candida orthopsilosis, and 31 Candida parapsilosis; Table 2). The C. parapsilosis group was identified by using specific primers based on the intron of the RPSO gene (Canton et al., 2010). Among the 6 C. albicans echinocandin-resistant (CLSI MIC, N1 μg/mL) and 16 non-WT FKS mutant isolates (CLSI micafungin MICs N0.03 μg/mL), there were 3 well-documented heterozygous and one homozygous strains (Douglas et al., 1997; Jacobsen et al., 2007); one caspofungin-resistant C. krusei isolate was also included (Canton et al., 2009). C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 isolates were tested as QC; micafungin MICs were within the established QC limits (CLSI 2008a, 2008b).
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and Etest strips (0.002–32 μg/mL). MICs were the lowest drug concentration at which the border of the elliptical inhibition intercepted the strip scale, ignoring trailing growth or microcolonies inside the ellipse. CLSI MICs were the lowest micafungin concentration that showed ≥50% inhibition. 1.3. Definitions The WT is the population of organisms/MICs in a species/ antifungal agent combination with no acquired resistance mechanisms (Turnidge et al., 2006). The high WT MIC has been defined as the WT cutoff value or the ECV (Pfaller et al., 2010a; Turnidge et al., 2006) in contrast to the non-WT microorganisms with acquired or mutational resistance mechanisms (Dalhoff et al., 2009). On the other hand, the drug concentration that classifies the organisms as treatable (susceptible) or nontreatable (resistant) or the predictor of clinical outcome is now defined as the CBP (Dalhoff et al., 2009, Turnidge and Patterson, 2007). CBPs are established based on clinical trial data, global susceptibility surveillance, resistance mechanisms, and PK/PD parameters from model systems. 1.4. Data analysis For the comparison between the 2 methods, MICs from the 3 laboratories were pooled. Micafungin MICs were in EA when the discrepancies between the 2 methods were within 2 log2 dilutions. CA between the 2 methods was evaluated as follows: errors were calculated according to each available species-specific ECV and CBP (Table 1); they were considered very major errors when the reference MIC indicated resistance (according to the species CBP) or nonWT (an isolate with changed susceptibility phenotype or with an MIC above the corresponding ECV) and the Etest susceptibility or WT. Errors were considered major errors when the Etest categorized the isolate as resistant or non-WT and the reference method as susceptible or WT. Since intermediate CBPs are also available for some of the species, errors were considered minor when there was a single shift between the 2 results (e.g., susceptible to intermediate). A linear regression analysis using the least-square method (Pearson correlation coefficient; MS Excel software) was performed by plotting Etest versus reference MICs for correlation between the methods.
1.2. Antifungal susceptibility testing
2. Results and discussion
Each of the 3 participant laboratories tested their set of isolates; 2 of the 3 laboratories tested the C. albicans mutants. Micafungin (Astellas Pharma, Tokyo, Japan) MICs were determined by both CLSI M27-A3 and Etest methods after 24 h at 35 °C (CLSI, 2008a). Etest MICs were determined according to the manufacturer's instructions (AB BIODISK, Solna, Sweden) using RPMI agar (2% glucose)
A new or commercial method such as the Etest should identify resistant strains to the drug being evaluated in the same manner as the reference method or yield similar MICs as those by this method. Therefore, both CA and EA between Etest and CLSI reference methods were evaluated by using available ECVs as well as CBPs as listed in Table 1. To evaluate such performance, our set of isolates included
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echinocandin-resistant (micafungin MICs N1 μg/mL) and non-WT isolates (micafungin MICs above the CLSI microdilution species-specific ECV) among the C. albicans, C. glabrata, and C. krusei isolates; however, only 4 of our isolates (among the C. albicans) have known fks gene mutations. Echinocandin resistance for C. albicans, C. glabrata, C. krusei, and other species has been associated with high MICs (N1 μg/mL), mutations in the FKS gene, and therapeutic failure (Baixench et al., 2007; Desnos-Ollivier et al., 2008; Garcia-Effron et al., 2009a, 2009b; Kahn et al., 2007). To our knowledge this is the first evaluation of the suitability of Etest micafungin MICs for Candida spp. by using the newly species-specific proposed CBPs. Our CLSI MIC data for most species were the same or one dilution higher than those previously published (Pfaller et al., 2010a) as demonstrated by our MIC90s (encompassing 90% of isolates, Table 2). The susceptibilities of C. metapsilosis, C. orthopsilosis, and C. parapsilosis were variable by both methods as previously described (MIC ranges 0.03–0.5, 0.25–2, and 0.06-4 μg/mL, respectively (Canton et al., 2010). The overall EA was 94.7% (R = 0.814) and it was N90% for most species, except for C. tropicalis (89.7%), C. parapsilosis (87.1%), and the less common species listed as miscellaneous in Table 2. The EA in the present study was similar to a prior comparison of the 2 methods (EA, N90%) (Pfaller et al., 2010b), but superior to that reported for anidulafungin Etest and CLSI MICs (Espinel-Ingroff et al., 2010). In the latter study, EA agreement was b90% for most species, but 100% for C. tropicalis. The acceptable EA is
N90% (CLSI, 2007). Our micafungin Etest results usually were the same or one dilution lower than reference results, while anidulafungin Etest MICs have been reported as mostly higher (Espinel-Ingroff et al., 2010). These differences could be attributed to anidulafungin (non–watersoluble agent) diffusion problems. Table 3 depicts the CA between CLSI and Etest for the species for which both susceptible and resistant speciesspecific microdilution CBPs are available (C. albicans, C. guilliermondii, C. krusei, C. parapsilosis, C. tropicalis and more recently C. glabrata [not listed in Table 3]) (CLSI, 2010; Pfaller et al., 2011). The CLSI subcommittee has not yet defined the susceptible CBP for the less common species listed in Table 2 as miscellaneous. There were no very major errors for any of 2 species that had micafungin-resistant isolates (C. albicans and C. krusei). That means that both methods were able to identify 6 of the 7 micafungin-resistant isolates (MICs N1 μg/mL). The Etest MIC for one of the C. albicans laboratory mutants was 1 μg/mL, while the CLSI MIC was 0.25 μg/mL (major error). This strain was moderately resistant to caspofungin in the mouse model of disseminated candidiasis (Douglas et al., 1997) and was categorized as non-WT. The overall number of minor and major errors was small (2% and 0.8%, respectively), and most of them were found among the isolates of C. albicans (2 major or 1.6%, and 3 minor or 2.4%). There was only one minor error each among C. krusei, C. grabrata and C. parapsilosis isolates, where the resistant C. krusei isolate (MIC 1 μg/mL), the susceptible C. glabrata (MIC
Table 2 Susceptibilities of 337 isolates of Candida spp. to micafungin as determined by the CLSI broth microdilution (M27-A3) and Etest methods Species (no. tested)
C. albicans (125) C. glabrata (52) C. guilliermondii (11) C. krusei (39) C. metapsilosis (10) C. orthopsilosis (16) C. parapsilosis (31) C. tropicalis (39) Miscellaneous speciesd (14) Overall (337) a
Methoda
BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest
MIC (μg/mL)b
EA (%)c
MIC range
MIC50
MIC90
≤0.016–2 ≤0.016–2 ≤0.016–0.062 ≤0.016–0.125 0.032–0.5 ≤0.016–1 0.032–1 ≤0.016–0.5 0.032–0.5 0.12–0.25 0.25–2 0.25–1 0.06–4 0.03–1 ≤0.016–0.125 ≤0.016–0.062 ≤0.016–0.125 ≤0.016–4 ≤0.016–4 ≤0.016–2
0.016 0.016 ≤0.016 ≤0.016 0.25 0.25 0.062 0.062 0.25 0.25 1 0.25 1 0.5 0.016 0.016 0.016 0.06 0.032 0.03
0.25 0.25 0.032 0.032 0.5 1 0.25 0.062 0.5 0.25 2 1 2 1 0.125 0.032 0.062 0.5 1 0.5
96.8 100 90.9 94.9 90 100 87.1 89.7 78.6 94.7
BMD = CLSI M27-A3 broth microdilution MICs (50% inhibition); MICs by both tests were determined after 24 h of incubation. MIC50 and MIC90 were the MICs encompassing 50% and 90% of the isolates tested, respectively. c Agreement between BMD and Etest MICs. d Including C. famata (5 isolates), C. dubliniensis, C. kefyr (2 isolates), C. lusitaniae, C. lipoltyca, C. pelliculosa, C. rugosa, and Saccharomyces cerevisae (1 isolate each). b
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Table 3 CA agreement between micafungin CLSI broth microdilution and Etest MIC pairs of Candida spp. using species-specific CBPs Species (no. values)
C. albicans (125) C. krusei (39) C. guilliermondii (11) C. parapsilosis (31) C. tropicalis (39) Total (245)
Methoda
BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest BMD Etest
% of MIC by categoryb,c
%CAd
% of errors
S
I
R
92.8 92 94.8 94.9 100 100 96.8 100 100 100 95.1 95.1
2.4 1.6 2.6 5.1 0 0 3.2 0 0 0 2 1.6
4.8 6.4 2.6 0 0 0 0 0 0 0 2.9 3.3
Major
Very Major
Minor
1.6
0
2.4
96
0
0
2.6
97.4
0
0
0
0
0
3.2
0
0
0
0.8
0
2
100 96.8 100 97.2
a
BMD = CLSI M27-A3 broth microdilution MICs (50% inhibition); MICs were determined by both tests after 24 h of incubation (CLSI, 2008a; 2008b). CBP or category by microdilution method: (i) S = susceptible MICs ≤0.25 μg/mL for C. albicans, C. krusei, and C. tropicalis and MICs ≤2 μg/mL for C. guilliermondii and C. parapsilosis; R = resistant MICs of ≥1 μg/mL for the former 3 species and MICs of ≥8 μg/mL for the latter 2 species; I = intermediate (CLSI, 2010; Pfaller et al., 2011). c Percentages of BMD and Etest MICs that were within each CBP. d Percentages of BMD and Etest MIC pairs that were in agreement regarding each CBP. b
0.03 μg/mL; not listed in Table 3) and the intermediate C. parapsilosis isolate (MIC 4 μg/mL) were categorized by Etest as either intermediate or susceptible, respectively (MICs 0.5, 0.12 and 0.06 μg/mL). In contrast to a prior anidulafungin Etest evaluation (Espinel-Ingroff et al., 2010), the CA was excellent for C. parapsilosis (96.8%) in the present study. Our highest MICs for this species were mostly 1 μg/mL by both methods, while the susceptible CBP is ≤2 μg/mL and the resistant ≥8 μg/mL for this species; Etest anidulafungin MICs were unusually higher than reference values in that earlier evaluation. The CA agreement was 100% for C. tropicalis and C. guilliermondii, but we did not have echinocandin-resistant or echinocandin-intermediate
isolates among these species. The Food and Drug Administration target for major errors is b3% and b1.5% for very major errors (FDA, 2003). Therefore, according to the available CBPs for the 6 Candida spp., Etest could be considered suitable for testing the antifungal susceptibility of these species to micafungin, but a future evaluation with more resistant isolates of the most common Candida species is necessary. By using micafungin ECVs, we were able to evaluate the performance of Etest for testing C. glabrata since ECVs are available for this species and the other 5 species listed in Table 4 (Pfaller et al., 2010a). However, ECVs are not available for C. metapsilosis, C. orthopsilosis, and the less
Table 4 CA agreement between micafungin CLSI broth microdilution and Etest MIC pairs of Candida spp. using ECVs Species (no. of values)
Methoda (ECV)b
C. albicans (125)
BMD (0.03) Etest BMD (0.03) Etest BMD (4) Etest BMD (0.12) Etest BMD (4) Etest BMD (0.12) Etest BMD (NA) Etest
C. glabrata (52) C. guilliermondii (11) C. krusei (39) C. parapsilosis (31) C. tropicalis (39) Total (297) a b c d
% of MIC by categoryc
% of errors
%CA agreementd
≤ECV
N ECV
Major
Very major
87.2 86.4 96.2 98.1 100 100 89.7 94.9 100 100 100 100 92.6 93.3
12.8 13.6 3.8 1.9 0 0 10.3 5.1 0 0 0 0 7.4 6.7
1.6
0.8
97.6
1.9
3.8
94.3
0
0
0
5.1
0
0
100
0
0
100
1
1.7
BMD = CLSI M27-A3 broth microdilution MICs (50% inhibition); MICs were determined by both tests after 24 h of incubation. ECV by microdilution method (species-specific ECVs in μg/mL by the CLSI BMD method as defined by Pfaller et al., 2010a). Percentages of BMD and Etest MICs that were within each ECV. Percentages of BMD and Etest MIC pairs that were in agreement regarding each ECV.
100 94.9
97.3
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common species listed as miscellaneous under Table 1; with the exception of C. orthopsilosis, MICs for those species were ≤0.5 μg/mL. The CA according to Etest's ability to separate non-WT (or isolates with MICs above the ECV) from WT (or isolates with MICs equal or below the ECV) isolates was also excellent (94.3–100%, species dependent). There were 16 C. albicans with MICs above the ECV (CLSI MICs N0.03 μg/mL); one of these isolates was categorized as WT by the Etest (0.8% very major error) and 2 WT as nonWT (1.6 % major errors). Similar discrepant results were obtained among isolates with MICs above the ECV of C. glabrata (2 isolates, 1.9% and 3.8% major and very major errors, respectively) and of C. krusei (4 isolates, 5.1% very major errors), however, the CA agreement according to the recently developed CBPs was 98.1% (results not shown in Table 3). None of our isolates of C. parapsilosis and C. guilliermondii were outside the ECV of 4 μg/mL; these results are in agreement with the WT distribution of Pfaller et al., (2010a), where the highest micafungin MIC for the 1238 C. parapsilosis isolates was 2 μg/mL and there was only one isolate of C. guilliermondii for which the micafungin MIC was N4 μg/mL among their 88 isolates. It is interesting that in a prior comparison of echinocandin Etest and CLSI MICs using ECVs, the CA was excellent with the 3 echinocandins for most of the species; the exception was for C. krusei and caspofungin (81.8%) (Pfaller et al., 2010b). Seven isolates harboring fks1 and fks2 mutations were included among their isolates of C. albicans, C. glabrata, and C. tropicalis; both Etest and CLSI were able to differentiate WT strains from the 7 isolates harboring gene mutations, especially when using both micafungin and caspofungin as the antifungal agent. Although we had only 4 C. albicans mutants, our results corroborate those previous results. While the FDA's target for very major errors applies mostly to CBPs, percentages of these errors were unsuitably high regarding the identification of non-WT isolates among C. glabrata and C. krusei. Therefore, caution should be used doing interpretations of Etest data for these 2 species since Etest could miss some of the non-WT isolates. In conclusion, our data indicated suitable percentages of both EA and CA (the latter by using both CBPs and ECVs for the analysis) for most of the species evaluated. In addition, Etest has not been evaluated in multicenter studies to assess its reliability and ability to identify either echinocandin-resistant or non-WT isolates. Such studies are essential with large numbers of isolates, including welldocumented resistant isolates. References Baixench MT, Aoun N, Desnos-Ollivier M, Garcia-Hermoso D, Bretagne S, Ramires S, Piketty C, Dannaoui E (2007) Acquired resistance to echinocandins in Candida albicans: case report and review. J Antimicrob Chemother 59:1076–1083. Cantón E, Espinel-Ingroff A, Pemán J, del Castillo L (2010) In vitro fungicidal activities of echinocandins against Candida metapsilois,
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