Assessing in vitro combinations of antifungal drugs against yeasts and filamentous fungi: comparison of different drug interaction models.

Contains fulltext : 48405.pdf (publisher's version ) (Closed access)Non-parametric and parametric approaches of two competing zero-interaction theories--the Loewe additivity and the Bliss independence - were evaluated for analyzing the in vitro interactions of various antifungal drugs. Fifty-one data sets, derived from three drug combinations, tested in triplicate against 17 clinical yeast and mold isolates with a two-dimensional checkerboard microdilution technique, were selected to span from strong synergy to strong antagonism. These were analyzed with the standard FIC index model and modern concentration-effect response surface models: the fully parametric model developed by Greco et al. and the 3-D analysis developed by Prichard et al. The FIC index model is subjective, sensitive to experimental errors and resulted in approximated results and variable conclusions depending on the MIC endpoints determined and interpretation endpoints used. By using the MIC-2 endpoint (lowest drug concentration showing 50% of growth) for calculating the FIC indices, problems due to trailing phenomena were reduced and weak interactions could be detected; higher levels of reproducibility and agreement with the other models were achieved using the MIC-0 and MIC-1 (lowest drug concentration showing 10 and 25% of growth, respectively). High reproducibility was achieved in interpreting the FIC indices when the cutoffs of 0.25 and 4 (for single experiments) and the cutoff of 1 (for replicates) were used for defining the limits of additivity/indifference. Although the fully parametric Greco model did not describe precisely the entire response surface of all antifungal drug interactions, it was able to differentiate synergistic from non-synergistic interactions with a non-unit, reproducible, concentration-independent interaction parameter, including its uncertainty, without requiring replication. The Bliss independence based models resulted in mosaics of synergistic and antagonistic combinations, raising questions about the concentration-dependent nature of antifungal drug interaction. The sum of all statistically significant interactions were used as a summary interaction parameter for the entire response surface, concluding synergy or antagonism when it was positive or negative, respectively. The cutoffs of 100% and 200% were used to distinguish weak and moderate interactions, respectively in 12-16 x 8-12 checkerboard formats. Semi-parametric approaches need particular care as experimental errors are not eliminated from the entire response surface

Current Status Of Antifungal Susceptibility Testing Methods

Antifungal susceptibility testing is a very dynamic field of medical mycology. Standardization of in vitro susceptibility tests by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST), and current availability of reference methods constituted the major remarkable steps in the field. Based on the established minimum inhibitory concentration (MIC) breakpoints, it is now possible to determine the susceptibilities of Candida strains to fluconazole, itraconazole, voriconazole, and flucytosine. Moreover, utility of fluconazole antifungal susceptibility tests as an adjunct in optimizing treatment of candidiasis has now been validated. While the MIC breakpoints and clinical significance of susceptibility testing for the remaining fungi and antifungal drugs remain yet unclear, modifications of the available methods as well as other methodologies are being intensively studied to overcome the present drawbacks and limitations. Among the other methods under investigation are Etest, colorimetric microdilution, agar dilution, determination of fungicidal activity, flow cytometry, and ergosterol quantitation. Etest offers the advantage of practical application and favorable agreement rates with the reference methods that are frequently above acceptable limits. However, MIC breakpoints for Etest remain to be evaluated and established. Development of commercially available, standardized colorimetric panels that are based on CLSI method parameters has added more to the antifungal susceptibility testing armamentarium. Flow cytometry, on the other hand, appears to offer rapid susceptibility testing but requires specified equipment and further evaluation for reproducibility and standardization. Ergosterol quantitation is another novel approach, which appears potentially beneficial particularly in discrimination of azole-resistant isolates from heavy trailers. The method is yet investigational and requires to be further studied. Developments in methodology and applications of antifungal susceptibility testing will hopefully provide enhanced utility in clinical guidance of antifungal therapy. However, and particularly in immunosuppressed host, in vitro susceptibility is and will remain only one of several factors that influence clinical outcome.Wo