Diligent global surveillance of influenza strains is a critical element of the U.S. National Strategy and the World Health Organization's (WHO) strategy for Pandemic Influenza. History has shown the need for rapid recognition of emerging viruses with pandemic potential. Current surveillance relies on slow virus culture, antigen recognition, or molecular assays that are designed to detect known circulating subtypes, although some assays may detect novel viruses as "untyped." Untyped viruses can be either single nucleotide variants or other variants of seasonal subtypes or novel reassortants with pandemic capabilities. The ability to easily and accurately identify and individually track these variants is lacking in current approaches. The objective of the proposal is the development of a field-deployable surveillance system that provides rapid and accurate detection and sequence recognition of current and emerging influenza strains that may present a serious risk to human populations. This system integrates Seattle Childrens Research Institute's (SCRI's) Consensus- Degenerate Hybrid Oligonucleotide Primer (CODEHOP)-based PCR and a fluorescent molecular beacon detection method on Micronics'advanced point-of-care (POC) microfluidics diagnostic platform, called the PanNAT". The integrated PanNAT Flu-CODEHOP system will expand and improve capabilities for in-field and near patient monitoring of influenza infections and emerging pandemics. This technology will detect and discriminate influenza hemagglutinin (HA) and neuraminidase (NA) subtypes and strains by means of CODEHOP-based PCR amplification and "sloppy" molecular beacon (SMB) fluorescent melt curve (Tm) signatures. The assay is incorporated into Micronics'PanNAT disposable cartridge, which is processed by the PanNAT instrument for sample-to-result point of information testing. Current influenza virus strains, both seasonal and pandemic, are identified by comparison with a library of known Tm signatures. Novel viruses resulting in new unique Tm signatures can be monitored for multiple occurrences and geographical spread. For novel viruses, the CODEHOP-derived PCR product contained in the PanNAT cartridge can be directly sequenced to identify the sequence tag corresponding to the new Tm signature. Virus in clinical samples identified with new Tm signatures can be completely sequenced via targeted Next-Gen sequencing, allowing a comprehensive analysis of its evolutionary and pathogenic properties guiding possible therapeutic approaches and other public health measures for the novel emerging strain.
Specific aims and milestones include: the development and full integration of nasal swab extraction, cDNA conversion, and amplicon detection and discrimination in the PanNAT Flu-CODEHOP molecular assay (Aim 1);the completion of the PanNAT instrument and cartridges including instrument modification for melt curve analysis and analytical performance testing with standardized synthetic plasmids and clinical samples (Aim 2);and prospective testing of clinical samples and demonstration of substantial equivalence to predicate tests (Aim 3). The successful development of the Flu-CODEHOP assay will further validate the foundational technology of the PanNAT microfluidics POC diagnostic platform providing the basis for additional molecular diagnostic tests for a wide range of respiratory and enteric pathogens or other agents that pose a biological threat to humans.
Diligent global surveillance of influenza strains is a critical element of the U.S. National Strategy and the World Health Organization's strategy for Pandemic Influenza. The objective of the proposal is an easy-to-use fieldable system that provides rapid and accurate detection and sequence recognition of current and novel emerging influenza strains at point-of-care, in order to expand and improve molecular diagnostic capabilities for global monitoring of influenza infections and pandemics.