Rapid Point-of-Care Molecular Test for the Differentiation of Dengue and Chikungunya Viruses

DESCRIPTION provided by applicant Increased human travel urbanization and climate changes have resulted in a wide geographic spread of febrile illness caused by both Dengue Virus DENV and Chikungunya Virus CHIKV These two infections show a substantial overlap in clinical presentation In addition both are also co prevalent in many countries This poses a significant challenge to clinicians as the two illnesses require different clinical management A recent infection can only be confirmed by detection of the virus in a blood sample as positive results in serological assays may instead be indicative of prior infection However as antibodies develop in response to the infection the viral load in the blood decreases Thus sensitive and specific detection of these viruses is imperative for an accurate diagnosis Typically molecular methods that employ reverse transcriptase real time polymerase chain reaction rtRT PCR based detection of viral RNA require nucleic acid extraction of whole blood samples In this Phase I plan we propose to develop a multiplexed molecular assay for the direct detection of either DENV or CHIKV in blood samples and then to incorporate this multiplexed assay into a Collect to Test C T system in order to overcome the need for complex sample processing The C T system includes a simple blood collection and processing method that requires no pipetting or sample manipulation combined with a cartridge containing the dried down assay reagents for simultaneous sample preparation and template amplification The sample preparation and cartridge loading are equivalent to methods currently used to run lateral flow devices This system will allow for simultaneous detection of DENV and CHIKV by rtRT PCR from a minimal volume of whole blood i e a finger prick without the need for centrifugation or time consuming extraction methods Critical to this is the development of a rtRT PCR based assay optimized for the detection of viral RNA directly from blood samples and requiring no further sample extraction These reagents together with the C T system will be compatible with the T CORandquot portable thermocycler Tetracore has extensive experience in the development of highly sensitive and specific dried down room temperature stable assays for real time PCR including a DENV specific assay previously developed with Naval Medical Research Center This assay was systematically evaluated and successfully tested using clinical samples on a commercial laboratory rtRT PCR platform Based on our preliminary data and experience we are confident to propose this plan to develop a multiplex rtRT PCR assay for differential detection of DENV and CHIKV that can be combined for use with the C T cartridge and the T COR portable thermocycler to create an integrated point of care system that can be equally effectively in both public health labs and low resource settings PUBLIC HEALTH RELEVANCE Dengue and Chikungunya fevers are both mosquito borne viral diseases with similar early clinical presentations but requiring distinctly different medical management Thus accurate diagnosis is critical to ensure optimal patient outcomes In Phase I of this project we propose to develop a rapid molecular diagnostic multiplex assay for direct detection and differentiation of Dengue and Chikungunya virus in whole blood compatible with a point of care POC device designed for use in low resource settings In Phase II we plan to further validate this assay in public health labs endemic areas and low resource diagnostic settings such as remote test labs and physiciansandapos offices using real world samples