Rapid Antifungal Susceptibility Testing Based on Single-Cell Metabolism Analysis Using Stimulated Raman Scattering Imaging

Rapid antifungal susceptibility testing (AFST) is urgently needed in clinics to treat invasive fungal infections with the appropriate antifungal drugs and to slow the emergence of antifungal resistance. However, current AFST methods are time-consuming (24-48 h) due to the slow growth of fungal cells and the methods not being able to work directly for clinical samples. Here, we demonstrate rapid AFST by measuring the metabolism in single fungal cells using stimulated Raman scattering imaging and deuterium probing. Distinct metabolic responses were observed in Candida albicans to different classes of antifungal drugs: while the metabolism was inhibited by amphotericin B, it was stimulated by azoles (fluconazole and voriconazole) and micafungin. Accordingly, we propose metabolism change as a biomarker for rapid AFST. The results were obtained in 4 h with 100% categorical agreement with the gold standard broth microdilution test. In addition, a protocol was developed for direct AFST from positive blood cultures. This method overcomes the limitation of slow growth in conventional methods and has the potential for the rapid diagnosis of candidemia and other clinical fungal infections.

Automated summary

This study demonstrates rapid antifungal susceptibility testing (AFST) by measuring the metabolism in single fungal cells. The results show that metabolism change can serve as a biomarker for rapid AFST, with a 100% categorical agreement with the gold standard broth microdilution test. Additionally, a protocol is developed for direct AFST from positive blood cultures, overcoming the limitation of slow growth in conventional methods and providing potential for rapid diagnosis of fungal infections.