Susceptibility testing of pathogenic fungi with itraconazole: a process analysis of test variables

A 2(10-5) fractional factorial model was used to investigate the influence of 10 process variables in broth microdilution susceptibility tests with it raconazole against eight isolates of Candida species and six isolates of fi lamentous fungi in two growth media. An analysis of variance (ANOVA) indica ted that glucose concentration and incubation time both significantly influ enced control turbidity optical density (OD) values for most of the Candida spp. isolates, while incubation in > 10% CO2 versus ambient air, incubatio n temperature and inoculum size significantly influenced these OD values fo r about half of the yeast isolates. Control OD values for the mould isolate s were most influenced by incubation time and temperature, and by occlusion of the wells with an adhesive sticker. Three statistical approaches, ANOVA , rank transformation and Mann-Whitney U-test, were used to assess the infl uence of the variable combinations on MIC, determined with a 50% growth red uction end-point. Incubation temperature and time, glucose concentration an d inoculum size were the variables that most often affected susceptibility results to the level of statistical significance; however, the supplier of RPMI 1640 medium, the use of adhesive stickers and the atmosphere of incuba tion significantly influenced the MIC for some isolates. The medium used to prepare the test inoculum, the solvent used to prepare the stock solution and the shape of the microdilution plate wells significantly affected outco me, but only sporadically. A principal component analysis of the data matri x confirmed this order of relative influence of the test variables on the M IC. Since each fungal isolate responded differently to combinations of proc ess variables in the test, we conclude that any unified method for antifung al susceptibility determination represents a compromise, rather than an ide alized system.