Development of a Rapid Diagnostic Test to Distinguish between Emerging Viruses That Cause Hemorrhagic Fever

Viruses that cause the clinical syndrome referred to as viral hemorrhagic fever (VHF) are responsible for numerous infectious disease outbreaks. High-priority emerging viruses include orthoebolaviruses, orthomarburgviruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). Many of these viruses cause a similar clinical presentation in infected humans and have an overlapping geographic distribution with a risk of coemergence. As such, an antigen rapid diagnostic test to distinguish between these viruses would be beneficial in low-resource settings. In this study, we developed single-plex and multiplex antigen detection lateral flow immunoassays (LFIs) to rapidly detect and distinguish between emerging viruses that can cause VHF. We evaluated two antibody-labeling methods, colloidal gold nanoparticles and cellulose nanobeads (CNBs), to determine which approach would increase assay performance and multiplexing capabilities. Assay performance was evaluated by determining their sensitivity, specificity, matrix evaluation, and stability testing. All assays were highly specific, with no crossreactivity observed for the single-plex assays. Several of the assays performed better with the CNBs, including the DENV, YFV, RVFV, and orthomarburgvirus LFIs. No matrix effect was observed with most of the assays except that serum did impact the RVFV and DENV assays. In general, the multiplex assays were less sensitive compared with their respective single-plex assay. The most successful assays were the single-plex CNB LFIs assembled into an eight-plex cartridge, which allows for rapid and simultaneous testing of antigen to seven viruses.