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.

Objective:

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).

Methods:

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.

Results:

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.

Conclusions:

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.

References

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: 609–618.