Rapid and simultaneous detection of Candida albicans and Cryptococcus neoformans using a duplex real-time RPA assay: a potential point-of-care tool for invasive fungal infections

Introduction Invasive fungal infections (IFIs) caused by Candida albicans and Cryptococcus neoformans are associated with high mortality, necessitating rapid and accurate diagnostic tools for timely antifungal therapy. Methods We developed and optimized a duplex real-time recombinase polymerase amplification (RPA) assay targeting the internal transcribed spacer (ITS) regions of both fungi. Analytical performance, including specificity, sensitivity, limit of detection (LOD), repeatability and reproducibility, was systematically evaluated. The assay’s diagnostic applicability was assessed using 26 clinical specimens and compared with culture and a duplex qPCR assay. Results The assay demonstrated high specificity with no cross-reactivity against non-target fungi or human DNA. The LODs were 559.2 fg/reaction (~34.7 copies) for C. albicans and 51.1 fg/reaction (~2.4 copies) for C. neoformans , with excellent repeatability and reproducibility (all CV <5%). Performance evaluation on clinical samples showed 100% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) compared with culture, and exhibited 100% overall agreement with qPCR. The entire process was completed within 20 minutes at 39 °C using a standard real-time PCR instrument. Conclusion This rapid, sensitive, and specific duplex real-time RPA assay represents a potential point-of-care testing (POCT) tool for the early and simultaneous diagnosis of two major IFIs-causing pathogens.