Concentration-dependent effects of caspofungin on the metabolic activity of Aspergillus species

Abstract

The minimum effective concentration (MEC) used to assess the in vitro antifungal activity of caspofungin against Aspergillus spp. is a qualitative endpoint requiring microscopic examination of hyphae. We therefore developed a tool for the quantitative assessment of caspofungin activity against Aspergillus spp. at clinically applicable concentrations. Susceptibility to caspofungin (0.008 to 8 microg/ml) was studied for 9 A. fumigatus, 8 A. flavus, and 12 A. terreus isolates based on the Clinical and Laboratory Standards Institute M38-A protocol. After 48 h of incubation, the MEC was defined microscopically, and metabolic activity assessed with a modified XTT assay, using 100 microg of the tetrazolium salt XTT/ml and 6.25 muM menadione. A significant reduction in metabolic activity was demonstrated at the MEC (0.25 to 0.5 microg/ml) for all Aspergillus spp. and was more pronounced for A. flavus (median metabolic activity, 25% of control) compared to A. fumigatus and A. terreus (median metabolism, 42 and 53%, respectively), allowing determination of MEC with the XTT assay (93 to 100% agreement with microscopic MEC). Fungal metabolism tended to reach the lowest levels (median, 17 to 38% of control) one to two dilutions higher than the MEC, at the minimum metabolic activity concentration (MMC). For 5 of 9 A. fumigatus isolates, 6 of 12 A. terreus isolates, and 1 of 8 A. flavus isolates, a paradoxical increase in metabolism was observed at concentrations greater than the MMC. Sigmoid (E(max)) or bell-shaped models described accurately (median R(2) = 0.97) the concentration-dependent metabolic changes in the absence or presence, respectively, of paradoxical response. Assessment of metabolic activity may provide useful quantitative endpoints for in vitro studies of caspofungin against Aspergillus spp.

FIG. 1.

Concentration-dependent changes in metabolic activity of Aspergillus spp. in the presence of increasing concentrations of caspofungin, as assessed by the XTT method using different concentrations of menadione (MEN). With 6.25 μM menadione, a significant reduction in fungal metabolism was detected at caspofungin concentrations greater than or equal to the MEC. Measurements from a single replicate per isolate are presented.

FIG. 2.

Concentration-effect curves of metabolic activity in the presence of increasing concentrations of caspofungin, generated with the sigmoid E_max model for A. flavus (A) and the bell-shaped model for A. fumigatus (B). The parameters calculated by the two models (with the exception of the slope factors) are presented in the graph. The proximity of the EC50 (or EC50-1) and microscopic MEC values (<1 dilution) can be seen. The data represent the mean of three replicates.

FIG. 3.

Comparison of the bell-shaped (solid line) and Gaussian (dashed line) models for description of the concentration-dependent effects of caspofungin on the metabolic activity of an A. terreus isolate demonstrating the paradoxical effect. The asymmetric nature of the biphasic response in the case of caspofungin is not adequately described by the symmetric Gaussian curve.