Determination of Minimum Effective Concentration (MEC) and Demonstration of Paradoxical Effect of Caspofungin against Aspergillus spp with XTT Assay.

C. ANTACHOPOULOS, J. MELETIADIS, T. SEIN, E. ROILIDES, T. J. WALSH;

Author address: 

Natl. Cancer Inst., Bethesda, MD.

Abstract: 

Background: Determination of MEC for assessment of in vitro activity of caspofungin (CAS) against Aspergillus spp is complicated by the need for microscopic examination of hyphal structures. We therefore developed an XTT assay of metabolic activity for MEC determination. Methods: Susceptibility to CAS (0.008-8 μg/ml) was studied for 29 clinical isolates of Aspergillus spp (9 A. fumigatus, 8 A. flavus, 12 A. terreus) using the broth microdilution method based on CLSI M38-A protocol. After 48h of incubation, the MEC was defined microscopically as the lowest CAS concentration that produced short, stubby and highly branched hyphae. Subsequently 100 μg/ml XTT and 6.25 μM menadione were added and absorbance was measured at dual wave length (450/630 nm, in order to subtract the absorbance of hyphae) after further incubation at 37 oC for 2 h. Results: A significant reduction in metabolic activity was associated with the formation of aberrant hyphae. The microscopically defined MEC (0.5-1 μg/ml for all isolates) corresponded to the lowest CAS concentration showing metabolic activity 8804; 60% compared to drug-free control for A. fumigatus, 40% for A. flavus and 65% for A. terreus. By using these cutoff levels the relative agreement (± 1 dilution) between the XTT and microscopic determination of MEC was 100% for A. fumigatus and A. flavus and 93% for A. terreus. For 5/9 of A. fumigatus and 6/12 of A. terreus isolates, a gradual increase in metabolic activity was observed at CAS concentrations higher than the MEC, suggesting a paradoxical effect. For A. fumigatus the average metabolic activity at 8 μg/ml of CAS was increased by 1.82 compared to that of MEC, while for A. terreus the corresponding increase was 1.47 (p
2006

abstract No: 

M-365

Full conference title: 

46th Interscience Conference on Antimicrobial Agents and Chemotherapy
    • ICAAC 46th