JAC Advance Access originally published online on December 21, 2006
Journal of Antimicrobial Chemotherapy 2007 59(2):230-237; doi:10.1093/jac/dkl488
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Use of high inoculum for early metabolic signalling and rapid susceptibility testing of Aspergillus species
1 Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA 2 Third Department of Pediatrics, Aristotle University, Hippokration Hospital, Thessaloniki, Greece
Received 14 July 2006; returned 29 September 2006; revised 3 November 2006; accepted 6 November 2006
* Corresponding author. Tel: +1-301-402-0023; Fax: +1-301-480-2308; E-mail: walsht{at}mail.nih.gov
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 Emax model and compared with visual (lowest drug concentration showing no growth) and spectrophotometric MIC determination at 48 h (CLSI M38-A method).
RESULTS: The Emax 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.
Keywords: XTT , metabolic activity , Aspergillus spp
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