Methods for antifungal combination studies in vitro and in vivo in animal models

Deep-seated fungal infections are life-threatening diseases and despite treatment, mortality remains high. New antifungal drugs, already marketed or still under development, and possessing different mechanisms of action highlight the fact that antifungal combination is an interesting approach among the new therapeutic strategies for the treatment of fungal infections. Currently, there are no standardized methodologies for the study of antifungal combination in vitro or in vivo in animal models. Various techniques have been used such as agar diffusion or agar dilution methods, broth micro- or macro-dilution techniques, or time-kill methods. The most commonly used in vitro methodology in the recent literature is the broth micro-dilution checkerboard technique. This technique has been adapted from the standardized technique for susceptibility testing of antifungals alone. The aim of these studies is to determine the mode of interaction between antifungals that can be either synergistic, additive, or antagonistic. Because different methods can be used to analyze the data and to define the mode of interaction between antifungals, interpretation of the results remains difficult. The technique most commonly used to determine the mode of interaction between antifungal drugs is the calculation of fractional inhibitory concentrations, that can be graphically expressed as isobolograms. Nevertheless, a large number of other methods of calculation are available and it is noteworthy that opposite conclusions can sometimes be drawn with the same data set depending on the method used for analysis. For these reasons, studies in animal models, which are probably more clinically relevant than the in vitro studies, are of interest. In particular, pharmacokinetics and toxicological properties of the drugs are taken into account in animal models beside the interaction of the molecules at the cell level. The main drawbacks in animal studies are the cost and the duration of the experiments. Different animal models have been used to study antifungal combinations. Mortality and fungal burden of target organs are the most commonly used criteria to evaluate efficacy of the antifungal drugs. Nevertheless, clinical studies will be the only means to assess whether combinations that appear interesting in vitro or in animal models are effective in humans.