Laboratory diagnosis of human seasonal and pandemic influenza virus infection

Recovery of virus from nasopharyngeal aspirates, nasal washes, and bronchoalveolar lavages is superior to that from nasopharyngeal and throat swabs and expectorated sputum,5-7 as the latter generally contain less columnar and more squamous epithelial cells. Single-use swabs containing viral transport media are readily available commercially. Specimen requirements for antigen and nucleic acid testing are similar to those for virus isolation.

Early experience with human H5N1 infections suggests that virus is more readily detectable in lower respiratory tract samples. Upper respiratory tract samples are easier to collect, with the rate and load of detectable H5N1 RNA higher in samples collected from the pharynx (and the trachea) than the nose. Virus has been detected in serum, faeces and cerebrospinal fluid, reflecting the systemic nature of H5N1 infection, with viraemia associated with increased mortality.8

Serum specimens for serology should be collected during the acute phase within 7–10 days of symptom onset, and the convalescent phase 14–21 days after symptom onset, and tested in parallel.

The traditional method of influenza virus recovery from clinical specimens is by cell culture. However, the relatively slow turn-around time (the time from receipt of specimens by a laboratory to a result being available to the clinician) of at least 4–5 days has led to the use of more rapid assays, such as nucleic acid testing or immunofluoresence, and fewer laboratories isolating virus. Nevertheless, virus isolation is usually more sensitive than the rapid culture and antigen detection assays, it recovers novel or highly divergent strains missed by other tests, it provides an isolate for subsequent characterisation and consideration as potential vaccine strains, and it allows the simultaneous recovery of other respiratory viruses if an appropriate range of cell lines is used.

Influenza virus replication within cell culture, often using Madin Darby canine kidney cells, is detected by observing the cytopathic effect, generally manifested by 5 days.1 Rapid culture techniques that detect influenza virus replication using labelled monoclonal antibodies within 1–3 days (before the development of cytopathic effect) have a sensitivity of 56%–100% compared with conventional cell culture.9 Virus is readily isolated (PC3 facilities are recommended) from the nose and throat of humans with H5N1 infection, as well as from blood and rectal swabs.8

Virus isolation in the allantoic and amniotic cavities of 10–11-day-old embryonated chicken eggs is labour intensive and not commonly performed by diagnostic laboratories. However, it yields higher viral titres, and remains vital for vaccine production.

Initial typing of influenza virus isolates is most rapidly and conveniently accomplished by immunofluoresence using commercially available type-specific monoclonal antibodies, and should be performed as soon as possible after isolation. The reference virus subtyping method is the haemagglutination inhibition assay using specific antisera, a technique that characterises both antigenic drift and shift. More rapid subtyping techniques using monoclonal antibodies that differentiate between influenza A/H1N1, A/H3N2, and B virus subtypes have been developed for use on virus isolates, in rapid culture assays, and directly on clinical specimens.10,11 Subtyping using reverse transcription polymerase chain reaction (RT-PCR) with primers specific for various human and avian influenza strains can be performed on virus isolates or directly on clinical specimens.12-14 DNA microarrays have been used to detect type- and subtype-specific amplification sequences.15

Sequencing of amplified haemagglutinin and neuraminidase genes is an important subtyping method, as it allows rapid identification of novel or highly divergent strains, the analysis of strain variation, and the determination of the origin of outbreaks.16 Rapid sequencing of avian and human strains has been used to monitor the evolution of H5N1 viruses around the world, allowing an understanding of its spread, drug resistance and likely candidate vaccine strains.17

Immunofluoresence or enzyme immunoassay using commercial type-specific monoclonal antibodies directed against conserved influenza antigens are the most common rapid assays performed directly on clinical specimens (Box 5), although they are starting to be replaced by nucleic acid testing. Indirect or direct immunofluoresence is 60%–100% sensitive compared with cell culture.1,2 As specimens are examined with a fluorescent microscope, an assessment of specimen quality can be made (and reported to the clinician): where few columnar epithelial cells are seen, the sensitivity of immunofluoresence is low and repeat specimen collection should be requested.

Monoclonal antibodies to other common respiratory pathogens, such as parainfluenza viruses types 1–3, adenovirus and respiratory syncytial virus, can be applied to the same specimen. H5N1-specific immunofluoresence reagents are not yet routinely available.

The availability of the neuraminidase inhibitors has added impetus to the development of simple, rapid antigen detection assays, sometimes called point-of-care tests.1,2 Typically, these tests produce a visual result on an immunochromatographic strip using influenza A or B nucleoprotein-specific monoclonal antibodies within about 15 minutes of adding an extracted specimen (Box 6). Evaluations of these assays for H5N1 infection are limited, but for A/H3N2, A/H1N1 or B virus infections, they are 56%–100% sensitive and 73%–99% specific compared with cell culture or immunofluoresence, and are 56%–74% sensitive compared with RT-PCR.1,2,18 As with other techniques, specimen quality is a major determinant of their performance. Ideally, when using rapid antigen tests, specimens for cell culture and immunofluoresence or nucleic acid testing should be submitted in parallel to improve sensitivity and to obtain viral isolates for further typing and vaccine analysis, although this strategy increases costs.

Rapid antigen tests are attractive where virology laboratory support is limited or remote from clinical services, or where laboratories may be overwhelmed (for example, at the height of seasonal outbreaks or during a pandemic). They may also have a role in doctors’ surgeries, emergency departments, or “fever clinics”, to guide the early use of neuraminidase inhibitors, both for individuals or in outbreaks in closed environments, or during a pandemic.19 They can also be used in community-based surveillance programs.

Influenza virus RNA may be detected in clinical specimens by nucleic acid testing techniques such as RT-PCR. A variety of “in-house” nucleic acid testing assays using primers specific for a range of influenza virus genes, and various extraction, amplification and product detection methods have been described.12-14,18,20 Depending on primer selection, these assays may be type- or subtype-specific. Primers that target nucleoprotein or matrix gene sequences are popular, as they detect all influenza subtypes.

Although some studies documented a similar sensitivity of nucleic acid testing to cell culture, others have reported 5%–15% more influenza virus detections using RT-PCR.12,13,18 Specimen quality, timing, and transportation conditions may be less critical for nucleic acid testing than for culture or antigen detection, as viable virus and intact infected cells need not be preserved. Influenza virus RNA is detectable for several days longer into the clinical course than is cultivable virus, potentially allowing a diagnosis to be made in late-presenting patients. When specimen quality is limited, such as with community-based surveillance programs involving postal submission of specimens, yields from RT-PCR are significantly higher than from cell culture.12,13,21 Nucleic acid testing using H5N1-specific primers is the most sensitive assay for the diagnosis of human H5N1 infection.8

Although the turnaround time for nucleic acid testing is intermediate between cell culture and direct antigen detection, newer techniques can reduce this to 4–5 hours or less.20 Multiplex RT-PCR assays have been developed, and these can simultaneously detect influenza and other viral and bacterial respiratory pathogens directly in clinical specimens.1,20 RT-PCR can quantify the viral load in clinical specimens, as has been done in human H5N1-infected patients to assess prognosis and measure antiviral efficacy.8,22 Nucleic acid testing can subtype influenza viruses using subtype-specific primers, and analyse strain variation through genetic sequencing, thus complementing the traditional role of antigenic characterisation of viral isolates.

Serology can be used where specimens for virus isolation or antigen detection are negative, inadequate, or unavailable. It also provides useful, if delayed, surveillance data. Serological diagnosis of influenza is retrospective, as it requires acute and convalescent serum samples. Furthermore, it generally does not provide information on the antigenic composition of circulating strains.

As influenza virus infection generally represents reinfection, most people have some pre-existing immunity. Thus, the detection of influenza-specific IgG or total antibodies on a single serum specimen is not diagnostic of recent infection. The definitive serological diagnosis of acute influenza requires the demonstration of increasing antibody titres on paired acute and convalescent serum samples. Unfortunately, acute 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 provides presumptive evidence of recent infection.

A variety of influenza-specific assays are available, including complement fixation, haemagglutination inhibition assay, single radial haemolysis, neutralisation, immunofluorescence and enzyme immunoassay. The traditional “gold standard” techniques for detecting influenza-specific antibodies are neutralisation and the haemagglutination inhibition assay, as they can differentiate subtype-specific (including H5N1) and strain-specific serological responses. Antibody levels correlate accurately with protection from (or susceptibility to) influenza, and with vaccination responses. Complement fixation is more commonly used as it is easier to perform than the haemagglutination inhibition assay and neutralisation, but it does not distinguish between subtypes. Recent influenza infection is suggested by a fourfold rise in complement fixation titre or by a single high titre.1,23

Two classes of drugs are effective against influenza virus infection: the adamantane M2 channel inhibitors (amantadine and rimantadine), and the neuraminidase inhibitors (zanamivir and oseltamivir).24,25

Amantadine resistance emerges within a few days of treatment onset in 25%–35% of patients, and is mediated by amino acid substitutions in the M2 transmembrane region. Adamantane resistance is increasing around the world.24,25 Neuraminidase inhibitor resistance is most commonly associated with a variety of amino acid substitutions in the conserved residues of the neuraminidase enzyme active site. To date, however, viruses with these mutations have shown impaired replication in cell culture and animal models. Mutations in the haemagglutinin receptor-binding site can occur, leading to reduced viral dependence on neuraminidase function.24 Resistance to the neuraminidase inhibitors was rarely seen in immunocompetent humans during clinical trials, although more recent reports of children infected with influenza A in Japan, and some Vietnamese human H5N1 patients treated with neuraminidase inhibitors, showed that resistance may be more common than initially supposed.22,25 Resistant mutations have been detected after treatment of immunocompromised individuals, a situation that requires further study.

Drug resistance is tested using genotyping (sequence analyses of the neuraminidase gene) or phenotyping assays.24,25 These assays are not routinely available, but after therapeutic failure or for surveillance purposes, isolates can be referred to the appropriate reference laboratory. Newer molecular methods that allow rapid resistance determination in a clinically relevant time frame (such as during a pandemic when neuraminidase inhibitors may be used extensively) are under development.