A rapid multiplex platform for simultaneous detection of chikungunya virus, dengue virus, and dengue serotyping based on isothermal amplification and lateral flow dipsticks

Background: The expeditious and precise diagnosis of dengue virus (DENV) and chikungunya virus (CHIKV) is paramount for effective patient management and the control of outbreaks. In this study, a duplex reverse transcription multi-enzyme isothermal amplification (RT-MIRA) assay was established for the simultaneous detection of DENV and CHIKV, followed by a nested RT-MIRA assay for DENV serotyping (DENV-1 to -4). Methods: Specific primers and probes targeting the DENV 3'-UTR, CHIKV E1 gene, and four DENV serotypes were designed. The duplex RT-MIRA and nested DENV RT-MIRA serotyping reaction systems were optimized at 39 °C with portable fluorescence or lateral flow dipstick readouts. For methodological validation, specificity was evaluated against 35 related pathogens, and the 95% limit of detection (LOD95) was determined via probit regression. For clinical validation, serum samples from 236 suspected patients were tested, benchmarking against RT-qPCR and serology. Statistical analyses included the Wilson score method for calculating 95% confidence intervals (CIs) and Cohen's kappa (κ). For external verification, 12 CHIKV-positive clinical samples and 5 artificially simulated co-infection samples were retrospectively analyzed to validate assay accuracy. Results: The duplex RT-MIRA assay exhibited no cross-reactivity with other pathogens. The LOD95 values were 13.47 copies/μl for DENV and 10.49 copies/μl for CHIKV. Clinical validation demonstrated sensitivities of 96.15% (95% CI: 89.28%-98.67%) for DENV and 88.89% (95% CI: 67.20%-96.90%) for CHIKV. Specificity was 100% (95% CI: 92.87%-100%) for both. Agreement with RT-qPCR was strong for DENV (κ = 0.96) and CHIKV (κ = 0.92). The nested RT-MIRA serotyping assay showed high sensitivity (LOD95: 1.6-18.7 copies/μl) without cross-reactivity, accurately differentiating 75 DENV-positive samples into 71 DENV-1 and 4 DENV-2. In the external verification, the assay accurately detected 10 CHIKV mono-infections and 2 CHIKV/DENV co-infections, and distinguished four DENV serotypes in simulated matrices. Conclusions: A rapid and sensitive integrated method has been developed that combines duplex RT-MIRA for detecting DENV and CHIKV, and nested RT-MIRA for serotyping DENV. The simplicity and speed of the amplification and detection steps demonstrate this platform's potential for use in point-of-care testing and surveillance in areas with limited resources, particularly when used alongside portable extraction methods.