Sensitive and rapid detection of Candida tropicalis using closed dumbbell-mediated isothermal amplification

Candida tropicalis is responsible for a substantial proportion of invasive candidiasis cases and is recognized as the second most prevalent causative agent, only behind Candida albicans. A documented rise in invasive systemic infections and sepsis due to C. tropicalis has contributed to the increased mortality among vulnerable patient populations, highlighting the critical need for heightened clinical vigilance. Early detection of C. tropicalis is essential for guiding appropriate antifungal therapy and is associated with improved clinical outcomes.

To address the need for rapid and reliable detection of C. tropicalis, this research aimed to develop a novel closed dumbbell-mediated isothermal amplification (CDA) assay characterized by high speed, specificity, and efficiency. A set of specific CDA primers targeting the conserved ITS2 gene of C. tropicalis was successfully designed, selected, and validated, resulting in a simple and efficient detection method. The C. tropicalis CDA assay for in vitro diagnostic applications demonstrated robust performance, with its sensitivity, robustness, stability, and specificity systematically evaluated using 364 batches of diverse DNA samples.

The C. tropicalis CDA assay demonstrated perfect diagnostic performance with apparent 100% sensitivity (95%CI = 97.0%-100%), specificity (95%CI = 98.5%-100%), and accuracy (95%CI = 99.0%-100%) in a primary evaluation using 364 batches of diverse DNA samples, with a limit of detection of 5.6 × 10^-6 ng/μl (approximately 10 copies/μl), which is not significantly different from that of real-time quantitative PCR (qPCR), indicating comparable analytical sensitivity.

Validated in both real-time fluorescence and endpoint visual formats, the C. tropicalis CDA assay demonstrated high specificity, sensitivity, and accuracy, along with robust stability and reproducibility, supporting its future application potential.