Comparative in vitro pharmacodynamics of caspofungin, micafungin, and anidulafungin against germinated and nongerminated Aspergillus conidia

Abstract

The concentration-dependent effects of echinocandins on the metabolic activity of Aspergillus spp. were comparatively studied by using nongerminated and germinated conidia. The susceptibilities of 11 Aspergillus fumigatus, 8 A. terreus and 8 A. flavus isolates to caspofungin, micafungin, and anidulafungin were studied by a CLSI (formerly NCCLS) M38-A broth microdilution-based method. After 48 h of incubation the minimum effective concentration (MEC) was defined microscopically. Metabolic activity was assessed by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay and modeled by using the sigmoid (E max) or "bell-shaped" model. The median MEC values of caspofungin (0.5 to 1 microg/ml), micafungin (0.06 to 0.12 microg/ml), and anidulafungin (0.03 microg/ml) against nongerminated conidia increased by 0 to 1, 1 to 2, and 2 to 3 twofold dilutions, respectively (depending on the species), over those against germinated conidia. A similar shift to the right was demonstrated for the corresponding curves of metabolic activity. There was a significant correlation between the degrees of maximal metabolic inhibition caused by different echinocandins at both the species level (greater inhibition for A. flavus) and the strain level (r = 0.84 to 0.93; P < 0.0001). Paradoxical increases in metabolism in the presence of higher concentrations of caspofungin, micafungin, and anidulafungin were detected in 6, 2, and 5 of the A. fumigatus isolates, respectively; 5, 1, and 2 of the A. terreus isolates, respectively; and 1, 0, and 0 of the A. flavus isolates, respectively. Based on the model, 50% of the maximal paradoxical increase was detected with 4.2, 11.1, and 10.8 microg/ml of caspofungin, micafungin, and anidulafungin, respectively. All echinocandins therefore exerted comparable levels of maximal metabolic inhibition against Aspergillus spp. at concentrations that were differentially increased for germinated versus nongerminated conidia. The paradoxical increase in metabolism occurred more frequently and at lower concentrations with caspofungin than with micafungin and anidulafungin.

FIG. 1.

Representative concentration-effect curves of the metabolic activity of an A. fumigatus isolate (nongerminated [▵] and germinated [▿] conidia) in the presence of increasing concentrations of caspofungin, micafungin, and anidulafungin. For caspofungin and anidulafungin, the curves were generated with the bell-shaped model, and for micafungin, the curves were generated with the E_max model. The parameters calculated by the models, as well as the microscopically defined MECs, are presented.

FIG. 2.

Comparative concentration-effect curves for two A. terreus isolates (nongerminated conidia of isolates A [▵] and B [▿]) in the presence of echinocandins. There is a marked correlation in the degree of metabolic inhibition caused by these agents; the Y_min values obtained for isolate A are consistently and significantly higher than those obtained for isolate B for all three echinocandins.

FIG. 3.

Correlation of Y_min values obtained for all Aspergillus isolates (nongerminated conidia) in the presence of caspofungin, micafungin, and anidulafungin. r, correlation coefficient (P < 0.0001).

FIG. 4.

Photomicrographs of wells after 48 h of incubation of an A. fumigatus isolate (nongerminated conidia) demonstrating a paradoxical increase in metabolic activity at higher concentrations of anidulafungin. Anidulafungin was used at concentrations of 0.03 μg/ml (MEC) (A), 2 μg/ml (B), and 16 μg/ml (C). The short, stubby hyphae observed at the MEC (A) tend to become progressively more elongated at the higher concentrations (B and C), without, however, restoration of the normal hyphal mat that would be observed in the absence of drug. Magnifications, ×10.