Interim Recommendations for Clinical Use of Influenza Diagnostic Tests During the 2009-10 Influenza Season

September 29, 2009, 6:00 PM ET

Objective

To provide updated interim recommendations on influenza diagnostic testing for clinicians treating patients with suspected 2009 H1N1 influenza virus infection and to assist clinicians with testing decisions for the 2009-10 influenza season ¹. These recommendations may be further revised as more information becomes available. These recommendations also can be adapted according to local epidemiologic and surveillance data and other state and local considerations.  Clinical judgment is always an important part of testing and treatment decisions. 

Summary Points

• Most patients with 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.
• Patients who should be considered for influenza diagnostic testing include:
• Hospitalized patients with suspected influenza
• Patients for whom a diagnosis of influenza will inform decisions regarding clinical care, infection control, or management of close contacts.
• Patients who died of an acute illness in which influenza was suspected.
• When a decision is made to use antiviral treatment for influenza, treatment should be initiated as soon as possible without waiting for influenza test results. Antiviral treatment is most effective when administered as early as possible in the course of illness.
• Clinicians should be aware that the sensitivities of rapid influenza diagnostic tests (RIDTs) and direct immunofluorescence assays (DFAs) are lower than real-time reverse transcriptase polymerase chain reaction (rRT-PCR) tests and viral culture. A negative RIDT or DFA result does not rule out influenza virus infection. Further, these tests cannot distinguish between 2009 H1N1 and seasonal H1N1 or H3N2 influenza A viruses.
• If most circulating influenza viruses have similar antiviral susceptibilities (as is the case currently in the United States), information on the influenza A subtype may not be needed to inform clinical care.
• If identification of 2009 H1N1 influenza virus infection is required, testing with a rRT-PCR assay specific for 2009 H1N1 influenza or viral culture should be performed. 
• Laboratory tests to diagnose 2009 H1N1 influenza, such as rRT-PCR, should be prioritized for hospitalized patients and immunocompromised persons with suspected influenza where RIDT or DFA testing is negative or to determine influenza A virus subtype in patients who have died from suspected or confirmed influenza A virus infection.
• Information on testing of pathology specimens for suspected 2009 H1N1 influenza virus infection is available.

Background

As of September 19, 2009, more than 99% of circulating influenza viruses identified in the United States were 2009 H1N1 influenza (previously referred to as novel influenza A (H1N1)).  The clinical presentation of patients with uncomplicated 2009 H1N1 influenza virus infection is generally similar to seasonal influenza and includes abrupt onset of fever, cough, sore throat, myalgias, arthralgias, chills, headache and fatigue.  Vomiting and diarrhea have been reported more often with 2009 H1N1 influenza than with seasonal influenza².  As with seasonal influenza, some patients with 2009 H1N1 influenza will present without fever. Clinical judgment and local surveillance data for influenza and other respiratory pathogens are important in considering the differential diagnosis of patients presenting with influenza-like illness.

CDC recommends early empiric antiviral treatment for suspected or confirmed influenza in hospitalized patients and in outpatients at higher risk for complications from influenza (see complete antiviral recommendations and list of high risk conditions. Empiric antiviral treatment, when indicated, should be initiated as early as possible and should not be delayed pending the results of influenza testing.

Influenza Diagnostic Tests

A number of diagnostic tests (Table 1) are available to detect the presence of influenza viruses in respiratory specimens. 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).

Rapid influenza diagnostic tests (RIDTs) are widely available but have variable sensitivity³ (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 specificity⁵ (>95%⁶).  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. More information on sensitivity, specificity and interpretation of RIDT results is available.

Like RIDTs, direct immunofluorescence assays (DFAs) are widely available, have variable sensitivity (range 47 – 93%) for 2009 H1N1 influenza virus, and a high specificity (≥96%⁷). 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, the positive predictive value of the RIDT and DFA tests are generally high and a positive test result indicates that influenza virus infection is likely.  However, as stated above, a negative test does not rule out influenza virus infection.

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 evaluated and authorized by the Food and Drug Administration (FDA) under an emergency use authorization (EUA)⁸ to diagnose 2009 H1N1 influenza virus infection. Public health laboratories in the United States have been provided with reagents and procedures from CDC to perform the rRT-PCR testing under Emergency Use Authorizations (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 currently FDA authorized but have the potential to detect 2009 H1N1 influenza with appropriate validation.

Use of Influenza Diagnostic Tests during the 2009-10 Influenza Season

State and national influenza surveillance information on circulating influenza virus types and influenza A virus subtypes can be helpful in determining the most likely influenza viruses circulating and their antiviral susceptibilities. National information on circulation of influenza viruses is updated weekly.

Outpatients

Once influenza activity has been documented in a community or geographic area, most patients with an uncomplicated illness consistent with influenza can be diagnosed clinically and do not require influenza testing for clinical management, including antiviral treatment decisions.

In certain situations, influenza diagnostic testing of patients who are not severely ill may help inform decisions regarding clinical care, infection control, or management of close contacts.  Clinicians should use their clinical judgment in these situations to decide when to test for influenza in patients who are not severely ill. When interpreting the test results, clinicians should consider the following factors:

• sensitivity of the influenza diagnostic test used (Table 1)
• 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 that can cause influenza-like illness 

Given the lower sensitivity of RIDT and DFA relative to rRT-PCR, a negative test result does not rule out influenza virus infection. A positive RIDT or DFA result, however, 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.

If identification of  2009 H1N1 influenza is required, testing with an rRT-PCR assay specific for 2009 H1N1 influenza or viral culture should be performed.  For example, specific influenza diagnostic testing for 2009 H1N1 influenza with rRT-PCR may be important for patients with certain conditions, such as pregnancy or severe immunosuppression.

Hospitalized Patients

Hospitalized patients with suspected influenza should receive immediate empiric antiviral treatment and be tested with an available influenza diagnostic test (Table 1).  Identification of influenza infection can improve clinical care and infection control in hospitalized patients.  Appropriate antiviral treatment and infection control measures should not be delayed pending diagnostic testing results. 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 a nucleic acid amplification test, such as rRT-PCR, if influenza infection is clinically suspected.  For patients with severe lower respiratory tract disease in whom the diagnosis of 2009 H1N1 influenza has not been confirmed, collection of lower respiratory tract specimens may be useful for rRT-PCR testing although the performance of rRT-PCR assays specific for 2009 H1N1 influenza has not been established for bronchoalveolar lavage and tracheal aspirate specimens.  Testing and treatment for bacterial pathogens and other respiratory viruses should be conducted as appropriate. Influenza subtype testing using rRT-PCR or viral culture also should be prioritized for use in patients who have died from suspected or confirmed influenza.

^{1 - Serologic testing on paired acute- (within 1 week of illness onset) and convalescent-phase (collected 2-3 weeks later) sera is limited to epidemiological and research studies,  is not routinely available through clinical laboratories, and should not inform clinical decisions.
2 - The amount of time needed from specimen collection until results are available.
3 - Compared with rRT-PCR tests; rRT-PCR tests are compared to other testing modalities including other rRT-PCR assays.
4 - Rapid Influenza Diagnostic Tests include tests that are CLIA waived (can be performed in an outpatient setting) and tests that are moderately complex (can be performed only in a laboratory). Clinical specimens approved for RIDTs vary by test, and may not include all respiratory specimens.
5 - Performance of these assays relies heavily on laboratory expertise and requires a fluorescent microscope
6 - Requires additional testing on the viral isolate
7 - The performance of rRT-PCR assays specific for 2009 H1N1 influenza have 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.
8 - See discussion above on available rRT-PCR assays}

For further information regarding influenza diagnostic tests, please see:

• Rapid influenza diagnostic tests
• Seasonal influenza diagnostic testing

Information on Specimen Collection and Storage

Preferred respiratory specimens

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)
• combined nasopharyngeal or nasal swab with oropharyngeal swab.

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 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.

Specimens should be collected according to instructions in the test’s package insert as soon as possible after illness onset; additional specimens may aid in confirmatory testing for some patients.   Specimens to be tested by rRT-PCR or viral culture should be placed into sterile viral transport medium (VTM) (or diluted 1:1 in VTM) and immediately placed on ice or cold packs or at 4°C (refrigerator) for transport to the laboratory.   Recommended infection control guidance is available for persons collecting clinical specimens and for laboratory personnel.

Swabs

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 1-3ml of VTM (e.g., containing protein stabilizer, antibiotics to discourage bacterial and fungal growth, and buffer solution).

Storing clinical specimens

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

¹These recommendations address the clinical use of influenza diagnostic testing. Information regarding influenza testing for ongoing surveillance activities is provided elsewhere [link when available].

²Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, CDC. Emergence of a Novel Swine-Origin Influenza A (H1N1) Virus in Humans. N Engl J Med 2009;360:2605-15.

³Sensitivity: the proportion of people with influenza infection who are correctly identified with a positive influenza test result

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

⁵Specificity: The proportion of people without influenza infection who are correctly identified as not having influenza with a negative test result

⁶Faix DJ, Sherman SS, Waterman SH. Rapid-Test Sensitivity for Novel Swine-Origin Influenza A (H1N1) Virus in Humans. N Engl J Med. 2009 Aug 13;361(7):728-9.

⁷Pollock, 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.

⁸Subject to the terms and conditions of the EUA.