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 g/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 g of the tetrazolium salt XTT/ml and 6.25 M menadione. A significant reduction in metabolic activity was demonstrated at the MEC (0.25 to 0.5 g/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.Echinocandins are lipopeptide antifungal compounds that inhibit the (1,3)-␤-D-glucan synthase activity of Candida spp. and Aspergillus spp. Caspofungin has shown efficacy in the treatment of invasive candidiasis and aspergillosis in vivo and in clinical trials and is currently the most widely used echinocandin (1,11,13,16,21).Assessment of in vitro activity of caspofungin against Aspergillus spp. is complicated by the fact that the MIC often exceeds safely achievable plasma concentrations, which range approximately between 1 and 11 g/ml in adults receiving 50 mg/day and between 1 and 20 g/ml in children receiving 50 mg/m 2 /day (11,12,25,27). The minimum effective concentration (MEC), defined as the lowest drug concentration at which short, stubby, and highly branched hyphae are observed on microscopic examination has been shown to generate more consistent susceptibility results than the MIC and is currently the suggested endpoint for determining the in vitro activity of caspofungin against Aspergillus spp. (3, 11, 12, 20).The MEC endpoint, however, still represents a subjective assessment of the appearance of growth and cannot be automated. Its determination requires laborious microscopic examination of the hyphal structures or sufficient...

Concentration-Dependent Synergy and Antagonism within a Triple Antifungal Drug Combination againstAspergillusSpecies: Analysis by a New Response Surface Model

“…This hypothesis is consistent with the observation that deletion of ERG genes increased caspofungin susceptibility (18). The antagonistic effects of the double combinations voriconazole-caspofungin and amphotericin B-caspofungin at high caspofungin concentrations may be associated with the paradoxical increase of fungal growth observed recently with an in vitro colorimetric assay of fungal metabolic activity at high caspofungin concentrations similar to those found in the present study (1). Thus, the antagonistic interactions within the triple combination observed at high amphotericin B and voriconazole concentrations are due to the antagonism between amphotericin B and voriconazole, while the synergistic interactions within the triple combination observed at lower amphotericin B and voriconazole concentrations are due to the synergistic interactions of voriconazolecaspofungin and amphotericin B-caspofungin combinations.…”

“…However, combinations with higher doses of caspofungin (2.5 mg/kg) may result in higher CFU per gram than combinations with lower caspofungin doses (1 mg/kg) (14), consistent with the concentration-dependent interactions that we found in the present study. This apparent increase in fungal burden in vivo may be caused by dosage-dependent hyphal fragmentation (1,32,41).…”

“…The complexity of these interactions was well described by an E max -based mixture-amount response surface model using full cubic canonical mixture polynomials (40). The sigmoid E max model has been used extensively to successfully describe the concentration-effect relationships of amphotericin B, caspofungin, and azoles (1,22,26,32,41). In addition, the double combination of polyene-azole was recently analyzed by isobolographic analysis, and the pattern of interactions was dependent on the proportions of antifungal drugs in the mixtures (26), which could be modeled with polynomial regression analysis.…”

Comparative Evaluation of Sensititre YeastOne and CLSI M38-A2 Reference Method for Antifungal Susceptibility Testing of Aspergillus spp. against Echinocandins

“…Using a different colorimetric marker (XTT), Antachopoulos et al also reported a proportionate species-dependent reduction in metabolic activity at the MEC for all Aspergillus isolates tested, which was more pronounced for A. flavus (median metabolic activity 25% of control) compared to A. fumigatus and A. terreus (median metabolisms of 42 and 53%, respectively) (17). Thus, the current alamarBlue concentration may be sufficient for the slower-growing A. fumigatus and A. terreus but not for the fast-growing A. flavus.…”

Role of Trehalose Biosynthesis in Aspergillus fumigatus Development, Stress Response, and Virulence

“…For most experiments, strains were grown on YEPD agar (1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar) at 37°C. Some experiments required the use of Aspergillus minimal medium containing trace elements (Na 2 4 ), 1% glucose, and NaNO 3 adjusted to pH 6.5. Media used in specific experiments included Aspergillus minimal medium containing 0.5% glucose (Aspergillus minimal glucose medium), Aspergillus minimal glucose medium supplemented with 0.5% trehalose, and Aspergillus minimal glucose medium supplemented with 1 M sorbitol.…”