Conidial viability assay for rapid susceptibility testing of Aspergillus species

Abstract

Antifungal susceptibility testing of filamentous fungi has become more important given the recognition of drug-resistant organisms and the availability of therapies other than amphotericin B (AMB). As current microdilution and E-test methods are limited by a 2 to 3 day incubation time required to obtain results, a more rapid method for susceptibility testing of fungi is needed. We report here a flow cytometric assay that relies on conidial metabolism of the viability dye FUN-1. Conidia are incubated in media containing increasing concentrations of AMB for 3 h, exposed to FUN-1, and then analyzed by flow cytometry. Relative susceptibility to AMB can be measured both by forward and side scatter characteristics of the conidial population and by mean fluorescence intensity (MFI) of the dye. MIC, calculated as the concentration of AMB to yield 90% reduction in MFI relative to growth controls, was determined for 27 clinical isolates Aspergillus species and correlated well with the standard (i.e., NCCLS) method. The results of these studies illustrate a method by which AMB susceptibility can be rapidly and reproducibly determined by measuring conidial viability.

FIG. 1.

(a) Light scatter characteristics of conidia. FSc and SSc characteristics of susceptible conidia (NCCLS; MIC, 0.5 μg/ml) incubated with 0.5 μg of AMB (right panel)/ml show an overall decrease in size compared to the growth control (left panel). (b) Conidia of the resistant isolate (NCCLS; MIC, >2 μg/ml) continue to grow when incubated with 2 μg/ml (right panel), showing no shift in FSc and SSc properties.

FIG. 2.

Conidial FUN-1 fluorescence. (a) FUN-1 orange fluorescence of live (open histogram) and ethanol-killed (solid histogram) A. fumigatus controls are shown. The gray shaded histogram (leftmost peak) represents conidia in the absence of FUN-1. (b and c) FUN-1 orange fluorescence in the absence (open histogram) and presence of 2 μg/ml AMB (bold histograms) is shown for the susceptible and resistant isolates.

FIG. 3.

Serial histogram profiles. The FUN-1 orange fluorescence results of conidia treated with increasing concentrations of AMB are shown for susceptible and resistant Aspergillus isolates. The percentage of cells within the FUN-1-negative gate is indicated.

FIG. 4.

Growth curves obtained by multiple methods. Growth curves for the AMB-susceptible (black squares) and AMB-resistant (gray diamonds) isolates relative to the growth control, as measured by MFI (a), OD (b), and viability (c), were determined.