Overview of Influenza Testing Methods
Influenza virus testing is not required to make a clinical diagnosis of influenza in outpatients with suspected influenza, particularly during increased influenza activity when seasonal influenza A and B viruses are circulating in the local community. However, influenza virus testing can inform clinical management when the results may influence clinical decisions such as whether to initiate antiviral treatment, perform other diagnostic testing, or to implement infection prevention and control measures for influenza. Influenza virus testing is recommended for all patients with suspected influenza who are being admitted to hospital. Most importantly, clinicians should understand the limitations of influenza virus tests and how to properly interpret the results, particularly negative results. During a respiratory illness outbreak in a closed setting (e.g., hospitals, long-term care facility, cruise ship, boarding school, summer camp) testing for influenza virus infection can be very helpful in determining if influenza is the cause of the outbreak.
Diagnostic tests available for detection of influenza viruses in respiratory specimens include molecular assays (including rapid molecular assays, reverse transcription polymerase chain reaction (RT-PCR) and other nucleic acid amplification tests); and antigen detection tests (including rapid influenza diagnostic tests and immunofluorescence assays). Viral culture is important for public health purposes, but does not provide timely results to inform clinical management. Sensitivity and specificity of any test for influenza viruses in respiratory specimens might vary by the type of testing method and specific test used, the time from illness onset to specimen collection, the quality of the specimen collected, the respiratory source of the specimen, handling and processing of the specimen, and the time from specimen collection to testing. The post-test probability or predictive values (positive and negative predictive values) of an influenza virus test depend upon the prevalence of circulating seasonal influenza viruses in the patient population, and the specific test characteristics (sensitivity and specificity) compared to a “gold standard” comparison test (molecular assay or viral culture). As with any diagnostic test, results should be evaluated in the context of other clinical and epidemiologic information available to health care providers. Serological testing does not provide timely results to inform clinical management decisions.
The Infectious Diseases Society of America (IDSA) recommends use of rapid influenza molecular assays over rapid influenza diagnostic tests (RIDTs) for detection of influenza viruses in respiratory specimens of outpatients. IDSA recommends use of RT-PCR or other molecular assays for detection of influenza viruses in respiratory specimens of hospitalized patients. Consult the IDSA Influenza Clinical Practice Guidelines for recommendations on influenza testing and information on interpretation of testing results.
Rapid molecular assays are a kind of molecular influenza diagnostic test to detect influenza virus nucleic acids in upper respiratory tract specimens with high sensitivity (90-95%) and specificity. FDA-cleared rapid molecular assays are available that produce results in approximately 15-30 minutes. Some of these rapid molecular assays are CLIA-waived for point-of-care use.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and other molecular assays can identify the presence of influenza viral RNA or nucleic acids in respiratory specimens with very high sensitivity and specificity. Some molecular assays are able to detect and discriminate between infections with influenza A and B viruses; other tests can identify specific seasonal influenza A virus subtypes [A(H1N1)pdm09, or A(H3N2)]. These assays can yield results in approximately 45 minutes to several hours depending upon the assay. Notably, the detection of influenza viral RNA or nucleic acids by these assays does not necessarily indicate detection of viable infectious virus or on-going influenza viral replication. It is important to note that not all assays have been cleared by the FDA for diagnostic use. Some multiplex molecular assays are available that can detect influenza viral nucleic acids and distinguish influenza virus infection from other respiratory pathogens and may also be useful for management of severely immunosuppressed patients, or for use in identifying the cause of an institutional outbreak of respiratory illness.
Rapid influenza diagnostic tests (RIDTs) are antigen detection assays that can detect influenza viral antigens in 10-15 minutes with moderate sensitivity (50-70%) and high specificity. Some tests are CLIA-waived and approved for use in any outpatient setting, whereas others must be used in a moderately complex clinical laboratory. Some RIDTs utilize an analyzer reader device to standardize results to improve sensitivity (75-80%). FDA now requires RIDTs to achieve 80% sensitivity. Detection of influenza virus antigen does not necessarily indicate detection of viable infectious virus or on-going influenza viral replication.
None of the rapid influenza diagnostic tests provide any information about influenza A virus subtypes. The types of specimens acceptable for use (i.e., nasopharyngeal, or nasal aspirates, swabs, or washes) also vary by test. The specificity and, in particular, the sensitivity of rapid influenza diagnostic tests are lower than for viral culture and RT-PCR and vary by test. Most of the rapid influenza diagnostic tests that can be done in a physician’s office are approximately 50-70% sensitive for detecting influenza virus antigens and greater than 90% specific. Recently the FDA has reclassified the RIDTs and published requirements for improved accuracy, including higher sensitivity. Tests with low to moderate sensitivity and high specificity can produce false negative results more commonly than false positive results, especially during peak influenza activity in the community. Because of the lower sensitivity of the rapid influenza diagnostic tests, clinicians should consider confirming negative test results with molecular assays, especially during periods of peak community influenza activity and/or during suspected institutional influenza outbreaks because of the possibility of false-negative RIDT results. In contrast, false-positive RIDT results are less likely, but can occur and are more common during periods of low influenza activity. Therefore, when interpreting results of a rapid influenza diagnostic test, clinicians should consider the of the test in the context of the level of influenza activity in their community ( See Algorithm to assist in the interpretation of influenza testing results and clinical decision-making during periods when influenza viruses are circulating in the community and Algorithm to assist in the interpretation of influenza testing results and clinical decision-making during periods when influenza viruses are NOT circulating in the community for more information). Package inserts and the laboratory performing the test should be consulted for more details regarding use of rapid influenza diagnostic tests.
Immunofluorescence assays are antigen detection assays that generally require use of a fluorescent microscope to produce results in approximately 2-4 hours with moderate sensitivity and high specificity. Both direct (DFA) and indirect fluorescent antibody (IFA) staining assays are available to detect influenza A and B viral antigens in respiratory tract specimens. Subtyping or further identification of influenza A viruses is not possible by immunofluorescence assays. One rapid immunofluorescence assay is an RIDT and utilizes an analyzer device to produce results in approximately 15 minutes.
Viral culture results do not yield timely results to inform clinical management. Shell-vial tissue culture results may take 1-3 days, while traditional tissue-cell viral culture results may take 3-10 days. However, viral culture allows for extensive antigenic and genetic characterization of influenza viruses. The collection of some respiratory samples for viral culture is essential for for surveillance and antigenic characterization of new seasonal influenza A and B virus strains that may need to be included in the next year’s influenza vaccine.
Serological testing for influenza is not recommended for clinical decision-making. Although offered by some commercial laboratories, serological testing results for antibodies to influenza A or B viruses on a single serum specimen cannot be reliably interpreted. Proper serological testing for diagnosis of influenza requires paired acute and convalescent sera collected 2-3 weeks apart, with reliable testing at a limited number of public health or research laboratories to assess a 4-fold or greater rise in influenza virus strain-specific antibodies. Therefore, serological testing for influenza does not provide timely results to help with clinical decisions-making and is not recommended except for research and public health investigations.
If human infection with a novel influenza A virus of animal origin (e.g. avian influenza A virus or swine influenza A virus) is suspected, the local and state health department should be contacted to perform RT-PCR for seasonal influenza viruses and novel influenza A viruses. Commercially available influenza diagnostic tests do not specifically detect novel influenza A viruses and a positive result for influenza A virus cannot distinguish seasonal influenza A virus from avian or swine influenza A virus infections. Information about novel influenza A viruses is available at:
- Testing, Reporting & Lab Information
- Interim Guidance for Clinicians on Human Infections with Variant Influenza Viruses
- Table 1: Influenza Virus Testing Methods
Merckx J, Wali R, Schiller I, Caya C, Gore GC, Chartrand C, Dendukuri N, Papenburg J. Diagnostic Accuracy of Novel and Traditional Rapid Tests for Influenza Infection Compared With Reverse Transcriptase Polymerase Chain Reaction: A Systematic Review and Meta-analysis. Ann Intern Med. 2017 Sep 19;167(6):394-409.
Vos LM, Bruning AHL, Reitsma JB, Schuurman R, Riezebos-Brilman A, Hoepelman AIM, Oosterheert JJ. Rapid molecular tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review of diagnostic accuracy and clinical impact studies. Clin Infect Dis. 2019 Jan 28. doi: 10.1093/cid/ciz056. [Epub ahead of print]