Overview of Influenza Testing Methods

Overview of Influenza Testing Methods

Overview

Influenza virus testing is not mandatory for diagnosing influenza in outpatients with suspected cases, particularly during periods of heightened influenza activity when seasonal influenza A and B viruses are prevalent. Nevertheless, influenza virus testing can aid in clinical management, informing decisions on antiviral treatments, additional diagnostic testing, and implementing infection prevention strategies. Testing is advised for all patients with suspected influenza who are being admitted to the hospital. It is crucial for clinicians to comprehend the limitations of influenza virus tests and to interpret the results accurately, especially negative outcomes. During respiratory illness outbreaks in confined environments (such as hospitals, long-term care facilities, cruise ships, boarding schools, and summer camps), testing can be beneficial in identifying the cause of the outbreak.

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Influenza Virus Tests

Diagnostic tests for detecting influenza viruses in respiratory specimens include molecular assays (such as 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). While viral culture is essential for public health, it does not yield timely results for clinical management. The sensitivity and specificity of any influenza test can vary based on the testing method, the specific test used, the interval from the onset of illness to sample collection, specimen quality, the respiratory source, handling and processing timelines, and the duration from sample collection to testing. The predictive values (both positive and negative) of an influenza virus test are influenced by the prevalence of circulating seasonal influenza viruses in the population and the test's characteristics when benchmarked against a "gold standard" (like molecular assays or viral culture). As with any diagnostic test, results should be considered alongside other clinical and epidemiologic information. It is worth noting that serological testing does not provide timely results for guiding clinical decisions.

The Infectious Diseases Society of America (IDSA) recommends rapid influenza molecular assays over rapid influenza diagnostic tests (RIDTs) for diagnosing influenza viruses in outpatient respiratory specimens. For hospitalized patients, IDSA suggests employing RT-PCR or other molecular assays. Consult the IDSA Influenza Clinical Practice Guidelines for detailed recommendations on testing and interpreting results.

Rapid Molecular Assays

Rapid molecular assays are a type of molecular test that detects influenza virus nucleic acids in upper respiratory tract specimens with high sensitivity (90-95%) and specificity. FDA-cleared rapid molecular assays provide results in approximately 15-30 minutes, and some are CLIA-waived for point-of-care usage.

Other Molecular Assays

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and other molecular assays can reveal the presence of influenza viral RNA or nucleic acids in respiratory specimens with very high sensitivity and specificity. Certain molecular assays can detect and differentiate between influenza A and B infections, while others can identify specific seasonal influenza A virus subtypes [A(H1N1)pdm09 or A(H3N2)]. The turnaround time for these assays ranges from approximately 45 minutes to several hours. However, it's important to note that detecting influenza viral RNA or nucleic acids does not necessarily indicate viable infectious virus or ongoing viral replication. Not all assays have received FDA clearance for diagnostic purposes. Some multiplex molecular assays can detect influenza viral nucleic acids and differentiate influenza infections from other respiratory pathogens, which may be beneficial in managing severely immunosuppressed patients or understanding the etiology of respiratory illness outbreaks.

Rapid Influenza Diagnostic Tests

Rapid influenza diagnostic tests (RIDTs) are antigen detection assays capable of detecting influenza viral antigens within 10-15 minutes, with moderate sensitivity (50-70%) and high specificity. Some RIDTs are CLIA-waived and can be used in outpatient settings, while others require use in moderately complex labs. A few RIDTs incorporate an analyzer reader device to enhance result standardization, which improves sensitivity (75-80%). Currently, the FDA mandates that RIDTs achieve a minimum sensitivity of 80%. Detection of influenza virus antigens does not imply the presence of viable infectious virus or ongoing viral replication.

RIDTs do not provide subtype information for influenza A viruses. The acceptable specimen types can differ by test (nasopharyngeal swabs, nasal aspirates, or washes). The sensitivity and specificity of RIDTs are generally lower than viral culture and RT-PCR, and their performance can vary by individual test. Most rapid tests conducted in a physician's office exhibit a sensitivity of approximately 50-70% and specificity exceeding 90%. Recently, the FDA reclassified the RIDTs and created stipulations for enhanced accuracy, including increased sensitivity. Tests with low to moderate sensitivity and high specificity tend to result in false negatives more frequently than false positives, particularly during peak influenza activity. Therefore, clinicians should consider confirming negative results with molecular assays during high influenza activity, given the risk of false-negative RIDT results. Conversely, while false positives are less likely, they can still arise and are more frequent during low influenza activity periods. Thus, when interpreting RIDT results, the existence of influenza activity in the community must be considered (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 further guidance). Package inserts and the laboratory conducting the test should be consulted for additional details regarding the use of RIDTs.

Immunofluorescence

Immunofluorescence assays are antigen detection tests that generally require a fluorescent microscope, providing results in approximately 2-4 hours with moderate sensitivity and high specificity. Both direct (DFA) and indirect fluorescent antibody (IFA) staining assays are available for detecting influenza A and B antigens in respiratory specimens. Subtyping or further identification of influenza A viruses is unattainable through immunofluorescence assays. A rapid immunofluorescence assay, which is an RIDT, can produce results in about 15 minutes using an analyzer device.

Viral Culture

Viral culture results are not timely enough for informing clinical management. Shell-vial tissue culture results can take 1-3 days, while traditional tissue-cell cultures may take 3-10 days. However, viral culture allows for comprehensive antigenic and genetic characterization of influenza viruses. The collection of some respiratory samples for viral culture is vital for surveillance and understanding the antigenic characteristics of new seasonal influenza A and B strains that may require inclusion in the forthcoming year's vaccine.

Serologic Testing

Serological testing for influenza is not recommended for clinical decision-making. Although some commercial laboratories offer this testing, results for antibodies to influenza A or B viruses based on a single serum specimen are not reliably interpretable. Accurate serological testing for diagnosing influenza necessitates paired acute and convalescent sera collected 2-3 weeks apart, with reliable testing performed at select public health or research labs to assess a four-fold or greater rise in influenza virus strain-specific antibodies. Hence, serological testing does not yield timely results for clinical decision-making and is advised only for research and public health investigations.

Novel Influenza A Virus Infections

If there is suspicion of human infection with a novel influenza A virus from animal sources (such as avian or swine influenza viruses), local and state health departments should be contacted to conduct RT-PCR for seasonal and novel influenza A viruses. Commercial influenza diagnostic tests do not specifically detect novel influenza A viruses, and a positive result for influenza A cannot differentiate between seasonal and zoonotic virus infections. Further information on novel influenza A viruses is available at:

References

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. 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. Jan 28. doi: 10./cid/ciz056. [Epub ahead of print]