title: Use of high inoculum for early metabolic signalling and rapid susceptibility testing of Aspergillus species.
abstract: OBJECTIVE
  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 microg/mL of the tetrazolium salt XTT and 25 microM menadione were added and absorbance measured at 450 nm after 2 h of incubation at 37 degrees C. Inocula used were 10(6) conidia/mL for A. fumigatus and A. terreus and 10(5) 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 r(2): 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.
authors.affiliation: Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Charalampos
authors.initials: C
authors.lastname: Antachopoulos
authors.forename: Joseph
authors.initials: J
authors.lastname: Meletiadis
authors.forename: Tin
authors.initials: T
authors.lastname: Sein
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Roilides
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Walsh
mesh_terms: D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D001230:Aspergillus / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D018533:Biomass; D003124:Colorimetry; D008826:Microbial Sensitivity Tests / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013778:Tetrazolium Salts / Q000378:metabolism; D013997:Time Factors; D024483:Vitamin K 3 / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; D024483:Vitamin K 3