Influenza other diagnostic studies

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1] Associate Editor(s)-in-Chief: Mohammad Braizat, M.S. [2]

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Other Diagnostic Studies

Overview

This microchapter covers influenza diagnostic modalities beyond routine antigen-based point-of-care testing: nucleic acid amplification tests (NAATs, including RT-PCR and rapid molecular assays), multiplex respiratory panels, immunofluorescence, viral culture, serology, influenza A subtyping (including expedited H5 subtyping), and antiviral-resistance genotyping. Emphasis is on test selection by care setting, specimen choice and timing, interpretation of results in the context of community prevalence, and preanalytic factors affecting diagnostic yield.

Molecular Assays (NAAT) as Reference Standard

The 2024 IDSA/ASM Guide to Utilization of the Microbiology Laboratory states that NAAT is the gold standard for detecting influenza virus in clinical samples, with multiple FDA-cleared platforms including multiplex respiratory panels that differentiate influenza A from B. Rapid antigen tests have sensitivity of only 50–75% (office-based RIDT sensitivity is typically 50–70%), may perform poorly against certain strains (pandemic H1N1, swine-origin H3N2), and negative antigen results should be confirmed by NAAT or culture before ruling out influenza. Specificities for all influenza tests are generally >90%.

Performance and turnaround by category, per the AAP 2025–2026 technical report:

Test category	Method	Subtyping of influenza A	Time to result	Performance	CLIA status	References
Rapid molecular assay (POC NAAT)	Nucleic acid amplification	No	15–30 min	High sensitivity, high specificity	CLIA-waived (most)
RT-PCR / other NAAT	Nucleic acid amplification	Yes, if subtype primers used	1–8 h	Very high sensitivity and specificity	Moderate/high complexity
Multiplex molecular panel	Nucleic acid amplification (flu, SARS-CoV-2, RSV ± bacterial targets)	Yes, if subtype primers used	1–2 h	High sensitivity, high specificity	Moderate/high complexity
Rapid influenza diagnostic test (antigen)	Antigen detection	No	10–15 min	Moderate sensitivity (50–70% in office setting), high specificity	CLIA-waived
Immunofluorescence (DFA/IFA)	Antigen detection	No	2–4 h	Moderate sensitivity, high specificity	Moderate complexity
Rapid (shell vial) culture	Virus isolation	Yes	1–3 d	High sensitivity and specificity	Moderate/high complexity
Conventional viral culture	Virus isolation	Yes	3–10 d	High sensitivity and specificity	High complexity

Note: Negative results may not rule out influenza; sensitivity varies by assay, specimen type, and timing of collection.

Head-to-head pediatric data show CLIA-waived point-of-care NAATs (ID NOW Influenza A & B 2, cobas Liat, Xpert Xpress Flu) achieve 93–100% sensitivity for influenza A and 92–97% for influenza B against CDC RT-PCR, with specificity >97% — substantially better than the BD Veritor antigen test (79.5% for A, 66.7% for B). Invalid-result rates differ across platforms (0.5% ID NOW, 3.0% Xpert, 5.5% Liat), which is operationally relevant for high-throughput settings.

Specimen Selection, Timing, and Handling

• Nasopharyngeal swab has the highest yield; mid-turbinate nasal swab or combined nasal plus throat swabs are acceptable depending on the assay's validated specimen types.
• Collect specimens as close to illness onset as possible — ideally within 4 days of symptom onset, though viral RNA may persist longer in young children and immunocompromised patients.
• In hospitalized patients with respiratory failure, test lower respiratory tract specimens (endotracheal aspirate, BAL) if upper respiratory specimens are negative — a common source of missed diagnoses in influenza pneumonia. Note that many assays are not cleared for lower respiratory specimens; laboratory validation is required.
• Preanalytic factors — use viral transport medium as specified by the assay manufacturer; specimens should be processed within 24–72 hours if refrigerated; freezing may reduce viability for culture but is acceptable for molecular assays. Refer to CDC and manufacturer instructions for specific transport and storage conditions.

Indications for Testing

Diagnostic testing is beneficial when results will be used to inform clinical management or infection prevention measures and to distinguish influenza from other respiratory viruses.

Multiplex Testing During Co-Circulation

Influenza cannot be distinguished clinically from SARS-CoV-2 or from coinfection, so testing is needed when both circulate. Multiplex assays detecting influenza A/B, SARS-CoV-2, and RSV simultaneously identify coinfection and support divergent treatment pathways; turnaround ranges from 15–40 minutes to 8 hours depending on platform. Over-the-counter at-home combined influenza/SARS-CoV-2 tests are available under FDA emergency use authorization for symptomatic individuals ≥2 years; patients require counseling that a negative home test does not exclude influenza and should prompt contact with the clinician, particularly for high-risk patients.

Influenza A Subtyping and Expedited H5 Testing

Most clinical influenza assays do not distinguish avian influenza A(H5) from seasonal influenza A. Amid the ongoing A(H5) animal outbreak and sporadic human cases, in January 2025 the CDC recommended expedited subtyping of influenza A–positive specimens from hospitalized patients, particularly those in the ICU. Specimens positive for influenza A but negative for seasonal subtypes A(H1) and A(H3) must be forwarded to a public health laboratory as soon as possible and within 24 hours for A(H5) evaluation.

Operational considerations for H5 subtyping (CDC advisory):

• Obtain thorough history of animal exposures (poultry, cattle, raw milk) and travel.
• Initiate standard and droplet precautions; contact precautions if exposure to animals or contaminated environments.
• Subtype in the hospital's own clinical laboratory when available; commercial laboratory referral is an alternative.
• Notify the health department immediately upon identification of an unsubtypeable influenza A specimen.
• Current A(H5) human cases remain rare; subtyping is primarily a preparedness and surveillance activity. In a 2025 validation study of 740 influenza A–positive specimens, zero H5 positives were detected, underscoring that H5 subtyping is not a high-yield diagnostic activity in most clinical settings.

Antiviral Resistance Genotyping

Genotypic resistance testing (e.g., NA-H275Y for oseltamivir resistance, PA-I38X for baloxavir resistance) can be performed by sequencing or allele-specific RT-PCR on positive specimens. This is not routinely recommended for clinical management but may be considered in:

• Immunocompromised patients with prolonged viral shedding and clinical failure on antiviral therapy.
• Institutional outbreaks with suspected resistance.
• Public health surveillance during pandemics or when resistance rates rise.

Resistance testing is generally performed in public health or reference laboratories.

Serology

Serology is not recommended for clinical diagnosis. It requires paired acute and convalescent sera, specialized laboratories, and is confounded by high background rates of vaccination and prior exposure; its role is limited to vaccine studies and epidemiologic investigation.

Clinically Actionable Recommendations

Setting	Recommendation	Evidence strength	References
Hospitalized patients	Test with a molecular assay, not an antigen test.	Strong recommendation; moderate-quality evidence (observational)
Ambulatory setting	Prefer rapid molecular assays over RIDTs; test only when the result changes management.	Strong recommendation; moderate-quality evidence
High community influenza activity	Confirm a negative RIDT with a molecular assay before excluding influenza in a patient in whom the diagnosis would alter management.	Strong recommendation; moderate-quality evidence (test-performance data)
Low community influenza activity	Be skeptical of positive antigen results — positive predictive value falls and false positives increase.	Strong recommendation; moderate-quality evidence (Bayesian reasoning)
Intubated/critically ill with negative upper respiratory NAAT	Escalate to lower respiratory sampling (endotracheal aspirate, BAL).	Strong recommendation; low-quality evidence (observational/case series)
Unsubtypeable influenza A	Flag for immediate public health laboratory referral for A(H5).	Strong recommendation; expert opinion (CDC advisory)

Interpretation Algorithms

CDC provides algorithms for interpreting positive and negative influenza test results when influenza is and is not circulating: CDC Influenza Test Result Interpretation Algorithm.

Uncertainty and Controversy

• Detection of viral RNA does not equal replication-competent virus or infectivity; NAAT positivity can persist after clinical recovery, particularly in children and immunocompromised hosts, and there is no validated Ct threshold for defining infectiousness or for de-isolation.
• Whether multiplex panels improve patient-centered outcomes relative to targeted influenza NAAT — cost and stewardship implications are unsettled (expert opinion).
• Test-of-cure and serial testing in immunocompromised patients, where prolonged shedding and antiviral resistance emergence occur, lacks guideline-level direction (expert opinion).
• Guideline wording differs in emphasis: IDSA/ASM allows either NAAT or culture to confirm a negative antigen test, whereas AAP frames confirmation in molecular terms.

Clinical Pearls

• Pretest probability drives interpretation. The same result means different things in December versus July.
• A negative antigen test never justifies withholding antivirals in a hospitalized or high-risk patient with a compatible syndrome. (Refer to Influenza medical therapy for treatment recommendations.)
• Positive rapid tests support antimicrobial stewardship by reducing unnecessary antibacterial use and further workup.
• Influenza A positive but H1/H3 negative is an actionable alert, not a technical failure.
• Culture retains value for antigenic characterization, vaccine strain selection (via WHO/GISRS), and phenotypic susceptibility testing when NAAT is positive.

Common Pitfalls

• Sampling with a swab type or anatomic site not validated for the assay in use.
• Applying an upper-respiratory-only strategy to intubated patients with influenza pneumonia.
• Using an assay off-label on lower respiratory specimens without laboratory validation (expert opinion).
• Treating an OTC home test negative as definitive in a high-risk symptomatic patient.
• Ordering acute serology for diagnosis.