Dengue is the most rapidly spreading mosquito-borne virus in the world and currently infects 390 million people annually. Studies have shown that existing point-of-care tests have inadequate sensitivity to identify many individuals infected with dengue. None have the quantitative capability necessary to aid in the detection of severe dengue, especially in secondary dengue infections. Our approach will provide a low-cost yet quantitative and sensitive system. Our lateral flow immunoassay (LFIA) test will analyze and quantitate the presence of dengue virus non-structural protein (DENV NS1), an analyte that is frequently used to determine a diagnosis of dengue. Fluorescent dyes will be used to improve the limit of detection compared to existing rapid tests. A smart phone is utilized for imaging, analysis and automated reporting;the latter feature will simplify disease tracking. A long Stokes shift dye, R-phycoerythrin (PE) will be used as the fluorescent reporter, either singly or bundled into a cluster. The optical properties of PE allow the use of an inexpensive illumination device;the minimal non-specific binding of PE results in sensitive detection with a wide dynamic range. Additional gains in sensitivity will be achieved through an integrated test feature designed to dissociate immune complexed protein for improved access by the test reagents. In Phase I we will: 1) Optimize the PE reporter (cluster) on anti-NS1 antibody;2) Develop a smartphone application to allow automated analysis;3) Develop an on-LFIA strip """"""""zone of dissociation"""""""" to aid in analysis of complexed NS1;4) Develop the use of the control line on the lateral flow strip to allow absolute quantitation of NS1;5) Design, fabricate and test a prototype of a low cost fluorescence system designed with a materials cost less than $35. 6) Validate the NS1 LFIAs by comparing results with commercially available ELISA and LFIA tests using patient samples. These investigations will demonstrate the performance advantage of our system for detection and analysis of NS1. For phase II we hope to include multiple analytes, such as DENV IgG and IgM, or a multiplex febrile disease test strip (e.g. malaria and DENV).
Dengue infects 390 million people per year, yet the tests available for point-of-care use often fail due to inadequate sensitivity. We plan to develop a very low cost, easily used fluorescence system and lateral flow immunoassay where the detector, analysis and automated reporting are provided by a smartphone. Our system will provide greater sensitivity for more accurate and informative diagnosis of dengue in resource- limited settings.
