METHODS: MINIMAL INHIBITORY AND FUNGICIDAL CONCENTRATION AND TIME-KILLING STUDIES. Gobernado b. Fe, Valencia 46009, Spain.

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1 AAC Accepts, published online ahead of print on 20 April 2009 Antimicrob. Agents Chemother. doi: /aac Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 IN VITRO ACTIVITY OF ECHINOCANDINS AGAINST CANDIDA KRUSEI BY THREE METHODS: MINIMAL INHIBITORY AND FUNGICIDAL CONCENTRATION AND TIME-KILLING STUDIES Emilia Cantón* a, Javier Pemán b, Amparo Valentín a, Ana Espinel-Ingroff c and Miguel Gobernado b a Unidad de Microbiología Experimental, Centro de Investigación, Hospital Universitario La Fe, Valencia 46009, Spain. b Servicio de Microbiología, Hospital Universitario La Fe, Valencia 46009, Spain. c Division of infectious Disease c, VCU Medical Center, Richmond, Virginia. *Corresponding author: Unidad de Microbiología Experimental Centro de Investigación Hospital Universitario La Fe Avenida Campanar 21, Valencia. Spain. Phone: ; Fax: address: canton_emi@gva.es Coauthors addresses: Javier Pemán: peman_jav@gva.es Amparo Valentín: valentin_amp@gva.es Ana Espinel-Ingroff: avingrof@verizon.net Miguel Gobernado: gobernado_mig@gva.es Key words: Anidulafungin, Caspofungin, Micafungin, Candida krusei, Time-killing, MIC, MFC. Running title: Echinocandins kill-curves for C. krusei Abstract: 74. Body of the text: 997.

2 2 ABSTRACT We evaluated the in vitro activity of anidulafungin, micafungin and caspofungin by MIC, minimum fungicidal concentration (MFC) and time-kill methods against Candida krusei. Geometric mean (GM) MIC/GM-MFC was 0.1/0.34, 0.25/0.44, and 1/2.29, respectively. Mean time to reach 99.9% growth reduction was 19.1±18.2h (2 mg/l anidulafungin), 37.4±8.8h (2 mg/l caspofungin), and 30.7±12.2h (1 mg/l micafungin). Anidulafungin exhibited the greatest killing rate followed by micafungin. The three echinocandins showed fungicidal activity at concentrations reached in serum. Downloaded from on April 29, 2018 by guest

3 3 Candida krusei is isolated mainly in immunocompromised and those who have received fluconazole treatment. The isolation (2 to 60%) and mortality rates (30 to 60%) depend on the institution and on the unit of admission (10, 13-14, 17, 20). Treatment of invasive C. krusei infections can be difficult due to its intrinsic resistance to fluconazole and its reduced susceptibility to amphotericin B and flucytosine (2, 15). In immunocompromised patients, fungicidal agents may be required to eradicate infection since the major host defence (phagocytic killing of neutrophils and monocytes/macrophages) is reduced (9). Anidulafungin, caspofungin and micafungin are novel antifungals that share the same spectrum of activity but differ in MIC values. Although the fungicidal activity of echinocandins and killing rate have been previously evaluated (3, 7-8, 16), none of these studies performed a head to head comparison. However, these agents also may differ in their fungicidal activity. This study aimed to determine the antifungal activity against C. krusei by three methods: i) MIC values, ii) minimal fungicidal concentrations (MFCs), and iii) time-kill studies. Anidulafungin (Pfizer España, Madrid, Spain), caspofungin (Merck Sharpe & Dome, Madrid, Spain) and micafungin (Fugisawa Pharmaceutical Company, Japan) were provided by their manufacturers. Caspofungin and micafungin were dissolved in water and anidulafungin in dimethyl sulfoxide; further dilutions were prepared in standard RPMI-1640 medium (Sigma-Aldrich, Madrid, Spain). Final anidulafungin and micafungin drug concentrations ranged from 0.03 to 16 mg/l and caspofungin from 0.12 to 64 mg/l. MIC and MFC values were obtained for 20 C. krusei bloodstream isolates, the caspofunginresistant CY-118 strain (MIC >8 mg/l) (11) and C. krusei ATCC MICs were performed following the CLSI M27-A3 microdilution method (4). MIC 2 and MIC 0 (minimum concentrations that produce 50 and 100% growth reduction, respectively) were determined at 24 and 48h. As previously described but using a higher inoculum size (1), MFCs were

4 4 obtained by plating 0.1 ml from 48h-clear MIC wells onto Sabouraud dextrose agar plates (SDA) (1). The MFC was the lowest drug concentration that resulted in 1 colony ( 99% killing). C. krusei ATCC 6258 was included in each batch of experiments (5). Time kill studies were performed for six isolates (randomly selected from the 20 blood isolates), CY-118 and ATCC 6258 isolates. Antifungal carryover effect for each agent and time killing curve were performed as previously described (RPMI-1640 medium, CFU/ml inoculum, and 5-mL volume) (2). Anidulafungin and caspofungin drug concentrations evaluated were: 0.03, 0.12, 0.5, 2 and 8 mg/l, and an additional 32 mg/l for caspofungin. Micafungin concentrations assessed were: 0.06, 0.25, 1, 8 and 16 mg/l. These concentrations are within the range achieved clinically (6, 18, 19). At 0, 2, 4, 6, 12, 24, and 48h, aliquots of 0.1-mL were removed to determine the number of. The lowest limit of accurate and reproducible detectable colony counts was 100. All experiments were performed twice with three replicates for every dilution of each time-point. To our knowledge, this is the first study comparing the killing activity of anidulafungin, caspofungin and micafungin head-to-head against six bloodstream C. krusei isolates, the caspofungin-resistant and the ATCC 6258 strains. Geometric mean (GM) MIC 2, GM-MIC 0, GM-MFC, and the concentration that inhibited and killed 90% of isolates (MIC 90, and MFC 90, respectively) were calculated. Time kill data were fitted to an exponential equation: N t =N 0 e kt (N t, viable cells at time t; N 0, starting inoculum; k, killing rate; t, incubation time). The goodness of fit for each isolate/drug was assessed by the R 2 value. The time (hours) to achieve 50, 90, 99, and 99.9% reductions in growth, compared to starting inoculum, was calculated from the k value as described elsewhere (2). MICs for C. krusei ATCC 6258 were within the established ranges (5); anidulafungin, micafungin and caspofungin MFCs for this strain were 0.25, 0.5 and 2 mg/l, respectively.

5 5 Table 1 summarizes MIC and MFC determinations for the 20 blood isolates. Anidulafungin was the most active followed by micafungin and caspofungin. MFC/MIC 0 ratio was 2 for 90, 90 and 100% of isolates, respectively. Anidulafungin and micafungin MFCs were one to seven dilutions lower than those of caspofungin, while anidulafungin MFCs were only one to two dilutions lower than those of micafungin. The kill-curve data for the six isolates were averaged and the means and standard deviations are depicted in Figure 1A. Table 2 shows the MICs and MFCs for these isolates, and Figure 2A, the relationship between killing rate and concentration. During the first 6 hours, the killing activity of the three drugs was isolate-dependent, but concentrationindependent (killing rate did not increase substantially by increasing concentration). However, after 6 hours, little killing or re-growth was observed depending on the concentration. In general, killing activity began at the MIC, but the 3 log decrease (killing/fungicidal endpoint) was achieved with 0.5 mg/l at h with anidulafungin; h with caspofungin; and h with micafungin (Table 3). Against the caspofungin-resistant CY-118 isolate, anidulafungin and micafungin exhibited killing activity, reaching the fungicidal endpoint with 8 mg/l of anidulafungin at 24h and 16 mg/l of micafungin at 48h (Figure 1B). Against C. krusei ATCC 6258, killing kinetics was similar to that exhibited for the blood isolates (Figure 1C) and that reported by Ernst et al. (7). The relationship between killing rate (k) and concentration for each echinocandin was lineal (Figure 2). Anidulafungin showed the greatest killing rate against blood isolates and CY-118 and ATCC In contrast, micafungin killing rate was slightly lower but parallel to that of anidulafungin against CY-118 and ATCC Our results are similar to those reported by other authors; although they only evaluated two C. krusei isolates (7-8, 16). Recently, our results and the utility of killing studies have been confirmed in a murine model of C. krusei infection treated with anidulafungin (12).

6 6 In summary, the three echinocandins have demonstrated fungicidal activity by both MFC and time-killing studies at total drug concentrations reached in serum ( 1 mg/l) throughout the dosing interval, which is an advantage when treating infections caused by C. krusei mainly in immunocompromised patients. AKNOWLIDGEMENTS This study was supported in part by research grant from Pfizer. Downloaded from on April 29, 2018 by guest

7 7 Figure 1. Time kill assay curves of anidulafungin (AND), caspofungin (CAS), and micafungin (MCF) for the six blood isolates (A), the isolate CY-118 (B) and the ATCC 6258 (C). Data plotted are the means and the standard deviations from two separate experiments for each growth curve, including the control. Dotted line, 3 log decrease. Figure 2. Means and standard deviations of the killing rate vs. drug concentrations obtained for six C. krusei blood isolates (A), isolate CY-118 (B) and the ATCC 6258 (C). K values are the slope of the regression line obtained from the kill curve. Positive and negative K values correlate with increases and decreases in viable cell numbers, respectively. Anidulafungin (AND), continuous line; caspofungin (CAS), discontinuous line; and micafungin (MFC), dotted line References 1. Cantón, E., J., Pemán, A., Viudes, G., Quindós, M., Gobernado, A., Espinel- Ingroff Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species. Diagn. Microbiol. Infect. Dis. 45: Cantón E., J. Pemán, M., Gobernado, A., Viudes, A., Espinel-Ingroff Patterns of amphotericin B killing kinetics against seven Candida species. Antimicrob. Agents Chemother. 48: Clancy, C. J., Huang, H., Cheng, S., Derendorf, H., Nguyen, M. H Characterizing the effects of caspofungin on Candida albicans, Candida parapsilosis, and Candida glabrata isolates by simultaneous time-kill and postantifungal-effect experiments. Antimicrob Agents Chemother. 50:

8 8 4. Clinical and Laboratory Standards Institute: Reference method for broth dilution antifungal susceptibility testing of yeasts, approved standard. CLSI Document M27-A3. Clinical and Laboratory Standards Institute, Wayne, PA. 5. Clinical and Laboratory Standards Institute Reference method for broth dilution antifungal susceptibility testing of yeasts. Third informational supplement. CLSI Document M27-S3. Clinical and Laboratory Standards Institute,Wayne, PA. 6. Dowell, J. A., W., Knebel, T., Ludden, M., Stogniew, D., Krause, T., Henkel Population pharmacokinetic analysis of anidulafungin, an echinocandin antifungal. J. Clin. Pharmacol. 44: Ernst, E., J., E. E., Roling, C. R., Petzold, D. J., Keele, M. E., Klepser In vitro activity of micafungin (FK-463) against Candida spp.: microdilution, time-kill, and postantifungal-effect studies. Antimicrob. Agents Chemother. 46: Karlowsky, J. A., G. A., Harding, S. A., Zelenitsky, D. J., Hoban, A. Kabani, T. V., Balko, M., Turik, G. G., Zhanel In vitro kill curves of a new semisynthetic echinocandin, LY , against fluconazole-sensitive and -resistant Candida species. Antimicrob. Agents Chemother. 41: Maródi, L., J. R., Forehand, R. B., Johnston Jr Mechanisms of host defense against Candida species. II. Biochemical basis for the killing of Candida by mononuclear phagocytes. J. Immunol. 15; 146: Muñoz, P., M., Sánchez-Somolinos, L., Alcala, M., Rodríguez-Créixems, T., Peláez, E., Bouza Candida krusei fungaemia: antifungal susceptibility and clinical presentation of an uncommon entity during 15 years in a single general hospital. J. Antimicrob. Chemother. 55: Odds, F. C., M., Motyl, R., Andrade, J., Bille, E., Cantón, M., Cuenca-Estrella, A., Davidson, C., Durussel, D., Ellis, E., Foraker, A. W., Fothergill, M. A.,

9 9 Ghannoum, R. A., Giacobbe, M., Gobernado, R., Handke, M., Laverdière, W., Lee-Yang, W. G., Merz, L., Ostrosky-Zeichner, J., Pemán, S., Perea, J. R., Perfect, M. A., Pfaller, L., Proia, J. H., Rex, M. G., Rinaldi, J. L., Rodriguez- Tudela, W. A., Schell, C., Shields, D. A., Sutton, P. E., Verweij, D. W., Warnock Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species. J. Clin. Microbiol. 42: Ostrosky-Zeichner, L., V. L., Paetznick, J., Rodriguez, E.,Chen, and D. J., Sheehan Activity of Anidulafungin in a Murine Model of Candida krusei Infection: Evaluation of Mortality and Disease Burden by Quantitative Tissue Cultures and Measurement of Serum (1,3)-{beta}-D-Glucan Levels. Antimicrob. Agents Chemother. 53: Pemán J, E., Cantón, A., Orero, A., Viudes, J., Frasquet, M., Gobernado Estudio multicéntrico sobre la epidemiología de las candidemias en España. Rev. Iberoam. Micol. 19: Pemán, J., E., Cantón, M., Gobernado, and the Spanish ECMM Working Group on Candidaemia Epidemiology and antifungal susceptibility of Candida species isolated from blood: results of a 2-year multicentre study in Spain. Eur. J. Clin. Microbiol. Infect. Dis. 24: Pfaller. M., A., D. J., Diekema, D. L., Gibbs, V. A., Newell, E., Nagy, S., Dobiasova, M., Rinaldi, R., Barton, A Veselov; and Global Antifungal Surveillance Group Candida krusei, a multidrug-resistant opportunistic fungal pathogen: geographic and temporal trends from the ARTEMIS DISK Antifungal Surveillance Program, 2001 to J. Clin. Microbiol. 46: Roling, E. E., M. E., Klepser, A., Wasson, R. E., Lewis, E. J., Ernst, M. A., Pfaller Antifungal activities of fluconazole, caspofungin (MK0991), and

10 10 anidulafungin (LY303366) alone and in combination against Candida spp. and Crytococcus neoformans via time-kill methods. Diagn. Microbiol. Infect. Dis. 43: Sandven, P Epidemiology of candidemia. Rev. Iberoam. Micol. 17: Seibel, N. L., C., Schwartz, A., Arrieta, P., Flynn, A., Shad, E., Albano, J., Keirns, W. M., Lau, D. P., Facklam, D. N., Buell, T. J. Walsh Safety, tolerability, and pharmacokinetics of Micafungin (FK463) in febrile neutropenic pediatric patients. Antimicrob. Agents Chemother. 49: Stone J. A., S. D., Holland, P. J., Wickersham, A., Sterrett, M., Schwartz, C., Bonfiglio, M., Hesney, G. A., Winchell, P. J., Deutsch, H., Greenberg, T. L., Hunt, S. A., Waldman Single- and multiple-dose pharmacokinetics of caspofungin in healthy men. Antimicrob. Agents Chemother.46: Viudes, A., J., Pemán, E., Cantón, P., Ubeda, J. L., López-Ribot, M. Gobernado Candidemia at a tertiary-care hospital: epidemiology, treatment, clinical outcome and risk factors for death. Eur. J. Clin. Microbiol. Infect. Dis. 21: Downloaded from on April 29, 2018 by guest

11 11 Table 1. Echinocandin in vitro activity of against 20 C. krusei bloodstream isolates Drug MIC 2 MIC 0 MFC Time (h) Range MIC 50 MIC 90 GM Range MIC 50 MIC 90 GM Range MFC 50 MFC 90 GM Anidulafungin Micafungin Caspofungin MIC 2 and MIC 0, 50 and 100% reduction in growth, respectively. MFC, minimal fungicidal concentration. GM, geometric mean. MIC 50 and MIC 90, concentration that inhibited 50 and 90% of isolates, respectively. Downloaded from on April 29, 2018 by guest

12 12 Table 2. Echinocandin MIC and MFC values for the eight isolates evaluated by the killing curve method Isolate Anidulafungin Caspofungin Micafungin MIC 2 MFC MIC 2 MFC MIC 2 MFC CK CK CK CK EU EU CY > ATCC MIC 2 50 reduction in growth (24h); MFC, minimal fungicidal concentration Downloaded from on April 29, 2018 by guest

13 13 Table 3. Time to achieve 50 (T50), 90 (T90),99 (T99)and 99.9% (T99.9) growth reduction from the starting inoculum Echinocandin Mean time (h) at the following concentrations (mg/l): Agent AND T ± > ± ± ± 1.6 T ± > ± ± ± 5.5 T99 >48 > ± ± 10.9 T99.9 >48 > ± ± 16.4 CAS T50 No killing 8.3 ± ± ± ± 0.6 T90 No killing 27.6 ± ± ± ± 1.9 T99 No killing > ± ± ± 3.9 T99.9 No killing > ± ± ± 5.9 MCF T ± ± ± ± 1.1 T ± ± ± ± 3.7 T ± > ± ± ± 7.5 T ± > ± ± ± 11.2

14 A B C AND CT 8 µg/ml 2 µg/ml 0,5 µg/ml 0,12 µg/ml 0,03 µg/ml CY-118 ANID Figure 1 1,E+10 1,E+09 ATCC 6258 ANID CT 8 µg/ml 2 µg/ml 0,5 µg/ml 0,12 µg/ml 0,03 µg/ml µg/ml 2 µg/ml CT 0,5 µg/ml 0,12 µg/ml 0,03 µg/ml 1,E+09 CT 32µg/ml 8 µg/ml 2 µg/ml CAS 0,5 µg/ml 0,12 µg/ml 0,03 µg/ml 1,E+10 1,E+09 CY-118 CT CAS 8mg/mL CAS 2 mg/ml CAS ATCC 6258 CAS CT 32µg/ml 8 µg/ml 2 µg/ml 0,5 µg/ml 0,12 µg/ml 1,E+09 UFC/mL MCF CT 16 µg/ml 4 µg/ml 1 µg/ml 0,25 µg/ml 0,06 µg/ml 1,E+10 1,E+09 CY-118 CT 16 µg/ml 4 µg/ml MCF 1 µg/ml 0,25 µg/ml 0,06 µg/ml ATCC 6258 MCF CT 16 µg/ml 4 µg/ml 1 µg/ml 0,25 µg/ml 0,06 µg/ml 1,E+09

15 C FU /m L/H 0,30 0,20 0,10 0,00-0,10 Figure 2 AND MCF CAS 0,3 0,3 CAS AND MCF AND CAS MCF A B C AND; y = x ; R 2 =0.91 CAS; y = x ; R 2 =0.81 MCF; y = -0,018x + 0,054; R 2 =0.97 K ( /h ) 0,2 0,1 0-0,1-0,20-0,2 AND; y = x ; R 2 =0.88-0,2 CAS; y = x ; R 2 =0.22 MCF; y = -0,012x + 0,091; R 2 =0.89-0,30-0,3-0,3-0,40-0,4-0,4 0, mg/l mg/l mg/l K (CFU /ml/h) 0,2 AND; y = x ; R 2 =0.84 0,1 CAS; y = x ; R 2 =0.78 MCF; y = -0,019x + 0,024; R 2 = ,1