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Antimicrobial Agents and Chemotherapy, March 2001, p. 696-700, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.696-700.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Sensitive Bioassay for Determination of Fluconazole Concentrations in Plasma Using a Candida albicans Mutant Hypersusceptible to Azoles

Oscar Marchetti,1 Paul A. Majcherczyk,1 Michel P. Glauser,1 Jacques Bille,2 Philippe Moreillon,1 and Dominique Sanglard2,*

Division of Infectious Diseases1 and Institute of Microbiology,2 University Hospital, Lausanne, Switzerland

Received 21 August 2000/Returned for modification 3 October 2000/Accepted 20 November 2000

The antifungal agent fluconazole (FLC) is widely used in clinical practice. Monitoring FLC levels is useful in complicated clinical settings and in experimental infection models. A bioassay using Candida pseudotropicalis, a simple and cost-effective method, is validated only for FLC levels ranging from 5 to 40 mg/liter. An extension of the analytical range is needed to cover most yeast MICs. A new bioassay in RPMI agar containing methylene blue was developed using C. albicans DSY1024, a mutant rendered hypersusceptible to FLC constructed by the deletion of the multidrug efflux transporter genes CDR1, CDR2, CaMDR1, and FLU1. Reproducible standard curves were obtained with FLC concentrations in plasma ranging from 1 to 100 mg/liter (quadratic regression coefficient > 0.997). The absolute sensitivity was 0.026 µg of FLC. The method was internally validated according to current guidelines for analytical method validation. Both accuracy and precision lied in the required ±15% range. FLC levels measured by bioassay and by high-performance liquid chromatography (HPLC) performed with 62 plasma samples from humans and rats showed a strong correlation (coefficients, 0.979 and 0.995, respectively; percent deviations of bioassay from HPLC values, 0.44% ± 15.31% and 2.66% ± 7.54%, respectively). In summary, this newly developed bioassay is sensitive, simple, rapid, and inexpensive. It allows nonspecialized laboratories to determine FLC levels in plasma to within the clinically relevant concentration range and represents a useful tool for experimental treatment models.

* Corresponding author. Mailing address: Institut de Microbiologie, Centre Universitaire Hospitalier Vaudois, Rue du Bugnon 44, CH-1011 Lausanne, Switzerland. Phone: 0041 21 3144083. Fax: 0041 21 3144060. E-mail: Dominique.Sanglard{at}chuv.hospvd.ch.

Antimicrobial Agents and Chemotherapy, March 2001, p. 696-700, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.696-700.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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