Concentration-Dependent Effects of Caspofungin on the Metabolic Activity of Aspergillus Species

Abstract

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 μg/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 μg of the tetrazolium salt XTT/ml and 6.25 μM menadione. A significant reduction in metabolic activity was demonstrated at the MEC (0.25 to 0.5 μg/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.