Comparison of posaconazole and voriconazole in vitro killing against Candida krusei

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Diagnostic Microbiology and Infectious Disease 62 (2008) 177 – 181 www.elsevier.com/locate/diagmicrobio

Mycology

Comparison of posaconazole and voriconazole in vitro killing against Candida krusei Emilia Cantóna,⁎, Javier Pemánb , Amparo Valentína , Maria Boschb , Ana Espinel-Ingroffc , Miguel Gobernadob a

Unidad de Microbiología Experimental, Centro de Investigación, Hospital Universitario La Fe, 46009 Valencia, Spain b Servicio de Microbiología, Hospital Universitario La Fe, 46009 Valencia, Spain c Division of Infectious Diseases, VCU Medical Center, Richmond, VA 23298-0049, USA Received 19 May 2008; accepted 1 July 2008

Abstract The in vitro activity of posaconazole and voriconazole was evaluated by MIC, minimum fungicidal concentration (MFC), and time-kill methods. MFCs were determined for 15 Candida krusei and time-killing curves for 5 of these isolates. MFCs were obtained transferring 100 μL from clear MIC wells onto Sabouraud dextrose agar. Time-kill studies were performed in RPMI 1640 medium (5 mL, inoculum ≈105 colonyforming unit [CFU]/mL). Geometric mean (GM) MIC and GM-MFC were 0.2 and 0.72 mg/L for posaconazole and 0.4 and 2.64 mg/L for voriconazole. The killing rate was isolate and concentration dependent; reductions in CFUs start at ≥0.12 mg/L (posaconazole) and ≥0.5 mg/L (voriconazole). The mean time to reach a 90% growth reduction was 41.5 ± 14.2 h (8 mg/L posaconazole) and 48.7 ± 61.3 h (32 mg/L voriconazole). By time kill, a 99% killing rate was not reached by either agent. Both methods demonstrated that posaconazole (more active and greater killing rate) and voriconazole have fungicidal activity against C. krusei. © 2008 Elsevier Inc. All rights reserved. Keywords: Posaconazole; Voriconazole; Candida krusei; Time killing; MIC; MFC

1. Introduction Candida krusei is a species that is intrinsically resistant to fluconazole and has decreased susceptibility to amphotericin B and flucytosine (Pfaller et al., 2008). It is isolated primarily in cancer patients and in settings where fluconazole is used as prophylaxis (Girmenia et al., 2005; Wingard et al., 1991). The frequency of candidemia caused by this species ranges from 2% to 60% (Pemán et al., 2005; Sandven, 2000; Viudes et al., 2002), depending on the institution and, within an institution, on the admission unit. Compared with infections caused by other Candida spp., those caused by C. krusei are associated with the highest mortality rate (30–60%) (Muñoz et al., 2005; Pemán et al., 2002). Posaconazole and ⁎ Corresponding author. Tel.: +34-961973111; fax: +34-96-3868718. E-mail addresses: [email protected] (E. Cantón), [email protected] (J. Pemán), [email protected] (A. Valentín), [email protected] (M. Bosch), [email protected] (A. Espinel-Ingroff), gobernado_mig @gva.es (M. Gobernado). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.07.001

voriconazole are newly available triazole agents that have in vitro activity against C. krusei isolates as determined by MIC values (Cantón et al., 2008; Pemán et al., 2006; Sabatelli et al., 2006), but scarce information is available about their fungicidal activity and killing kinetics. The aim of this study was to determine the in vitro activity of posaconazole and voriconazole against C. krusei by 3 methods: i) MIC values, ii) minimal fungicidal concentrations (MFCs), and iii) time-kill studies. 2. Materials and methods 2.1. Isolates MIC and MFC end points were determined for 15 C. krusei blood isolates collected from patients hospitalized in different units (intensive care, hematology, and oncology) in the hospital La Fe, Valencia, Spain, from 2000 to 2006. The isolates were identified by VITEK system (bioMérieux, Madrid, Spain). For time-kill studies, a subset of 5 from the

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15 isolates was randomly selected; C. krusei ATCC 6258 also was evaluated. 2.2. Drugs Voriconazole (Pfizer España, Madrid, Spain) and posaconazole (Schering-Plough, Kenilworth, NJ), provided as pure powder by the manufacturers, were dissolved in dimethyl sulfoxide, and final drug dilutions were prepared in the standard RPMI 1640 medium (Sigma-Aldrich, Madrid, Spain). The final drug concentration range was 0.06 to 32 mg/L. 2.3. MIC and MFC procedure Broth microdilution MICs were performed following the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical Laboratory Standards [NCCLS]) M27-A2 document (NCCLS, 2002). Both MIC2 and MIC0 (minimum concentration that produces ≥50 and 100% growth reduction, respectively) end points were determined. MFC values were obtained at 48 h by plating 0.1-mL volumes from the optically clear MIC wells onto Sabouraud dextrose agar plates as described elsewhere (Cantón et al., 2003). The MFC was the lowest drug concentration that resulted in either no growth or less than 2 colonies (99% killing). Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 isolates were included as controls (NCCLS, 2002). 2.4. Time-kill curves Antifungal carryover effect and time-kill studies were performed as described elsewhere (Cantón et al., 2004). Briefly, these studies were carried out in RPMI 1640 medium (5 mL) by using an inoculum size of 1 to 7 × 105 colonyforming unit (CFU)/mL and concentrations of 0.03, 0.12, 0.5, 2, 8 (posaconazole and voriconazole), and 32 (voriconazole) mg/L. At predetermined time points (0, 2, 4, 6, 12, 24, and 48 h), 100 μL of aliquots were removed from each control (drug free) and test solution tube and then serially diluted in sterile water. Volumes of 30 to 100 μL from serially dilute aliquots, depending on dilution and concentration of the drug, were spread onto Sabouraud dextrose agar plates to determine CFUs after 24 to 48 h of incubation times at 35 °C. The lower limit of accurate and reproducible detectable colony counts was 100 CFU/mL. All experiments were performed twice (on different days), and 3 replicates were also evaluated for every dilution at each time point. 2.5. Data analysis Geometric mean (GM) MIC2, GM-MIC0, and GM-MFC results were calculated, as well as the concentration that inhibited 90% of the isolates (MIC90) for both MIC end points (MIC290 and MIC090) and the concentration that killed 90% of the isolates (MFC90). Time-kill data were fitted to an exponential equation: Nt = N0 × e−kt (Nt, viable cells at time t; N0, starting inoculum; k, killing rate; t, incubation

time). The goodness of the fit for each isolate/drug combination was assessed by the R2 value (N0.8). The time (hours) to achieve 50%, 90%, 99%, and 99.9% (T50, T90, T99, and T99.9, respectively) reductions in growth, as compared with the initial inoculum, was calculated from the K value as described elsewhere (Cantón et al., 2004). 3. Results and discussion MICs for the quality control (QC) isolates were within the established range (CLSI, 2008). The MFC values of posaconazole for the QC isolates C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were 0.5 mg/L for both strains, and those of voriconazole were 1 and 0.12 mg/L, respectively. Table 1 shows posaconazole and voriconazole MICs and MFCs for the 15 C. krusei blood isolates. Posaconazole and voriconazole MIC2 ranged from 0.12 to 4 and from 0.25 to 2 mg/L, respectively, whereas posaconazole MICs0 ranged from 0.12 to 4 and voriconazole from 0.5 to 2 mg/L; posaconazole fungicidal activity was also higher (MFCs, 0.5–4 mg/L) than that of voriconazole (MFCs, 0.5– 16 mg/L). For both posaconazole and voriconazole, the GMMIC0 was 2-fold higher than the GM-MIC2; the GM-MFC was 1.67 and 2.9 times greater than their respective MIC0 values. The posaconazole MFC was ≤2× MIC0 for 93.3% of the isolates, whereas this value was obtained for only 53.3% of the isolates with voriconazole. The kill-curve data for 5 of the 15 clinical isolates were averaged (Fig. 1). For both agents, reductions in CFUs were observed after 12 h of incubation at concentrations ≥0.12 mg/L (posaconazole) and ≥0.5 mg/L (voriconazole); at lower drug concentrations, there were slight increases in CFUs, but these increases were

Table 1 In vitro activity (mg/L) of voriconazole and posaconazole against 15 clinical C. krusei isolates Isolate

CK-01 CK-02 CK-03 CK-04 CK-05 CK-06 CK-07 CK-08 CK-09 CK-10 CK-11 CK-12 CK-13 CK-14 CK-15 GM MIC90

Voriconazole

Posaconazole

MIC2

MIC0

MFC (99%)

MIC2

MIC0

MFC (99%)

0.5 2 0.5 0.5 0.5 0.25 0.25 0.25 0.5 0.5 0.5 0.5 0.25 0.25 0.25 0.42 0.5

1 2 1 1 1 0.5 1 1 1 1 1 1 0.5 1 0.5 0.91 1

8 4 4 4 16 2 4 2 4 2 2 1 0.5 2 1 2.64 16

0.5 0.25 0.12 0.12 0.25 0.25 0.25 0.12 0.5 0.25 0.25 0.12 0.12 0.25 0.12 0.20 2

0.5 0.5 0.25 0.25 0.5 0.25 0.25 0.25 0.5 0.25 2 0.5 0.5 4 0.12 0.43 2

0.5 0.5 0.5 0.5 1 0.5 0.5 0.5 0.5 1 2 1 0.5 4 0.5 0.72 2

MIC2 and MIC0, ≥50 and 100% reduction in growth; MIC90, a concentration that inhibits 90% of isolates.

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Fig. 1. Time-kill plots for voriconazole (VO) and posaconazole (POS) against 5 C. krusei clinical isolates (CK-1, CK-2, CK-6, CK-8, and CK-15) and C. krusei ATCC 6258. Each data point represents the average log CFU/mL provided for the 5 blood isolates; error bars indicate the SD. CT is the drug-free control.

less than those seen in the drug-free control. After 48 h, the greatest reduction in CFU with respect to the control was 3.9 log10 (range, 3.2–4.4) for posaconazole and 2.57 log10 (range, 1.7–3.1) for voriconazole. Although the extent of killing was isolate and concentration dependent, reductions in CFU started at concentrations ≥0.12 mg/L (posaconazole) and ≥0.5 mg/L (voriconazole) (Fig. 2). In these experiments, the maximum killing percentage with respect to the initial inoculum size was 90.6% (range, 80–98.3%) for posaconazole and 72.9% (range, 40–90%) for voriconazole. The mean time to reach 90% (T90) reduction in growth was 41.5 ± 14.2 h (range, 27.2–50.5 h) with 8 mg/L of posaconazole and 48.7 ± 61.3 h (range, 25.1–132.6 h) with 32 mg/L of

voriconazole. However, a 99% mean killing rate was not reached by this time-kill method with either agent probably because of the influence of the test conditions (inoculum size and final volume) on the fungicidal activity. The killing kinetics of both agents against C. krusei ATCC 6258 was similar to that obtained for the 5 blood isolates (Fig. 1). Prior studies on the fungicidal activity of voriconazole have been limited to Candida albicans, Candida glabrata, Candida tropicalis, Candida lusitaniae, and Cryptococcus neoformans. For these species, the CFUs at 1 to 16 × MIC increased when compared with the initial inoculum size but were lower than in the drug-free controls (Ernst et al., 2002; Klepser et al., 2000). However, fungicidal activity was

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Fig. 2. Mean and SD of the killing rate versus drug concentration obtained for 5 C. krusei blood isolates. 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.

demonstrated (decreased CFUs versus the initial inoculum size) with both agents against C. krusei in our study. The fungicidal activity of posaconazole against C. krusei among other species has been recently examined by MFC and time-killing methodologies (Sóczó et al., 2007). In contrast with our results, these authors obtained a greater rate of killing, perhaps because of the agitation used in their experiments, and reached the fungicidal end point after 48 h. On the other hand, MFC results are similar to ours. Voriconazole fungicidal activity against C. krusei has only been measured by MFC determinations by Rubio et al. (2005). Our MIC results are similar but we obtained a wider range of MFC values. To our knowledge, this is the 1st study that has evaluated the killing kinetics of voriconazole against this species. Although both MFC values and rate of killing were isolate dependent, kill-curves demonstrated that the number of viable counts were significantly lower (P b 0.05) than those of the control from 12 h onward; counts also decreased as the drug concentration increased. The mean maximum killing rate (with respect to initial inoculum size) was 90% for posaconazole and 73% for voriconazole at concentrations ≥2× MIC. Other authors have reported killing activity of voriconazole against phagocytized C. krusei cells at 5× MIC, which could be an advantage when treating invasive yeast infections (Bopp et al., 2006). Both methods (MFC and kill-

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