High-Throughput Monitoring of Pathogenic Fungal Growth Using Whole Slide Imaging for Rapid Antifungal Susceptibility Assessment.

Invasive fungal infections are a major health threat with high morbidity and mortality, highlighting the urgent need for rapid diagnostic tools to detect antifungal resistance. Traditional culture-based antifungal susceptibility testing (AFST) methods often fall short due to their lengthy process. In our previous research, we developed a whole-slide imaging (WSI) technique for the high-throughput assessment of bacterial antibiotic resistance. Building on this foundation, this study expands the application of WSI by adapting it for rapid AFST through high-throughput monitoring of the growth of hundreds of individual fungi. Due to the distinct "budding" growth patterns of fungi, we developed a unique approach that utilizes specific cell number change to determine fungi replication, instead of cell area change used for bacteria in our previous study, to accurately determine the growth rates of individual fungal cells. This method not only accelerates the determination of antifungal resistance by directly observing individual fungal cell growth, but also yields accurate results. Employing Candida albicans as a representative model organism, reliable minimum inhibitory concentration (MIC) of fluconazole inhibiting 100% cells of Candida albicans (denoted as MIC100) was obtained within 3h using the developed method, while the modified broth dilution method required 72h for the similar reliable result. In addition, our approach was effectively utilized to test blood culture samples directly, eliminating the need to separate the fungi from whole blood samples spiked with Candida albicans. These features indicate the developed method holds great potential serving as a general tool in rapid antifungal susceptibility testing and MIC determination.