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 that is caused by distinct virus types and subtypes. Early diagnosis is crucial for disease treatment and control as it reduces the inappropriate use of antibiotics and provides the indication for antiviral therapy. Rapid diagnosis is also a key component of surveillance activity. This requires the ability to detect and accurately diagnose infection at or close to the source/outbreak with minimum delay, a tactic consistent with the global experience during the SARS epidemic in 2003. This experience underlines the need for specific, sensitive point-of-care testing capable of discriminating between influenza subtypes. None of the available influenza diagnostic assays combines a point-of-care format with the multiplex capability to identify a large repertoire of human and animal viruses. 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. The system will utilize three components: 1) a microarray immunoassay that distinguishes influenza A and B virus as well as A subtypes; 2) an innovative self-contained disposable lateral-flow device that allows the addition of specimens and reagents in a temporally-controlled manner; 3) a robust automated processing reading instrument of novel conception that employs a low cost, high performance optical module. This project will provide small laboratories, health offices, veterinary clinics and outposts (airports) with the diagnostic capability of major research institutions and reference centres, thus providing better care for patients and most importantly, facilitating the implementation of surveillance activities and guiding response measures that are being built to face a possible influenza pandemic caused by a highly virulent virus.[+] Read More
Early diagnosis of influenza is increasingly recognized as a crucial instrument for disease treatment and control of transmission. A correct diagnosis can reduce the inappropriate use of antibiotics and provide the indication for using antiviral therapy that, if given within the first days of infection, can significantly reduce both morbidity and mortality particularly in susceptible individuals. Rapid diagnosis is also a key component of disease surveillance activity carried out by health authorities to monitor the presence of influenza viruses in the community. Diagnosis based on clinical examination is neither obvious nor rapid because the initial symptoms of influenza, such as high fever, headache, generalized malaise and respiratory symptoms, are similar to those caused by other infectious agents. Furthermore clinical diagnosis is also inadequate to implement surveillance measures as these require the identification of the predominant circulating virus types, subtypes, and possibly strains. There is an urgent need to develop technically innovative solutions for portable, robust, discriminatory devices which allow type and subtype influenza virus detection in low skill settings with little or no laboratory infrastructure.
A number of in vitro assays are currently utilized to help in the diagnosis of influenza. Among these, immunoassays detecting viral antigens, particularly those that utilise a lateral flow dipstick format (rapid tests), have become increasingly popular because they give rapid results and are easy to perform. These assays vary in their ability to distinguish influenza virus types. Different tests can detect 1) only influenza A viruses; 2) both influenza A and B viruses, but not distinguish between the two types; or 3) both influenza A and B and distinguish between the two. These tests do not have the capability to distinguish influenza A subtypes. None of the available influenza diagnostic assays combines the two key features that are deemed essential to effectively combat the disease: 1) a format suitable for point-of-care use and 2) a multiplex capability to identify a large repertoire of human and animal viruses.
During the last years a number of influenza in vitro diagnostic tests known as "Rapid influenza tests" have been developed. Most of them utilize a lateral flow dipstick format and produce results in about 30 minutes. Most of the commercially available rapid tests have a sensitivity of approximately 70% and a specificity of 90%. Furthermore, their use is linked to their capability to distinguish, in the best case, only between A and B viruses. New formats and technologies are emerging and microarray technology, in particular, offers an opportunity to develop a new generation of in vitro diagnostic assays, capable of assessing multiple parameters simultaneously.
Develop an automated portable microarray assay system to distinguish influenza virus subtypes at point-of-care.
The SMEs participating in this project have developed the knowledge and expertise 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 diagnostic system capable of executing, over a small surface area, hundreds of distinct parallel immunoassays, with minimal sample and no loss of performance. This technology has been validated in a number of different diagnostic applications including allergy, infections and autoimmune diseases where a number of different parameters must be evaluated to reach a correct diagnosis. We propose here to exploit such proprietary SME technology to develop a high throughput, point-of-care diagnostic system that is capable of unifying in a single format a number of assays for the detection of influenza viruses infecting man and animals. Micro-deposition, immunoassay chemistry, design moulding, optoelectronics, robotics and computational technologies will be employed to develop and assess a functional fully automated point-of-care system for the diagnosis of influenza consisting of three components: 1) a microarray immunoassay for the detection of viral antigens; 2) an innovative self-contained disposable lateral-flow device enabling automation of reagents incubation and washing steps; 3) a robust, affordable and simple automated equipment incorporating a novel optical reading module that overcomes the complexity of existing microarray reading instrumentation. To match the throughput capability provided by the this system, using current influenza diagnostic test, any laboratory would otherwise need the resources to manage a panoply of costly instruments, kits and reagents with different expiry dates, each requiring specific storage conditions. The proposed rapid test will offer significant benefits beyond the primary impact of improved diagnostic capability. Its portable nature and capacity to rapidly discriminate between influenza types and subtypes will also offer wider benefits for patient management, outbreak response management and broader virological surveillance.
i. Define immunoassay, device and instrument specification
The objective is to set the specifications of a rapid point-of-care influenza multi-parametric diagnostic system. We aim to align our activities with ongoing international efforts to improve influenza diagnostic assays and to build a response to the threat of a pandemic. International experts, veterinarians and clinicians as well as representatives of international agencies will be consulted and asked to provide feedback on the different components of the diagnostic system, the assay, the device and the instrument.
ii. Assay concept
The assay will be designed to detect in clinical specimens conserved antigens, such as nucleoproteins (NPs), which can discriminate between the Influenza A and B types, as well as the type A subtypes. The assay aims at identifying virus subtypes and not virus strains and as such it is anticipated that the targeting of conserved antigens will offer suitable stability to justify this approach and the diagnostic value of the end product. The assay key analytical parameters such as sensitivity, specificity, reproducibility and accuracy will be assessed.
iii. Tooling and optimization of a lateral flow device
The objective is to optimize the proposed lateral flow device and assess its performance in detecting influenza antigens as well its compatibility with clinical specimens (i.e., throat, nasopharyngeal, or nasal aspirates, swabs, or washes).
iv. Instrument development and optimization
The aim is to develop, from the stage of proof-of-principle/prototype, a small, simple, robust and easy to operate microarray reading/processing instrument that can be utilized in small laboratories (point-of-care) and if necessary in non-medical environments such as airports. It has been estimated that the validation of the assay system will require the initial production of 10 instruments.
iv. Point-of-care system performance
We have planned to carry out a clinical validation of the integrated point-of-care system (assay, device and instrument) utilising both reference and clinical samples in comparison with available diagnostic procedures (virus culture, PCR and rapid diagnostic test).
The commercial opportunities arising from the exploitation of the technology are potentially very large with the current acute respiratory diagnostics market standing at $400 million per annum and exhibiting 10-20% annual growth. How the market will respond to the proposed point-of-care system can only be gauged when such products are launched, however several critical issues can be identified such as the impact of multiplex testing on other diagnostic markets as well as political, economic and regulatory pressures to control spending in the current healthcare industry.