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


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.
Balajee
S Arunmozhi
SA
Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N. D3-100, Seattle, WA 98109, USA. abalajee@fhcrc.org
Imhof
Alexander
A
Gribskov
Jennifer L
JL
Marr
Kieren A
KA
D000666:Amphotericin B / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology*; D001230:Aspergillus / Q000145:classification / Q000187:drug effects*; D025141:Drug Resistance, Fungal; D005452:Fluoresceins / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D017964:Itraconazole / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000295:instrumentation / Q000379:methods; D011743:Pyrimidines / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology; D065819:Voriconazole
D000935:Antifungal Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011743:Pyrimidines; D014230:Triazoles; C069306:5-chloromethylfluorescein; D017964:Itraconazole; D000666:Amphotericin B; D065819:Voriconazole