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JAC Advance Access published online on November 16, 2004
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkh489
© 2004 by The British Society for Antimicrobial Chemotherapy
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Received April 19, 2004
Revised September 30, 2004
Accepted October 1, 2004

Brief report

Determination of antifungal drug susceptibilities of Aspergillus species by a fluorescence-based microplate assay

S. Arunmozhi Balajee 1*, Alexander Imhof 1, Jennifer L. Gribskov 1, and Kieren A. Marr 2

1 Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N. D3-100, Seattle, WA 98109, USA
2 Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N. D3-100, Seattle, WA 98109, USA; Department of Medicine, University of Washington, Seattle, WA, USA

* To whom correspondence should be addressed.
S. Arunmozhi Balajee, E-mail: abalajee{at}fhcrc.org


  Abstract

Objectives: We have investigated the use of a viability dye, chloromethylfluorescein di-acetate (CMFDA), for antifungal susceptibility testing in a fluorescence microplate (FM) assay format.

Methods: For this FM assay, conidia were incubated in increasing concentrations of antifungal drug for 16 h and stained with CMFDA. Fluorescence, measured as mean fluorescence units (MFU) in a fluorescence microplate reader, was graphed relative to that of a drug-free control, and the MIC was defined as the lowest concentration of the drug that resulted in complete reduction (100%) in MFU for amphotericin B, or 90% reduction in MFU for itraconazole and voriconazole. Susceptibilities of 10 clinical isolates of Aspergillus fumigatus, Aspergillus terreus and Aspergillus niger to amphotericin B, itraconazole and voriconazole were tested in a blinded fashion using the FM and the NCCLS methods.

Results and conclusions: Reproducibility of the FM assay was excellent, and results correlated with those of the NCCLS microdilution method. The FM assay appears to be a rapid, objective method for testing fungal susceptibilities to itraconazole, voriconazole and amphotericin B.

Keywords: MICS; susceptibility testing; fluorescent dyes; Aspergillus spp..
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