Effect of 50% human serum on the killing activity of micafungin against eight Candida species using time–kill methodology ☆ , ☆☆ Richárd Földi, Judit Szilágyi, Gábor Kardos, Réka Berényi, Renátó Kovács, László Majoros ⁎ Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen, 4032 Debrecen, Hungary abstract article info Article history: Received 25 November 2011 Accepted 9 May 2012 Available online 20 June 2012 Keywords: Echinocandins Antifungal susceptibility Impaired killing Serum-based susceptibility testing Micafungin activity was determined against 24 wild-type clinical isolates and 5 American Type Culture Collection strains belonging to 8 Candida species in RPMI-1640 with and without 50% serum using broth microdilution and time–kill methodology. MIC values increased from 4- to 128-folds in 50% serum for all Candida species. Micafungin was not fungicidal against C. albicans, C. tropicalis, and against 2 of 3 C. metapsilosis at ≥0.25, 1, and 1 μg/mL, respectively, after 48 h with 50% serum, showing good fungistatic activity. Fungicidal activity at ≥2, 4, and 32 μg/mL was noticed against C. glabrata, C. inconspicua, and C. krusei isolates, respectively. Micafungin at 8–32 μg/mL showed fungistatic activity against C. parapsilosis and C. orthopsilosis. Serum decreased the in vitro activity of micafungin. With serum binding of echinocandins taken into account, safely fungistatic or fungicidal concentrations seem to require elevated doses against some Candida species, including C. parapsilosis, C. orthopsilosis, and C. krusei. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Echinocandins (caspofungin, micafungin, and anidulafungin) show excellent fungicidal or fungistatic activity against the Candida species at clinically attainable concentrations and are considered the cornerstone in the treatment of invasive candidiasis, especially among neutropenic patients (Chen et al., 2011; Ernst et al., 2002; Goto et al., 2010; Hiemenz et al., 2005; Ishikawa et al., 2009; Pappas et al., 2009; Pfaller et al., 2011; Undre et al., 2012). The majority of these species (i.e., C. albicans, C. glabrata, C. tropicalis, and C. krusei) are innately susceptible to echinocandins, while others (i.e., C. parapsilosis) show higher MIC 90 values to echinocandins as measured in RPMI-1640 medium (Chen et al., 2011; Ernst et al., 2002; Pfaller et al., 2011). Echinocandins are highly protein-bound (from 96.5% to 99.8%), which decreases the drug concentration available in the serum and tissues (Chen et al., 2011; Ernst et al., 2002; Ishikawa et al., 2009; Odabasi et al., 2007; Paderu et al., 2007). Increased MIC values were reported for all 3 echinocandins against Candida and Aspergillus species in RPMI-1640 plus 50% serum as test medium (Ernst et al., 2002; Garcia-Effron et al., 2011; Ishikawa et al., 2009; Odabasi et al., 2007; Paderu et al., 2007). Although MIC values increase in 50% serum, this increase is lower than extrapolated from high protein binding. Thus, it is supposed that some part of the protein-bound drug retains activity against Candida species (Garcia-Effron et al., 2011; Ishikawa et al., 2009). Despite the more frequent usage of serum-based susceptibility methods, information about the influence of serum on killing activity of echinocandins against Candida species is scant (Baltch et al., 2011, Stein et al., 2010). Therefore, the aim of our study was to compare the activity of micafungin in RPMI-1640 and RPMI-1640 with 50% serum against 8 Candida species using time–kill methodology. 2. Materials and methods The 24 wild-type clinical isolates and the 5 American Type Culture Collection (ATTC) strains used in this study are listed in Table 1. All C. albicans, C. tropicalis, C. glabrata, C. parapsilosis, C. krusei, and 1 of 3 C. inconspicua isolates were derived from bloodstream. Isolates were identified using CHROMagar Candida and API ID32C; species identity of C. parapsilosis, C. orthopsilosis, C. metapsilosis, and C. inconspicua was confirmed by molecular biological methods (Majoros et al., 2003; Tavanti et al, 2005). All clinical isolates were first isolate (1 patient per 1 isolate). No patients received any echinocandins previously. MICs were determined using the Clinical and Laboratory Standards Institute (CLSI) standard broth microdilution method (CLSI, 2008). Micafungin pure powder (Astellas, Ibaraki, Japan) was dissolved in sterile distilled water; final concentration ranged between 0.015– 8 and 0.06–32 mg/L in RPMI-1640 and in RPMI-1640 plus 50% human serum (from a human male, type AB; Sigma, Budapest, Hungary), Diagnostic Microbiology and Infectious Disease 73 (2012) 338–342 ☆ Transparency declaration: L. Majoros received conference travel grants from MSD and Pfizer. ☆☆ Funding: R. Földi was supported by a Richter Gedeon Pharma PhD scholarship; L. Majoros was supported by a Bolyai Research Scholarship of the Hungarian Academy of Sciences. ⁎ Corresponding author. Tel.: + 36-52-255-425; fax: + 36-52-255-424. E-mail address: major@med.unideb.hu (L. Majoros). 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2012.05.011 Contents lists available at SciVerse ScienceDirect Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio