Rapid flow cytometric susceptibility testing of Candida albicans

Abstract

A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed by adapting the proposed reference method for broth macrodilution testing of yeasts. Membrane permeability changes caused by the antifungal agent were measured by flow cytometry using propidium iodide, a nucleic acid-binding fluorochrome largely excluded by the intact cell membrane. We determined the in vitro susceptibility of 31 Candida albicans isolates and two quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) to amphotericin B and fluconazole. Amphotericin B MICs ranged from 0.03 to 2.0 g/ml, while fluconazole MICs ranged from 0.125 to 128 g/ml. This method results in clear-cut endpoints that were reproducible. Four-hour incubation was required for fluconazole, whereas a 2-h incubation was sufficient for amphotericin B to provide MICs comparable to the reference macrodilution method developed by the National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Tests. Results of these studies show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of C. albicans.

In Vitro and In Vivo Activity of a Novel Antifungal Small Molecule against Candida Infections

“…Propidium iodide, a membrane-impermeable fluorescent dye that binds to nucleic acids, is widely used to differentiate cells that have damaged plasma membranes from healthy ones [42,43]. To evaluate the effect of SM21 on the fungal plasma membrane, C. albicans SC5314 cells (approximately 1610 3 CFUs/ml) were obtained from logarithmic phase cultures and suspended in RPMI 1640 medium, as described above.…”

Antimicrobial effects of α-MSH peptides

“…In related experiments, to examine the role of cAMP in inhibition of colony formation, we used dideoxyadenosine (ddAdo, Sigma) a potent inhibitor of adenylyl cyclase [23]. C. albicans (1 ϫ 10 6 cells) were exposed to different concentrations of ddAdo (25,50, and 100 ϫ 10 -5 M) for 2 h and to ␣-MSH 10 -5 M or HBSS for another 2 h. At the end of the incubation period, cells were washed in saline solution and diluted with HBSS to a concentration of approximately 100 organisms/mL.…”

In Vitro Antifungal Susceptibility Testing

“…It was evaluated with Candida sp and C. neoformans. [54][55][56][57] The method measures permeability of the fungal cell membrane after contact with various concentrations of antifungal agent. A membrane-impermeable fluorescent DNA-intercalating dye is placed in the standardized yeast suspension 2-6 hours after exposure to the drug.…”