Portable Duplex Diagnostic Platform for Point-of-Care Differentiation of Ebola and Lassa Virus

Ebola virus (EBOV) and Lassa virus (LASV) infections present with indistinguishable early symptoms, creating a persistent diagnostic gap in coendemic regions. This challenge is compounded in resource-limited settings, where existing lateral flow assays are single-target, qualitative, and reliant on subjective interpretation. We developed a duplex colorimetric lateral flow immunoassay (LFIA) for simultaneous detection of EBOV VP40 and LASV nucleoprotein (NP), integrated with the Portable Rapid Onsite Versatile Image-based Detection System (PROVIDS), a compact optical reader that automates line segmentation, trichannel deconvolution, and test-to-control ratio quantification in under 5 s. The assay achieved limits of detection of 44 ng/mL for EBOV and 69 ng/mL for LASV, with intra-assay CVs below 10%, retained full analytical performance after 8 weeks of ambient storage, and showed high specificity for Zaire-lineage EBOV and LASV lineages II (Macenta) and IV (Josiah) without cross-reactivity to Marburg virus (MARV). Meeting WHO Target Product Profile (TPP) benchmarks, this PROVIDS-enabled duplex LFIA provides a portable, quantitative, and multiplexed platform for outbreak triage and establishes a scalable foundation for future syndromic surveillance of viral hemorrhagic fevers (VHFs).