Antifungal Susceptibility Test via Single-Cell Morphology, Development, and Metabolism

Antifungal susceptibility test (AFST) is essential for guiding effective therapy, but it remains challenged by long turnaround times and the limited generalizability of single-parameter phenotypic readouts across diverse fungal species and antifungal classes. Here, we present a multifeature AFST (MAFST) platform that integrates single-cell Raman spectroscopy and brightfield imaging to enable rapid and pleiotropic assessment of antifungal responses. By unifying complementary metabolic and morphological features into a composite inhibition index (CII), MAFST captures coordinated cellular responses to antifungal exposure that cannot be reliably resolved by individual metrics alone. Benchmarking against the broth microdilution demonstrates that CII-based MAFST achieves high concordance across multiple <i>Candida</i> species and antifungal classes while substantially reducing the time required for susceptibility determination. Thus, by capturing single-cell morphological, developmental, and metabolic drug responses in a fast, accurate, and widely applicable fashion, MAFST can contribute to tackling fungal infections.