Susceptibility of Aspergillus fumigatus to voriconazole by flow cytometry
Abstract number: 10.1111/j.1198-743X.2004.902_o401.x
The reference method for susceptibility testing of moulds NCCLS M38-A is cumbersome and time-consuming. Flow cytometry (FC) has proved to be a valuable alternative for susceptibility testing of Candida spp (1) and it would be of interest to evaluate its role in susceptibility testing of moulds.
To study the antifungal activity of Vor on clinical isolates of A. fumigatus, using FC with the probes FUN-1 (a marker of metabolic integrity) and propidium iodide (PI) (a marker of cell death by cell membrane lesion).
Two clinical strains of A. fumigatus were employed. MIC values were determined according the NCCLS M38-A protocol. Conidial suspensions (2 × 106 conidia/mL) were incubated at 37°C with different concentrations of Vor (MIC, 2 × MIC, 4 × MIC, MIC/2, MIC/4), for 8 h. Conidia were washed (to prevent interference between antifungal agents and the probe), suspended in PBS with 2% glucose and stained with 5 mg/mL of FUN-1 or 1 mg/mL of PI, for 30 min. Heat-treated conidia (90°C, for 30 min) were used as the control for cell death. The conidial cells were analysed by FC (Beckman-Coulter Corp., Hialeah, FL, USA): the morphology (scattergram) and the intensity of fluorescence of the stained cells (FL2 (green) for FUN-1and FL3 (red) for PI) were determined.
It was possible to establish a MIC for Vor after 4 h by FC. At this time, virtually all conidia were metabolically impaired, as shown by FUN-1 staining. Similar cytometric results were seen with 2 × MIC and 4 × MIC. Dead conidia were detected with PI staining after 5 h, at MIC concentration.
FC can be used to determine the susceptibility of A. fumigatus to Vor. FC is a rapid and reliable alternative to the standard NCCLS M-38A method. Moreover, FC provides key information regarding the mechanism of antifungal activity of Vor and allows the analysis of a large amount of conidia.
1.Pina-Vaz, C, Sansonetty, F & Rodrigues, AG et al. (2001) Cytometric approach for rapid evaluation of susceptibility of Candida strains to antifungal. Clinical Microbiology and Infection 11: 609618."
|Session name:||XXIst ISTH Congress|
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