Table 2.

Laboratory diagnosis of influenza infections in the clinical setting55, 57

Testing method	Advantages	Disadvantages
Influenza virus
RIDT (immunoassay for antigens)	Results available in <30 min Applicable to a range of upper respiratory samples Differentiates between influenza A and B High specificity (90–95%)	Low sensitivity (40–70%); performance affected by virus subtype/strain, sampling time, specimen type and pre‐test probability Cannot distinguish between virus subtypes
Immunofluorescence, direct (DFA) or indirect (IFA) antibody staining	Results available within hours Applicable to upper and lower respiratory samples Detects influenza A, B and other viruses simultaneously (e.g. RSV) if included in the panel High specificity (90–95%)	Moderate sensitivity (50–85%) Quality specimen containing adequate epithelial cells (e.g. NP aspirate and flocked swab) and laboratory expertise is essential Cannot distinguish between virus subtypes
Viral cell culture (conventional or shell vial)	High specificity Allows virus subtyping, strain identification, titre assay/quantification and resistance testing Provides information on infectiousness	Moderate sensitivity (7–20% lower than PCR) Requires optimal sampling (e.g. prior to antiviral use) and post‐sampling processing conditions Results too slow to guide patient care (conventional: >3 to 10 days; shell vial: >2 to 3 days)
Reverse transcription PCR (rRT‐PCR)	Results available within hours High sensitivity and specificity (gold standard) Applicable to a wider range of specimen types Able to distinguish between virus subtypes (e.g. H1, H3, H5 and H7) and detect genotypic resistance (e.g. H275Y) Multiplex PCR methods may allow simultaneous detection of other respiratory pathogens	Accessibility, technical demands and cost are the practical concerns Unable to distinguish non‐viable from viable viruses ‘False‐negative’ upper tract samples (~10%) may result from site differential viral kinetic changes in pneumonia cases

Most RIDT are chromatographic immunoassays (some are fluorescence‐based immunoassays); applicable to NP swabs, nasal and/or throat swabs and NP aspirates/washes (training and protection equipment are required). Performance is best if applied within 48–72 h from onset before a significant drop in viral load (up to 4–5 days in selected populations). Lower sensitivity for A(H1N1)pdm09 virus has been reported.

Viral cell culture detects viable viruses, including those contained in the live‐attenuated influenza vaccines (LAIV). Isolates can be subjected to phenotypic resistance assays (e.g. neuraminidase enzyme inhibition assay). Viral load, specimen quality, transport, storage and processing techniques may affect test performance.

PCR assays can either provide universal detection of influenza A virus by targeting the matrix (M) gene or subtype‐specific virus detection (e.g. H1N1pdm09, H3N2, H5N1 and H7N9) by targeting the haemagglutinin (HA) gene. Viruses that cannot be subtyped may indicate a novel strain. Newer molecular‐based point‐of‐care tests may improve accessibility and reduce processing time and technical demands; some may allow detection of multiple viruses. Cost‐effectiveness of PCR is variable, depending on the circumstances.

Some multiplex PCR platforms may provide detection of >14 respiratory viruses (e.g. RSV, human metapneumovirus, parainfluenza virus, rhinovirus and coronavirus) and atypical pathogens (e.g. Mycoplasma pneumoniae and Chlamydophila pneumoniae).

DFA, direct fluorescent antibody test; IFA, immunofluorescence assay; NP, nasopharyngeal; RIDT, rapid influenza diagnostic test; RSV, respiratory syncytial virus.