Deuterium Oxide Labeling and Single-Cell Stimulated Raman Scattering Imaging for Rapid Antifungal Response Assessment in Seven Yeast Species from China

Background: Fungal infections cause over 1.5 million deaths worldwide annually, and timely antifungal susceptibility testing (AST) is important for guiding treatment of invasive fungal infections. Methods: We evaluated deuterium oxide (D2O) labeling combined with single-cell stimulated Raman scattering (SRS) imaging in seven clinical yeast isolates of non-albicans Candida and non-Candida yeasts from the China Hospital Invasive Fungal Surveillance Net, one each of Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida haemulonii, Cryptococcus neoformans, Saccharomyces cerevisiae, and Wickerhamomyces anomalus. Twelve isolate–drug combinations were examined using fluconazole, voriconazole, micafungin, or amphotericin B. An exploratory Raman-based MIC was defined as the lowest tested concentration showing a statistically significant change in intracellular C–D signal relative to the drug-free control. Test concentrations were selected around MICs obtained using Sensititre YeastOne (SYO). Results: Eleven of 12 Raman-based MICs were within one twofold dilution of the corresponding SYO MIC (91.7%). The C. guilliermondii–micafungin Raman-based MIC differed by two dilutions. This numerical agreement is distinct from categorical agreement. Azole exposure increased C–D signals, whereas micafungin and amphotericin B decreased them in the isolates tested. The complete workflow includes overnight culture followed by a 6–8 h analytical interval; prior isolation requires additional time. Conclusions: These observations support further evaluation of an exploratory metabolic-response assay, but do not establish equivalence to growth-based MICs or clinical validity. Independent-replicate analysis and blinded testing of larger isolate collections are required.