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 itraconazole against eight isolates of Candida species and six isolates of filamentous fungi in two growth media. An analysis of variance (ANOVA) indicated that glucose concentration and incubation time both significantly influenced control turbidity optical density (OD) values for most of the Candida spp. isolates, while incubation in >10% CO(2) versus ambient air, incubation temperature and inoculum size significantly influenced these OD values for about half of the yeast isolates. Control OD values for the mould isolates 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 influence of the variable combinations on MIC, determined with a 50% growth reduction end-point. Incubation temperature and time, glucose concentration and 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 incubation 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 outcome, but only sporadically. A principal component analysis of the data matrix confirmed this order of relative influence of the test variables on the MIC. Since each fungal isolate responded differently to combinations of process variables in the test, we conclude that any unified method for antifungal susceptibility determination represents a compromise, rather than an idealized system.