Portable High surety Detection of Zika Virus Directly in Mosquitoes and Humans

PROJECT SUMMARY ABSTRACT Zika virus ZIKV an emerging arbovirus transmitted by the Aedes mosquitoes has been correlated with newborn microcephaly and Guillain Barre syndrome in adults The World Health Organization has declared Zika a public health emergency and is calling for new diagnostics and enhanced surveillance In particular point of care POC diagnostics that enable rapid sharing of clinical and epidemiological data will facilitate international control and research efforts Real time reverse transcriptase polymerase chain reaction RT PCR analysis of patient blood is the current standard for ZIKV diagnosis Unfortunately RT PCR requires technical expertise complex instruments and infrastructure typically only available in clinical laboratories Accurate inexpensive diagnostic tools for patient care and epidemiological surveillance of insect vectors in remote or resource limited areas are sorely lacking To fill this critical need we propose to develop a POC nucleic acid diagnostic that will identify ZIKV directly from mosquito vectors and from patient saliva that can not only be non invasively sampled but is more frequently positive for ZIKV compared to blood Paratus Diagnostics has previously design developed and demonstrated a point of care system the PreparedNOW system that can meet the demanding challenges outlined above As described in further detail below the Paratus Diagnostics POC device has previously been shown to be capable of detecting ZIKV genes using a highly advanced and innovative molecular multiplexed assay developed by our partner Prof Andy Ellington of the University of Texas Two variants of the basic Paratus PreparedNOW system have been demonstrated one version is capable of processing actual mosquitos the second version will accept human patient saliva as the specimen from which to perform a diagnostic test to determine if an individual has been infected with Zika Our proposed system under this project will be capable of detecting biologically relevant concentrations of viral RNA in human saliva or infected mosquitoes within minutes Aim Objective Our platform leverages our patented and well tested portfolio of devices and several components have been reduced to practice and successfully tested Our approach relies on a consumable cartridge for sample collection preparation and molecular assay processing all within a housing that fits in the palm of a hand and requires only a few intuitive user operated steps Development of this simple but robust approach to fast and accurate pathogen detection can be readily diversified to a wide array of pathogens and vectors Our point of care diagnostic solution will be powered by the unique one pot assay being developed by our partner Dr Andrew Ellington University of Texas Austin This assay incorporates several innovations First the unique amplification assay RT LAMPdeg will be adapted for co amplifying four ZIKV genes in a single assay Aim Objective Second false positives will be nearly eliminated by coupling the amplification assay with a unique nucleic acid probe GO that will sequence specifically transduce up to four ZIKV genes into single channel fluorescence readable by the Paratus system Aim Objective This will allow a single assay to accurately detect known ZIKV variants fully accounting for most currently circulating Asian and African lineages Third fluorescence will be readout using our smartphone based system and the assay will be proofed with clinical and biological samples obtained from Dr Grant Hughes University of Texas Medical Branch Aim Objective PROJECT NARRATIVE Rapid methods to accurately detect Zika virus directly in patient saliva and also in mosquito vectors would significantly facilitate more ubiquitous screening of patients and better surveillance of pathogen prevalence We propose to develop a portable low cost low complexity detection platform built around the patented Paratus Diagnostics PreparedNOW system and capable of running a highly sophisticated multiplexed molecular amplification assay for Zika virus We will further adapt the device and assay to accept patient saliva or mosquitoes and transduce only a few copies of viral nucleic acids into fluorescent signals that can be read using our smartphone enabled instrument Our device would be used at point of care to streamline the time required for epidemiological monitoring and identification of infected individuals allowing timely and appropriate management and will also pave the way to developing point of care assays and devices for a much wider variety of pathogens and vectors