ACCEPTED JCM REVISED. In Vitro Susceptibility of Invasive Isolates of Candida spp. to Anidulafungin, Caspofungin, and

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1 JCM Accepts, published online ahead of print on 1 November 00 J. Clin. Microbiol. doi:./jcm.01-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved JCM-01-0 REVISED In Vitro Susceptibility of Invasive Isolates of Candida spp. to Anidulafungin, Caspofungin, and Micafungin: Six Years of Global Surveillance M.A. Pfaller 1 *, L. Boyken 1, R.J. Hollis 1, J. Kroeger 1, S.A. Messer 1, S. Tendolkar 1, and D.J. Diekema 1, Departments of Pathology 1 and Medicine, Roy J. and Lucille A. Carver College of *Corresponding author: Medicine, University of Iowa, Iowa City, Iowa Michael A. Pfaller, M.D. Medical Microbiology Division, C0 GH Department of Pathology University of Iowa College of Medicine Iowa City, Iowa Phone:(1) -1 Fax: (1) -1 michael-pfaller@uiowa.edu Key Words: echinocandin, surveillance, Candida Short running title: Candida susceptibility to the echinocandins 1

2 ABSTRACT The echinocandins are being used increasingly as therapy for invasive candidiasis. Prospective sentinel surveillance for the emergence of in vitro resistance to the echinocandins among invasive Candida spp. isolates is indicated. We determined the in vitro activity of anidulafungin, caspofungin and micafungin against, invasive (bloodstream or sterile-site) isolates of Candida collected from over 0 medical centers worldwide from 1 January 001 to 1 December 00. We performed susceptibility testing according to the CLSI M-A method and used RPMI 10 broth, h incubation, and a prominent inhibition endpoint for determination of the MIC. Of, invasive Candida spp. isolates, species distribution was: % C. albicans, 1% C. parapsilosis, 1% C. glabrata, 1% C. tropicalis, % C. krusei,1% C. guilliermondii, and % other Candida spp. Overall, all echinocandins were very active against Candida: MIC 0 /MIC 0, anidulafungin (0.0/ µg/ml), caspofungin (0.0/0. µg/ml), micafungin (0.01/1 µg/ml); >% of isolates were inhibited < µg/ml of all agents. Results by species (expressed as the % inhibited by < µg/ml of anidulafungin, caspofungin and micafungin, respectively) were as follows: C. albicans (./0/0), C. parapsilosis (././0), C. glabrata (././0), C. tropicalis (0/./0), C. krusei (0/0/0), C. guilliermondii (0./.1/0). There was no significant change in activity of the echinocandins over the -yr study period, and no difference in activity by geographic region. All echinocandins have excellent in vitro activity against invasive isolates of Candida from centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging echinocandin resistance among invasive clinical isolates of Candida.

3 The echinocandin class of antifungal agents acts by inhibition of the synthesis of 1,-β-Dglucan in the fungal cell wall (1, ). All three available echinocandins-anidulafungin (Pfizer), caspofungin (Merck) and micafungin (Astellas)-possess fungicidal activity against most species of Candida, including those resistant to polyenes () and to azoles (1,,,,, 1,,, -,, ). Caspofungin and anidulafungin have been approved by the U.S. Food and Drug Administration (FDA, years 00 and 00, respectively) for the treatment of invasive candidiasis (IC), including candidemia (1, 1) and micafungin has been approved for the treatment of esophageal candidiasis (year 00) (). Although FDA approval for the treatment of candidemia is pending, micafungin has been shown to be safe and efficacious in the treatment of candidemia in recently published open-label (1, ) and randomized (1, 1) clinical trials. These agents all provide excellent clinical efficacy coupled with low toxicity for the treatment of serious candidal infections. Collaborative studies conducted by the Clinical and Laboratory Standards Institute (CLSI) Antifungal Subcommittee have resulted in the development of a standardized broth microdilution (BMD) method for determining echinocandin MICs for Candida spp (). The method employs RPMI 10 broth medium, incubation at C for h, and a MIC endpoint criterion of prominent reduction in growth (MIC- or >0% inhibition relative to control growth). The CLSI method provides reproducible MIC results with good separation of the wild-type MIC distribution from isolates with mutations in the FKS1 gene, for which reduced susceptibility to echinocandins has been demonstrated (, 1, ). The availability of this standardized method has facilitated the performance of large-scale surveillance studies that have documented the potency and spectrum of echinocandin drugs against clinical isolates of Candida spp. (,, -). Recently, the CLSI Antifungal Subcommittee has taken into consideration the MIC distributions generated by the in

4 vitro surveys, the mechanisms of action and of resistance known for the echinocandins, the pharmacokinetic and pharmacodynamic data available, and the clinical efficacy of each agent as it relates to the MIC for the infecting strain, to arrive at a consensus MIC breakpoint for susceptibility of < µg/ml for all three echinocandins and Candida (CLSI June, 00; manuscript in preparation). This breakpoint encompasses more than % of all Candida isolates tested against each agent and reliably discriminates the susceptible wild-type strains from those with target site mutations (1, ). As patient exposure to echinocandins broadens, the number of infecting strains with reduced susceptibility may increase (1). Indeed, data from in vitro surveys document the presence of rare strains of otherwise highly susceptible species that exhibit unusually high MICs for one or more echinocandins (, -). Furthermore, they highlight the presence of less-susceptible species such as C. parapsilosis and C. guilliermondii, the MICs for which may be -0-fold higher than those observed for C. albicans, C. glabrata, or C. tropicalis (,, ). Notably, sporadic treatment failures consistent with clinical resistance have been documented in association with so-called high MIC isolates (i.e. MIC > µg/ml) (,, 1, 1, 1, 0,, 0, 0). In each case the MIC of the echinocandin used in treatment was shown to increase progressively during the course of therapy and where investigated, a mutation in FKS1 was demonstrated in the high MIC isolate (1, 1, 0). In most (but not all) instances, resistance to all three echinocandins was demonstrated, consistent with known mechanisms of action and resistance of echinocandins in Candida (1). These observations underscore the importance of antifungal susceptibility testing of echinocandins in detecting unusual resistance profiles as these agents are used more broadly worldwide. In the present study we provide a unique head-to-head comparison of all three clinically available echinocandins using CLSI reference BMD against a global collection of, BSI isolates

5 of Candida spp. We examine geographic trends in both species distribution and echinocandin activity and discuss issues of cross-resistance among the three agents. MATERIALS AND METHODS Organisms. A total of, clinical isolates obtained internationally from 1 medical centers from 001 to 00 were tested. The collection included, strains of Candida albicans, of Candida parapsilosis, of Candida glabrata, of Candida tropicalis, 1 of Candida krusei, 1 of Candida guilliermondii, of Candida lusitaniae, of Candida kefyr, of Candida famata, of Candida pelliculosa, of Candida lipolytica, of Candida dubliniensis, of Candida rugosa, of Candida zeylanoides, and 1 of Candida intermedia. All isolates were obtained from blood or other normally sterile sites and represented individual infectious episodes. The isolates were collected at individual study sites and were sent to the University of Iowa (Iowa City) for identification and susceptibility testing as described previously (-). The isolates were identified by standard methods () and stored as water suspensions until used in the study. Prior to testing, each isolate was passaged at least twice onto potato dextrose agar (Remel) and CHROMagar Candida (Becton Dickinson and Company, Sparks, MD) to ensure purity and viability. Antifungal agents. Reference powders of anidulafungin, caspofungin, and micafungin were obtained from their respective manufacturers. Stock solutions were prepared in water (caspofungin, micafungin) or DMSO (anidulafungin), and serial twofold dilutions were made in RPMI 10 medium (Sigma, St. Louis, Mo.) buffered to ph.0 with 0.1M morpholinepropanesulfonic acid (MOPS) buffer (Sigma). Antifungal susceptibility testing. BMD testing was performed in accordance with the guidelines in CLSI document M-A () using RPMI 10 medium, an inoculum of 0.x to

6 x cells/ml, and incubation at C. MICs were determined visually after h of incubation as the lowest concentration of drug that caused a significant diminution (MIC- or >0%) of growth below control levels (1, -). Quality Control. Quality control was performed by testing CLSI-recommended strains C. krusei ATCC and C. parapsilosis ATCC 01 (). RESULTS AND DISCUSSION Table 1 demonstrates the species distribution of Candida BSI isolates according to the geographic region of origin. A total of, isolates were obtained from 1 different medical centers in the Asia-Pacific region (1 sites), Latin America (1 sites), Europe ( sites), and North America ( sites). Consistent with previous reports (, -), the distribution of Candida species isolated from blood and other sterile sites varied considerable across the different regions. C. albicans constituted well over 0% of BSI isolates in Europe (.%) and the Asia-Pacific region (.1%) whereas only.% of isolates from Latin America and 0% of isolates from North America were C. albicans. Likewise, C. parapsilosis and C. tropicalis were prominent in the Asia- Pacific and Latin American regions but less so in both Europe and North America. Whereas C. glabrata was the most common non-albicans species in both North America and Europe, it was distinctly less common than either C. parapsilosis and C. tropicalis in Latin America and the Asia- Pacific region. Finally, C. krusei was considerably more common in Europe than in the other three regions, whereas the same was true for C. guilliermondii in Latin America. These latter two species are especially notable for their propensity toward multi-drug resistance compared to other more common species of Candida (, ).

7 Table summarizes that in vitro susceptibility of, isolates of Candida spp. to anidulafungin, micafungin, and caspofungin when tested in RPMI 10 medium with h incubation and the prominent reduction endpoint criteria. First of all, it should be noted that all three echinocandins demonstrate excellent potency and spectrum with. to 0% of all isolates susceptible at the MIC breakpoint of < µg/ml. The degree of susceptibility of isolates to all three echinocandins did not change over the duration of the study (data not shown). An MIC of > µg/ml for any echinocandin was observed for only six (0.1%) of the, isolates tested: isolates of C. guilliermondii (caspofungin MIC, > µg/ml), and 1 isolate each of C. glabrata (caspofungin MIC, > µg/ml), C. tropicalis (caspofungin MIC, > µg/ml), and C. rugosa (anidulafungin MIC, > µg/ml). As noted previously (-), the MIC distribution for each of the echinocandins defined two broad groups among the nine major species tested (Tables 1 and ). C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr were all highly susceptible to each of the echinocandins (modal MIC, µg/ml; MIC 0, µg/ml), whereas C. parapsilosis (modal MIC, 0.-1 µg/ml; MIC 0, 1- µg/ml), C. guilliermondii (modal MIC, 0.-1 µg/ml; MIC 0, 1- µg/ml), C. lusitaniae (modal MIC, µg /ml; MIC 0, µg/ml) and C. famata (modal MIC, µg/ml; MIC 0, 1- µg/ml) were significantly less susceptible to all three agents. These differences may be due to differences in the sensitivity of the glucan synthesis enzyme complex to echinocandin inhibition (, 1,, 1). Importantly, 0-0% of the latter four species are classified as susceptible to all three echinocandin based on the < µg/ml breakpoint. Although the reduced susceptibility of species such as C. parapsilosis and C. guilliermondii relative to that of C. albicans, C. glabrata, and C. tropicalis has not proven to influence outcome in the various clinical trials (1,

8 , 1,, 1), it may come into play when infections with these species involve the eye or central nervous system, where adequate free drug levels cannot be readily obtained (0, 0). The echinocandin susceptibilities of isolates stratified by geographic region and by species are shown in Table. Despite the difference in species distribution noted previously, the same overall and species-specific activity was observed for each echinocandin in each of the four regions. One important observation that can be made from this large data set is that although rare, isolates of C. albicans for which anidulafungin MICs were µg/ml were detected in all geographic regions (Table ). Although such isolates would still be considered to be susceptible based on the newly described CLSI breakpoints, they must be recognized as distinctly unusual given that the modal MIC for this species is 0.0 µg/ml (Table ). Notably, of the 1 isolates of C. albicans with this high MIC anidulafungin phenotype, all were found to have micafungin MICs of 0.-1 µg/ml (modal MIC for micafungin and C. albicans is 0.01 µg/ml) and caspofungin MICs of µg/ml (modal MIC for caspofungin and C. albicans is 0.0 µg/ml). Although susceptible, these isolates are clearly outside of the normal wild-type distribution of echinocandin MICs for C. albicans. Isolates with this abnormal phenotype warrant further study and although they may respond clinically to echinocandin treatment they could pose problems under conditions of decreased drug penetration. An analogous situation can be seen with C. tropicalis where three isolates for which anidulafungin MICs of µg/ml (modal MIC for anidulafungin and C. tropicalis is 0.0 µg/ml) were detected (Table ). The caspofungin MICs for these isolates were 0., 1, and 1 µg/ml (modal MIC for caspofungin and C. tropicalis is 0.0 µg/ml) and the micafungin MICs were 0.,1, and 1 µg/ml (modal MIC for micafungin and C. tropicalis is µg/ml). Again, these three strains exhibit a high MIC phenotype for all three echinocandins relative to the wild type MIC

9 distribution. Similarly, one isolate of C. glabrata was noted to be non-susceptible to both caspofungin (MIC= µg/ml) and anidulafungin (MIC= µg/ml) with a micafungin MIC that was susceptible at 1 µg/ml but still high compared to the modal MIC of 0.01 µg/ml. Although C. guilliermondii is well known for its reduced susceptibility to caspofungin (, ), MICs for this agent are generally <1 µg/ml with a modal MIC of 0. µg/ml (Tables and ). In the present study we detected three isolates, all from Latin America, for which the caspofungin MICs were > µg/ml (Table ). Similar to that described for high MIC isolates of C. albicans, C. glabrata, and C. tropicalis, these isolates were found to have anidulafungin MICs of 1,, and µg/ml (modal MIC for anidulafungin and C. guilliermondii is 1 µg/ml) and micafungin MICs of 0., 1, and µg/ml (modal MIC for micafungin and C. guilliermondii is 0. µg/ml). Thus, although isolates of Candida with a high MIC echinocandin phenotype are very rare, they may be detected worldwide, encompass several different species, and generally exhibit MICs outside of the wild type distribution for all three echinocandins. Notably, we did not detect any isolates of C. parapsilosis that exhibited the multi-azole, multi-echinocandin-resistant phenotype described by Moudgal et al () and by Vazquez et al (), suggesting that this phenotype has not spread beyond the initially reported environment. The results of this study clearly demonstrate the comparable and excellent spectrum and potency of all three available echinocandin antifungal agents against a large collection of clinically important Candida spp. We have shown that the activity of all three agents remains consistent over time and broad geographic regions and that species-specific differences in echinocandin activity against Candida are apparent worldwide. Although slight differences in potency in vitro may be observed among the three echinocandins for a given species of Candida, such differences have been shown to be normalized by the addition of serum to the in vitro test system () and do not appear to

10 1 be significant in vivo (1). In addition to highlighting the presence of species such as C. parapsilosis and C. guilliermondii that exhibit decreased susceptibility to all three echinocandins, this large survey provides additional documentation of the presence of rare strains of otherwise highly susceptible species of Candida that exhibit unusually high, but not necessarily non- susceptible (MIC > µg/ml), MICs for all three echinocandins. These high MIC strains are sufficiently rare that they have not been encountered with any frequency in clinical trials (1, 1, 1,, 1), although several isolates with echinocandin MICs > µg/ml have recently been associated with clinical resistance to echinocandin therapy in published case reports (,, 1, 1, 1, 0,, 0, 0). Thus far the rare high MIC echinocandin phenotypes appear to exhibit a class-specific resistance profile. These observations underscore the importance of antifungal susceptibility testing of echinocandins in detecting unusual resistance profiles. Further investigation and monitoring is warranted.

11 ACKNOWLEDGEMENTS Linda Elliot and Tara Schroder provided excellent support in the preparation of this manuscript. This work was supported in part by grants from Pfizer and Astellas.

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19 Table 1. Species distribution of Candida isolates by geographic region % of isolates a APAC LAM EU NAM Total Candida species (n=) (n=1,) (n=1,) (n=1,) (n=,) C. albicans C. parapsilosis C. glabrata C. tropicalis C. krusei C. guilliermondii C. lusitaniae C. kefyr C. famata Candida spp. b a Regions: APAC, Asia-Pacific (1 study sites); LAM, Latin America (1 study sites); EU, Europe ( study sites); NAM, North America ( study sites). For each region the number of isolates is given in parentheses. b Includes C. pelliculosa ( isolates), C. lipolytica ( isolates), C. dubliniensis ( isolates), C. rugosa ( isolates), C. zeylanoides ( isolates), and C. intermedia (1 isolate).

20 Table. In vitro susceptibilities of, clinical isolates of Candida spp. to anidulafungin, caspofungin and micafungin No. Antifungal Cumulative % susceptible at a MIC (µg/ml) of: Organism tested agent > C. albicans, Anidulafungin.... a Caspofungin Micafungin C. parapsilosis Anidulafungin Caspofungin Micafungin C. glabrata Anidulafungin Caspofungin Micafungin C. tropicalis Anidulafungin Caspofungin Micafungin C. krusei 1 Anidulafungin Caspofungin Micafungin C. guilliermondii 1 Anidulafungin Caspofungin Micafungin

21 C. lusitaniae Anidulafungin Caspofungin Micafungin C. kefyr Anidulafungin Caspofungin Micafungin C. famata Anidulafungin Caspofungin Micafungin Candida spp. 0 Anidulafungin Caspofungin Micafungin Total, Anidulafungin Caspofungin Micafungin a. Minimum inhibitory concentration at which at least 0% of isolates are inhibited is listed in bold type.

22 Table. Variation in echinocandin MIC profiles by geographic region Region(s) and No. Antifungal No. of isolates for which MIC (µg/ml) was: Species tested agent > Asia-Pacific C. albicans Anidulafungin Caspofungin Micafungin 1 1 C. parapsilosis 1 Anidulafungin 1 1 Caspofungin 1 Micafungin 1 1 C. glabrata 1 Anidulafungin 1 Caspofungin 1 1 Micafungin 1 C. tropicalis 1 Anidulafungin Caspofungin 1 Micafungin 0 C. krusei Anidulafungin Caspofungin Micafungin 1 C. guilliermondii Anidulafungin Caspofungin 1 1

23 Micafungin 1 1 C. lusitaniae Anidulafungin 1 Caspofungin Micafungin 1 1 C. kefyr 1 Anidulafungin 1 Caspofungin 1 Micafungin 1 C. famata Anidulafungin 1 1 Caspofungin Micafungin 1 Latin America C. albicans Anidulafungin Caspofungin 1 1 Micafungin C. parapsilosis 0 Anidulafungin Caspofungin Micafungin C. glabrata Anidulafungin 1 1 Caspofungin 1 Micafungin C. tropicalis Anidulafungin 1 Caspofungin 1 1

24 Micafungin 1 1 C. krusei 1 Anidulafungin 1 Caspofungin Micafungin 1 1 C. guilliermondii 0 Anidulafungin 0 Caspofungin 1 1 Micafungin C. lusitaniae Anidulafungin Caspofungin Micafungin C. kefyr Anidulafungin Caspofungin Micafungin C. famata Anidulafungin 1 Caspofungin Micafungin Europe C. albicans 1,0 Anidulafungin 1 1 Caspofungin Micafungin 1 1 C. parapsilosis Anidulafungin 0 1 1

25 Caspofungin Micafungin 1 1 C. glabrata Anidulafungin Caspofungin Micafungin C. tropicalis 1 Anidulafungin Caspofungin 1 Micafungin 0 1 C. krusei Anidulafungin 1 Caspofungin Micafungin 1 0 C. guilliermondii Anidulafungin 1 1 Caspofungin 1 Micafungin C. lusitaniae Anidulafungin Caspofungin 1 Micafungin C. kefyr Anidulafungin 1 Caspofungin 1 1 Micafungin 1 C. famata Anidulafungin 1 1 Caspofungin 1 1

26 Micafungin 1 1 North America C. albicans Anidulafungin 1 1 Caspofungin Micafungin 0 1 C. parapsilosis 0 Anidulafungin 1 1 Caspofungin 1 Micafungin C. glabrata Anidulafungin Caspofungin Micafungin C. tropicalis Anidulafungin 1 Caspofungin Micafungin 1 1 C. krusei Anidulafungin 1 Caspofungin Micafungin C. guilliermondii Anidulafungin Caspofungin 1 Micafungin 1 C. lusitaniae Anidulafungin 1 1 Caspofungin 1

27 Micafungin 1 1 C. kefyr Anidulafungin 1 Caspofungin Micafungin C. famata Anidulafungin 1 1 Caspofungin 1 1 Micafungin