The common respiratory viruses (influenza, parainfluenza, respiratory syncytial virus or RSV, and rhinovirus) cause millions of cases of upper and lower respiratory tract disease every year. The spectrum of illness induced directly by the viruses is very broad, ranging from mild colds and simple ear infections to more serious and sometimes life-threatening diseases such as bronchiolitis, pneumonia, and asthma. In spite of the importance of diagnosing and treating these viral infections, current diagnostic methods of physical exam or rapid immunoassays or culture in reference laboratories do not provide the accuracy, sensitivity, or timely information that the primary care physician needs for treatment. Antiviral therapeutics are becoming increasingly viable, but they require accurate identification of the infectious virus(es). While sensitive, newer assays and instrumentation based on nucleic acid technologies are not cost effective or practical for point of care in a physician's office. To provide a cost effective, point of care system for diagnosing respiratory viruses, this application describes an integrated and unique device that will begin with a nasal wash sample and use nucleic acid-based technologies to accurately identify eight respiratory viruses: RSV A and B; parainfluenza 1, 2, and 3; influenza A and B; and rhinovirus. After loading the sample, proprietary steps of sample preparation, RNA extraction, multiplexed amplification, and detection by lateral flow strips will be automatically performed. These processing steps will be directed on a disposable, microfluidic card that both minimizes the reagent costs and provides an enclosed container for convenient, biosafe disposal. To develop this diagnostic device, a series of technical milestones will be accomplished. First, a novel method of multiplexed target amplification will be optimized on respiratory viral targets. Second, nasal wash samples will be directly prepared for multiplexed amplification by flow through extraction and unique solid phase analysis. Sample preparation and amplification will be managed by developing disposable, laminated microfluidics cards with channels, reagents, and valves. Using a protoype workstation, the described system will be tested with patient samples and spiked samples to verify the utility, specificity, sensitivity, and reproducibility of the system. ? ?
