Abstract
The rise in antifungal resistance has limited treatment options for serious fungal infections, emphasizing the need for effective combination therapies. However, low-cost and rapid systems to evaluate synergy and antagonism in antifungal combinations are lacking. Here, we introduce a novel in vitro testing method for assessing antifungal interactions in C. albicans, enabling the simultaneous testing of three antifungal agents in a single agar plate with overnight results. This method, validated against the checkerboard assay, provides consistent fractional inhibitory concentration (FICi) measurements with reduced variability and workload. We applied this method in a comprehensive screen of 92 clinical C. albicans isolates for three antifungals-amphotericin B, fluconazole, and anidulafungin-yielding assessments of a total of 276 distinct combinations of antifungals and isolates. Results revealed isolate-specific interaction patterns, with amphotericin B and fluconazole showing synergy in 1% of isolates, anidulafungin and fluconazole in 19.5%, and amphotericin B and anidulafungin in 23.9%. These findings underscore the need for isolate-specific testing in clinical settings. This proposed assay aims to present a solution to that as a scalable high throughput approach to this clinical problem.