Clinical Breakpoints for the Echinocandins and Candida Revisited

G Model YDRUP-474; No. of Pages 13 ARTICLE IN PRESS

Drug Resistance Updates xxx (2011) xxx–xxx

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Drug Resistance Updates

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Clinical breakpoints for the echinocandins and Candida revisited: Integration of molecular, clinical, and microbiological data to arrive at species-speciﬁc interpretive criteria

M.A. Pfaller a,∗, D.J. Diekema a, D. Andes b, M.C. Arendrup c, S.D. Brown d, S.R. Lockhart e, M. Motyl f, D.S. Perlin g, the CLSI Subcommittee for Antifungal Testing a University of Iowa, Iowa City, Iowa, United States b University of Wisconsin, Madison, WI, United States c Statens Serum Institute, Copenhagen, Denmark d The Clinical Microbiology Institute, Wilsonville, OR, United States e Centers for Disease Control and Prevention, Atlanta, GA, United States f Merck and Company, Inc, Rahway, New Jersey, United States g Public Health Research Institute, New Jersey Medical School–UMDNJ, Newark, NJ, United States article info abstract

Article history: The CLSI established clinical breakpoints (CBPs) for caspofungin (CSF), micafungin (MCF) and anidulafun- Received 28 June 2010 gin (ANF) versus Candida. The same CBP (susceptible (S): MIC ≤ 2 mcg/ml; non-S: MIC > 2 mcg/ml) was Received in revised form 17 January 2011 applied to all echinocandins and species. More data now allow reassessment of these CBPs. Accepted 20 January 2011 We examined cases of echinocandin failure where both MICs and fks mutations were assessed; wild type (WT) MICs and epidemiological cutoff values (ECVs) for a large Candida collection; molecular analysis Keywords: of fks hotspots for Candida with known MICs; and pharmacokinetic and pharmacodynamic (PK/PD) data. Candida We applied these findings to propose new species-specific CBPs for echinocandins and Candida. Echinocandins Susceptibility testing Of 18 candidiasis cases refractory to echinocandins and with fks mutations, 28% (CSF), 58% (ANF) and 66% (MCF) had MICs in the S category using CBP of ≤2 mcg/ml, while 0–8% would be S using CBP of ≤0.25 mcg/ml. WT MIC distributions revealed ECV ranges of 0.03–0.25 mcg/ml for all major species except C. parapsilosis (1–4 mcg/ml) and C. guilliermondii (4–16 mcg/ml). Among Candida tested for fks mutations, only 15.7–45.1% of 51 mutants were detected using the CBP for NS of >2 mcg/ml. In contrast, a cutoff of >0.25 mcg/ml for C. albicans, C. tropicalis, C. krusei, and C. dubliniensis detected 85.6% (MCF) to 95.2% (CSF) of 21 mutant strains. Likewise, a cutoff of >0.12 mcg/ml for ANF and CSF and of >0.06 mcg/ml for MCF detected 93% (ANF) to 97% (CSF, MCF) of 30 mutant strains of C. glabrata. These data, combined with PK/PD considerations, support CBPs of ≤0.25 mcg/ml (S), 0.5 mcg/ml (I), ≥1 (R) for CSF/MCF/ANF and C. albicans, C. tropicalis and C. krusei and ≤2 mcg/ml (S), 4 mcg/ml (I), and ≥8 mcg/ml (R) for these agents and C. parapsilosis. The CBPs for ANF and CSF and C. glabrata are ≤0.12 mcg/ml (S), 0.25 mcg/ml (I), and ≥0.5 mcg/ml (R), whereas those for MCF are ≤0.06 mcg/ml (S), 0.12 mcg/ml (I), and ≥0.25 mcg/ml (R). New, species-specific CBPs for Candida and the echinocandins are more sensitive to detect emerging resistance associated with fks mutations, and better able to predict risk for clinical failure. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction and exhibit concentration-dependent fungicidal activity against most species of Candida (Cappelletty and Eiselstein-McKitrick, The echinocandins (anidulafungin [ANF], caspofungin [CSF], 2007; Chandrasekar and Sobel, 2006; Deresinski and Stevens, 2003; and micafungin [MCF]) are lipopeptide antifungal agents that Dodds-Ashley et al., 2006; Messer et al., 2006a,b; Pfaller, 2004; inhibit the synthesis of ␤-1, 3-d-glucan in the fungal cell wall Vazquez, 2005; Zaas and Alexander, 2005). All three agents have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of esophageal candidiasis and invasive candidiasis

∗ (IC), including candidemia (DeWet et al., 2004; Kuse et al., 2007; Corresponding author at: Medical Microbiology Division, C606 GH, Department Mora-Duarte et al., 2002; Ostrosky-Zeichner et al., 2005; Pappas of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242, United States. Tel.: +1 319 356 8615; fax: +1 319 356 4916. et al., 2007; Mycamine [MCF] package insert, 2005; Astellas Pharma E-mail address: [email protected] (M.A. Pfaller). US, Deerﬁeld, IL; Cancidas [CSF] package insert, 2001, Merck and

Please cite this article in press as: Pfaller, M.A., et al., 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. (2011), doi:10.1016/j.drup.2011.01.004 G Model YDRUP-474; No. of Pages 13 ARTICLE IN PRESS 2 M.A. Pfaller et al. / Drug Resistance Updates xxx (2011) xxx–xxx

Co., Whitehouse Station, NJ; and Eraxis [ANF] package insert, 2006, et al., 2009a; Arendrup et al., 2010; Baixench et al., 2007; Desnos- Pfizer, Inc., New York, NY), and are now recognized as the preferred Ollivier et al., 2008; Garcia-Effron et al., 2008a,b; Garcia-Effron systemically active antifungal agents for the treatment of IC (Pappas et al., 2009a,b, 2010; Laverdiere et al., 2006; Pfaller et al., 2010a,b; et al., 2009). When used in the treatment of IC (e.g., bloodstream Thompson et al., 2008). Furthermore, kinetic studies of the glucan infections [BSI], deep tissue sites, other normally sterile site infec- synthase (GS) enzyme complex suggest that a lower MIC cutoff of tions), ANF is administered as an initial intravenous loading dose of 0.25–0.5 mcg/ml may be more sensitive in detecting those strains 200 mg followed by a daily dose of 100 mg; CSF is administered as a with fks1/fks2 mutations (Garcia-Effron et al., 2009a,b; Wiederhold loading dose of 70 mg, followed by a daily dose of 50 mg; and MCF is et al., 2008). These observations call into question the ability of administered as a daily dose of 100 mg without the requirement of the current CBPs to reliably identify isolates with resistance mech- a loading dose (Cappelletty and Eiselstein-McKitrick, 2007; Dodds- anisms associated with treatment failure (Arendrup et al., 2010; Ashley et al., 2006; Zaas and Alexander, 2005). Doses of ANF and Garcia-Effron et al., 2009a,b). MCF in excess of 300 mg/d have been shown to be well-tolerated In this review, we readdress the issue of echinocandin break- (Chandrasekar and Sobel, 2006; Vazquez, 2005), and a recent mul- points for Candida spp. by using the available published molecular, ticenter, double-blind trial of CSF at a daily dose of 150 mg/d versus microbiologic, pharmacodynamic (PD), and clinical data in an effort the standard dosing regimen found that the high-dose regimen was to optimize the ability of in vitro susceptibility testing to detect safe and efficacious in the treatment of IC (Betts et al., 2009). emerging echinocandin resistance and to ensure the safe and effi- There is now a broad clinical experience using the echinocandins cacious use of these agents in the treatment of IC. These analyses to treat both mucosal and invasive forms of candidiasis (Bal, 2010; are summarized below. Glockner et al., 2009; Lichtenstein et al., 2008; Ortega et al., 2010; Sipsas et al., 2009a,b; Zaas et al., 2006). Despite the expanding use of these agents, clinical failures remain uncommon, although reports 2. Mechanisms of action and resistance to echinocandins in of echinocandin resistance among Candida spp. are becoming more Candida spp. prevalent (Baixench et al., 2007; Ghannoum et al., 2009; Kofteridis et al., 2010; Perlin, 2007; Perlin, 2009; Pfeiffer et al., 2010; Sipsas Echinocandins inhibit ␤-1, 3-d-glucan synthase (GS), which et al., 2009b; Sun and Singh, 2010). The application of in vitro sus- catalyzes the biosynthesis of ␤-1, 3-d-glucan, the major glucan ceptibility testing and the use of molecular methods have served component of Candida cell walls (Douglas, 2001; Onishi et al., to detect potentially resistant strains of Candida and to character- 2000). GS is an enzyme complex with at least two subunits, Fksp ize the various mechanisms of resistance to the echinocandin class and Rho1p. The latter is a regulatory element involved in a num- among clinical isolates of Candida spp. (Arendrup et al., 2010; Cleary ber of cellular processes (Kondoh et al., 1997). Fksp, encoded by et al., 2008; Garcia-Effron et al., 2009a,b; Perlin, 2007; Perlin, 2009; three related genes, fks1, fks2, and fks3, contains the active site, Pfaller et al., 2008a,b, 2010a,b; Wiederhold et al., 2008). which catalyzes the transfer of sugar moieties from activated donor The Clinical and Laboratory Standards Institute (CLSI) Subcom- molecules to specific acceptor molecules forming glycosidic bonds mittee for Antifungal Testing has developed and standardized (Kondoh et al., 1997; Sawistowska-Schroder et al., 1984; Tang and broth microdilution (BMD) and disk diffusion methods for in vitro Parr, 1991). The inhibition of GS by echinocandin drugs disrupts susceptibility testing of Candida spp. against the echinocandins the structure of the growing cell wall, resulting in osmotic instabil- (Clinical and Laboratory Standards Institute, 2008a; Clinical and ity and the death of susceptible yeast cells (Bowman et al., 2002; Laboratory Standards Institute, 2008b; Clinical and Laboratory Kartsonis et al., 2003). Standards Institute, 2008c). In addition to standardized testing Echinocandin resistance in susceptible species such as C. albi- methods, the CLSI Subcommittee has approved quality control (QC) cans, C. tropicalis, and C. krusei is uncommon, but it has been limits for BMD testing of all three echinocandins and for disk dif- associated with amino acid substitutions in Fks1p (Perlin, 2007). fusion testing of CSF and MCF (Clinical and Laboratory Standards Likewise, it is now understood that amino acid substitutions in Institute, 2008b; Clinical and Laboratory Standards Institute, 2007). Fks1p and Fks2p are responsible for clinical echinocandin resis- These methods have been applied worldwide to generate a detailed tance in C. glabrata (Cleary et al., 2008; Garcia-Effron et al., 2009a; and comprehensive understanding of the in vitro susceptibility pro- Garcia-Effron et al., 2010; Katiyar et al., 2006; Thompson et al., file of Candida spp. to ANF, CSF, and MCF (Arendrup et al., 2009a; 2008). These mutations, which result in elevated MICs (4- to 30- Baixench et al., 2007; Espinel-Ingroff, 2003; Messer et al., 2006a; fold MIC increases for CSF and 90- to 110-fold increases for ANF Ostrosky-Zeichner et al., 2003; Perlin, 2009; Pfaller et al., 2005, and MCF), reduce the sensitivity of GS to inhibition by drug by 30- 2006, 2008a, 2010a; Pfaller and Diekema, 2007). to more than a thousand fold (Garcia-Effron et al., 2009a,b, 2010; In 2007, the CLSI Subcommittee for Antifungal Testing used the Park et al., 2005)(Table 1). Among isolates of C. albicans, the most accumulated clinical and microbiological data to propose clinical significant MIC increases have been shown to be related to amino interpretive breakpoints (CBP) for MIC testing of the echinocandins acid changes at Ser 645 (S645P, S645F, and S645Y), whereas the against Candida spp. (Pfaller et al., 2008b). The CBPs, which were other mutations account for smaller increases (Garcia-Effron et al., subsequently incorporated into CLSI documents M27-A3 and M27- 2009b)(Table 1). As shown in Table 1, a relatively narrow spectrum S3 (Clinical and Laboratory Standards Institute, 2008a; Clinical and of fks1 mutations in strains of C. albicans confer reduced susceptibil- Laboratory Standards Institute, 2008b), were as follows: suscepti- ity across the entire class of echinocandin agents. Likewise, these ble (S), MIC ≤ 2 mcg/ml for all three echinocandins and all species of mutations alter the GS enzyme kinetics resulting in significantly Candida. Due to the lack of echinocandin resistance in the popula- higher 50% inhibitory concentrations (IC50), as well as the kinetic tion of Candida isolates at that time, the Subcommittee decided not inhibition constant (Ki), for the mutant enzymes when compared to to define a resistant (R) breakpoint and recommended that isolates corresponding enzymes from wild-type strains (Garcia-Effron et al., for which the MIC exceeded 2 mcg/ml be called non-susceptible 2009b)(Table 1). Furthermore, this pattern of decreased enzyme (NS) and be referred to a reference laboratory for confirmation sensitivity to inhibition (increased IC50) extends across all three of of species identification and susceptibility testing (Pfaller et al., the echinocandins. 2008b). Recently, however, it has become apparent that clini- Similar to that seen with C. albicans, amino acid substitutions in cally resistant Candida infections involving strains with mutations both Fks1p and Fks2p of C. glabrata and in Fks1p of C. tropicalis and in fks 1 and/or fks 2 (encodes glucan synthase, the echinocandin C. krusei have been linked with increases in echinocandin MIC and target) do not necessarily have MICs above the CBP (Arendrup IC50 values, supporting the contention that changes in fks represent

Please cite this article in press as: Pfaller, M.A., et al., 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. (2011), doi:10.1016/j.drup.2011.01.004 G Model YDRUP-474; No. of Pages 13 ARTICLE IN PRESS M.A. Pfaller et al. / Drug Resistance Updates xxx (2011) xxx–xxx 3

Table 1 a Echinocandin MIC and ␤1,3-d-glucan synthase inhibition proﬁles (IC50) for C. albicans and C. glabrata fks mutant strains.

b b Species Mutation MIC (␮g/ml) IC50 (ng/ml)

Fks1p Fks2p ANF CSF MCF ANF CSF MCF

C. albicans WT – 0.08 0.42 0.05 0.89 3.88 58.2 WT – 0.03 0.21 0.03 1.51 0.9 10.2 F641L – 0.16 2.0 0.33 11.3 10.03 183.6 F641S – 0.83 4.0 1.0 2622 1091 1441 F641S – 1.0 3.33 1.0 1085 910 1693 S645P – 1.33 8.0 4.0 989 245.4 1085 S645P – 2.0 8.0 4.0 1152 689.1 1254 F645Y – 2.0 8.0 4.0 2739 2075 2533 S645F – 2.0 4.0 2.67 787 459.1 449.8 D648Y – 0.83 2.67 0.83 93.3 68.22 375.5 P649H – 0.67 4.0 0.83 491 82.66 924.6 R1361H – 0.25 2.0 0.25 75.99 33.72 498.2 S645F – 4.0 4.0 2.67 782 530.7 1765 R1361 R/H – 0.25 1.0 0.5 40.6 20.43 77.39 S645 S/P – 0.5 4.0 0.5 128.7 218.1 177.3 S645P – 4.0 8.0 4.0 901 322.4 1194

C. glabrata WT WT 0.06 0.06 0.03 2.71 1.92 0.71 F625S WT 2.0 8.0 0.5 110.6 667.8 126.3 S629P WT 4.0 8.0 2.0 3855.4 4987.5 854.5 D632G WT 1.0 4.0 0.5 101.8 756.3 343.4 D632E WT 2.0 2.52 2.52 151.5 844.5 444.7 D632Y R1377STOP 4.0 8.0 1.0 2543.5 5248.5 413.4 WT F659del 2.0 2.0 4.0 160 117.2 123.5 WT F659V 1.0 4.0 1.0 86.6 111.3 113.5 WT F659S 4.0 16.0 4.0 858.1 1064.0 1067.0 WT S663P 1.0 16.0 1.0 4003.5 5115.5 2569.0 WT D666G 2.0 4.0 0.12 175.8 506.6 100.6 WT D666E 1.0 4.0 0.25 127.4 257.9 88.7 WT D667T 2.0 4.0 0.25 99.5 166.2 110.9 WT W1375L 0.25 4.0 0.25 85.2 608.4 50.7

a Data compiled from Garcia-Effron et al. (2009a) and Garcia-Effron et al. (2009b). b ANF, anidulafungin; CSF, caspofungin; MCF, micafungin; IC50, 50% inhibitory concentration for ␤ 1,3-d-glucan synthase. MIC values are the geometric mean of 3 replicate determinations. IC50 values are the arithmetic mean of three replicate determinations. a universal echinocandin resistance mechanism among Candida and Singh, 2010), provide compelling examples of the relationship spp. (Desnos-Ollivier et al., 2008; Garcia-Effron et al., 2008b, 2009a, between high or increasing echinocandin (caspofungin) MICs and a 2010; Kahn et al., 2007; Park et al., 2005; Perlin, 2007)(Table 1). poor clinical outcome, they also underscore the inability of the cur- As demonstrated in Table 1 for both C. albicans and C. glabrata, rent CBP to identify many of these strains with acquired resistance while the MICs for the fks mutants are elevated above those of mechanisms. Among the 18 cases listed in Table 3 where clinical WT strains for all three echinocandins, the highest MICs are seen failure was associated with fks mutations, 72% would be classified with caspofungin. Although there is a clear linkage between fks as NS to CSF, 42% (5 of 12 where ANF was tested) as NS to ANF, and mutations, increased echinocandin MICs and IC50s, and treatment 33% (4 of 12 where MCF was tested) as NS to MCF at the CBP for S of failure (Garcia-Effron et al., 2009a,b; Perlin, 2007), it is notable that ≤2 mcg/ml. In contrast, 92% to 100% would be correctly identified whereas 10 of 12 (83%) clinical fks mutants of C. albicans and 11 using a lower cutoff of 0.25 mcg/ml for all three echinocandins. of 13 (85%) fks mutants of C. glabrata are captured at CSF MICs of Recently the ability of the CLSI BMD method to differentiate >2 mcg/ml, only 16% to 50% of these mutants would be detected wild-type (WT) from fks1/fks2 mutant strains of Candida spp. was at the same cutoff for both ANF and MCF (Table 2). Similar results examined in three independent studies (Arendrup et al., 2010; were reported by Wiederhold et al. (2008) for a different set of C. Pfaller et al., 2010b; Zimbeck et al., 2010). The data from these albicans isolates (Table 2). three studies is combined in Table 4 to provide a more robust look Thus, the CBP of ≤2 mcg/ml is clearly inadequate when test- at potential echinocandin breakpoints. The isolates were selected ing ANF or MCF despite comparably altered enzyme inhibition from global and population-based surveillance and reference col- kinetics for all three agents. As suggested by Garcia-Effron et al. lections to represent both WT and non-WT MIC results for each (2009a,b), one may need to lower the breakpoint to as low as of the three echinocandins and all isolates were further character- 0.25 mcg/ml for all three echinocandins in order to ensure the cor- ized regarding the presence or absence of mutations in the hot-spot rect classification of mutant strains of Candida. Based on the data (HS) region of fks1 and fks2 (C. glabrata)(Table 5). A total of 51 shown in Tables 1 and 2, a breakpoint of 0.25 mcg/ml for all three isolates were found to have fks1/fks2 mutations (Tables 4 and 5): agents would capture 79–100% of fks mutant isolates of C. albicans 11 C. albicans,30C. glabrata,6C. tropicalis,3C. krusei, and 1 C. whereas a breakpoint of 0.12 mcg/ml would capture 92–100.0% of dubliniensis. Using a cutoff of >0.25 mcg/ml for C. albicans, C. tropi- fks mutants of C. glabrata. calis, C. krusei, and C. dubliniensis, the CLSI method distinguished 19 FKS mutations conferring resistance to caspofungin and other (90.5%) of 21 mutant strains from WT strains using ANF as the test echinocandins have been identified in several strains of C. albicans, reagent, 20 (95.2%) of 21 using CSF, and 18 (85.6%) of 21 using MCF C. glabrata, C. tropicalis and C. krusei from patients with infections (Table 4). The data for the three echinocandins and C. glabrata sug- refractory to echinocandin therapy (17 of 18 cases treated with gest that an even lower cutoff of >0.12 mcg/ml for ANF and CSF and CSF and 1 each with ANF and MCF) (Table 3). Whereas these and of >0.06 mcg/ml for MCF may be necessary to allow maximal detec- other case reports, for which studies to document mutations were tion of the mutant strains (Table 4). This lower cutoff distinguished not performed (Baixench et al., 2007; Pfaller et al., 2008b; Sun 93% (ANF), 97% (CSF) and 97% (MCF) of the 30 mutant strains of C.

Please cite this article in press as: Pfaller, M.A., et al., 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. (2011), doi:10.1016/j.drup.2011.01.004 G Model YDRUP-474; No. of Pages 13 ARTICLE IN PRESS 4 M.A. Pfaller et al. / Drug Resistance Updates xxx (2011) xxx–xxx

Table 2 Echinocandin MICs predictive of C. albicans and C. glabrata strains with FKS1 mutations and glucan synthase enzyme insensitivity to inhibition by drug.

Reference Species (no. tested) Antifungal agenta MIC (␮g/ml)b No. for which MIC (␮g/ml)

Range 50% 90% >0.25 >0.5 >1 >2

Garcia-Effron et al. (2009b) C. albicans (12) ANF 0.25–4 1 2 11 10 6 2 CSF 2–8 4 8 12 12 12 10 MCF 0.25–4 1 4 11 10 6 6

Wiederhold et al. (2008) C. albicans (12) ANF 0.12–1 0.25 1 6 2 0 0 CSF 2–8 4 8 12 12 12 9 MCF 0.5–4 1 4 12 9 3 2

Garcia-Effron et al. (2009a) C. glabrata (13) ANF 0.25–4 2 4 12 12 8 3 CSF 2–16 4 16 13 13 13 11 MCF 0.12–4 0.5 4 9 7 4 3

a ANF, anidulafungin; CSF, caspofungin; MCF, micafungin. b 50% and 90%, MIC encompassing 50% and 90% of isolates tested, respectively.

Table 3 Published cases of Candida spp. infections with fks1/fks2 mutations and increased MICs of echinocandins as determined by the CLSI reference method.a

Species (ref.) Year reported Infection type Antifungal Agents (MIC in ␮g/ml) Comments treatmentb

C. albicans (Park et al., 2005) 2005 Disseminated CSF CSF (>8) S645F and S645P mutations C. albicans (Park et al., 2005) 2005 Disseminated CSF CSF (4) S645F mutation C. krusei (Park et al., 2005) 2005 Fungemia CSF CSF (32) R1361G mutation C. glabrata (Dodgson et al., 2005) 2005 Fungemia AMB, VRC, CSF CSF (>8), ANF (>8), MCF (>8) S663P mutation C. albicans (Miller et al., 2006) 2006 Esophagitis AMB, FLC, VRC, CSF CSF (8) S645P mutation C. albicans (Laverdiere et al., 2006) 2006 Esophagitis AMB, FLC, VRC, CSF, CSF (2), ANF (1), MCF (2) S645F and R1361H mutations ITZ, MCF C. krusei (Hakki et al., 2006;Kahn et al., 2007) 2006 Fungemia CSF, AMB, VRC CSF (8), ANF (4), MCF (4) F655C mutation C. albicans (Baixench et al., 2007) 2007 Esophagitis FLC, VRC, CSF, AMB CSF (2), MCF (1) F641S mutation C. glabrata (Cleary et al., 2008) 2008 Fungemia CSF CSF (>4), ANF (>4), MCF (>4) D632E mutation C. glabrata (Thompson et al., 2008) 2008 Fungemia CSF CSF (2), ANF (0.5), MCF (0.25) F659V mutation

C. tropicalis (Garcia-Effron et al., 2008b) 2008 Fungemia CSF, VRC CSF (4), ANF (2), MCF (2) Mutation, 50× increase in IC50 C. tropicalis (Garcia-Effron et al., 2008b) 2008 Fungemia CSF, AMB CSF (4), ANF (1), MCF (2) Mutation, 50× increase in IC50 C. tropicalis (Garcia-Effron et al., 2008b) 2008 Fungemia CSF, FLC CSF (1), ANF (0.5), MCF (0.5) Mutation, 50× increase in IC50 C. albicans (Arendrup et al., 2009a) 2009 Fungemia CSF, FLC CSF (1), ANF (0.5) S645P mutation C. glabrata (Chapeland-Leclerc et al., 2010) 2010 Fungemia FLC, AMB, 5FC, VRC, CSF (>32) S663P mutation in Fks2 CSF C. tropicalis (Garcia-Effron et al., 2010) 2010 Fungemia CSF, FLC CSF (4), ANF (1), MCF (1) F76S mutation C. glabrata (Garcia-Effron et al., 2010) 2010 Fungemia CSF, AMB CSF (8), ANF (4), MCF (2) S629P and S663P mutations C. glabrata (Garcia-Effron et al., 2010) 2010 Fungemia ANF, AMB CSF (>16), ANF (8), MCF (>16) S629P and S663P mutations

a Abbreviations: CLSI, Clinical and Laboratory Standards Institute; AMB, amphotericin B; ANF, anidulafungin; CSF, caspofungin; FLC, ﬂuconazole; ITZ, itraconazole; MCF, micafungin; VRC, voriconazole; 5FC, 5-ﬂucytosine. b Antifungal agents administered to patient.

Table 4 MIC distributions of three echinocandins versus Candida spp. strains tested for the presence of fks1/fks2 mutations using the CLSI broth microdilution method.a

Species (no. tested) Antifungal agentb No. of isolates at MIC (␮g/ml; no. showing mutation)

≤0.03 0.06 0.12 0.25 0.5 1 2 4 ≥8

C. albicans (52) ANF 31 7 4 (1) – 2 (2) 6 (6) 2 (2) – – CSF 15 23 3 – 2 (2) 1 (1) 3 (3) 2 (2) 3 (3) MCF 31 7 3 2 (2) 1 (1) 2 (2) 5 (5) 1 (1) –

C. glabrata (169) ANF 35 52 (1) 48 (1) 4 (1) 3 (3) 8 (5) 12(12) 7 (7) – CSF 19 55 55 (1) 5 (3) 3 (2) 10 (5) 7(6) 3 (1) 12(12) MCF 124 10(1) 6 (3) 8 (6) 5 (4) 2 (2) 7 (7) 7 (7) –

C. tropicalis (31) ANF 18 6 1 (1) 1 1 (1) 4 (4) – – – CSF 8 12 3 3 (1) – 1 (1) 2 (2) 2 (2) – MCF 9 6 7 3 (1) 3 (2) 3 (3) – – –

C. krusei (27) ANF 4 16 3 1 – 1 (1) 1 (1) 1 (1) – CSF 1 4 4 8 3 4 (1) 1 – 2 (2) MCF – 8 5 10 1 – 2 (2) 1 (1) –

C. dubliniensis (2) ANF – 1 – – – 1 (1) – – – CSF – – 1 – – – – 1 (1) – MCF– – 1 ––– 1(1)––

C. parapsilosis (44) ANF – – – 2 3 9 22 7 1 CSF – – – 5 16 18 1 1 – MCF– – – 237 293–

a Data compiled from Arendrup et al. (2010), Pfaller et al. (2010b) and Zimbeck et al. (2010). b ANF, anidulafungin; CSF, caspofungin; MCF, micafungin.

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Table 5 inactive compound, with approximately 30% excreted in the feces Amino acid substitutions within the Fks protein from the Candida isolates in Table 4. and <1% in urine (Vazquez, 2005). CSF is metabolized by hydrolysis Species Mutations (no. strains) and N-acetylation with 35% recovered in feces and 41% recovered in urine as primary degradation products (Cappelletty and Fks1p Fks2p Eiselstein-McKitrick, 2007). MCF is metabolized to M-1 (cate- C. albicans F641S (2) chol form) by arylsulfatase, with further metabolism to the M-2 F641Y S645Y (methoxy form) by catechol-o-methyltransferase (Cappelletty and S645F Eiselstein-McKitrick, 2007). M-5 is formed by hydroxylation at the S645P (3) side chain (w-1 position) of MCF catalyzed by cytochrome P450 S645F and R1361 R/H (CYP) isozymes (Mycamine, Package insert, 2005). Seventy-one D648Y percent of the dose is recovered in the feces with an additional P649H 11% in the urine. C. dubliniensis S645P ANF, CSF, and MCF are similar in terms of their dose- C. glabrata F625S NM proportional linear PK following intravenous administration with S629P (2) NM peak serum concentrations of 10 mcg/ml and area under the S645P NM concentration curve (AUC) values of approximately 110 mg h/L D632G NM R631G NM (Cappelletty and Eiselstein-McKitrick, 2007; Dodds-Ashley et al., NM F659V 2006). NM F659S (2) PD investigations of the echinocandins and Candida have been NM F659Y undertaken, and both in vitro and in vivo models have demon- NM S663P (11) NM S663F (2) strated a correlation between drug dose, organism MIC, and NM R665G outcome (Andes et al., 2003, 2008a,b; Louie et al., 2005). All three NM D666G agents exhibit concentration-dependent killing and a prolonged NM D666E (12- to 24-h) post antifungal effect (Andes et al., 2008a,b; Ernst NM P667T et al., 1999, 2000, 2002). Dose-fractionation studies have shown NM P667H NM L644W that echinocandin fungicidal activity in vivo is maximized against NM S645P (2) C. albicans isolates when the ratio of total serum drug concentration to organism MIC (Cmax/MIC) approaches 10 (Andes et al., C. krusei R1361G F655F/C 2003), if the unbound serum 24-h AUC versus MIC ratio (AUC/MIC) L658W and L701M exceeds 10–20 (Andes et al., 2008a,b), or total drug tissue AUC/MIC

C. tropicalis F641S (2) is greater than 250 (Louie et al., 2005). PK/PD studies with ANF F76S (Andes et al., 2008a), CSF (Louie et al., 2005), and MCF (Andes et al., S80P (3) 2008b) have shown these characteristics to be relatively consistent for all echinocandins against Candida. glabrata from the WT population. By comparison only 15.7–45.1% of the fks1/fks2 mutants would be classified as NS using the current 4. Echinocandin WT MIC distribution profile and CLSI CBP. Among the 91 strains of C. albicans, C. tropicalis, C. kru- epidemiological cutoff values (ECVs) for Candida spp. sei, and C. dubliniensis without fks1/fks2 mutations, 100.0% would be correctly classified as WT using ANF, 92.3% using CSF, and 97.8% The MIC profile for the three echinocandins and each of eight ≤ using MCF when the 0.25 mcg/ml cutoff value was applied. Among different species of Candida (8271 isolates) is shown in Table 6. The the 139 strains of C. glabrata without fks1/fks2 mutations, 95.7% MIC distributions in this study were all determined in a single ref- would be classified as WT using ANF, 92.1% using CSF, and 95.7% erence laboratory (University of Iowa) by CLSI-recommended BMD ≤ ≤ using MCF when the 0.12 mcg/ml (ANF, CSF) or the 0.06 mcg/ml methods (Pfaller et al., 2010a), and thus may be less broad with a (MCF) cutoffs were applied. lower modal MIC than distributions generated by multiple labora- The WT isolates of C. parapsilosis are separated clearly from tories. This is recognized as a potential limitation in the assignment those of the other species for all three echinocandins as previously of the ECVs. This large data set represents recent (2003–2007), clini- described (Arendrup et al., 2010; Pfaller et al., 2010b). The MIC was cally important (blood and normally sterile site) isolates from more greater than 0.25 mcg/ml for 95% of isolates with ANF and MCF and than 100 different medical centers throughout the world (Pfaller ≤ for 89% with CSF. Conversely, the MIC was 2 mcg/ml for 82% of the et al., 2010a). The MIC distributions clearly show the very low MICs isolates with ANF, 98% with CSF, and 93% with MCF. C. parapsilosis typical of WT strains of C. albicans, C. glabrata, C. tropicalis, C. kru- has a naturally occurring polymorphism at the edge of HS1 in fks1, sei, and C. kefyr and the higher MICs typical of C. parapsilosis, C. accounting for the MIC level being in the same range as those of HS guilliermondii, and C. lusitaniae for all three echinocandins. mutant isolates of the other Candida species (Garcia-Effron et al., In assessing MIC distributions such as those in Table 6,aWT 2008a)(Table 4). organism is defined as a strain which does not harbor any acquired resistance to the particular antimicrobial agents being examined 3. Pharmacokinetic (PK) and pharmacodynamic (PD) (Turnidge et al., 2006; Turnidge and Paterson, 2007). The typical considerations MIC distribution for WT organisms covers three to five 2-fold dilution steps surrounding the modal MIC (Arendrup et al., 2009b; All three echinocandins are lipopeptide antifungal agents with Kahlmeter et al., 2003; Kahlmeter and Brown, 2004). Inclusion of half-lives ranging from 10 h (CSF) to 24 h (ANF). Protein binding is WT strains in Table 6 was ensured by testing only the incident high (96–99%), and the distribution of these compounds is high- isolate for each infectious episode. est in the liver, kidneys, large intestines, spleen and lungs with The ECV for each echinocandin and species of Candida was low or undetectable levels in the urine, cerebrospinal and vit- obtained as described by the European Committee on Antimicrobial reous fluids (Wiederhold and Lewis, 2007). ANF undergoes slow Susceptibility Testing (EUCAST) (Kahlmeter et al., 2003; Kahlmeter chemical degradation at physiologic temperature and pH to an and Brown, 2004), by considering the WT MIC distribution, the

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Table 6 Echinocandin Wild Type MIC distribution and epidemiological cutoff values (ECVs) for Candida spp.a

Species (no. tested) Antifungal agent MIC (␮g/ml) % of isolates at ≤MIC

Range Mode ECV (%b) 0.12 0.25 0.5 1 2

C. albicans (4,283) Anidulafungin 0.007–1 0.03 0.12 (99.7) 99.6 99.7 99.9 100.0 100.0 Caspofungin 0.007–0.5 0.03 0.12 (99.8) 99.8 99.9 100.0 100.0 100.0 Micafungin 0.007–0.5 0.015 0.03 (97.7) 99.9 99.9 100.0 100.0 100.0

C. glabrata (1,236) Anidulafungin 0.015–4 0.06 0.25 (99.4) 97.3 99.4 99.6 99.8 99.9 Caspofungin 0.015–8 0.03 0.12 (98.5) 98.5 99.2 99.8 99.8 99.8 Micafungin 0.007–2 0.015 0.03 (98.2) 99.5 99.7 99.8 99.9 100.0

C. tropicalis (996) Anidulafungin 0.007–2 0.03 0.12 (98.9) 98.9 99.6 99.7 99.7 100.0 Caspofungin 0.007 to >8 0.03 0.12 (99.4) 99.4 99.8 99.8 99.9 99.9 Micafungin 0.007–1 0.015 0.12 (99.1) 99.1 99.7 99.8 100.0 100.0

C. kefyr (61) Anidulafungin 0.015–0.12 0.06 0.25 (100.0) 100.0 100.0 100.0 100.0 100.0 Caspofungin 0.007–0.03 0.015 0.03 (100.0) 100.0 100.0 100.0 100.0 100.0 Micafungin 0.015–0.06 0.06 0.12 (100.0) 100.0 100.0 100.0 100.0 100.0

C. krusei (270) Anidulafungin 0.015–0.5 0.03 0.12 (99.3) 99.3 99.6 100.0 100.0 100.0 Caspofungin 0.015–1 0.06 0.25 (96.3) 81.5 96.3 99.3 100.0 100.0 Micafungin 0.015–0.25 0.06 0.12 (97.8) 97.8 100.0 100.0 100.0 100.0

C. lusitaniae (99) Anidulafungin 0.06–1 0.5 2 (100.0) 19.2 52.5 95.9 100.0 100.0 Caspofungin 0.03–1 0.25 0.5 (98.0) 47.5 93.9 98.0 100.0 100.0 Micafungin 0.007–1 0.12 0.5 (99.0) 66.7 98.0 99.0 100.0 100.0

C. parapsilosis (1,238) Anidulafungin 0.015–4 2 4 (100.0) 0.4 1.5 5.5 31.3 93.1 Caspofungin 0.015–4 0.25 1 (98.6) 13.2 57.3 89.5 98.6 99.9 Micafungin 0.015–2 1 4 (100.0) 1.2 6.5 27.6 82.2 100.0

C. guilliermondii (88) Anidulafungin 0.06–4 2 16 (100.0) 6.8 14.8 20.5 55.7 92.0 Caspofungin 0.03 to >8 0.5 4 (95.5) 20.5 44.3 80.7 94.3 95.5 Micafungin 0.015 to >8 0.5 4 (98.9) 17.0 35.2 70.5 96.6 98.9

a Data compiled from Pfaller et al. (2010a). b Percentage of isolates for which MIC is less than or equal to the ECV. modal MIC for each distribution, and the inherent variability of the as the test reagent the CLSI BMD method differentiated 49 of test (usually ±1 log2 dilution). In general the ECV should encompass 50 (98%) mutant strains from WT strains (Table 4). The ECVs for at least 95% of isolates in the WT distribution (Turnidge et al., 2006; MCF were 0.03 mcg/ml for both C. albicans and C. glabrata and Turnidge and Paterson, 2007). Statistical determination of ECVs for 0.12 mcg/ml for both C. tropicalis and C. krusei. Using these ECVs, the each species and antifungal agent was performed as described by CLSI method with MCF identified all 50 mutant strains (Table 4). Turnidge and colleagues (Turnidge et al., 2006). It is evident that only a small number (<4%) of isolates of C. Organisms with acquired resistance mechanisms may be albicans, C. glabrata, C. tropicalis and C. krusei fall outside of the included among those for which the MICs are higher than the ECV respective ECVs for each of the three echinocandins (Table 6). (Kahlmeter et al., 2003; Kahlmeter and Brown, 2004, Rodriguez- Almost all would be classified as susceptible using the CBP criteria Tudela et al., 2008). Whereas CBPs are used to indicate those isolates despite the possibility that they may have an acquired resistance that are likely to respond to treatment with a given antimicrobial mutation. The insensitivity of the CBP to detect the emergence of agent administered at the approved dosing regimen for that agent, potential resistance to the echinocandins is demonstrated further the ECV can be used as the most sensitive measure of the emergence in Table 7 where both the CBP and the CSF ECVs for each species of strains with reduced susceptibility to a given agent (Kahlmeter is applied to a collection of Candida isolates spanning the years et al., 2003; Kahlmeter and Brown, 2004; Simjee et al., 2008). from 2001 to 2006 (Pfaller et al., 2008a). Whereas the prevalence of Compared to the CBP value of ≤2 mcg/ml, the ECVs are between decreased susceptibility to CSF is 0–0.5% for any given species with 8- and 66-fold lower for the three echinocandins and C. albicans, C. no change over time using the CBP, it is approximately 0.1% for C. glabrata, C. tropicalis, C. krusei and C. kefyr (Table 6). Whereas the albicans, 0.5–2% for C. glabrata and C. tropicalis, 5–18% for C. krusei CBP encompasses 99.8–100% of the isolates of these five species, the and 1–2% for C. parapsilosis using the ECVs for each species. ECVs of each agent encompasses 96–100% of the isolates, highlight- Although in vitro susceptibility testing is often used to select ing the small number of isolates of each species that fall outside of antimicrobial agents that are most likely to be active clinically, the WT distribution yet remain susceptible to each agent accord- perhaps the most important role of such testing is in detecting resis- ing to the CBP. In contrast, the ECVs for the three less susceptible tance (e.g. determining those agents that will not work) (Turnidge species, C. lusitaniae, C. parapsilosis and C. guilliermondii, are similar and Paterson, 2007). As such, one would like to use the most sensi- to the CBP for all three of the echinocandins. tive means available to detect emerging resistance. Thus, the ECVs The ability of the ECVs to differentiate strains of Candida spp. for C. albicans, C. glabrata, C. tropicalis, and C. krusei will be impor- with acquired resistance mechanisms for the echinocandins can be tant in detecting the emergence of decreased susceptibility to the seen when one applies the ECVs for each species and echinocandin echinocandins in surveys of antifungal resistance. The CBPs for to the data in Table 4. As shown in Table 6, the ECVs for ANF and C. these agents may serve the same purpose for C. parapsilosis and albicans, C. glabrata, C. tropicalis, and C. krusei were 0.12 mcg/ml, C. guilliermondii but appear to be too insensitive to be of epidemi- 0.25 mcg/ml, 0.12 mcg/ml, and 0.12 mcg/ml, respectively. Using ological value in monitoring the more susceptible species. Taken these cutoffs, the CLSI method distinguished 45 of the 50 mutant together the data in Tables 1–4, 6 and 7 provide compelling evi- strains (90%) from WT strains (Table 4). The ECVs for CSF and C. dence for the need to lower the CBPs for the echinocandins and albicans, C. glabrata, C. tropicalis, and C. krusei were 0.12 mcg/ml, species such as C. albicans, C. glabrata, C. tropicalis, and C. krusei in 0.12 mcg/ml, 0.12 mcg/ml and 0.25 mcg/ml, respectively. With CSF order to provide a more sensitive means to detect the emergence of

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Table 7 Application of epidemiological cutoff values (ECVs) and clinical breakpoints (CBPs) to follow trends in decreased susceptibility of Candida spp. bloodstream infection isolates to caspofungin.a

Species (ECV in ␮g/ml) Year No. tested MIC range (␮g/ml) % of MICsb

>ECV >CBP (2 ␮g/ml)

C. albicans (0.12) 2001 1000 0.007–0.25 0.1 0.0 2002 1208 0.007–0.12 0.0 0.0 2003 1178 0.007–0.25 0.1 0.0 2004 1119 0.007–0.25 0.4 0.0 2005 1045 0.007–0.12 0.0 0.0 2006 869 0.007–0.5 0.2 0.0

C. glabrata (0.12) 2001 287 0.015–4 1.4 0.3 2002 332 0.007–0.5 1.5 0.0 2003 291 0.015–0.5 0.3 0.0 2004 252 0.015–0.5 1.2 0.0 2005 291 0.015–8 2.1 0.3 2006 238 0.015–0.5 1.3 0.0

C. tropicalis (0.12) 2001 174 0.007–0.25 0.6 0.0 2002 265 0.007–1 1.9 0.0 2003 268 0.007–0.12 0.0 0.0 2004 211 0.007 to >8 1.9 0.5 2005 227 0.007–0.25 0.9 0.0 2006 203 0.007–0.12 0.0 0.0

C. krusei (0.25) 2001 50 0.06–1 12.0 0.0 2002 73 0.06–1 8.2 0.0 2003 65 0.03–2 18.5 0.0 2004 53 0.015–0.5 7.5 0.0 2005 59 0.06–1 5.1 0.0 2006 34 0.06–0.25 0.0 0.0

C. parapsilosis (1) 2001 206 0.06–2 0.5 0.0 2002 275 0.06–4 0.7 0.4 2003 360 0.015–2 0.8 0.0 2004 284 0.06–4 2.1 1.8 2005 243 0.06–2 2.5 0.0 2006 209 0.06–2 1.4 0.0

a Data compiled from Pfaller et al. (2006) and Pfaller et al. (2008a). b The ECVs are those described by Pfaller et al. (2010a) and the CBP is that of the CLSI (2008a;2008b). acquired resistance. In approaching the development of CBPs from 6. Cross-resistance among echinocandins and between the standpoint of ECVs one can be assured of a maximum detection echinocandins and fluconazole of strains with acquired resistance mechanisms and a minimum impact on the WT strains (low rate of false-resistance). It is now well established that cross-resistance between echinocandins and fluconazole does not exist (Messer et al., 5. Echinocandin MICs for Candida spp. Isolated in 2006a,b; Niimi et al., 2006; Pfaller et al., 2005; Pfaller and Diekema, randomized trials of echinocandins in the treatment of 2007; Richards et al., 2008; Silver et al., 2008). CSF and the other candidiasis echinocandins are poor substrates for most multidrug efflux trans- porters, and the results of studies involving fluconazole-resistant There have been four phase II or III studies of esophageal can- strains of C. albicans expressing high levels of CDR1, CDR2, and/or didiasis treated with CSF (Arathoon et al., 2002; Kartsonis et al., MDR1 demonstrated full susceptibility to caspofungin (Bachmann 2005; Villanueva et al., 2001; Villanueva et al., 2002), two phase III et al., 2002). Furthermore, 96–98% of 315 clinical isolates of studies of IC treated with CSF (Kartsonis et al., 2005; Mora-Duarte fluconazole-resistant Candida spp. were susceptible to all three et al., 2002; Walsh et al., 2004), one phase III study of IC with ANF echinocandins at MICs ≤ 0.25 mcg/ml (Messer et al., 2006a; Pfaller (Reboli et al., 2007), and two phase III studies of IC treated with et al., 2008a). MCF (Kuse et al., 2007; Pappas et al., 2007). The isolates from these Given the mechanism of action that is shared among the clinical trials were tested for susceptibility to ANF, CSF and MCF echinocandins, it is not surprising that they demonstrate a simi- by CLSI BMD methods (Table 8). The six major species from these lar spectrum and potency (Perlin, 2007). Scatterplots of ANF and studies are the same as those shown in Table 6 and in other multi- MCF versus CSF and versus one another show high degrees of center surveys. Among these more common species (1163 isolates), correlation (r = 0.85, 0.84, and 0.89, respectively) (Pfaller et al., the relative susceptibility to ANF, CSF, and MCF was similar to that 2008b). The essential agreement (MIC ±2 dilutions) for the shown in Table 6 with C. albicans, C. glabrata, C. tropicalis and C. comparisons is striking at 93% for ANF versus CSF, 97% for krusei representing the most susceptible species and C. parapsilo- MCF versus CSF, and 92% for ANF versus MCF. These find- sis the least susceptible species (Tables 8 and 9). Using the ECVs ings support the observations of Perlin and colleagues (Cleary from Table 6, 99% of isolates showed ANF and MCF MICs and 94% et al., 2008; Garcia-Effron et al., 2008b, 2009a,b, 2010) indicat- show CSF MICs that were less than or equal to their respective ing that when breakthrough infections with high MIC strains ECVs (Table 9). Thus, large scale surveys such as that shown in occur, mechanism-specific resistance has been reported. Resis- Table 6 produce both MICs and species distribution profiles that tant isolates show cross-resistance across the entire class of are entirely representative of those seen in more formal clinical echinocandin drugs. Such cross-resistance is clearly depicted in trials of echinocandin efficacy. Tables 1 and 4.

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Table 8 Relationship between MIC and outcome for candidiasis and the echinocandins.

Species MIC (␮g/ml)a Successful outcome by MICb

Anidulafungin Caspofungin Micafungin

n/N % n/N % n/N %

C. albicans 0.007 79/84 94 4/6 67 0.015 14/16 88 25/35 71 0.03 10/11 91 62/79 79 135/170 79 0.06 6/7 86 96/117 82 1/1 100 0.12 2/2 100 80/107 75 0.25 1/1 100 21/23 91 0.5 1/1 100

Total 112/121 93 289/368 79 136/171 80

C. glabrata 0.007 11/12 92 0.015 2/2 100 1/1 100 0.03 6/8 75 11/12 92 28/32 88 0.06 20/23 87 14/16 88 0.12 2/2 100 12/13 92 0.25 1/1 100 5/5 100 0.5 1/1 100 1/1 100 1 1/1 100

Total 42/48 88 45/49 92 29/33 88

C. tropicalis 0.007 9/9 100 1/1 100 0.015 1/2 50 8/10 80 0.03 3/4 75 21/27 78 54/70 77 0.06 3/3 100 11/20 55 0.12 2/2 100 7/8 88 1/1 100 0.25 0/1 0 1/1 100 0.5 1/1 100 0/1 0 1/1 100

Total 19/21 90 48/68 71 57/73 78

C. krusei 0.015 0/1 0 0/1 0 0.03 1/1 100 1/2 50 0.06 2/4 50 4/5 80 0.12 3/4 75 4/5 80 0.25 6/8 75 0.5 1/1 100

Total 3/6 50 9/13 69 10/13 77

C. parapsilosis 0.015 1/1 100 0.03 1/1 100 1/1 100 2/3 67 0.06 1/2 50 1/1 100 0.12 7/10 70 3/3 100 0.25 1/1 100 10/15 67 8/9 89 0.5 5/6 83 20/27 74 26/33 79 1 13/15 87 14/17 82 2 3/3 100 1/1 100 1/1 100 4 5/6 83

Total 15/17 88 54/72 75 55/67 82

C. guilliermondii 0.03 1/1 100 2/2 100 0.06 0.12 1/2 50 0.25 1/1 100 3/4 75 1/1 100 0.5 1/1 100 3/3 100 4/4 100 1 4/4 100

Total 2/2 100 11/12 92 8/9 89

a MICs were determined according to the standards in CLSI document M27-A3 (CLSI, 2008a). Data compiled from Pfaller et al. (2008b). b n/N, number of patients successfully treated/total number treated.

7. Clinical correlation and development of species-specific Although PK/PD data and resistance mutations were taken into clinical breakpoints consideration in establishing the CBP, the data for each of these important considerations was not nearly as robust as it is now. Fur- The previously established CLSI MIC interpretive breakpoints thermore, the CLSI Antifungal Subcommittee lumped all species for Candida spp. tested against the echinocandins were based on an of Candida together, assigning a single breakpoint for all species analysis of MIC distributions and the clinical relationship between despite clear evidence that echinocandin MICs were significantly MIC and efficacy indicating that standard dosing regimens of ANF, lower for some species than others. Since that time significant CSF, and MCF may be used to treat infections due to Candida spp. progress has been made in relating MICs to resistance mutations for which MICs are as high as 2 mcg/ml (Pfaller et al., 2008b). An (Table 1) with case reports and case series clearly showing that MIC predictive of resistance could not be defined based on the data clinical resistance is related to fks1/fks2 mutations in strains for from clinical trials (Table 8). which the MICs are greater than those of WT strains, but not nec-

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Table 9 Echinocandin epidemiological cutoff values (ECVs) applied to clinical trials data.a

Species Antifungal agents No. infections ECV (␮g/ml) % of isolates for which MICs are ≤ECV % success at ≤MIC (␮g/ml)

ECV 0.25 0.5 1 2

C. albicans Anidulafungin 121 0.12 99.2 92.5 92.6 92.6 92.6 92.6 Caspofungin 368 0.12 93.5 77.6 78.5 78.5 78.5 78.5 Micafungin 171 0.03 99.4 79.4 79.5 79.5 79.5 79.5

C. glabrata Anidulafungin 48 0.25 100.0 87.5 87.5 87.5 87.5 87.5 Caspofungin 49 0.12 85.7 90.5 91.5 91.7 91.8 91.8 Micafungin 33 0.03 97.0 87.5 87.5 87.9 87.9 87.9

C. tropicalis Anidulafungin 21 0.12 95.2 90.0 90.0 90.5 90.5 90.5 Caspofungin 68 0.12 97.1 72.7 71.6 70.6 70.6 70.6 Micafungin 73 0.12 97.3 77.5 77.8 78.1 78.1 78.1

C. krusei Anidulafungin 6 0.12 100.0 50.0 50.0 50.0 50.0 50.0 Caspofungin 13 0.25 100.0 69.2 69.2 69.2 69.2 69.2 Micafungin 13 0.12 92.3 75.0 75.0 76.9 76.9 76.9

C. parapsilosis Anidulafungin 17 4 100.0 88.2 100.0 87.5 87.5 90.9 Caspofungin 72 1 98.6 74.6 69.0 71.4 74.6 75.0 Micafungin 67 4 100.0 82.1 87.5 81.6 81.8 82.1

C. guilliermondii Anidulafungin 2 16 100.0 100.0 100.0 100.0 100.0 100.0 Caspofungin 12 4 100.0 91.7 80.0 87.5 91.7 91.7 Micafungin 9 4 100.0 88.9 80.0 88.9 88.9 88.9

a Data compiled from Pfaller et al. (2008b, 2010a). essarily greater than the CBP (Table 3). These findings coupled and C. krusei. This breakpoint would capture 99% to 100% of the clin- with an improved understanding of the PD indices associated with ical trials isolates of C. albicans, 95% to 99% of C. tropicalis, and 92% efficacy for the echinocandins provide the rationale to develop to 100% of C. krusei (Table 8). The clinical efficacy associated with species-specific CBPs for both susceptibility and resistance (Andes that breakpoint is comparable to that observed with the previous et al., 2008a,b, 2010; Cleary et al., 2008; Garcia-Effron et al., 2008b, CBP of ≤2 mcg/ml (Table 9). With regard to the three echinocandins 2009a,b, 2010). and C. glabrata the data in Tables 1, 2, 4 and 9 support a susceptible The establishment of ECVs for the major species of Candida and breakpoint of ≤0.06 mcg/ml for MCF and of ≤0.12 mcg/ml for ANF each echinocandin and the relationship of those cutoffs to clini- and CSF in order to maximize the detection of fks mutants and still cally resistant strains with defined fks1/fks2 mutations provides a provide good clinical utility. reasonable indication of possible CBPs for the various species of This low CBP clearly is not appropriate for either C. parapsilosis Candida. As discussed previously, the work of Garcia-Effron et al. or C. guilliermondii (Tables 8 and 9). Despite the fact that the ECVs (2008b, 2009a,b) and of Wiederhold et al. (2008) have identified for these two species range from 1 to 16 mcg/ml, the clinical effi- cutoff values of around 0.25 mcg/ml that would improve the sensi- cacy of all three echinocandins in treating infections is comparable tivity of BMD testing of ANF, CSF, and MCF to detect fks mutants of C. to that seen with the more susceptible species (Tables 8 and 9). albicans, C. glabrata and C. tropicalis (Tables 1 and 2). We have shown The data from the clinical trials indicate that all three echinocan- that a cutoff value of 0.25 mcg/ml for ANF, CSF, and MCF and C. albi- dins may be used to treat infections due to either of these two cans, C. tropicalis, C. krusei, and C. dubliniensis and of 0.06 mcg/ml species for which MICs are as high as 2 mcg/ml (Tables 8 and 9). (MCF) or 0.12 mcg/ml (ANF, CSF) for C. glabrata reliably differen- In the case of ANF, 5 of 6 (83%) infections due to C. parapsilosis for tiates WT strains of these species from those with fks mutations which the MIC was 4 mcg/ml were also treated successfully. These (Table 4). The relationship between the MIC and outcome for can- data confirm the applicability of the previous CBP of ≤2 mcg/ml didiasis and the echinocandins is shown by species in Table 8.It for these less susceptible species. Greater than 90% of clinical iso- is immediately apparent that for C. albicans, C. glabrata, C. tropi- lates of C. parapsilosis and C. guilliermondii are encompassed by a calis, and C. krusei, the MICs for each echinocandin are almost all breakpoint of ≤2 mcg/ml (Table 6). Having said this, it is notable ≤0.25 mcg/ml and none are greater than 1 mcg/ml. Thus these clin- that current Infectious Diseases Society of America guidelines do ical trial isolates for the most part represent WT strains of each not recommend the echinocandins as first-line therapy for C. para- species regarding their susceptibility to echinocandins. This is seen psilosis fungemia (Pappas et al., 2009). more clearly in Table 9 where it is apparent that, with the exception Given these considerations, it is reasonable to propose a CBP of C. glabrata and CSF, more than 90% of the MICs of each echinocan- for susceptibility (S) to ANF, CSF and MCF of ≤0.25 mcg/ml for C. din are below the respective ECVs for the individual species of albicans, C. tropicalis, and C. krusei, one of ≤0.06 mcg/ml (MCF) or Candida. Furthermore, the proportion of cases with a successful ≤0.12 mcg/ml (ANF,CSF) for C. glabrata, and one of ≤2 mcg/ml for C. outcome is the same whether one uses the organism/echinocandin- parapsilosis and C. guilliermondii (Table 10). Given the data shown specific ECV or a higher value of 0.25 mcg/ml. It is apparent from in Tables 1, 3 and 4, strains of C. albicans, C. tropicalis, and C. kru- these data that for these species the CBP of ≤2 mcg/ml is unneces- sei for which the MIC of each echinocandin is ≥1 mcg/ml (≥0.25 sarily high and that a lower CBP would not only provide a reliable mcg/ml [MCF] or ≥0.5 mcg/ml [ANF,CSF] for C. glabrata) are almost predictor of efficacy, but would also serve as a more sensitive means always clinically resistant (R) and possess an acquired fks mutation of detecting strains with acquired resistance mechanisms. (Tables 1 and 4). Those isolates for which the MIC is 0.5 mcg/ml It is now evident that the PD index that is predictive of efficacy (0.12 mcg/ml [MCF] or 0.25 mcg/ml [ANF,CSF] for C. glabrata) fall in the treatment of candidiasis is a free drug AUC to MIC ratio of just outside of the ECV for these species (Table 6)andmayormay approximately 10–20 (Andes et al., 2008a,b, 2010). Given the MIC not contain an fks mutation (Tables 1 and 4). These strains may distributions shown in Table 6, this would support an MIC break- respond clinically to echinocandin therapy (Table 8) and may be point for susceptibility of ≤0.25 mcg/ml for C. albicans, C. tropicalis, best categorized as intermediate (I). The use of an I category pro-

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Table 10 MIC and zone diameter (ZD) interpretive breakpoints (BP) for the echinocandins and Candida species using CLSI BMD and DD methods.

Antifungal agent Species MIC BP (␮g/ml) ZD BP (mm)

SIRSIR

Anidulafungin C. albicans ≤0.25 0.5 ≥1 C. glabrata ≤0.12 0.25 ≥0.5 C. tropicalis ≤0.25 0.5 ≥1 C. krusei ≤0.25 0.5 ≥1 C. parapsilosis ≤24≥8 C. guilliermondii ≤24≥8

Caspofungin C. albicans ≤0.25 0.5 ≥1 ≥17 15–16 ≤14 C. glabrataa ≤0.12 0.25 ≥0.5 ≥20 18–19 ≤17 C. tropicalis ≤0.25 0.5 ≥1 ≥17 15–16 ≤14 C. krusei ≤0.25 0.5 ≥1 ≥17 15–16 ≤14 C. parapsilosis ≤24≥8 ≥13 11–12 ≤10 C. guilliermondii ≤24≥8 ≥13 11–12 ≤10

Micafungin C. albicans ≤0.25 0.5 ≥1 ≥22 20–21 ≤19 C. glabrataa ≤0.06 0.12 ≥0.25 ≥28 26–27 ≤25 C. tropicalis ≤0.25 0.5 ≥1 ≥22 20–21 ≤19 C. krusei ≤0.25 0.5 ≥1 ≥22 20–21 ≤19 C. parapsilosis ≤24≥8 ≥16 14–15 ≤13 C. guilliermondii ≤24≥8 ≥16 14–15 ≤13

a Disk diffusion testing of caspofungin and micafungin versus C. glabrata not recommended at this time due to technical problems. vides a buffer zone for antimicrobial susceptibility testing that is zone diameter and MIC categories is minimized (Tables 10 and 11). necessary in order to avoid very major and major errors that may The overall categorical agreement between the disk diffusion and occur given the inherent variability of the BMD method. In addition BMD MIC results was determined for CSF and MCF with the MIC such a category may also be used to designate strains with elevated interpretive categories used as the reference. Major errors (ME) MICs that may respond clinically to a higher than standard dose of were identified as a classification of R by the disk diffusion test and drug or in situations where drug penetration is maximized. S by BMD, very major errors (VME) were identified as a classifica- Assignment of a resistant (R) breakpoint for C. parapsilosis and tion of S by the disk diffusion method and R by BMD, and minor C. guilliermondii may be a bit more difficult given the fact that these errors (M) occurred when the result of one of the tests was S or R two species may be less fit as pathogens given their naturally occur- and that of the other test was I. ring fks polymorphism (relative to WT C. albicans) coupled with the With zone diameter breakpoints of ≥17 mm (S), 15 to 16 mm (I), fact that clinically resistant strains with acquired resistance mech- and ≤14 mm (R) (Table 10), the categorical agreement (CA) between anisms have not yet been described (Garcia-Effron et al., 2008a; the CSF disk diffusion test results and the MIC test results for C. Katiyar et al., 2006; Pfeiffer et al., 2010). Regardless, it would be albicans, C. tropicalis, and C. krusei was excellent (97.2%, 96.0% and prudent to consider isolates of these two species for which MICs 91.2%, respectively) with very few VME or ME (Table 11). Like- are ≥8 mcg/ml to be R to all three echinocandins. Such a break- wise the zone diameter breakpoints for CSF and C. parapsilosis point exceeds the ECV for both species and all echinocandins with and C. guilliermondii of ≥13 mm (S), 11–12 mm (I), and ≤10 mm the exception of ANF and C. guilliermondii. Furthermore, an MIC of (R) also provided excellent CA (95.5% and 100.0%, respectively). 8 mcg/ml is not supported by the PD target for efficacy for these The CSF zone diameter breakpoints for C. glabrata differed slightly agents and C. parapsilosis (Andes et al., 2010). from those of C. albicans, C. tropicalis and C. krusei: ≥20 mm (S), 18–19 mm (I) and ≤17 mm (R). Using these criteria the CA for C. 8. Development of disk interpretive breakpoints glabrata was very low at 60.4% with 3.3% VME and 36.3% minor errors (Table 11). Whereas the CSF disk diffusion test may be used The CLSI has standardized agar disk diffusion methods for CSF to test isolates of C. albicans, C. tropicalis, C. krusei, C. parapsilosis and and MCF and Candida spp. (Clinical and Laboratory Standards C. guilliermondii, it can not be recommended for testing C. glabrata. Institute, 2008c). The methods employ a 5-mcg CSF disk and a The greater disk mass of MCF produced larger zone diameters 10-mcg MCF disk and Mueller-Hinton agar supplemented with for this agent and C. albicans, C. tropicalis and C. krusei (≥22 mm 2% glucose and 0.5 mcg of methylene blue per ml (Clinical and [S], 20–21 mm [I], and ≤19 mm [R]) and a high level of CA (99.6%, Laboratory Standards Institute, 2008c). Similar to the BMD test for 99.0% and 98.2%, respectively) (Tables 10 and 11). Zone diameter these agents, disk diffusion results may be determined after only breakpoints for MCF and C. glabrata were ≥28 mm (S), 26–27 mm 24-h of incubation (Brown and Traczewski, 2008). Quality control (I), and ≤25 mm (R) with a CA of 92.0% and for C. parapsilosis and C. ranges have been approved by CLSI for both CSF and MCF disk dif- guilliermondii were ≥16 mm (S), 14–15 mm (MS), and ≤13 mm (R) fusion tests (Clinical and Laboratory Standards Institute, 2008c; with a CA of 98.8% and 81.3%, respectively (Tables 10 and 11). Clinical and Laboratory Standards Institute, 2007). Both CSF and MCF disk diffusion methods correctly identified a The relationship between CSF and MCF MICs and zone diameters small collection of 27 strains of fks mutants as either I or R with has been determined for six species of Candida (Table 10). Using the 6 false-susceptible (VME) results with MCF and 3 with CSF. The MIC breakpoints for CSF and MCF of ≤0.25 mcg/ml(S), 0.5 mcg/ml results by species (no. of mutants detected/total no. of mutants (I), and ≥1 mcg/ml (R) for C. albicans, C. tropicalis, and C. krusei;of tested) were as follows: C. albicans, 9/10 (CSF) and 8/9 (MCF); C. ≤0.06 mcg/ml (S), 0.12 mcg/ml (I), and ≥0.25 mcg/ml (R) (MCF) and glabrata, 9/10 (CSF) and 6/10 (MCF); C. tropicalis, 3/4 (CSF) and 3/4 ≤0.12 mcg/ml (S), 0.25 mcg/ml (I), and ≥0.5 mcg/ml (R) (CSF) for C. (MCF); and C. krusei, 3/3 (CSF) and 3/3 (MCF). On the basis of these glabrata; and ≤2 mcg/ml (S), 4 mcg/ml (I), and ≥8 mcg/ml (R) for C. findings, it appears that disk diffusion testing is a useful method for parapsilosis and C. guilliermondii, one can then derive zone diam- testing the activity of CSF and MCF against C. albicans, C. tropicalis, eter breakpoints by the error-rate bounded method (Metzler and C. krusei, C. parapsilosis, and C. guilliermondii. Although the CSF disk DeHaan, 1974), whereby the number of discrepancies between the diffusion test was able to correctly identify 9 of 10 mutant strains

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Table 11 Interpretive agreement between results of CLSI broth microdilution (BMD) and disk diffusion (DD) test results for caspofungin (5 ␮g disk mass) and micafungin (10 ␮g disk mass) versus Candida.a

Antifungal Agent Species (no. tested) Test method % by categoryb % CA % errors

S I R VME ME M

Caspofungin C. albicans (550) DD 97.3 0.7 2.0 97.2 0.4 0.0 2.4 BMD 95.8 1.6 2.5 C. glabratav (91) DD 73.6 19.8 6.6 60.4 3.3 0.0 36.3 BMD 70.3 16.5 13.2 C. tropicalis (75) DD 96.0 2.7 1.3 96.0 0.0 0.0 4.0 BMD 94.7 1.3 4.0 C. krusei (34) DD 85.3 2.9 11.8 91.2 0.0 0.0 8.8 BMD 76.4 11.8 11.8 C. parapsilosis (66) DD 95.5 3.0 1.5 95.5 0.0 0.0 4.5 BMD 98.5 1.5 0.0 C. guilliermondii (27) DD 100.0 0.0 0.0 100.0 0.0 0.0 0.0 BMD 100.0 0.0 0.0

Micafungin C. albicans (889) DD 99.1 0.1 0.8 99.6 0.2 0.0 0.2 BMD 98.9 0.1 1.0 C. glabrata (99) DD 94.0 2.0 4.0 92.0 2.0 0.0 6.0 BMD 88.0 4.0 8.0 C. tropicalis (197) DD 98.5 0.5 1.0 99.0 0.0 0.0 1.0 BMD 98.0 0.5 1.5 C. krusei (56) DD 94.6 0.0 5.4 98.2 0.0 0.0 1.8 BMD 92.8 1.8 5.4 C. parapsilosis (161) DD 99.4 0.6 0.0 98.8 0.0 0.0 1.2 BMD 99.4 0.6 0.0 C. guilliermondii (16) DD 81.3 18.7 0.0 81.3 0.0 0.0 18.7 BMD 100.0 0.0 0.0

a Abbreviations: S, susceptible; I, intermediate; R, resistant; CA, categorical agreement; VME, very major error; ME, major error; M, minor error. b Interpretive criteria by antifungal agent and species (MIC/zone diameter)as shown in Table 10. of C. glabrata, it misclassified a large proportion of WT (S) strains Acknowledgements as non-WT (either I or R) (36.3% minor errors) and so is not recommended for testing this species. The MCF disk diffusion test for Caitlin Howard provided excellent support in the preparation of C. glabrata demonstrated acceptable CA with BMD results (92.0%); the manuscript. however, it did not reliably distinguish the mutant strains from WT This work was supported in part by grants from Astellas, Merck, strains and thus is not recommended for testing of this species at and Pfizer. the present time. The findings and conclusions of this article are those of the authors and do not necessarily represent the views of the Centers 9. Summary and conclusions for Disease Control and Prevention.

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Please cite this article in press as: Pfaller, M.A., et al., 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. (2011), doi:10.1016/j.drup.2011.01.004 G Model YDRUP-474; No. of Pages 13 ARTICLE IN PRESS M.A. Pfaller et al. / Drug Resistance Updates xxx (2011) xxx–xxx 13

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