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Antimicrobial Agents and Chemotherapy, October 2000, p. 2752-2758, Vol. 44, No. 10
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Flow Cytometry Antifungal Susceptibility Testing of Pathogenic Yeasts other than Candida albicans and Comparison with the NCCLS Broth Microdilution Test

Rama Ramani1 and Vishnu Chaturvedi1,2,*

Mycology Laboratory, Wadsworth Center, New York State Department of Health,1 and Department of Biomedical Sciences, School of Public Health, State University of New York---Albany,2 Albany, New York

Received 7 January 2000/Returned for modification 14 February 2000/Accepted 12 July 2000

Candida species other than Candida albicans frequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing of Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired t test values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.

* Corresponding author. Mailing address: Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12208-2002. Phone: (518) 474-4177. Fax: (518) 486-7971. E-mail: vishnu{at}wadsworth.org.

Antimicrobial Agents and Chemotherapy, October 2000, p. 2752-2758, Vol. 44, No. 10
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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