There is a significant probability that in the next decade there will be either an influenza (flu) pandemic, a major epidemic of a novel respiratory virus (RV) [e.g., SARS], or a bioterrorism attack that results in respiratory symptoms. In addition, as many as 500,000 people die each year of flu and there is no method to clinically distinguish flu from the many other common RV infections. There is an urgent need for sensitive, highly specific, inexpensive, portable, low complexity devices/assays that allow for the multiplex detection and identification of flu and other common RV's. We have a long record of developing multiplex commercial assays for flu and other RVs and developed the first commercial multiplex molecular assay for flu A, B, RSV A, B, and parainflu 1,2,3 in the world (Hexaplex, 1996) and the first assay for SARS in the US (2003). We have now developed novel fully integrated technology based on a unique sample collection device (SCD), stable europium fluorescent microparticles (EFM), and binding chemistry based on pRNA that allows for sensitive (400x >than FDA approved assays) bedside or in-the-field multiplex POC antigen testing of patients. This technology runs on batteries, takes 15 minutes, and can detect multiple viruses simultaneously. Even more importantly the SCD collects and stabilizes a sample of nucleic acid (RNA and DNA) for use in molecular assays as needed (allowing for testing of negative samples or further analysis of positives while maintaining biocontainment). In this application we propose the following specific aims:! Select and optimize the chemistry, substrate, and hardware to produce a SCD-EFM-pRNA assay to detect 13 common RV's causing LRI or significant disease in children and adults (flu A, B, RSV, [paraflu 1,2,3, hMPV], corona- viruses [NL63, HKU1, 229E, OC43], adenovirus, and enterovirus). 2. Develop a multiplex-RT-PCR electronic microarray (eMR) assay to these same 13 viruses as a confirmatory molecular assay (also as a product), determine and compare analytical LOD, sensitivity and specificity with multiplex immunoassay developed in SA 1 and other established molecular assays. 3. Determine optimal parameters required by FDA. (e.g., ensure design of all components, ease of use guidelines, and results interpretation by non-professional laboratory personnel meets CLIA waived standards for assay in SA#1);4. Process Development (methods to ensure manufacturability of the diagnostic components and commercialization at a reasonable cost). 5. Clinical sample testing (frozen samples);To determine if the multiplex POC assays developed in SA#1 &2 to the majority of RV that produce """"""""Flu-like"""""""" symptoms and LRI can demonstrate improved clinical sensitivity, specificity, and cost effectiveness compared to currently available standard diagnostic assays. After completion of these SAs, clinicians and public health officials will have two important new tools to fight flu and viral respiratory infections, whether from their hospital, office or the most remote locations on the planet.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI077988-02
Application #
7651250
Study Section
Special Emphasis Panel (ZAI1-TP-M (J2))
Program Officer
Salomon, Rachelle
Project Start
2008-07-08
Project End
2012-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
2
Fiscal Year
2009
Total Cost
$475,444
Indirect Cost
Name
Medical College of Wisconsin
Department
Pediatrics
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
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Beck, Eric T; He, Jie; Nelson, Martha I et al. (2012) Genome sequencing and phylogenetic analysis of 39 human parainfluenza virus type 1 strains isolated from 1997-2010. PLoS One 7:e46048
Rebuffo-Scheer, Cecilia; Bose, Michael; He, Jie et al. (2011) Whole genome sequencing and evolutionary analysis of human respiratory syncytial virus A and B from Milwaukee, WI 1998-2010. PLoS One 6:e25468
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Bose, Michael E; Beck, Eric T; Ledeboer, Nate et al. (2009) Rapid semiautomated subtyping of influenza virus species during the 2009 swine origin influenza A H1N1 virus epidemic in Milwaukee, Wisconsin. J Clin Microbiol 47:2779-86