ArticleInfectious diseases of poverty2026
A rapid multiplex platform for simultaneous detection of chikungunya virus, dengue virus, and dengue serotyping based on isothermal amplification and lateral flow dipsticks.
Article in Infectious diseases of poverty, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
backgroundThe 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).
methodsSpecific 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.
resultsThe 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.
conclusionsA 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.
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