Laboratory diagnosis of influenza virus infection

Pathology (2002 ) 34, pp. 115– 125

BROADSHEET

Laboratory diagnosis of influenza virus infection E. GEOFFREY PLAYFORD* AND DOMINIC E. DWYER† *Microbiology Registrar and †Medical Virologist, Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia This broadsheet is published on behalf of the Board of Education of the Royal College of Pathologists of Australasia

Key words: Influenza, diagnosis, diagnostic techniques and procedures , laboratory techniques and procedures, surveillance.

INTRODUCTION Influenza is an acute febrile respiratory illness of global importance. It is a major cause of morbidity and mortality, especially in the elderly and in those with chronic cardiac and pulmonary conditions, and causes social disruption and financial losses through workplace absenteeism and hospital admissions. Recent advances in antiviral chemotherapy and the ever-present potential of pandemic influenza emphasise the importance of accurate and timely diagnostic techniques, global and regional influenza surveillance, and influenza virus vaccines. Influenza viruses belong to the family Orthomyxoviridae and contain a single-stranded, negative-sense, segmented RNA genome.1 Influenza A and B viruses are members of the same genus and contain eight RNA segments, whereas influenza C virus is within a separate genus and contains seven RNA segments. The influenza A, B and C viruses are distinguishable by antigenic differences in the nucleocapsid ( NP) and matrix ( M) proteins. Subtypes of influenza A viruses are further characterised by antigenic differences in haemagglutinin ( HA) and neuraminidase ( NA) glycoproteins. Although HA and NA antigenic variability occurs among influenza B viruses, distinct subtypes are not recognised. The important structural proteins of influenza A viruses include the HA and NA glycoprotein spikes that radiate from the viral envelope, the M2 transmembrane ion channel, and the abundant M1 protein that underlies the envelope.1 The HA glycoprotein, named for its ability to agglutinate erythrocytes, mediates viral attachment to N-acetylneuraminic ( sialic) acid-containing glycoprotein receptors on the surface of host cells, membrane fusion, and internalisation. It is the major target of neutralising antibodies, and alterations in its antigenic structure underlie influenza virus epidemics and pandemics. The other major glycoprotein spike, NA, removes sialic acid residues from receptors, and is important for release of virus from infected cells and for preventing viral aggregation. Although antibodies to NA are not neutralising, they attenuate illness through inhibiting viral release and spread. Inhibitors of NA have been recently developed as therapeutic agents.

Subtypes of influenza A virus are determined by antigenic variation in HA and NA: 15 distinct HA types ( H1-H15 ) and nine distinct NA types ( N1-N9 ) are recognised, which all occur in avian hosts. Only a restricted range of subtypes ( H1-H3, N1 and N2) have adapted to humans, although occasionally others, such as H5N1 and H9N2, have infected humans. The nomenclature of virus strains recommended by the World Health Organization ( WHO) defines the virus type, geographical origin, serial number of strain from that location, year of isolation and, in the case of influenza A viruses, subtype of the HA and NA antigens ( e.g., A/Sydney/5/97 [H3N2] ). Antigenic variation of the HA and NA glycoproteins of influenza A viruses occur by two mechanisms: antigenic shift and antigenic drift. Antigenic shift involves major antigenic changes through the reassortment of avian and mammalian genomic segments; this results in a new HA and/or NA subtype of influenza A virus with the potential to produce pandemic influenza. Antigenic drift involves minor antigenic alterations in the HA and/or NA glycoproteins resulting from pressures of host immune responses and replicative mutations. Depending upon the degree of antigenic divergence, these strains may evade immune recognition resulting from past infection and/or vaccination, and produce epidemic influenza. In most respects, influenza B viruses are similar to influenza A viruses. Although HA and NA subtypes are not recognised, influenza B viruses exhibit HA and/or NA antigenic variation through antigenic drift. On the other hand, influenza C viruses differ from both influenza A and B viruses structurally; a single surface glycoprotein, with receptor binding, fusion, and receptor destroying activity, is expressed. They are also of considerably less clinical and epidemiological significance. Influenza A and B virus infection encompasses a broad range of clinical manifestations, from asymptomatic or minimally symptomatic infection to severe pneumonitis and multisystem involvement.2 The severity of infection is, in part, related to host factors, such as age, underlying acute or chronic medical conditions, pregnancy, cigarette smoking, and the presence of pre-existing immunity to antigenicallyrelated strains. The latter may be induced by either past infection or vaccination. The burden of mortality and severe morbidity associated with influenza occurs in persons with chronic cardiovascular and pulmonary disorders and in

ISSN 0031–3025 printed/ISSN 1465– 3931 online/02/020115 –11 © 2002 Royal College of Pathologists of Australasia DOI:10.1080/003130201201117909

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those over 65 years of age. Although an acute febrile respiratory tract infection is fairly typical of influenza, other respiratory viruses often circulate at the same time as influenza viruses and these may produce an identical clinical syndrome. Hence influenza virus infection cannot be reliably diagnosed on clinical features alone. Influenza C viruses cause sporadic upper respiratory tract infection, generally amongst children, and are rarely associated with significant lower respiratory tract disease. The immune response to influenza virus infection involves the production of antibodies to the HA, NA, NP and M proteins.3 Antibodies to the HA glycoprotein are neutralising, and include mucosal IgA and serum IgG and IgM. Antibodies to NA, although not neutralising, also confer resistance to re-infection and/or illness. As these protective antibodies are directed against the surface glycoproteins of the influenza virus, they are relatively strain-specific, although they may confer partial protection for antigenically similar viruses. Cellular immune responses are also important in influenza infection: cytotoxic T lymphocytes eliminate virus-infected cells and CD4 + T cells regulate both the humoral and cytotoxic T lymphocyte response. 4 Influenza virus primarily infects the ciliated columnar epithelial cells of the respiratory tract and induces vacuolisation, cellular oedema, cilial loss, and desquamation.5 Loss of the tracheobronchial mucosa, which may be complete or near-complete, is associated with submucosal oedema and an inflammatory infiltrate involving both neutrophils and mononuclear cells. Regeneration of the mucosa may take up to a month. This loss of mucosal integrity, together with defects in phagocyte function, predisposes to secondary bacterial pneumonia. Although respiratory pathogens such as Streptococcus pneumoniae and Haemophilus influenzae are commonly isolated, a particular association between influenza and Staphylococcus aureus pneumonia exists. Influenza pneumonitis, which predominantly affects high-risk individuals, such as the elderly and those with underlying cardiopulmonary disease, is manifest by bronchiolitis, alveolar cell loss, intra-alveolar oedema, and infiltration with mixed inflammatory cells.5 Other occasional complications of influenza include an acute encephalopathy, postinfectious encephalitis, myocarditis, and myositis. Reye’s syndrome, a multisystem disorder involving encephalopathy and fatty liver degeneration, may complicate influenza or other viral infections in children, characteristically in association with aspirin use.

EPIDEMIOLOGY Influenza viruses are transmitted from person to person by respiratory droplets. Environmental survival may exceed 24 hours in droplets and on non-porous surfaces under conditions of low humidity. The rapid spread of influenza through human populations relates to the short incubation period ( 1–5 days) and the high concentrations of virus in respiratory secretions during the initial phase of the illness. Annual epidemics of influenza occur during the coldest months in temperate regions ( May to September in the southern hemisphere and December to March in the northern hemisphere), whereas influenza activity occurs

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year-round in tropical regions. Influenza A and/or B viruses are both responsible for influenza epidemics, but their relative proportions and their severity vary from year-to-year ( Fig. 1). Since 1977, two influenza A virus subtypes, H1N1 and H3N2, have co-circulated, although the latter have predominated over the past decade. Epidemics are typically manifested by an abrupt onset with rapidly increasing activity over a 2– 3 week period, followed by a gradual decline over 5–6 weeks. Epidemic influenza is usually heralded by increases in children with febrile respiratory illnesses, followed by adults; increases in hospitalisations and influenza-related mortality tend to lag by several weeks. In Australia, more than 2000 influenza-related hospital admissions and 1500 deaths are estimated to occur annually. However, other respiratory viruses, such as respiratory syncytial virus, also are significant contributors to the morbidity and mortality occurring during or around the time of the influenza season. Pandemic influenza is caused by the spread of a new influenza A virus subtype amongst an immunologically na¨õve population, which is both virulent and efficiently transmitted between persons. Pandemics are characterised by high rates of influenza-related morbidity and mortality and a high population attack rate. Pandemic influenza viruses usually arise from antigenic shift involving genetic reassortment between avian and human influenza subtypes. Other mammalian species, particularly pigs, appear to be important as intermediate hosts for this reassortment and for human adaptation. Phylogenetic and epidemiological evidence suggest that strains from the 1957 ( H2N2 subtype ) and 1968 ( H3N2 subtype) pandemics arose from China in the setting of close interactions between aquatic birds, pigs and humans.6 Pandemic influenza viruses may also arise directly from avian or non-human mammalian strains; the 1918 pandemic strain ( H1N1 subtype) was likely derived directly from a swine strain.6 However, pandemic influenza also requires efficient person-to-person transmission; the avian H5N1 influenza A virus subtype that affected humans in Hong Kong in 1997, although virulent, did not result in pandemic influenza, presumably the consequence of inefficient transmission between humans and intensive local public health interventions. Finally, human strains that have remained ‘dormant’ for prolonged periods may re-emerge with pandemic potential, as occurred in 1977 with the H1N1 subtype which had not circulated for more than 20 years. However, only younger people were significantly affected following its re-emergence, presumably the consequence of pre-existing immunity to homotypic strains in older persons. The mammalian host range of influenza A virus is broad, including humans, pigs, horses and sea mammals. Birds, particularly aquatic birds such as ducks, represent the most important reservoir of influenza A viruses and harbour all known HA and NA subtypes. In these hosts, influenza virus infection is generally but not invariably avirulent, with replication occurring predominantly within the intestinal epithelium and faecal excretion of high concentrations of virus. Animal influenza viruses can occasionally cause zoonotic infection of humans, including H1N1 subtype from swine, H7N7 subtype from seals, and H9N2 subtype from poultry. Both influenza B and C viruses, on the other hand, are largely restricted to humans.

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Fig. 1 Influenza A and B laboratory reports, Australia 1989–2000, by month/year of specimen collection. ( Reproduced with permission from Thomson et al. 74.

LABORATORY DIAGNOSIS A definitive diagnosis of influenza is made by the isolation of influenza virus or its detection by properly validated immunological or nucleic acid amplification (NAA) methods from a person with an influenza-like illness (ILI). A presumptive diagnosis can be made serologically or by a positive validated rapid point-of-care test.

Specimen requirements 1. Virus isolation The recovery of influenza viruses is optimal from high quality respiratory tract specimens that have been collected early in the clinical illness, transported to the laboratory and processed as rapidly as possible. The best indicator of specimen quality is the presence of columnar epithelial cells, the primary replicative site of influenza virus in humans.7 The recovery of influenza virus from nasopharyngeal aspirates, nasal washes and bronchoalveolar lavages is superior than from nasopharyngeal and throat swabs and expectorated sputum,8–13 as these latter specimens generally contain less columnar and more squamous epithelial cells. Nasopharyngeal aspirates are usually performed in children and are obtained by passing a fine bore suction catheter through the nose and collecting nasopharyngeal cells in the suction trap. At the end of the procedure, aspirating 2–3 ml of viral transport medium ( VTM) recovers cells retained in the suction tube. Nasal washes are obtained by instilling 2–3 ml of VTM alternately into each nostril whilst the patient’s head is tilted back

against a closed airway and recollecting the fluid as the patient blows through the nostril. Although somewhat less sensitive than a nasopharyngeal aspirate, the combination of a throat swab and two nasal swabs is easier to collect from adults. Cotton, rayon, or Dacron (not calcium alginate) swab tips on aluminium or plastic ( not wooden) shafts should be used and placed together in VTM immediately following collection. Specimen desiccation significantly reduces virus recovery. Specimens are best collected within the first 96 hours of clinical illness when maximal viral shedding occurs, whereas virus recovery is unlikely after seven days from symptom onset.10,14 Specimens should be transported at 4°C and processed as quickly as possible; if processing is delayed for more than 72– 96 hours, they should be frozen at –70°C ( not – 20°C). If samples have not been properly transported and stored, they may still be suitable for NAA. 2. Antigen detection and nucleic acid amplification Specimen requirements for antigen and nucleic acid detection are similar to those for virus isolation; indeed specimens should be collected, transported and processed to allow the simultaneous or subsequent performance of virus isolation. Although otherwise suboptimal specimens may be positive using NAA techniques,15,16 high quality specimens should be collected wherever possible. 3. Serology Serum specimens should be collected during the acute phase ( i.e., within 10 days of symptom onset) and the

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convalescent phase ( i.e., 14–21 days after symptom onset) and tested in parallel. Virus isolation 1. Conventional virus isolation The traditional method of influenza virus recovery from clinical specimens is virus isolation using cell culture. The relatively slow turnaround time ( TAT) of cell culture, requiring at least 4– 5 days for a positive result, has lead to the use of more rapid diagnostic assays. However, conventional virus isolation remains an important technique for diagnostic laboratories. It offers significant advantages over other techniques, because it is more sensitive than rapid culture and antigen detection assays, it recovers novel or highly divergent influenza virus strains that may be missed by rapid assays, it provides an isolate for subsequent characterisation, and it allows the simultaneous recovery of other respiratory viruses. A variety of primary, diploid, and continuous cell lines are permissive for influenza viruses, although mammalian epithelioid cells lines, particularly Madin-Darby canine kidney ( MDCK) cells ( a continuous cell line), are generally most sensitive.7 Post-translational cleavage of HA and other influenza virus glycoproteins is required for virus replication, necessitating the addition of the proteolytic enzyme, trypsin, to MDCK cells.7 Primary monkey kidney (PMK) cells are acceptable and are widely used for influenza virus recovery, however, they produce lower viral titres, suffer from batch-to-batch variability in susceptibility, and are susceptible to foamy virus infection.7 Other cells, such as mink lung cells have also been shown to be highly sensitive to influenza A and B viruses.17,18 Human-derived epithelial cell lines have little utility for the isolation of influenza virus.7 Influenza virus replication within cell culture is detected by cytopathic effect ( CPE), haemadsorption ( HAd), or haemagglutination. The CPE is not specific for influenza viruses, and although it generally manifests by 5 days, 19 it may be limited or absent. HAd or haemagglutination demonstrate the property of influenza virus HA antigen to bind sialic acid residues on the surface of erythrocytes of several avian and animal species, and may detect virus replication in the absence of CPE. HAd is performed by washing guinea pig erythrocytes over the cell layer followed by incubation at 4°C; a positive test is indicated by erythrocyte adsorption to the cell layer. Haemagglutination generally requires higher viral titres in the culture and is performed by incubating turkey or guinea pig erythrocytes with culture media supernatant in tubes or microtitre wells; a positive test is indicated by the formation of a cell lattice that fails to sediment. However, positive HAd and/or haemagglutination reactions are also produced by parainfluenza and mumps viruses. Immunofluorescence ( IF) using influenza virus type-specific fluorescent-labelled monoclonal antibodies ( MAbs) should then be used to identify influenza A and B viruses in all cell cultures exhibiting CPE, HAd, or haemagglutination. A panel of MAbs to other common respiratory viruses may be required because of the non-specificity of CPE, HAd, and haemagglutination. Following typing using IF, influenza virus isolates should be subtyped by haemagglutination inhibition (HAI) or another method.

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Influenza virus isolation in the allantoic and amniotic cavities of 10–11-day-old embryonated chicken eggs is labour-intensive and uncommonly performed by diagnostic laboratories. It yields higher viral titres and so remains vital for influenza vaccine production. Unfortunately, as HAmutant strains may arise during isolation in embryonated eggs and exhibit antigenic heterogeneity, candidate vaccine strains require parallel primary isolation in eggs and in mammalian cell lines. Laboratories undertaking influenza virus culture should keep an aliquot of the original clinical sample frozen at –70°C to allow re-inoculation should novel virus strains be isolated or if strains are subsequently needed for vaccine development. 2. Rapid virus culture assays Rapid culture techniques detect influenza virus replication in cell culture within 1– 3 days prior to the development of CPE. These assays use permissive cells, such as MDCK or PMK cells, grown in shell-vials or multiwell plates to which specimen and trypsin are added. Following incubation for 16–72 hours, IF is used to detect influenza virus replication. Overall, these techniques are rapid and cost-effective, with a sensitivity of 56–100% compared with conventional cell culture.20–24 As with conventional cell culture, the sensitivity is likely to be affected by variables such as the quality and timing of specimen collection and transportation conditions. Centrifugation of the specimen onto the cell layer ( e.g., 700 ´ g for 30– 60 minutes) generally increases the sensitivity.20 Although early staining of the cells at 16–24 hours reduces the TAT, 6–32% more isolates are detected by delaying staining to 48 hours.20,22,25,26 The potential problem with rapid culture assays is that novel influenza virus strains with significant antigenic variation may fail to be recognised by MAbs. Furthermore, unless an aliquot of the original specimen or of the shell-vial assay cells is retained for subsequent expansion, isolates are not available for characterisation. Rapid diagnostic techniques As conventional virus isolation has a slow TAT, rapid diagnostic assays have been developed to provide timely support for clinical, therapeutic and infection control decisions. 1. Antigen detection assays Influenza virus in clinical specimens ( including those used for virus isolation) may be rapidly and directly detected by IF, enzyme immunoassay (EIA), radioimmunoassay, and time resolved fluoroimmunoassay, using type-specific MAbs directed against conserved antigens such as M and NP. The most widely used technique is IF using commercially available MAbs ( Fig. 2), being 60–100% sensitive compared with cell culture.15,27–34 Assays using indirect and direct IF staining techniques appear to be equally sensitive.7 As specimens are examined with fluorescent microscopy, an assessment of specimen quality can be made; where few columnar epithelial cells are seen, the sensitivity of IF is low34 and repeat specimen collection should be requested. IF has a TAT of approximately 2 hours and is relatively labour-intensive, requires considerable interpretative expertise, and cannot be automated. Microwave-accelerated and other rapid IF staining protocols with reduced

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Fig. 2 Influenza A infection: demonstrated by indirect immunofluorescence staining of nasopharyngeal specimen.

TATs compare favourably with conventional staining.35,36 MAbs to other common respiratory pathogens, such as parainfluenza types 1– 3, adenovirus, and respiratory syncytial virus can also be applied to the same clinical specimen, sometimes in combination. Influenza virus antigen may also be detected in specimens using EIA. A variety of assays, using different formats and monoclonal or polyclonal antibodies have been developed; reported sensitivities are moderate ( 50– 90%) when compared with cell culture37–39 and approximately equivalent when compared with IF.7,40,41 Technically, EIA is easier to perform and less subjective than IF, although an assessment of specimen adequacy cannot be given. Overall, given the commercial availability of fluorescent-labelled MAbs and their common use for identifying cell culture isolates, most laboratories use IF. However, simpler, more rapid and more accurate commercial antigen detection assays will continue to be developed. As technological advances allow assays to be miniaturised and technical performance simplified, these will increasingly be used within clinics and at patients’ bedsides as well as within laboratories. 2. Rapid point-of-care tests The availability of effective antiviral chemotherapy and the resulting need for rapid diagnostic techniques has driven the development of antigen detection assays that can be performed within the clinic or at the bedside. In general, these point-of-care tests ( POCTs) produce a visual result within approximately 15 minutes following the addition of specimen to a test strip or surface. An initial extraction step is usually required. The Directogen FLU-A assay (Becton Dickinson, USA), an enzyme immunomembrane filter assay that uses

enzyme-conjugated MAbs specific for a conserved epitope of the NP antigen of influenza A virus, has been most extensively evaluated. Compared with cell culture it is 59–100% sensitive and 92– 99% specific for influenza A virus infection,10,27–29,33,35,42 although it does not detect influenza B viruses. A new format of the assay ( Directogen Flu A + B) is claimed to detect both influenza virus types. Other POCTs, such as the QuickVue Influenza Test ( Quidel, Australia), the FLU OIA Test ( BioStar, USA), and the Zstat Flu test ( Zyme Tx, USA) detect both influenza A and B viruses. Evaluations of these assays are relatively limited: the FLU OIA test is 75–80% sensitive and 73% specific compared with cell culture,11,43 but only 56–74% sensitive compared with RT-PCR.43,44 The Zstat Flu and QuickVue Influenza tests are approximately 70–81% sensitive and 92– 99% specific.34,45 As with other techniques, specimen quality is likely a major determinant of assays’ performance characteristics; nasal washes and nasopharyngeal aspirates have been found to be generally more sensitive than swabs for at least one POCT.11 Despite the convenience of POCTs, they are expensive, only moderately sensitive, and their use risks the loss of important epidemiological data as well as missed opportunities for obtaining virus isolates. Thus, wherever possible, specimens for cell culture and/or IF should be submitted in parallel when using POCTs, although this strategy increases costs. However, POCTs are attractive where laboratory services are unavailable or remote from clinical services, and they may have a role in community-based surveillance programmes. Even with their current sensitivity, they may also have a role in primary care in guiding the appropriate use of neuraminidase inhibitors.46

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Nucleic acid amplification Influenza virus RNA may be detected in clinical specimens by reverse transcription-polymerase chain reaction (RTPCR). A variety of ‘in-house’ assays using primers specific for M, HA, NP or NS influenza virus gene sequences and various extraction methods, amplification techniques ( such as single-round or nested), and product detection methods ( such as agarose gel electrophoresis, probe hybridisation, EIA and fluorogenic probes) have been described.14–16,31,44,47–54 Depending on primer selection, these assays may be type- or subtype-specific. RT-PCR offers potential advantages over other methods. Although some studies documented comparable sensitivity of RT-PCR to cell culture,31,47,48,50 others have reported 5–15% more influenza virus detections using RTPCR.15,16,44,49,53 The true status of specimens with discrepant results, i.e., those positive by RT-PCR but negative by the ‘gold standard’ of cell culture, are difficult to resolve; overall, however, it is likely that RT-PCR is more sensitive than cell culture. Specimen quality, timing and transportation conditions may be less critical for RT-PCR than for cell culture or antigen detection. Influenza virus RNA is detectable for several days longer into the clinical course than is cultivable virus,14 which may potentially permit a virological diagnosis to be made in late-presenting patients. In situations where specimen quality is limiting, such as community-based surveillance programmes involving postal submission of specimens, yields from RT-PCR are significantly higher than from cell culture.15,16,55 Although influenza viral RNA may be more stable than cultivable virus in specimens, prompt specimen transportation and processing is important to minimise viral RNA degradation by ribonucleases. Technological advances have improved, and will continue to improve, the utility and applicability of NAA techniques to influenza diagnosis and surveillance. Although the TAT for RT-PCR is intermediate between cell culture and direct antigen detection, real-time PCR techniques can reduce this to 4–5 hours.54 Multiplex RT-PCR assays have been developed which are able to simultaneously detect a wide range of viral and bacterial respiratory pathogens directly in clinical specimens.52–54 NAA may also be used to subtype influenza virus strains through the use of subtype-specific primers and to analyse strain variation through genetic sequencing data. However, these advances in NAA techniques have not replaced the role of the primary isolation and antigenic characterisation of virus isolates from clinical specimens. As NAA assays are not available commercially at present, their development, validation and quality control needs to be performed ‘in-house’, which restricts their availability to larger laboratories. As with all NAA techniques, rigorous procedures are essential to prevent and detect false-positive results. Serology Serology is useful for diagnosing influenza where specimens for virus isolation or antigen detection are negative, inadequate or unavailable. It also provides useful, if delayed, surveillance data. The serological diagnosis of influenza is retrospective, requiring acute and convalescent serum samples, and it generally does not provide information on the antigenic composition of circulating strains.

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Influenza virus infection generally represents re-infection, so most persons have some degree of pre-existing immunity. Thus, the detection of influenza-specific antibodies on a single serum specimen is not diagnostic of recent infection; rather the definitive serological diagnosis of acute influenza requires the demonstration of increasing antibody titres on paired acute and convalescent serum samples. Unfortunately, convalescent specimens are frequently not collected. However, a single high antibody titre in the context of a compatible clinical illness during a period of circulating influenza activity does provide presumptive evidence of recent infection. A variety of serological assays have been applied to the diagnosis of influenza virus infection, including complement fixation ( CF), haemagglutination inhibition ( HAI), single radial haemolysis, neutralisation ( Nt), and EIA. 1. Neutralisation and haemagglutination inhibition The traditional Nt and HAI techniques remain ‘gold standard’ serological techniques for detecting influenza-specific antibodies; a 4-fold rise in titres indicates recent influenza infection. Results from these assays correlate accurately with protection from or susceptibility to influenza, and with vaccination responses. Subtype- and strain-specific antibodies can be demonstrated with these techniques. Nt involves plaque reduction in the presence of neutralising antibodies and is time consuming, technically demanding, and unsuitable for testing large numbers of specimens. HAI demonstrates inhibition of the haemagglutination reaction between virus and erythrocytes in the presence of specific antibodies; results correlate closely with those from Nt, but the technique is somewhat easier. However, HAI still requires considerable technical expertise and is complicated by false-positive results induced by non-specific haemagglutination inhibitors that are present in some serum samples. Serum pretreatment with Vibrio cholerae receptor-destroying enzyme at least partially destroys these inhibitors. Poor interlaboratory reproducibility occurs with HAI, in part related to inadequate destruction of haemagglutination inhibitors and variability of erythrocyte and antigen preparations.7 For these reasons, HAI is performed relatively infrequently for routine laboratory diagnosis. 2. Complement fixation The most commonly used serological test for influenza is CF, which although technically demanding, is somewhat easier to perform than HAI and Nt. Type-specific complement-fixing antibodies directed against NP antigens of either influenza A or B virus are detected, but subtype- or strain-specific antibodies are not distinguished. Recent influenza infection is suggested by a 4-fold rise in CF titres or by a single high titre ( generally  64, but this varies between laboratories). The major disadvantages of the CF test include the technical complexity of the assay, the interference by anticomplementary sera, the slow rise in complement-fixing antibodies following acute infection, and its relative insensitivity.7 3. Enzyme immunoassay EIA has potential advantages over other techniques in that it is technically simpler to perform and able to be at least partly automated, it is more sensitive at detecting influenza-specific antibody rises than other techniques, and it is able to detect individual antibody subclasses, such as IgG, IgM, and IgA.41 The detection of IgM, although specific for recent infection, has a sensitivity

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TABLE 1

Comparison of diagnostic techniques for influenza virus infection

Test

Conventional cell culture

Sensitivity

Turnaround time

~ 100% ( though less than RT-PCR)

At least 4–5 days

Advantages

j j j j j

Rapid cell culture ( shell vial with IF)

56–100% ( generally 70– 90% )

1–4 days

j j

Disadvantages

Highly sensitive/specific Relatively inexpensive Isolate for characterisation Recovers novel/divergent strains Recovers other respiratory viruses

j

Quicker TAT than conventional cell culture Relatively inexpensive

j

j j j j

j j

Immunofluorescence ( direct antigen detection )

60–100% ( generally 70– 90% )

RT-PCR

~ 100% ( greater than cell culture)

2–4 hours

j j

Rapid TAT Provides assessment of specimen quality

j j j j

< 1– 2 days

j j j j j j

Point-of-care tests

59– 93% ( generally ~ 70% )

15– 30 minutes

j j j

Highly sensitive/specific Less dependant on specimen quality/ transport Typing/subtyping possible Molecular analysis ( sequencing ) Detects other respiratory viruses ( multiplex assays) More rapid TAT with real time assays

j

Rapid TAT No technical skill required Specimen transportation not required

j

j j j j j

j j j j j

Serology

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Up to 100%

1–3 weeks

j

Useful where specimens unobtainabl e/ laboratory facilities limited

j j j j

generally less than 50%,56,57 which reflects the absent or blunted IgM response associated with re-infections. On the other hand, IgA responses are prominent following acute influenza; using capture EIA assays, up to 60% of patients have detectable serum IgA within the first week following symptom onset.57–59 IgA is also detectable by EIA in mucosal specimens, offering the potential for testing noninvasive specimens.7 Despite the potential advantages of EIA, there are as yet no satisfactory criteria for antibody levels that are diagnostic of recent infection or that correlate with protective immunity. Results from the Royal College of Pathologists of Australasia Quality Assurance Programme ( RCPA-QAP ) serology programme reveal that all 16 participating laboratories used CF for influenza serology, and that 75% reported results for quality assurance serum specimens within two dilutions of the issuing laboratory.60 The relative advantages and disadvantages of the different diagnostic techniques for influenza virus infection are summarised in Table 1. THE ROLE OF CLINICAL MICROBIOLOGY LABORATORIES IN THE DIAGNOSIS AND SURVEILLANCE OF INFLUENZA The rapid and accurate diagnosis of influenza likely has a positive impact upon medical management through the

Dependant on specimen quality/transport Slow TAT Labour-intensive Technical expertise Specialised equipment Dependant on specimen quality/transport Less sensitive than conventional cell culture May miss divergent strains Labour-intensive Interpretative skill required Fluorescent microscopy required No isolate for antigenic characterisation Expense Labour intensive ( depending on assay) Technical skill Specialised equipment Potential for cross-contaminatio n/falsepositives No isolate for antigenic characterisation Expense Lower sensitivity False-positive results ( misinterpretation of faint bands) No isolate for antigenic characterisation Documentation Potential loss of epidemiological data Delayed diagnosis Requires paired serum specimens Labour-intensive/technical skill ( HAI, CFT) No isolate for antigenic characterisation

timely provision of antiviral therapy and prophylaxis, the implementation of appropriate infection control strategies, and the limitation of unnecessary investigations and inappropriate antibiotic therapy.30,61,62 Clinical microbiology laboratories servicing paediatric and/or adult populations should thus offer, at a minimum, a validated rapid diagnostic assay, such as IF, during the influenza season. RT-PCR assays, particularly multiplex assays that detect influenza A and B viruses as well as other common respiratory pathogens, are an alternative to IF and other rapid assays. Wherever possible, conventional cell culture should be performed for improved diagnostic sensitivity and to obtain influenza virus isolates for characterisation. Laboratories that perform virus isolation or regional reference laboratories should type all isolates as influenza A/H1, A/H3 or B as rapidly as possible. All isolates should then be referred for subtyping, at least fortnightly, to the WHO Collaborating Centre in Melbourne, usually via a WHO National Centre or other reference laboratory. Suspicious isolates should be referred urgently, such as isolates acquired overseas or outside the usual influenza season, those associated with atypical or severe clinical presentations, or those persistently isolated despite appropriate antiviral therapy. Serology should be performed where timely specimens for influenza virus detection and isolation are delayed or

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otherwise unobtainable. As serology generally requires the relatively demanding CF and HAI techniques, these are generally performed by larger laboratories.

SUBTYPING INFLUENZA VIRUS ISOLATES Initial typing of influenza virus isolates is most rapidly and conveniently accomplished by IF using commercially available type-specific MAbs and should be performed as soon as possible after influenza virus isolation. The reference virus subtyping method is HAI using specific antisera raised in ferrets, sheep or chickens. One or more passages of isolates in cell culture or embryonated eggs are required to generate a sufficient haemagglutination titre to perform HAI. This technique characterises antigenic drift as well as antigenic shift, however, it is labourintensive and time-consuming, resulting in often lengthy delays. Due to these difficulties, other subtyping techniques have been developed. MAbs able to differentiate between influenza A/H1, A/H3 and B virus subtypes have been used on virus isolates, in rapid culture assays, and directly on clinical specimens.63–66 Although virus isolates over prolonged periods have been successfully subtyped using MAbs,66 the occurrence of, and the potential for, falsenegative results due to antigenic variation over time represents the major disadvantage. As subtyping using MAbs provides only limited antigenic information, isolates should still be characterised by HAI. Subtyping using RT-PCR with primers specific for influenza A/H1, A/H3 and B virus sequences is also possible, and can be performed on virus isolates or directly on clinical specimens.15,16,47,51,52 Recently, DNA microarrays ( DNA chips) have been used to detect type- and subtype-specific amplification sequences.67 Sequencing of amplified HA and NA genes is another subtyping method,7 potentially allowing the rapid identification of novel or highly divergent strains, the analysis of strain variation, and the determination of the origin of outbreaks ( Fig. 3). Correlation of genetic sequence and subtyping results is contained in an influenza sequence database.68

ANTIVIRAL THERAPY AND ANTIVIRAL SUSCEPTIBILITY TESTING Two classes of drugs are effective against influenza virus infection: the M2 channel inhibitors, amantadine and rimantadine, and the NA inhibitors, zanamivir and oseltamivir ( Table 2). Amantadine and rimantadine act by blocking the M2 protein ion channel that spans the influenza virus envelope, disrupting the dissociation of the M and NP proteins.69 However, as the efficacy of these agents is limited to influenza A viruses, and as their use is associated with the rapid emergence of resistance and prominent central nervous system effects, they are rarely used in clinical practice in Australia. The NA inhibitors are analogues of N-acetylneuraminic acid, and their development was based on the understanding of the three dimensional structure of neuraminidase. Inhibition of neuramindase prevents viral attachment to the surface of host cells and impairs the release of virus from infected cells, a process that decreases the spread of virus

Fig. 3 Phylogenetic analysis of haemagglutinins for human influenza A virus subtypes H1N1 ( Panel A) and H3N2 ( Panel B ). Unrooted neighbour-joining distance trees were constructed using database and local sequences. Bold type indicates samples collected from individual s in Sydney during 2000 and 2001 ( ICPMR, Westmead Hospital) . Reference sequences ( plain type) were obtained from the influenza database. Accession numbers for reference sequences include Aichi AB043497, New Caledonia ISDNAU0001, Yokahama AB043498, and Finland L33747 ( H1N1 sequences) and Panama ISDNCDA001, Nagasaki AB019354, Sydney 1997 ISDNASY97, and Oslo ISDNOS018 ( H3N2 sequences).

and the intensity of infection.70 Zanamivir is administered by inhalation, whereas oseltamivir is given orally and converted in the liver to its active component, oseltamivir carboxylate. 70 Adverse events reported in clinical trials with

LABORATORY DIAGNOSIS OF INFLUENZA

TABLE 2

123

Currently available antiviral agents for influenza virus infection

Class/agent

Trade name

Dose

M2 inhibitors Amantidine Rimantidine*

Symmetrel Flumadine

100 mg twice daily 100 mg twice daily

NI inhibitors Zanamivir Oseltamivir

Relenza Tamiflu

10 mg twice daily 75 mg twice daily

Route of administration

Oral Oral Inhaled ( by Diskhaler) Oral

* Not registered in Australia.

NA inhibitors are uncommon. Zanamivir has been associated with bronchial hyper-reactivity, usually in people with underlying asthma or other respiratory diseases. Oseltamivir has been associated with nausea and vomiting, which is relieved by co-administration with food. NA inhibitors are approved for the treatment of influenza virus infection within 48 hours of symptom onset; their clinical benefits have been reviewed elsewhere.69,70 Broadly, they significantly reduce the duration and severity of illness by approximately 30–40% and, depending on the study, reduce the frequency of influenza-related complications and antibiotic use. These drugs have also been used for long-term influenza prophylaxis (e.g., 4– 6 weeks during the peak influenza season) and for short-term prophylaxis in contacts of an index case with influenza, where they significantly reduce the risk of influenza in contacts by approximately 80–90%.69 Amantadine and rimantadine resistance emerges within a few days of treatment onset in approximately 25–35% of patients, and is mediated by amino acid substitutions in the transmembrane portion of M2.67 These isolates are crossresistant to each other. On the other hand, resistance to NA inhibitors is very rare: during clinical trials, resistance to zanamivir was not detected, whereas resistance to oseltamivir occurred in approximately 1.5% of treated persons.69 Where resistant viruses have been isolated from immunocompetent persons, these have occurred during, rather than following, treatment. However, resistant mutations have been observed after treatment of immunocompromised individuals, a situation that requires further study. NA inhibitor resistance has been selected in vitro following passage of virus in increasing concentrations of these agents, and is associated with a variety of amino acid substitutions in the conserved residues of the NA enzyme active site.71 Such mutations have also been detected in resistant mutants recovered from treated individuals. However, to date these mutations have demonstrated impaired replication in cell culture and animal models.71 NA inhibitor resistance has also occurred with mutations in the HA receptor-binding site, causing reduced dependence on NA function. 71 Screening for NA inhibitor resistance is based on sequence analyses of HA and NA, or on NA inhibition phenotyping. The absence of a reliable cell culture method for determining resistance makes routine testing for NA inhibitor resistance impractical, but laboratories should culture and refer isolates to the appropriate reference laboratory in the setting of therapeutic failure, a situation most likely to occur in immunocompromised individuals.

INFLUENZA SURVEILLANCE Influenza surveillance is a regional, national, and international public health priority. Surveillance involves collection of data on the incidence and impact of influenza and the isolation and characterisation of circulating influenza virus strains. The latter assists the preparation of the subsequent season’s vaccine and in the detection of emerging and potentially pandemic strains. During times of threatened or actual pandemic influenza, surveillance is essential to follow the introduction and spread of the new strain and to co-ordinate control measures and resource allocation. Global influenza surveillance involves four WHO Collaborating Centres (London, Atlanta, Melbourne and Tokyo) supported by 110 National Influenza Centres (including three in Australia: ICPMR in NSW, VIDRL in Victoria, and PathCentre in WA) and other reference laboratories. Together they definitively characterise circulating influenza virus strains, collate and disseminate data on regional and global influenza epidemiology, evaluate drug resistance, prepare and distribute reagents for influenza diagnosis and identification, and develop laboratory techniques.72 Within Australia, a variety of independent surveillance programmes are performed each year. Selected laboratories report laboratory-confirmed cases to the Commonwealth Department of Health (through the Lab-VISE programme) and sentinel general practices report rates of influenza-like illnesses ( through the ASPREN programme). Programmes organised by various State Departments of Health and pharmaceutical companies in selected regions also collect clinical and laboratory data. Surveillance of the clinical impact of influenza includes recording influenza-related deaths and pneumonia, seasonal hospital admissions, and workplace absenteeisms. Unfortunately, at this stage, Australia lacks a single, co-ordinated national surveillance programme using standardised surveillance methods, case definitions and laboratory methods. Since the beginning of 2001, laboratory-confirmed influenza has been notifiable to the State Departments of Health. The case definition for influenza includes the isolation by culture or the detection by NAA or IF of influenza virus from an appropriate respiratory tract specimen, or the demonstration of seroconversion or rising IgG titre to influenza virus. Australian influenza pandemic plan Laboratories also participate in planning for potential influenza pandemics. These activities encompass surveillance for potentially pandemic influenza strains, liaison

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with WHO National or Collaborating Centres, and planning for responses to pandemic influenza. The Influenza Pandemic Planning Committee of the Communicable Diseases Network Australia New Zealand has addressed these issues.73 ACKNOWLEDGEMENTS We gratefully acknowledge the advice and assistance provided by Ken McPhie, Mala Ratnamohan ( Department of Virology, Westmead Hospital) and Belinda Herring ( Centre for Viral Research, Westmead Millennium Institute). Address for correspondence: Dr E. G. Playford, Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW 2145, Australia. E-mail: [email protected] u

References 1. Lamb RA, Krug RM. Orthomyxoviridae: The viruses and their replication. In: Fields BN, Knipe DM, Howley PM, editors. Fields Virology. Philadelphia: Lippincott-Raven, 1996. 2. Nicholson KG. Human influenza. In: Nicholson KG, Webster RG, Hays AJ, editors. Textbook of influenza. Oxford: Blackwell Science, 1998. 3. Thomas DB, Patera AC, Graham CM, Smith CA. Antibody-mediate d immunity. In: Nicholson KG, Webster RG, Hays AJ, editors. Textbook of influenza. Oxford: Blackwell Science, 1998. 4. Stevenson PD, Doherty PC. Cell-mediated immune response to influenza virus. In: Nicholson KG, Webster RG, Hays AJ, editors. Textbook of influenza. Oxford: Blackwell Science, 1998. 5. Murphy BR, Webster RG. Orthomyxoviruses. In: Fields BN, Knipe DM, Howley PM, editors. Fields Virology. Philadelphia: LippincottRaven, 1996. 6. Webster RG. Predictions for future human influenza pandemics. J Infect Dis 1997; 176: S14–9. 7. Zambon M. Laboratory diagnosis of influenza. In: Nicholson KG, Webster RG, Hays AJ, editors. Textbook of influenza. Oxford: Blackwell Science, 1998. 8. Donaldson A, Lewis FA, Kennett ML, White J, Gust ID. The 1976 influenza epidemic in Melbourne. Med J Aust 1978; 2: 45–9. 9. Cruz JR, Quinonez E, Fernandez A, Devalte F. Isolation of viruses from nasopharyngeal secretions. Comparison of aspiration and swabbing as means of sample collection. J Infect Dis 1987; 156: 415– 6. 10. Kaiser L, Briones MS, Hayden FG. Performance of virus isolation and Directogen Flu A to detect influenza A virus in experimental human infection. J Clin Virol 1999; 14: 191–7. 11. Covalciuc KA, Webb KH, Carlson CA. Comparison of four clinical specimen types for detection of influenza A and B viruses by optical immunoassay ( FLU OIA test) and cell culture methods. J Clin Microbiol 1999; 37: 3971–4. 12. Schmid ML, Kudesia G, Wake S, Read RC. Prospective comparative study of culture specimens and methods in diagnosing influenza in adults. BMJ 1998; 316: 275. 13. Heikkinen T, Salmi AA, Ruuskanen O. Comparative study of nasopharyngeal aspirate and nasal swab specimens for detection of influenza. BMJ 2001; 322: 138. 14. Cherian T, Bobo L, Steinhoff MC, Karron RA, Yolken RH. Use of PCT-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus. J Clin Microbiol 1994; 32: 623– 8. 15. Ellis J S, Fleming DM, Zambon MC. Multiplex reverse transcriptionPCR for surveillance of influenza A and B viruses in England and Wales in 1995 and 1996. J Clin Microbiol 1997; 35: 2076–82. 16. Schweiger B, Zadow I, Heckler R, Timm H, Pauli G. Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. J Clin Microbiol 2000; 38: 1552–8. 17. Fong CKY, Lee MK, Griffith BP. Evaluation of R-Mix FreshCells in shell vials for detection of respiratory viruses. J Clin Microbiol 2000; 38: 4660–2. 18. Huang YT, Turchek BM. Mink lung cells and mixed mink lung and A549 cells for rapid detection of influenza virus and other respiratory viruses. J Clin Microbiol 2000; 38: 422–3. 19. Johnson SLG, Wellens K, Siegel C. Comparison of hemagglutination and hemabsorption tests for influenza detection. Diagn Microbiol Infect Dis 1992; 15: 131– 4.

Pathology (2002 ), 34, April 20. Espy MJ, Smith TF, Harmon MW, Kendal AP. Rapid detection of influenza virus by shell vial assay with monoclonal antibodies. J Clin Microbiol 1986; 24: 677– 9. 21. Stokes CE, Bernstein JM, Kyger SA, Hayden FG. Rapid diagnosis of influenza A and B by 24-h fluorescent focus assay. J Clin Microbiol 1988; 26: 1263– 6. 22. Guenthner SH, Linneman CC. Indirect immunofluorescence assay for rapid diagnosis of influenza virus. Lab Med 1988; 19: 581–3. 23. Bartholoma NY, Forbes BA. Successful use of shell vial centrifugation and 16 to 18-hour immunofluorescent staining for the detection of influenza A and B in clinical specimens. Am J Clin Pathol 1989; 92: 487– 90. 24. Mills RD, Cain KJ, Woods GL. Detection of influenza virus by centrifugal inoculation of MDCK cells and staining with monoclona l antibodies. J Clin Microbiol 1989; 27: 2505– 8. 25. Engler HD, Preuss J. Laboratory diagnosis of respiratory virus infections in 24 hours by utilizing shell vial cultures. J Clin Microbiol 1997; 35: 2165–7. 26. Shih SR, Tsao KC, Ning HC, Huang YC, Lin TY. Diagnosis of respiratory tract viruses in 24h by immunofluorescent staining of shell vial cultures containing Madin-Darby canine kidney ( MDCK) cells. J Virol Methods 21999; 81: 77–81. 27. Waner JL, Todd SJ, Shalaby H, Murphy P, Wall LV. Comparison of Directogen FLU-A with viral isolation and direct immunofluorescenc e for the rapid detection and identification of influenza A virus. J Clin Microbiol 1991; 29: 479– 82. 28. Johnston SLG, Bloy H. Evaluation of a rapid enzyme immunoassay for detection of influenza A virus. J Clin Microbiol 1993; 31: 1421– 3. 29. Dominguez EA, Taber LH, Couch RB. Comparison of rapid diagnostic techniques for respiratory syncytial and influenza A virus respiratory infections in young children. J Clin Microbiol 1993; 31: 2286– 90. 30. Leonardi GP, Leib H, Birkhead GS, Smith C, Costello P, Conron W. Comparison of rapid detection methods for influenza A virus and their value in health-care management of institutionalized geriatric patients. J Clin Microbiol 1994; 32: 70–4. 31. Claas ECJ, van Milaan AJ, Sprenger MJW, et al. Prospective application of reverse transcription polymerase chain reaction for diagnosing influenza infections in respiratory samples from a Children’s Hospital. J Clin Microbiol 1993; 31: 2218– 21. 32. Doing KM, Jerkofsky MA, Dow EG, Jellison JA. Use of fluorescentantibody staining of cytocentrifuge-prepared smears in combination with cell culture for direct detection of respiratory viruses. J Clin Microbiol 1998; 36: 2112–4. 33. Landry ML, Cohen S, Ferguson D. Impact of sample type on rapid detection of influenza virus A by cytospin-enhanced immunofluorescence and membrane enzyme-linked immunosorbent assay. J Clin Microbiol 2000; 38: 429– 30. 34. Noyola DE, Clark B, O’Donnell FT, Atmar RL, Greer J, Demmler G J. Comparison of a new neuraminidase detection assay with an enzyme immunoassay, immunofluorescence, and culture for rapid detection of influenza A and B viruses in nasal wash specimens. J Clin Microbiol 2000; 38: 1161– 5. 35. Todd SJ, Minnich L, Waner JL. Comparison of rapid immunofluorescence procedure with TestPack RSV and Directogen FLU-A for diagnosis of respiratory syncytial virus and influenza A virus. J Clin Microbiol 1995; 33: 1650–1. 36. Hite SA, Huang YT. Microwave-accelerated direct immunofluorescen t staining for respiratory syncytial virus and influenza A virus. J Clin Microbiol 1996; 34: 1819–20. 37. Duverlie G, Houbart L, Visse B, et al. A nylon membrane immunoassay for rapid diagnosis of influenza A infection. J Virol Methods 1992; 40: 77– 84. 38. Hornsleth A, Jankowski M. Sensitive immunoassay for the rapid diagnosis of influenza A virus infections in clinical specimens. Res Virol 1990; 141: 373– 84. 39. Chomel JJ, Thouvenot D, Onno M, Kaiser C, Gourreau JM, Aymard M. Rapid diagnosis of influenza infection of NP antigen using an immunoscapture ELISA test. J Virol Methods 1989; 25: 81– 92. 40. Grandien M, Pettersson CA, Gardner PS, Linde A, Stanton A. Rapid viral diagnosis of acute respiratory infections: comparison of enzymelinked immunosorbent assay and the immunofluorescence technique for detection of viral antigens in nasopharyngeal secretions. J Clin Microbiol 1985; 22: 757– 60. 41. Harmon MW. Influenza virus. In: Lennette EH, Smith TF, editors. Laboratory diagnosis of viral infections. 3rd ed. New York: Marcel Dekker, 1999.

LABORATORY DIAGNOSIS OF INFLUENZA

42. Ryan-Poirier KA, Katz JM, Webster RG, Kawaoka Y. Application of Directogen FLU-A for the detection of influenza A virus in human and nonhuman specimens. J Clin Microbiol 1992; 30: 1072– 5. 43. Boivin G, Hardy I, Kress A. Evaluation of a rapid optical immunoassay for influenza viruses ( FLU OIA test) in comparison with cell culture and reverse transcription-PCR. J Clin Microbiol 2001; 39: 730– 2. 44. Herrmann B, Larsson C, Zweygberg BW. Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcriptionPCR: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay ( FLU OIA). J Clin Microbiol 2001; 39: 134– 8. 45. Quidel. QuickVue Influenza test. Mt Gravatt East, Qld: Quidel, 2000 ( package insert). 46. Sintchenko V, Gilbert GL, Coiera E, Dwyer D. Treat or test first? Decision analysis of empirical antiviral treatment of influenza versus treatment based on rapid test results. J Clin Virol ( in press ). 47. Wright KE, Wilson GAR, Novosad D, Dimock C, Tan D, Weber JM. Typing and subtyping of influenza viruses in clinical samples by PCR. J Clin Microbiol 1995; 33: 1180–4. 48. Atmar RL, Baxter BD, Dominguez EA, Taber LH. Comparison of reverse transcription-PCR with tissue culture and other rapid diagnostic assays for detection of type A influenza virus. J Clin Microbiol 1996; 34: 2604– 6. 49. Pregliasco F, Mensi C, Camorali L, Anselmi G. Comparison of RTPCR with other diagnostic assays for rapid detection of influenza viruses. J Med Virol 1998; 56: 168–73. 50. Fan J, Henrickson KJ, Savatski LL. Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay ( Hezplex ). Clin Infect Dis 1998; 26: 1397– 402. 51. Stockton J, Ellis JS, Saville M, Clewley, Zambon M C. Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses. J Clin Microbiol 1998; 36: 2990– 5. 52. Gr¨ondahl B, Puppe W, K¨uhne I, Weigl JAI, Schnitt HJ. Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study. J Clin Microbiol 1999; 37: 1–7. 53. Magnard C, Valette M, Aymard M, Lino B. Comparison of two nested PCR, cell culture, and antigen detection for the diagnosis of upper respiratory tract infections due to influenza viruses. J Med Virol 1999; 59: 215– 20. 54. Van Elden LJR , Nijhuis PS, Schuurman R, van Loon AM. Simultaneous detection of influenza viruses A and B using real-time quantitative PCR. J Clin Microbiol 2001; 39: 196–200. 55. Carman WF, Wallace LA, Walker J, et al. Rapid virological surveillance of community influenza infection in general practice. BMJ 2000; 321: 736–7. 56. Harmon MW, Phillips DJ, Reimer CB, Kendal AP. Isotype-specific enzyme immunoassay for influenza antibody with monoclonal antibodies to human immunoglobulins. J Clin Microbiol 1986; 24: 913– 6.

125

57. Rothbarth PH, Groen J, Bohnen AM, de Groot R, Osterhaus ADME. Influenza virus serology-acomparative study. J Virol Methods 1999; 78: 163– 9. 58. Vikerfors T, Lindegren G, Grandien M, van der Logt J. Diagnosis of influenza A virus infections by detection of specific immunoglobulin s M, A, and G in serum. J Clin Microbiol 1989; 27: 453–8. 59. Voeten JTM, Groen J, van Alphen D, et al. Use of recombinan t nucleoproteins in enzyme-linked immunosorbent assays for detection of virus-specific immunoglobulin A ( IgA) and IgG antibodies in influenza virus A- or B-infected patients. J Clin Microbiol 1998; 36: 3527– 31. 60. Royal College of Pathologists of Australasia Quality Assurance Programmes. Influenza Serology. Surry Hills: RCPA– QAP, 1996. 61. Noyola DE, Demmler GJ. Effect of rapid influenza diagnosis on management of influenza A infections. Pediatr Infect Dis J 2000; 19: 303–7. 62. Woo PCY, Chiu SS, Seto WH, Peiris M. Cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients. J Clin Microbiol 1997; 35: 1579– 81. 63. Schmidt NJ, Ota M, Gallo D, Fox VL. Monoclonal antibodies for rapid strain-specific identification of influenza virus isolates. J Clin Microbiol 1980; 16: 763– 5. 64. Walls HH, Harmon MW, Slagle JJ, Stocksdale C, Kendal AP. Characterization and evaluation of monoclonal antibodies developed for typing influenza A and B viruses. J Clin Microbiol 1986; 23: 240– 5. 65. Tk´acov´a M, Varecov´a E, Baker IC, Love JM, Ziegler T. Evaluation of monoclonal antibodies for subtyping of currently circulating human type A inflenza viruses. J Clin Microbiol 1997; 35: 1196– 8. 66. Ziegler T, Hall H, S´anchez-Fauquier A, Gamble WC, Cox NJ. Typeand subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay. J Clin Microbiol 1995; 33: 318–21. 67. Li J, Chen S, Evans DH. Typing and subtyping using DNA microarrays and multiplex reverse transcriptase PCR. J Clin Microbiol 2001; 39: 696–704. 68. Influenza sequence database. University of California, Los Alamos National Laboratory. http://www.flu.lanl.gov/index.html. Accessed 21/3/01. 69. Couch RB. Prevention and treatment of influenza. New Engl J Med 2000; 343: 1778– 87. 70. Gubareva LV, Kaiser L, Hayden FG. Influenza virus neuraminidas e inhibitors. Lancet 2000; 355: 827– 35. 71. McKimm-Breschkin JL. Resistance of influenza viruses to neuraminidase inhibitors – a review. Antiviral Res 2000; 47: 1–17. 72. Flunet. Global Influenza Surveillance Network. World Health Organization. http://ms2.b3e.jussieu.fr/flunet /. Accessed 21/3/01. 73. Influenza Pandemic Planning Committee of the Communicable Diseases Network Australia New Zealand. A framework for an Australian Influenza Pandemic Plan. Technical Report Series No. 4, Communicable Diseases Intelligence. Canberra: Australian Government Publishing Service, 1999. 74. Thomson J, Lin M, Hampson A. Annual report of the National Influenza Surveillance Scheme, 1999. Commun Dis Intell 2000; 24: 145– 54.