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Development and validation of a microarray based automated diagnostic system for the detection of influenza virus types and subtypes at point-of-care

Final Report Summary - FLUARRAY (Development and validation of a microarray based automated diagnostic system for the detection of influenza virus types and subtypes at point-of-care)

Influenza is an extremely contagious infection of the upper and/or lower respiratory tract and is caused by distinct types and subtypes of influenza viruses. Depending on the pathogenicity of the virus and the susceptibility of the host, influenza can vary from a self resolving moderate disease to a life threatening illness. In Europe every year influenza viruses infect 10-15% of the population and account for about 100,000 cases of hospital admissions of which 20% die as either direct or indirect consequence of influenza infection, particularly the elderly and people suffering from chronic heart and respiratory diseases. Humans are infected by influenza types A, B, and C viruses. Influenza A viruses can be further distinguished in different subtypes because of amino acid differences in the surface proteins hemagglutinin (HA) and neuraminidase (NA). To date 16 HA and 9 NA subtypes have been identified occurring in many possible combinations each representing a distinct virus subtype. The viruses currently circulating among people worldwide causing season influenza include H1N1 and H3N2 subtypes and B virus. Influenza viruses also infect a wide range of domestic and wild animals, particularly birds, where all known subtypes of influenza A viruses can be found. Human infection from bird viruses (avian viruses) occurs only upon exposure to infected poultry, wild birds or contact with surfaces contaminated with secretion/excretions of infected animals. The transmission of avian influenza viruses from man-to-man has been reported very rarely. However, the rapid spread in wild bird species of a highly pathogenic virus subtype, H5N1, that can cause a deadly infection in man, has generated the concern that a mutated form of this virus could acquire the capability to spread in humans and cause a pandemic of catastrophic consequences.

Rapid diagnosis of influenza infection is a key component of disease surveillance activity carried out by health authorities to monitor the presence of these viruses in the community. During the last years a number of influenza in vitro diagnostic tests have been developed because of the need to make a timely diagnosis of influenza for the optimal use of available antiviral treatments. Most of them utilize a lateral flow dipstick format. This technical solution allows the development of assays that are robust, affordable, easy to perform and therefore suitable for point-of-care. In spite of such clear advantages, rapid tests have two major limitations that restrict their use only during influenza season. First, most of the commercially available rapid tests have a sensitivity of approximately 70% and a specificity of 90%. Second, available rapid tests are capable to distinguish, in the best case, only between A and B viruses and do not have the capability to distinguish influenza A subtypes. The limited multiplex capability is again intrinsically linked to the format. Only a few assays and controls can be accommodated on a test strip and discriminated with confidence without the help of an instrument. Available rapid influenza test are then already at the limit of their performance and multiplex capability. The development of a point-of-care assay system that combines high technical performance (in terms of sensitivity and specificity) with the ability to distinguish a repertoire of different subtype can only be achieved using different and innovative technology. In this regard, the establishment of automated robust micro-deposition technologies has recently allowed the development of high density ordered arrays of proteins and nucleic acids. Protein chip immunoassays represent novel and powerful immunological tools that dramatically differ from single immunoassays in the number of antigen-antibody reactions that can be detected at one time. They can perform thousands of distinct antigen antibody reactions simultaneously, thus having clear advantages in terms of cost within overall national healthcare programs. In the field of in vitro clinical diagnosis the utilization of Microarray technology offers an opportunity to develop a new generation of in vitro diagnostic assays, capable of assessing multiple parameters simultaneously using individual samples. In this regard, a point-of-care assay that distinguishes virus subtypes will provide small laboratories, health offices, veterinary clinics and outposts (airports) with the diagnostic capability of major research institutions and reference centres. Most importantly, it will significantly facilitate the implementation of surveillance activities and will prove invaluable in guiding response measures (mass vaccination, preventive drug treatment, and containment) that are being designed to face a possible influenza pandemic caused by a highly virulent virus. This capability will have a tremendous impact in providing patients with better care and appropriate therapy. This project exploits the knowledge and the expertise of the partners to convert microarray assays that have a powerful multiplex capability but are laborious, complex and expensive to perform, into a simple, robust and affordable automated point-of-care system for the diagnosis of influenza.