Diagnostic Influenza Tests

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.

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.

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)

*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)

*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)

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)

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

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)

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]

A literature review by Gentilotti et al indicated that for use as point-of-care tools in community settings, rapid antigen tests for influenza, respiratory syncytial virus, human metapneumovirus, and Streptococcus pneumoniae are not completely reliable, having high false-negative rates. Although specificity for such testing was high (>80%), sensitivity was considered to be suboptimal (49-84%).[17]

Employing a home-use rapid diagnostic test (RDT) for influenza, a study by Geyer et al found that the test’s accuracy was comparable to that associated with many clinically used RDTs. Compared with the laboratory reference test (a quantitative RT-PCR assay), the home test had an overall sensitivity and specificity of 61% and 95%, respectively. However, an increase in false-negative outcomes was found when the self test was used more than 72 hours after symptom onset.[18]

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.