The accuracy of Influenza A (H1N1) “swine flu” laboratory testing: A systematic review of diagnostic test accuracy.

Review question/objective The aim of this systematic review is to comprehensively search the available literature and to synthesise the best available evidence to determine the diagnostic accuracy of currently available laboratory tests for swine flu (H1N1), using viral culture as a reference test. Background The Influenza virus affects mainly the nose, throat, bronchi and, occasionally the lungs. Infection usually lasts for about a week and is characterised by sudden onset of high fever, aching muscles, headache and severe malaise, non-productive cough, sore throat and rhinitis. 1 The virus is transmitted easily from person to person via aerosol droplets and small particles produced when infected people cough or sneeze. Influenza tends to spread rapidly in seasonal epidemics. Most infected people recover within one to two weeks without requiring medical treatment. However, in the very young, the elderly, and those with other serious medical conditions, infection can lead to severe complications of the underlying condition, pneumonia and death. 1 In their simplest form, viruses are composed of an outer protein coat and an inner core of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), however, unlike bacteria, no virus has been shown to contain both 2 indicating they are unable to transcribe DNA to RNA, thus requiring host cells. A virus particle is present as either in either a replicating or defective form within the infected cell. The extracellular virus is defined as a virion. Influenza viruses belong to the family of Orthmyxoviridae. This family of viruses have a single stranded, negative sense, eight-segmented RNA genome.3 The eight segments of RNA encode 11 proteins, of which nine are assembled into the infectious virion.4 Three proteins are found on the surface of the virion, haemagglutinin (HA) and neuraminidase (NA), as well as the transmembrane ion channel protein (M2). This family of viruses are enveloped by a lipid envelope that is derived from proteins synthesised by the host. The lipid envelope confers some protection to the virus, not only from host immune cells but also from environmental factors. Influenza A, B and C viruses differ in the host species used and their pathogenicity 5 and can be distinguished on the basis of antigenic differences between proteins on their outer protein coat.6 Of the influenza viruses, only A and B cause frequent (and occasionally severe) diseases in humans 3 There is only one type of Influenza B, whereas Influenza A has multiple subtypes, based on a combination of genes that encode for surface proteins, 3 discussed further below. A single extracellular Influenza A virus particle (virion) has a characteristic spiky appearance when viewed under electron microscopy. 7 The spikes are due to glycoproteins that protrude beneath the lipid envelope. The major glycoproteins are Haemagglutinin and Neuraminidase. A minor component of the lipid envelope is the M2 protein which acts as an ion channel. The matrix protein (M1) lies beneath the lipid envelope and covers the ribonucleoprotein (RNP) complexes. The RNP complexes are comprised of the viral RNA, covered with nucleoprotein and associated with other protein complexes. The viral genome consists of eight separate RNA segments and the segments 1, 2 and 3 code for viral polymerase i.e., PB2, PB1, and PA respectively, segment 4 for haemagglutinin (HA), segment 5 for Nucleoprotein (NP), segment 6 for neuraminidase (NA), segment 7 for matrix protein (M1), and membrane channel proteins (M2), segment 8 for non-structural proteins (NS1 and NS2). 8 The sketch in Figure 1 is a sketch showing the main features of a typical influenza A virus.Figure 1 Schematic influenza A virion: 8The category of Influenza A viruses is further subdivided on the basis of haemagglutinin (H) and neuraminidase (N) proteins expressed on the outer surface of the virus. 5 Both of these proteins are important for the virus to be able to get into the host cell, reproduce and be released to infect other cells - making them potential targets for both diagnosis and therapy. Haemagglutinin allows the virus to enter host cells, such as epithelial cells lining the respiratory tract. This molecule mediates viral attachment to the host cell and its subsequent entry into cells by causing fusion of the cell membranes (endocytosis). Variation in haemagglutinin molecules can result in altered affinity for the different types of the cellular receptor (sialic acid) for binding either the human or the avian variant forms. Because of this, receptor specificity of the haemagglutinin molecule is thought to be able to determine the transmissibility of an influenza virus strain in a given species. 3 Once the virus is inside the host cell (such as epithelial cells), it uses the cell to synthesise copies of itself. Neuraminidase hydrolyses the bonds between haemagglutinin and sialic acid after the virus has reproduced, allowing it to leave the cell to continue infecting other cells. Neuraminidase activity is required for the release of the viral particles/virions and the activity of neuraminidase is the target of the current antiviral drugs, Oseltamivir and Zanamivir. 5 Treatment prevents the action of neuraminidase, resulting in a failure of the release the new virions being released and therefore prevents further spread of the virus. Influenza A viruses are given the denotation HxNy, where x can be 1-16, as there 16 known subtypes of haemagglutinin and y can be 1-9 as there are 9 known neuraminidase subunits, however only H 1, 2, 3 and N 1 and 2 are commonly found in humans. 5 The 2009 pandemic influenza H1N1 consists of genetic material from viruses from swine, human and avian origins. 9 Globally, at any one time there are likely to be several influenza viruses circulating in seasonal patterns. Influenza viruses gradually change over time by a process called antigenic drift. This process is characterised by sporadic point mutations in either the haemagglutinin or neuraminidase protein. 6 Occasionally, a major antigenic change in either the haemagglutinin or neuraminidase protein occurs by a process called antigenic shift, resulting in a virus that mutates much faster and with the ability to spread much more widely, quickly and replace the prevailing seasonal influenza strain e.g. the 2009 pandemic influenza H1N1. This may result in a new strain of virus. Antigenic shift occurs under rare conditions where a host is infected with a combination of two or more influenza A viruses of different origins or strains that are able to become mixed (or ressorted), leading to the generation of a new viral strain. The resulting virus has the ability to spread rapidly within a population (and also to other populations and subsequently globally), as people are likely to be immunologically naive to this new virus. The 2009 H1N1 virus was a triple ressortment influenza virus with swine human and avian genetic material that rapidly became pandemic. 3, 9, 10 However, although the 2009 H1N1 strain was novel, it shared features with other H1N1 strains which aided in its identification. 11 Pandemic influenza viruses are thought to arise when there is close and frequent contact between humans and other animal species that can be infected by Influenza viruses. The virus develops the ability to jump the species barrier to be able to infect humans. This “crossing” is made possible by certain genetic mutations that permit the binding of the animal viruses to surface proteins in the human respiratory tract. 3 Prior to the 2009 H1N1 pandemic, there have been three well-documented Influenza A pandemics in recent history. 6, 12 Table 1 summaries some of the major details of recent Influenza A pandemics, with the aim of framing the 2009 H1N1 pandemic in a historical context. 4, 5, 12Table 1: Recent Influenza A pandemicsTable: No Caption available.The first of these pandemics was caused by a H1N1 virus and coincided with the outbreak of World War I. It is estimated that roughly a third of the world's population was infected (approximately 500 million people) and resulted in approximately 50 million deaths. 3 The 1957 and 1968 pandemics resulted in a lower number of cases and fatalities; however, they still resulted in a significant global effect. The 2009 pandemic was caused by a H1N1 virus and although the number of fatalities was much lower than any of the previous pandemics, concern was raised by how quickly the virus spread. The outbreak began in the state of Veracruz, Mexico in March/April 2009 and spread rapidly across the world. Only a matter of weeks later (in June 2009), the World Health Organization (WHO) and US Centers for Disease Control (CDC) stopped counting cases (30,000 confirmed cases) and the WHO declared the outbreak a pandemic. 13 Subsequent estimates have reported millions of cases and at least 16,813 deaths. 5 Due to the availability of epidemiological records and preserved archival material, the viruses responsible for the recent pandemics have been identified, allowing patterns of infectivity to be modelled. 11 The haemagglutinin gene of 2009 H1N1 is derived from “classical swine H1N1” virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918. By using homology modelling, Igarashi 11showed that the two viral strains exhibit such a high degree of structural homology that prior exposure to the 1918 strain is likely to confer specific immunity against H1N1. Techniques that model the structure and homology of haemagglutinin and neuraminidase proteins are useful in identifying potential therapeutic targets for drug and/or vaccination development, 14, 15 as well as potential targets for diagnostic tests. H1N1 was first described in the 1918 pandemic and made a resurgence in April 2009 in the form of a triple-reassortant influenza A virus, which is composed of a combination of human, swine, and Eurasian avian strains. 16What was so unusual about this Influenza A strain was that it appeared to be comprised of several separate types of known flu strain and was described as “..an unusually mongrelised mix of genetic sequences” 10 In June 2009, the World Health Organisation declared an H1N1 pandemic - the first global pandemic since the 1968 Hong Kong flu. Infections with influenza H1N1 viruses are usually less severe in their impact than those with Influenza B viruses; however they appear to have more pandemic potential, possibly as a result of the type of haemagglutinin protein present. 3 Currently, there is no known cure for the influenza virus, therefore strategies have focussed on prevention (such as the use of vaccination programs) and reduction of symptoms (such as with antiviral drugs such as Oseltamivir and Zanamivir). The lack of a cure relates to the success of the influenza virus to adapt in order to evade the immune system. Distinguishing an influenza virus from other circulating pathogens on the basis of signs and symptoms alone is unreliable due to the large degree of similarity in presentation. For example, Mycoplasma pneumonia - causative agent of primary atypical pneumonia (also known as pleuro-pneumonia like organism or PPLO) causes similar signs and symptoms of those of respiratory tract infection. Laboratory testing of respiratory specimens offers a much improved method of identifying causative agents and therefore improving effectiveness of management. Diagnostic tests Diagnostic tests aid clinicians in making a diagnosis and a subsequent treatment plan for the patient based on the test results.17 Diagnostic test interpretation involves comparing features of the patient against known features of the suspected condition and generating a likelihood of the presence or absence of the condition. In order to determine how accurate a diagnostic test is, the test should be compared to a reference test/standard that has been proven as being reliable for the particular condition of interest and patient population. Systematic reviews of diagnostic tests are a recent development and aim to inform the clinician by critically appraising and synthesising the best available evidence concerning the accuracy of diagnostic tests, ideally in comparison with alternate techniques. 18 However, this type of review can be challenging as determining what is meant by accuracy in this context is more complex than with other study designs, such as studies of therapeutic interventions. 19 The accuracy of a test is determined by posing two questions: How often does the test give positive results for patients that have the condition? and How often does the test give negative results for patients that not have the condition? with any there are and of the major is that diagnostic tests are to and the clinician with evidence on which to a diagnosis within a useful a diagnostic a patient is compared against a reference or test therefore test accuracy can be the type of patient or reference is Diagnostic testing for flu that there are often different strains of influenza virus circulating at any one as well as other very similar identifying a viral strain responsible for an outbreak can be the diagnosis of influenza is based on symptoms of the more accurate laboratory 13 for this be the of time to laboratory test The reference or test to a virus is viral culture which can to 10 - 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