Use of high inoculum for early metabolic signalling and rapid susceptibility testing of Aspergillus species

Objectives: To develop and evaluate a new method for rapid susceptibility testing of Aspergillus spp. based on early metabolic signalling of high-inoculum biomass. Methods: Susceptibility to amphotericin B and voriconazole was studied in 39 clinical isolates of Aspergillus spp. (16 Aspergillus fumigatus, 11 Aspergillus flavus, 12 Aspergillus terreus. At 6 or 8 h after inoculation for A. fumigatus and A. flavus, and at 8 or 12 h after inoculation for A. terreus, 100 μg/mL of the tetrazolium salt XTT and 25 μM menadione were added and absorbance measured at 450 nm after 2 h of incubation at 37°C. Inocula used were 10⁶ conidia/mL for A. fumigatus and A. terreus and 10⁵ conidia/mL for A. flavus, as lower inocula exhibited very low metabolic activity at these time points. Data were analysed with the sigmoid E_max model and compared with visual (lowest drug concentration showing no growth) and spectrophotometric MIC determination at 48 h (CLSI M38-A method). Results: The E _max model described well the concentration-effect relationship for early metabolic activity and 48 h fungal biomass (median r²: 0.97 and 0.93, respectively). Use of the model allowed characterization and quantification of species- and drug-related differences in pharmacological inhibition of early metabolic activity as well as calculation of appropriate cutoff levels for MIC determination with the XTT assay. Using these cutoff levels, for A. fumigatus and A. flavus at both time points (6 and 8 h) and for A. terreus at 12 h, the agreement (± one dilution) of the XTT assay with the CLSI method was 91-100% and its reproducibility was 97-100%. Conclusions: This newly developed high-inoculum-based method provides rapid and reproducible MIC determinations for Aspergillus spp.