Development and validation of a duplex RPA-LFS assay for simultaneous detection of Candida tropicalis and Candida parapsilosis

Candida tropicalis and Candida parapsilosis are two common non-albicans Candida species implicated in invasive candidiasis. They differ markedly in antifungal susceptibility— C. tropicalis often resists fluconazole, whereas C. parapsilosis shows reduced echinocandin susceptibility—highlighting the need for rapid, species-level identification to guide therapy. Traditional culture methods are slow, and molecular techniques usually require complex instrumentation, limiting their use in resource-limited settings. In this study, we established a duplex recombinase polymerase amplification combined with lateral flow strip (RPA-LFS) assay for the simultaneous visual detection of these two pathogens. Species-specific primers and probes were designed against the ITS2 region of C. tropicalis and the FKS2 gene of C. parapsilosis , with probes labeled at the 5′ end with FITC or DIG and reverse primers biotinylated. After optimizing reaction conditions (39 °C, 15 min), the assay achieved a limit of detection of 10 genomic equivalents/reaction for C. tropicalis and 100 genomic equivalents/reaction for C. parapsilosis . It showed 100% inclusivity across 30 target strains and 100% specificity against 14 non-target pathogens, including related Candida species and common bacteria. Clinical evaluation with 374 clinical samples (predominantly respiratory specimens) showed perfect agreement with quantitative real-time PCR, yielding 100% sensitivity (95% CI 97.8–100%), 100% specificity (95% CI 98.2–100%), and a Cohen’s kappa of 0.99 (95% CI 0.99–1.00). The entire procedure takes less than 30 min and requires no specialized equipment. This duplex RPA-LFS assay offers a rapid, accurate, diagnostic tool for the simultaneous detection of C. tropicalis and C. parapsilosis , well-suited for point-of-care testing in resource-limited environments and for supporting timely antifungal stewardship.