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Interim Recommendations for Clinical Use of Influenza Diagnostic Testing During the 2009-2010 Influenza Season

September 29, 2009, 6:00 PM ET

For Health Care Providers

• What does CDC recommend this season regarding testing for influenza?
• What is the reason for these recommendations?
• Does CDC recommend diagnostic testing for patients with uncomplicated illness from suspected influenza infection?
• Should hospitalized patients with suspected influenza infection receive diagnostic testing?
• In what situations are influenza diagnostic tests useful for patients who are not severely ill?
• What considerations should clinicians consider when interpreting the results of influenza diagnostic tests?
• What are the different kinds of influenza diagnostic tests?
• What laboratory diagnostic tests should be done for hospitalized patients with suspected influenza?
• What does a positive/negative rapid influenza diagnostic test (RIDT) or direct immunofluorescence assay (DFA) test result mean?
• What are the sensitivities and availability of rapid influenza diagnostic tests?
• What are the sensitivities and availability of direct immunofluorescence assays (DFAs)?
• What are the sensitivities and availability of real-time reverse transcriptase polymerase chain reaction (rRT-PCR) diagnostic tests?
• What real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays have been authorized by the Food and Drug Administration (FDA) to diagnose 2009 H1N1 influenza virus infection?
• Which laboratories can perform real-time reverse transcriptase polymerase chain reaction (rRT-PCR) testing in the United States?
• Which respiratory specimens are preferred for influenza virus diagnostic testing?
• How should swab specimens be collected?
• How should clinical specimens be stored?
• References

What does CDC recommend this season regarding testing for influenza?

This season CDC recommends that influenza diagnostic testing be prioritized for 1) hospitalized patients with suspected influenza; 2) patients for whom a diagnosis of influenza will inform decisions regarding clinical care, infection control, or management of close contacts; and 3) patients who died of an acute illness in which influenza was suspected. Most patients with a clinical illness consistent with uncomplicated influenza who reside in an area where influenza viruses are circulating do not require diagnostic influenza testing for clinical management.  Clinical judgment is another important factor in making decisions regarding testing. For more information, see Interim Recommendations for Clinical Use of Influenza Diagnostic Testing During the 2009-2010 Influenza Season.

What is the reason for these recommendations?

The purpose of the Interim Recommendations for Clinical Use of Influenza Diagnostic Testing During the 2009-2010 Influenza Season is to assist clinicians with influenza diagnostic testing decisions – including testing for 2009 H1N1 influenza (previously referred to as novel influenza A (H1N1)) and seasonal influenza – for the 2009-2010 influenza season.

Does CDC recommend diagnostic testing for patients with uncomplicated illness from suspected influenza infection?

Most patients with a clinical illness consistent with uncomplicated influenza who reside in an area where influenza viruses are known to be circulating do not require diagnostic influenza testing for clinical management. In certain situations, influenza diagnostic tests may provide additional information that is useful for clinical care decisions (see question: “In what situations are influenza diagnostic tests useful for clinical care decisions”). Clinicians should use their judgment in addition to these recommendations to decide whether to test for influenza in patients with uncomplicated illness.

Should hospitalized patients with suspected influenza infection receive diagnostic testing?

Yes. Hospitalized patients with suspected influenza should be tested with an available influenza diagnostic test. However, appropriate antiviral treatment and infection control measures should not be delayed pending diagnostic testing results. If influenza infection is clinically suspected, early empiric antiviral therapy should be initiated in hospitalized patients because antiviral medications are most effective when administered as early as possible.  

In what situations are influenza diagnostic tests useful for patients who are not severely ill?

Influenza diagnostic testing of patients who are not severely ill may help inform decisions regarding clinical care, infection control, or management of close contacts in certain situations. Clinicians should use their judgment to decide when to test for influenza in patients who are not severely ill. In addition, specifically testing for 2009 H1N1 influenza by rRT-PCR may be important for patients with certain conditions, such as pregnancy or severe immunosuppression, to improve their clinical care. However, if flu is suspected in these or other high risk patients, treatment should still be initiated while awaiting test results and should not be delayed since antiviral medications are most beneficial when started within the first 2 days of illness.

What considerations should clinicians consider when interpreting the results of influenza diagnostic tests?

When interpreting influenza diagnostic test results, clinicians should consider the following factors:

• Sensitivity of the influenza diagnostic test used
• The patient’s stage of illness (influenza diagnostic tests are more likely to be positive when performed in the first three days of illness when viral levels are highest)
• Local surveillance information on circulating influenza viruses and other respiratory viruses
• For information on interpreting a positive or negative test result, please see question “What does a positive/negative rapid influenza diagnostic test (RIDT) or direct immunofluorescence assay (DFA) test result mean?”

What are the different kinds of influenza diagnostic tests?

A number of diagnostic tests are available to detect the presence of influenza viruses in respiratory specimens. Diagnostic tests conducted by laboratories that are available for detecting influenza virus include viral culture, real-time reverse transcriptase polymerase chain reaction (rRT-PCR), rapid influenza diagnostic tests (RIDTs) and direct immunofluorescence assays (DFAs). These tests differ in their sensitivity and specificity for detecting influenza viruses, commercial availability, processing time, approved clinical setting, and ability to distinguish between different influenza virus types (A versus B) and influenza A subtypes (e.g., 2009 H1N1 versus seasonal H1N1 versus seasonal H3N2 viruses). Also, these tests vary in the chance that they will miss an influenza infection and sometimes falsely detect an infection. During an influenza outbreak, a positive test is likely to indicate influenza infection.

A description of the different types of influenza diagnostic tests is provided below:

• Rapid influenza diagnostic tests (RIDTs):

  Rapid influenza diagnostic tests (RIDTs) are widely available, commercial diagnostic tests that can detect influenza viruses in 30 minutes or less. Certain RIDTs can be performed outside of the laboratory in an outpatient setting (e.g., doctor’s offices or health clinics). These rapid tests differ in whether or not they can distinguish between influenza A and B viruses. These tests also vary in the chance that they will miss an influenza infection. For example, the sensitivity of these tests for detecting 2009 H1N1 varies from 10-70%¹. Therefore, a negative test does not exclude influenza infection. During an influenza outbreak, a positive test is likely to indicate influenza infection. Depending on which commercially available RIDT is used,the test can either 1) detect and distinguish between influenza A and B viruses; or 2) detect both influenza A and B but not distinguish between influenza A and B viruses. For more information on the sensitivity, specificity and interpretation of RIDT results, see Interim Guidance for the Detection of Novel Influenza A Virus Using Rapid Influenza Diagnostic Tests.

• Viral Culture:

  Viral culture is available in certain laboratories but often does not provide results in time to help with clinical decisions. However, viral culture is an important source of public health virologic data on influenza antigenic characteristics and antiviral susceptibilities. Viral culture is highly sensitive and specific.

• Direct immunofluorescence assays (DFAs) and Indirect immunofluorescence assays (IFAs):

  Immunofluorescence (fluorescent antibody staining) is available at many hospital laboratories and can generally yield test results in 2 to 4 hours, and sensitivities are generally higher than rapid tests, but lower than viral culture or real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Like RIDTs, direct immunofluorescence assays (DFAs) are widely available and have variable sensitivity for detecting 2009 H1N1 virus (range 47-93%)². DFAs detect and distinguish between influenza A and B viruses but do not distinguish among different influenza A subtypes. When influenza viruses are circulating in a community, a positive DFA test result is likely to indicate influenza virus infection.

• Real-time reverse transcriptase polymerase chain reaction (rRT-PCR):

  Nucleic acid amplification tests, including rRT-PCR, are the most sensitive and specific influenza diagnostic tests, but they may not be readily available, obtaining test results may take one to several days, and test performance depends on the individual rRT-PCR assay. As with any assay, false negatives can occur. Not all nucleic acid amplification assays can specifically differentiate 2009 H1N1 influenza virus from other influenza A viruses. If specific testing for 2009 H1N1 influenza virus is required, testing with an rRT-PCR assay specific for 2009 H1N1 influenza or viral culture should be performed.  Several rRT-PCR assays have been authorized by the Food and Drug Administration (FDA) under an emergency use authorization (EUA)³ to diagnose 2009 H1N1 influenza virus infection. For more information, see. Food and Drug Administration (FDA), Emergency situations (medical devices).

What laboratory diagnostic tests should be done for hospitalized patients with suspected influenza?

Hospitalized patients with suspected influenza should be considered for testing with an available influenza diagnostic test. Rapid influenza diagnostic tests (RIDTs) and direct immunofluorescence assays (DFAs) are widely available influenza diagnostic tests, but these tests have lower sensitivity than real-time reverse transcriptase polymerase chain reaction (rRT-PCR) tests. Since a negative RIDT or DFA test result does not exclude influenza virus infection, hospitalized patients with a negative RIDT or DFA result should have priority for further testing with rRT-PCR, if influenza infection is clinically suspected. For more information, see question “What does a positive/negative test result mean?”

What does a positive/negative rapid influenza diagnostic test (RIDT) or direct immunofluorescence assay (DFA) test result mean?

Given the lower sensitivity of RIDT and DFA relative to rRT-PCR* and viral culture, a negative test result using RIDT or DFA does not rule out influenza infection.  Hospitalized patients with a negative RIDT or DFA result should have priority for further testing with a nucleic acid amplification test, such as rRT-PCR, and should continue empiric antiviral treatment if influenza infection is clinically suspected. However, treatment with influenza antiviral medications should not be delayed pending test results if influenza is suspected and if treatment is indicated.

A positive RIDT or DFA result is informative because the specificity of these tests is high. These tests do not provide information on the influenza A subtype (e.g., 2009 H1N1 vs. seasonal H3N2), but if most circulating influenza A viruses have similar antiviral susceptibilities, influenza A subtype information may not be needed to inform clinical care. Under conditions where the majority of circulating influenza viruses are 2009 H1N1, a positive RIDT or DFA test result for influenza A virus can be assumed to be 2009 H1N1 influenza.

*For more information, see questions “What are the sensitivities and availability of rapid influenza diagnostic tests?” and “What are the sensitivities and availability of direct immunofluorescence assays (DFAs)?” and “What are the sensitivities and availability of real-time reverse transcriptase polymerase chain reaction (rRT-PCR) diagnostic tests?”

What are the sensitivities and availability of rapid influenza diagnostic tests?

Rapid influenza diagnostic tests (RIDTs) are widely available but have variable sensitivity (range 10 – 70%) for detecting 2009 H1N1 influenza when compared with real-time reverse transcriptase polymerase chain reaction (rRT-PCR), and a negative RIDT result does not rule out influenza virus infection⁴. RIDTs have a high specificity (>95%⁵). Depending on which commercially available RIDT is used,the test can either i) detect and distinguish between influenza A and B viruses; or ii) detect both influenza A and B, but not distinguish between influenza A and B viruses. For more information on the sensitivity, specificity and interpretation of RIDT results, see Interim Guidance for the Detection of Novel Influenza A Virus Using Rapid Influenza Diagnostic Tests.

What are the sensitivities and availability of direct immunofluorescence assays (DFAs)?

Like RIDTs, direct immunofluorescence assays (DFAs) are widely available, have variable sensitivity (range 47 – 93%) for 2009 H1N1 influenza virus, and have a high specificity (≥96% ⁶). DFAs detect and distinguish between influenza A and B viruses but do not distinguish among different influenza A subtypes. 

What are the sensitivities and availability of real-time reverse transcriptase polymerase chain reaction (rRT-PCR) diagnostic tests?

Nucleic acid amplification tests, including rRT-PCR, are the most sensitive and specific influenza diagnostic tests, but they may not be readily available, obtaining test results may take one to several days, and test performance depends on the individual rRT-PCR assay. Not all nucleic acid amplification assays can specifically differentiate 2009 H1N1 influenza virus from other influenza A viruses. If specific testing for 2009 H1N1 influenza virus is required, testing with an rRT-PCR assay specific for 2009 H1N1 influenza or viral culture should be performed. 

What real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays have been authorized by the Food and Drug Administration (FDA) to diagnose 2009 H1N1 influenza virus infection?

Several rRT-PCR assays have been authorized by the Food and Drug Administration (FDA) under an emergency use authorization (EUA)⁷ to diagnose 2009 H1N1 influenza virus infection. Information on these assays can be found at: Food and Drug Administration (FDA), Emergency situations (medical devices).

Which laboratories can perform real-time reverse transcriptase polymerase chain reaction (rRT-PCR) testing in the United States?

Public health laboratories in the United States have been provided with reagents and procedures from CDC to perform the rRT-PCR testing under an Emergency Use Authorization (EUA). State and local health department guidelines will determine which specimens can be submitted to public health laboratories for rRT-PCR testing. Several commercial and academic laboratories also are able to perform FDA-authorized rRT-PCR assays that can detect 2009 H1N1 influenza. In addition, other rRT-PCR assays have been developed that are not FDA authorized, but may detect 2009 H1N1 influenza with appropriate validation.

Which respiratory specimens are preferred for influenza virus diagnostic testing?

The package insert of influenza diagnostic tests contain manufacturer’s guidance on acceptable respiratory specimens. The following specimens are generally appropriate for influenza virus diagnostic testing: nasopharyngeal swab; nasal aspirate, wash or swab; endotracheal aspirate; bronchoalveolar lavage (BAL); and combined nasopharyngeal or nasal swab with oropharyngeal swab. The performance of rRT-PCR assays specific for 2009 H1N1 influenza has not been established for bronchoalveolar lavage and tracheal aspirates. If testing these specimens for 2009 H1N1 influenza, consider testing in parallel with a nasopharyngeal, nasal, or oropharyngeal swabs or a nasal aspirate.

In patients with severe lower respiratory disease (e.g., suspected viral pneumonia) who are intubated or undergoing bronchoscopy, lower respiratory tract specimens should be collected and tested to improve diagnostic yield. Consult with the laboratory regarding available testing options for lower respiratory specimens. If these specimens cannot be collected, a nasopharyngeal swab or combined nasal/oropharyngeal swabs can be tested but may yield negative results.

How should swab specimens be collected?

Ideally, swab specimens should be collected using sterile swabs with a synthetic tip (e.g., polyester or Dacron®) on an aluminum or plastic shaft. The swab specimen collection vials should contain 1-3 ml of VTM (e.g., containing protein stabilizer, antibiotics to discourage bacterial and fungal growth, and buffer solution).

How should clinical specimens be stored?

All respiratory specimens should be kept at 4°C for no longer than 72 hours before testing and ideally should be tested within 24 hours of collection. If storage longer than 72 hours is necessary, clinical specimens should be stored at -70°C.  Freezing at higher temperatures (e.g., -20°C) can reduce the likelihood of virus detection.

References

1. Hurt AC et al. Performance of influenza rapid point-of-care tests in the detection of swine lineage A(H1N1) influenza viruses. Influenza and Other Respiratory Viruses 2009;3(4):171-76
2. Pollock, NR, Duong S, et al, Ruling out novel H1N1 influenza virus infection with Direct Fluorescent Antigen testing. Clin Infect Dis. 2009 Sep 15;49(6):e66-8.
3. Subject to the terms and conditions of the EUA
4. Hurt AC et al. Performance of influenza rapid point-of-care tests in the detection of swine lineage A(H1N1) influenza viruses. Influenza and Other Respiratory Viruses 2009;3(4):171-76
5. Faix DJ, Sherman SS, Waterman SH. Rapid-Test Sensitivity for Novel Swine-Origin Influenza A (H1N1) Virus in Humans. N Engl J Med. 2009 Jun 29
6. Pollock, NR, Duong S, et al, Ruling out novel H1N1 influenza virus infection with Direct Fluorescent Antigen testing. Clin Infect Dis. 2009 Sep 15;49(6):e66-8.
7. Subject to the terms and conditions of the EUA