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Antimicrobial Agents and Chemotherapy, August 2007, p. 2985-2987, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00308-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Measurement of Voriconazole Activity against Candida albicans, C. glabrata, and C. parapsilosis Isolates Using Time-Kill Methods Validated by High-Performance Liquid Chromatography

Yanjun Li,¹ M. Hong Nguyen,^2,3,4 Hartmut Derendorf,¹ Shaoji Cheng,² and Cornelius J. Clancy^2,4*

Department of Pharmaceutics, University of Florida College of Pharmacy,¹ Departments of Medicine,² Molecular Genetics and Microbiology, University of Florida College of Medicine,³ North Florida/South Georgia Veterans Health System, Gainesville, Florida⁴

Received 5 March 2007/ Returned for modification 25 April 2007/ Accepted 12 May 2007

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We developed a high-performance liquid chromatography (HPLC) assay to validate time-kill and postantifungal-effect (PAFE) experiments for voriconazole against Candida albicans, Candida glabrata, and Candida parapsilosis isolates. Voriconazole exerted prolonged fungistatic activity but no PAFE at concentrations achievable in human sera. HPLC confirmed that experiments were conducted at the desired steady-state voriconazole concentrations.

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Voriconazole is a triazole agent that inhibits ergosterol synthesis by blocking the action of 14-demethylase. The drug is fungistatic and exhibits no postantifungal effect (PAFE) against Candida albicans (1, 3-5, 7, 9). Time-kill and PAFE data for Candida glabrata are limited (1, 7) and do not exist for Candida parapsilosis isolates. Moreover, standard time-kill and PAFE methodologies, although widely used, have not been validated for voriconazole or other antifungals by direct measurement of drug concentrations. In this study, we developed a high-performance liquid chromatography (HPLC) assay to validate the results of time-kill and PAFE experiments for voriconazole against C. albicans reference strains (ATCC 90029 and SC5314) and C. glabrata and C. parapsilosis bloodstream isolates (two each).

The MICs of all isolates were within the susceptible range, as measured by Etest and microdilution methods (Table 1) (10, 11). For time-kill and PAFE experiments, colonies from 48-hour cultures on Sabouraud dextrose agar were suspended in 9 ml sterile water (2, 7). One microliter of a 0.5 McFarland suspension was added to 10 ml of RPMI 1640 medium with or without voriconazole (0.25x, 1x, 4x, and 16x MICs), and the solution was incubated at 35°C with agitation. The maximal voriconazole concentration in these experiments was 3.04 µg/ml (16x MIC for C. glabrata isolate 1). For time-kill experiments, 100 µl from each solution was serially diluted at the desired time points (0, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h) and plated on Sabouraud dextrose agar for colony enumeration. For PAFE experiments, Candida cells were collected after 1 h of incubation, washed three times, and resuspended in warm RPMI 1640 medium (9 ml); colonies were enumerated at the desired time points. Voriconazole exhibited dose-response effects against all Candida isolates during time-kill experiments (Fig. 1; Table 1), as higher concentrations resulted in greater growth inhibition or killing. The ranges of maximal growth inhibition of isolates at 1x and 4x MICs were –0.61 to –2.78 log and –0.53 to –2.99 log, respectively, compared to those of controls (Table 1). At 16x MIC, the range of maximal growth inhibition was –0.58 to –4.15 log. Voriconazole did not demonstrate PAFEs (data not shown).

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TABLE 1. Voriconazole MICs and time-kill data for Candida isolates

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FIG. 1. Time-kill curves for voriconazole against Candida isolates. Representative curves are presented for each isolate. Experiments were performed in duplicate, without significant differences in results. N_CFU, number of CFU; Ctrl, control; 0.25*MIC, 0.25x MIC.

Voriconazole at 16x MIC was fungicidal against C. parapsilosis isolate 2, reducing the starting inoculum by –2.21 log at 24 h (fungicidal is defined as a >2-log reduction of starting inoculum). Although kills did not achieve fungicidal levels for other isolates, voriconazole at 4x and 16x MICs reduced the starting inocula of C. glabrata isolate 2 and C. parapsilosis isolate 1 (Table 1). Of note, voriconazole was consistently fungistatic at 1x to 16x MICs, and the effect persisted for 72 h. Indeed, maximal inhibition of the four C. albicans and C. glabrata isolates at 4x and 16x MICs (compared to starting inocula) was not evident until 48 to 72 h. The two C. parapsilosis isolates, on the other hand, were maximally inhibited by 24 to 36 h.

The time-kill curves of the C. parapsilosis isolates also differed from those of the other isolates at early time points. The C. parapsilosis isolates at 4x and 16x MICs were inhibited from entering the exponential growth phase, and dose-response effects were clearly evident by 8 h. The growth rates of C. albicans and C. glabrata isolates in the presence of voriconazole did not differ from those of controls during early exponential phase, but dose-response effects became increasingly apparent as exponential growth continued (8 to 24 h).

In our HPLC protocol for measuring voriconazole concentrations during time-kill and PAFE experiments, a 250- by 4.6-mm analytic column with a 10- by 3.2-mm guard cartridge (Hichrom, Reading, United Kingdom) was packed with a 5-µm-particle-size Kromasil column at 25°C in an Agilent 1100 series apparatus (6, 8). Mobile-phase acetonitrile-ammonium phosphate buffer (pH 6.0; 0.04 M; 1:1 [vol:vol]) was degassed by filtration through a 0.45-µm nylon filter under vacuum; the flow rate was 0.8 ml/min. Voriconazole concentrations were determined for peak areas detected by UV absorption at 255 nm with an 8.2-min retention time. For each isolate, we tested RPMI 1640 medium containing at least one dose of voriconazole between 1x and 16x MICs. Samples were diluted with 2 volumes of acetonitrile-ammonium phosphate buffer and centrifuged at full speed in a microcentrifuge for 10 min, and the supernatants (200 µl) were applied to the column. The maximum sensitivity was 0.025 µg/ml, and the method produced linear results over a range of 0.025 to 12.8 µg/ml (r² 0.9996). In each instance, we confirmed that voriconazole concentrations remained constant throughout the duration of time-kill experiments (Fig. 2), and the drug was fully removed during PAFE experiments (data not shown).

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FIG. 2. Measurements of voriconazole concentrations in culture media by HPLC. Representative data for a 48-h time-kill experiment with C. glabrata isolate 1 are presented. Concentrations of voriconazole against each Candida isolate were 85% of the starting concentration at all time points.

Our findings conclusively demonstrate that voriconazole exerts prolonged fungistatic activity against C. albicans, C. glabrata, and C. parapsilosis at concentrations that are achievable in human sera with routine dosing (the medians of the average and maximum voriconazole plasma concentrations in clinical trials were 2.51 and 3.79 µg/ml, respectively) (12). Our findings are potentially relevant clinically, since certain C. parapsilosis isolates exhibit diminished susceptibility to echinocandin antifungals and C. glabrata isolates can develop resistance to fluconazole and other antifungal agents. Although voriconazole caused a >2-log kill of one C. parapsilosis isolate, further studies will be needed to accurately define the extent to which the drug might be fungicidal against clinical isolates.

To our knowledge, this is the first study to verify standard time-kill and PAFE methodologies by directly measuring drug concentrations. We describe a simple and reproducible HPLC method that has a broad, clinically relevant dynamic range and does not require internal standards. The sensitivity of voriconazole measurements within liquid media was greater than that previously reported for human or guinea pig plasma (0.2 to 10 or 5 to 10 µg/ml, respectively) (6, 8). Based on our findings, we can assume that previous studies of azoles that showed fungistatic anticandidal activity and no PAFEs were conducted under the conditions of steady-state drug concentrations assumed by the investigators. This demonstration is crucial as efforts to use pharmacodynamic data to develop optimal antifungal treatment strategies move forward. In particular, HPLC methods will be essential to the design of dynamic in vitro models to assess the pharmacodynamics of voriconazole and other agents prior to the achievement of steady-state conditions.

 
We thank Vipul Kumar for his advice in developing the HPLC assay. Experiments were conducted in C. J. Clancy's laboratory at the North Florida/South Georgia Veterans Health System, Gainesville, FL.

This project was supported by the Medical Research Service of the Department of Veterans Affairs. It was conducted as part of the University of Florida Mycology Research Unit (NIH PO1 AI061537-01 to M. H. Nguyen and C. J. Clancy). C. J. Clancy has received research support from Pfizer.

 
* Corresponding author. Mailing address: University of Florida College of Medicine, Box 100277, JHMHC, 1600 SW Archer Rd., Gainesville, FL 32610. Phone: (352) 392-4058. Fax: (352) 271-4566. E-mail: clancyn{at}medicine.ufl.edu

Published ahead of print on 21 May 2007.

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Antimicrobial Agents and Chemotherapy, August 2007, p. 2985-2987, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00308-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Nguyen, K. T., Ta, P., Hoang, B. T., Cheng, S., Hao, B., Nguyen, M. H., Clancy, C. J. (2009). Anidulafungin Is Fungicidal and Exerts a Variety of Postantifungal Effects against Candida albicans, C. glabrata, C. parapsilosis, and C. krusei isolates. Antimicrob. Agents Chemother. 53: 3347-3352 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, Y. Articles by Clancy, C. J. Search for Related Content PubMed PubMed Citation Articles by Li, Y. Articles by Clancy, C. J.