Diagnostic Influenza Tests 

Updated: Sep 11, 2020
  • Author: Kenneth L Muldrew, MD, MPH, FCAP, FASCP, DABP-CP, MB, MGP; Chief Editor: Thomas M Wheeler, MD, FCAP  more...
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Reference Range

Diagnostic influenza tests aid with identification of influenza types A and B and influenza A subtypes 2009 H1N1, H1, H3, H5, N1, and N2. Variation exists among diagnostic methods for identification of types and subtypes of influenza (see Tables 1, 2, 3, and 4).

These tests can be used for any age or sex.

Qualitative: Positive or negative.

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Interpretation

If the value is positive, the patient may have influenza.

  • False-positive and true-negative test results occur more frequently when disease prevalence is low.

  • False-negative and true-positive test results occur more frequently when disease prevalence is high.

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Collection and Diagnostic Methods

Specimen: nasopharyngeal swab/aspirate, nasal swab/aspirate/wash, throat swab, bronchoalveolar lavage, sputum, in viral transport media, universal transport media, or test specific sample collection devices.

  • Acceptable specimens vary amongst diagnostic tests

  • Specimens from adults should be collected at the start of symptoms and usually no more than 4 or 5 days later. Very young children can shed influenza virus for more than 5 days.

  • Place specimen in viral transport medium.

  • Transport specimen on wet ice.

  • Specimens can be stored at 2-8º C and tested within 48 hours.

  • Specimens can be frozen at -80º for prolonged periods of time. Avoid multiple freeze-thaws since this results in a decrease in viral titer.

Table 1. Culture and Immunofluorescent Tests for Influenza [1, 2, 3, 4] (Open Table in a new window)

Detection Method

Influenza Types Identified

TAT*

CLIA Complexity**

 

Viral Culture

 

A & B

 

3-7 days

 

High

 

Rapid Culture (shell vial)

 

A & B

 

1-3 days

 

High

 

Direct Immunofluorescent Stain

 

A & B

 

2-3 hours

 

High

*TAT = turnaround time

**CLIA: Clinical Laboratory Improvement Amendments (amendments to the Public Health Service Act governing certification and oversight of clinical laboratory testing). CLIA complexity refers to the technical and interpretive complexity of performing the test, with each level requiring different levels of skill, aspects of training, supervision, and quality control.

Table 2. Immunofluorescent Tests (DFA & IFA) for Influenza [3, 5, 6, 7, 4] (Open Table in a new window)

 

 

Kit/Assay (manufacturer)

 

 

Sample Type

Influenza Types (Subtypes)

 

 

TAT

 

CLIA Complexity

D3 FastPointTM DFA Respiratory Virus Screening kit

(Diagnostic Hybrids, Inc.)

 

NA, NPA, NPS, NW, NS

 

A & B

 

30 min

 

High

 

 

D3 UltraTM DFA Respiratory Virus Screening & ID kit

(Diagnostic Hybrids, Inc.)

 

NA, NPA, NW & cell culture material

 

 

A & B

Direct specimen:

3 hr

 

Cell culture material: 3-7 days

High

 

 

High

ImagenTM Influenza Virus A and B DFA (ThermoFisher Scientific)

NPA and cell culture material

A & B

30 min

High

ImagenTM Respiratory Screen IFA (ThermoFisher Scientific)

 

NPA and cell culture material

 

A & B

 

30 min

 

High

*PathoDx® Respiratory Virus Panel (Remel)

Cell culture material

A & B

3-7 days

High

Light DiagnosticsTM Simulfluor® Viral Diagnostic Screen

(Millipore)

 

Cell culture material

 

A & B

 

3-7 days

 

High

Light DiagnosticsTM Respiratory Viral Screen IFA (Millipore)

Cell culture material

A & B

3-7 days

High

 

Light DiagnosticsTM Simulfluor® Flu A/Flu B DFA (Millipore)

 

BAL, NPA, NPS, NS, NW, TS, and cell culture material

 

 

A & B

Direct specimen: 30 min

 

Cell culture material: 3-7 days

High

Light DiagnosticsTM Simulfluor® RSV Flu A DFA (Millipore)

Cell culture material

A

3-7 days

High

*Note: these assays also detect parainfluenza, adenovirus, and respiratory syncytial virus. (DFA = direct fluorescent antibody, IFA = indirect fluorescent antibody, NPS = nasopharyngeal swab, NS = nasal swab, NPA = nasopharyngeal aspirate, NW = nasal wash, TS = throat swab, BAL = bronchoalveolar lavage)

Table 3. Rapid Antigen Tests (10-15 Minutes TAT) for Influenza [8, 5, 6, 7, 4] (Open Table in a new window)

 

Assay Name

 

Sample Types

CLIA Complexity

*Note: Same Assay Marketed by Different Companies:

1)  Biosign® Flu A+B (Princeton BioMeditech)

2)  Consult® Immunoassay Influenza A&B (McKesson)

3)  ImmunoCard STAT!® Flu A&B (Meridian Bioscience)

4)  OraSure Quick Flu Rapid Flu A+B Test (OraSure Tech)

5)  OSOM® Ultra Flu A&B (Sekisui Diagnostics)

6)  Status Flu A&B (Life Sign LLC)

NPS

NS

 

NW

NPA

Waived

 

 

Moderate

XpectTM Flu A&B (ThermoFisher Scientific)

NS

NW

TS

 

Moderate

QuickVue Influenza A+B Test(Quidel Corporation)

NS

NPA

Waived

Alere BinaxNOW® Influenza A&B Card 2 (Alere)

NPS

NS

Waived

BD VeritorTM (Becton Dickinson)

 

 

NPS

NS

 

Nasal Aspirate

NPA

NPS

Waived

 

 

 

Moderate

SofiaTM Influenza A+B FIA (Quidel Corporation)

NPA

NPS

NS

NW

 

Waived

AlereTM Influenza A & B (Alere)

NS

Waived

All are immunochromatographic except for the immunofluorescent SofiaTM Influenza A+B FIA assay. All tests detect both influenza A and B.

Table 4. Commercially Available, FDA-Cleared Molecular Tests for Influenza (not all-inclusive) [8, 5, 6, 7, 4] (Open Table in a new window)

 

Test Name (Manufacturer)

 

System

 

Method

Influenza Type (Subtype)

 

Sample Type

 

 TAT

CLIA Complexity

Xpert® Xpress Flu (Cepheid)

GeneXpert® Systems

Multiplex real-time RT-PCR

A & B

NPS in VTM

30 min

Waived

Xpert® Xpress Flu/RSV (Cepheid)

GeneXpert® Systems

Multiplex real-time RT-PCR

A & B

NPS in VTM

30 min

Waived

Xpert® Flu (Cepheid)

GeneXpert® Systems

Multiplex real-time RT-PCR

A & B

NPS, NA, NW in VTM

1.25 hr

Moderate

Xpert® Flu/RSV XC (Cepheid)

GeneXpert® Systems

Multiplex real-time RT-PCR

A & B

NPS, NA, NW in VTM

1 hr

Waived

cobas® Liat Influenza A/B

(Roche Molecular)

cobas® Liat System

Multiplex real-time RT-PCR

A & B

NPS

20 min

Waived

cobas® Liat Influenza A/B & RSV (Roche Molecular)

cobas® Liat System

Multiplex real-time RT-PCR

A & B

NPS

20 min

Waived

 

AlereTM i Influenza A&B (Alere)

 

AlereTM i

Isothermal nicking enzyme amplification reaction (NEAR)

 

A & B

NPS & NS direct or in VTM

 

15 min

 

Waived

 

AlereTM i Influenza A&B 2 (Alere)

 

AlereTM i

Isothermal nicking enzyme amplification reaction (NEAR)

 

A & B

NPS & NS direct or in VTM

 

15 min

 

Waived

AcculaTM Flu A/B Test

(Mesa Biotech, Inc)

AcculaTM Dock

Multiplex RT-PCR with colorimetric analysis

A & B

NS

30 min

Waived

 

Solana® Influenza A+B Assay (Quidel Corp)

 

Solana®

Isothermal reverse transcriptase helicase-dependent amplification

 

A & B

 

NPS, NS in VTM

 

45 min

 

Moderate

 

Lyra Influenza A+B (Quidel Corp)

ABI 7500 Fast and QuantStudioTM

Real-Time PCR Systems

Multiplex real-time RT-PCR

A & B

NS & NPS in VTM

1.25 hr

Moderate

Panther Fusion Flu A/B/RSV (Hologic, Inc)

Panther Fusion System

Multiplex real-time RT-PCR

A & B

NPS

2.5 hr

High

ARIES Flu A/B & RSV Assay (Luminex Corporation)

ARIES and ARIES M1 Systems

Multiplex real-time RT-PCR with melt curve analysis

A & B

NPS in VTM

2 hr

Moderate

artus® Influenza A/B RG Kit (Qiagen)

Rotor-Gene Q

Multiplex real-time RT-PCR

A & B

NPS in VTM

4 hr

High

SimplexaTM Flu A/B & RSV Direct (DiaSorin Molecular)

3M Integrated Cycler

Multiplex real-time RT-PCR

A & B

NPS in VTM

1 hr

Moderate

SimplexaTM Flu A/B & RSV

(DiaSorin Molecular)

3M Integrated Cycler

Multiplex real-time RT-PCR

A & B

NPS in VTM

4 hr

High

SimplexaTM Influenza A H1N1 (2009) Kit (DiaSorin Molecular)

3M Integrated Cycler

Multiplex real-time RT-PCR

A (2009 H1)

NPA, NPS, NS in VTM

4 hr

High

*FilmArray Respiratory Panel (BioFire Diagnostics)

FilmArray System, FilmArray System 2.0 or FilmArray Torch System

Multiplex RT-PCR with endpoint melt curve analysis

A (H1/H3/H1 2009) & B

 

NPS in VTM

 

1 hr

 

Moderate

*FilmArray Respiratory Panel 2.0 (BioFire Diagnostics)

FilmArray, FilmArray 2.0 or FilmArray Torch

Multiplex RT-PCR with endpoint melt curve analysis

A (H1/H3/H1 2009) & B

NPS in VTM

45 min

Moderate

 

*FilmArray Respiratory Panel EZ (BioFire Diagnostics)

FilmArray System,

FilmArray System 2.0 or

FilmArray Torch System

Multiplex RT-PCR with endpoint melt curve analysis

A (H1/H3/H1 2009) & B

 

NPS in VTM

 

1 hr

 

Waived

 

xTAGTM Respiratory Virus Panel (Luminex Corp)

Standard Thermal Cycler and Luminex 100 or 200 System

Multiplex RT-PCR with target-specific primer extension and bead-based array detection

 

A (H1/H3) & B

 

NPS in VTM

 

7.5 hr

 

High

xTAGTM Respiratory Virus Panel Fast (Luminex Corp)

Standard Thermal Cycler and Luminex

100 or 200 System

Multiplex RT-PCR with target-specific primer extension and bead-based array detection

 

A (H1/H3) & B

 

NPS in VTM

 

6 hr

 

High

NxTAGTM Respiratory Virus Panel (Luminex Corp)

 

MAGPIX

Multiplex RT-PCR with target-specific primer extension and bead-based array detection

 

A (H1/H3) & B

 

NPS in VTM

 

3.8 hr

 

High

*Verigene® Respiratory Pathogens Flex Nucleic Acid Test

(Luminex Corp)

 

Verigene® System

Multiplex RT-PCR & nanogrid microarray hybridization

with silver/gold nanoparticle photometric detection

 

A (H1/H3/H1 2009) & B

 

NPS in VTM

 

2 hr

 

Moderate

*ePlex® Respiratory Virus Panel (GenMark Diagnostics)

ePlex® System

Multiplex RT-PCR with probe detection using voltammetry

A (H1/H3/H1 2009) & B

NPS in VTM

1.75 hr

Moderate

*eSensor® Respiratory Virus Panel (GenMark Diagnostics)

eSensor® XT-8TM System

Multiplex RT-PCR with probe detection using voltammetry

A (H1/H3/H1 2009) & B

NPS in VTM

6 hr

High

*Note: panels include other respiratory viruses and bacteria.  (VTM = viral transport media, RT-PCR = reverse transcriptase PCR)

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Background

Description

In recent years, companies have introduced several rapid antigen assays, immunofluorescent reagents, rapid viral culture methods, and molecular tests for influenza virus. Some of the molecular methods can rapidly determine influenza A subtypes, which can be useful in determining the potential for resistance to some of the antiviral agents used in therapy. [4]

Indications/Applications

Diagnostic tests for influenza are useful in establishing a diagnosis and in the management of patients who have signs and symptoms compatible with influenza. They are also used to determine whether outbreaks of respiratory disease are due to influenza.

The susceptibility of influenza virus types and subtypes varies by antiviral (Table 4). Some of the molecular tests can rapidly provide the influenza type and subtype data.

Table 5. Susceptibility Profile of Influenza Types and Subtypes to Antiviral Agents [4] (Open Table in a new window)

 

Type/Subtype

 

Amantadine

 

Rimantadine

 

Oseltamivir

 

Peramivir

 

Zanamivir

Flu A 2009 H1N1

Resistant

Resistant

Sensitive

Sensitive

Sensitive

Seasonal flu A H1N1

Resistant

Resistant

Sensitive

Sensitive

Sensitive

Seasonal flu A H3N2

Resistant

Resistant

Sensitive

Sensitive

Sensitive

Flu B

Resistant

Resistant

Sensitive

Sensitive

Sensitive

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Considerations

Rapid antigen tests, immunofluorescent tests, and virus culture have been reported to often lack in assay specificity and/or sensitivity, testing time, and ability to subtype for influenza, compared with molecular methods. [2]  In a study of the nucleic acid amplification tests ID Now (Abbott), Cobas Influenza A/B Assay (Roche Molecular Diagnostics), and Xpert Xpress Flu (Cepheid), Kanwar et al found the three products to have comparable sensitivities for influenza A (93.2%, 100%, 100%, respectively) and B (97.2%, 94.4%, 91.7%, respectively) detection. In addition, each product had greater than 97% specificity for influenza A and B detection. [9]

Rapid antigen tests generally have a sensitivity of 50-70% and a specificity of 90-95%. Limited studies have demonstrated very low sensitivity for detection of 2009 H1N1 with some commercial brands. [10, 11, 12, 13, 14, 15]  A study by Fowlkes et al found that of 3681 patients who tested positive for influenza on real-time reverse transcription polymerase chain reaction (RT-RT-PCR) assay, 40% displayed negative results on rapid influenza diagnostic testing. PCR assays were run for influenza A (H1N1, H1N1pdm09, H3N2) and influenza B. [16]

In 2017, the US Food and Drug Administration (FDA) reclassified rapid antigen-based tests from class I to class II to improve the quality of these tests for influenza. [5] The FDA has required manufacturers to meet minimum sensitivity and specificity requirements compared with culture and/or molecular methods, [5] with these requirements being higher than previously used for FDA clearance of new products. To continue to market these antigen tests, companies had to modify them to meet these new specifications.

Immunofluorescent tests, with nasopharyngeal specimens, have been reported to have high sensitivity and specificity for identification of influenza A and B. [3]

Very early and very late in respiratory season, and when disease prevalence in the community is low, positive rapid antigen diagnostic test results should be confirmed by an alternate method, such as viral culture or a molecular test. [5]

Hospitalized patients with a serious respiratory condition should preferably be tested by a molecular method for influenza, and in many cases, institutions are shifting towards respiratory virus syndromic testing, which targets multiple pathogens in a single assay, including parainfluenza, adenovirus, respiratory syncytial virus, coronaviruses, and rhinoviruses/enteroviruses. [5]

Previously, only high-complexity tests that required a long turnaround time were performed using molecular methods, some of which are listed in Table 4. More recently, simple, rapid molecular tests have been developed and marketed for use as point-of-care devices (waived tests listed in Table 4). These tests are comparable to rapid antigen tests in fulfilling the needs of outpatient clinics in that they are rapid and easy to use as a point-of-care device. In addition, their sensitivity and specificity are similar to those of high-complexity molecular tests. The downside is that they are more expensive than rapid antigen tests.

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