Diagnostic Testing for Influenza

Diagnostic testing - work in progress

In the news:

• screening for influenza on plane flights

Entry screening is unlikely to be effective in preventing the importation of either SARS or influenza. The incubation period for SARS is too long to allow more than a small proportion of infected individuals to progress to symptomatic disease during a flight to the UK from any destination.

• FDA Approves New Bird Flu Test

rapid test (4 hours) for H5 in humans; requires confirmation of N1.

• testing FAQ from ECDC here

See CIDRAP’s section on lab testing for a synopsis. See CDC page: Lab Diagnosis and this .pdf from WHO. There are several widely used tests for influenza. Discussion below does not imply endorsement. They include:

• Viral culture

Viral culture remains the gold standard of influenza testing. However, there are unanswered questions about safety when culturing H5N1. For example, there was a worldwide recall of proficiency testing kits in April, 2005 with the suggestion that “the government should restrict the handling of flu virus to more secure labs, as thousands of scientists around the world destroyed a deadly flu strain that had been sent to thousands of labs for testing.” Viral culture, though definitive, also takes 5–10 days and requires lab expertise and facilities. From the CDC Interim Guidance for Influenza Diagnostic Testing During the 2004-05 Influenza Season:

Most importantly, the viruses acquired through isolation are used to characterize the subtype or strain of circulating influenza A viruses, including the degree of antigenic drift from vaccine strains, the emergence of antiviral resistance, and the presence of influenza A subtypes that show antigenic drift from the vaccine strain or antigenic shift and present a pandemic threat. Since influenza strains are continuously evolving, laboratory-based surveillance for influenza isolates is critically important to the selection of strains for the next season’s influenza vaccine.

• Rapid Diagnostic Tests

• Enzyme Immuno Assay (EIA)
• Immunofluorescence DFA Antibody Staining

From An Office-Based Approach to Influenza: Clinical Diagnosis and Laboratory Testing:

• Office-based techniques for detecting influenza A and B viruses include enzyme immunoassay and direct immunofluorescence. Using these sophisticated, yet relatively easy techniques, the physician can diagnose influenza A and B in 10 to 20 minutes.

• At present, five office-based assays for detecting influenza virus A and/or B are available commercially: BD Directigen Flu A+B, BD Directigen Flu A, FLU OIA, QuickVue Influenza A/B Test, and ZstatFlu. The assays vary in sensitivity, specificity, complexity, and skill and time required for performance and interpretation. Another assay, the Influenza A/B Rapid Test, cannot be obtained commercially but is available to physicians who are participating in influenza surveillance studies. However, one consumer reports having purchased a box of QuickVue Influenza A/B tests several years ago, and reports that apparently multiple online vendors are offering them at this time.

see Enders, et. al. below: “…commercially available rapid antigen tests such as Directigen Flu A+B (Becton Dickinson, Sparks, NJ, USA) or Binax NOW (Binax Inc., Portland, ME, USA) are rapid and simple, but subtyping of viruses is not feasible.”

From the CDC Interim Guidance for Influenza Diagnostic Testing:

• Most of the rapid influenza tests are approximately >70% sensitive for detecting influenza and approximately >90% specific compared with virus culture. Thus, most tests with positive results correctly identify infection, but as many as 30% of negative test results may be falsely negative. The predictive values of influenza tests depend on the level of influenza activity in the community, exposure of the patient to a contagious person, susceptibility of the patient, the characteristics of the tests (sensitivity and specificity), and the adequacy of specimen collection. Inadequate or inappropriate specimens are likely to yield false negative results. The tests are most reliable when there is known influenza activity in the community and when they are performed on patients who have signs and symptoms consistent with influenza (e.g., fever, cough, sore throat, muscle aches, headache, and malaise). However, the symptoms and signs of influenza can vary by age and underlying medical conditions, and not all patients manifest typical symptoms and signs of influenza.

• Reverse transcriptase polymerase chain reaction (RT-PCR)

see Effect Measure for a good description of RT-PCR.

Influenza A H5N1 Detection

Enders et al., Emerging Infectious Diseases, Aug. 2005

“We developed a sensitive and rapid real-time reverse transcription-polymerase chain reaction (RT-PCR) assay to detect influenza A H5N1 virus in clinical samples. This assay was evaluated with samples from H5N1-infected patients and demonstrated greater sensitivity and faster turnaround time than nested RT-PCR….the real-time assay not only reduces the risk for contamination but also reduces turnaround time to 1–2 hours…”

• Serology

This is a blood draw and is best suited for epidemiological evidence of exposure. Acute and convalescent blood samples are obtained from the patient (often done weeks apart) and compared for an increase in antibodies to influenza. The turn-around time is weeks, not days or hours. From An Office-Based Approach to Influenza: Clinical Diagnosis and Laboratory Testing:

In clinical practice, rapid viral tests would be most helpful in deciding whether to initiate treatment for influenza. However, antibody titers (viral serology) may provide some retrospective assessment of symptoms 10 to 14 days after the initial serologic specimen was obtained. A fourfold rise in titer provides confirmatory evidence of infection long after empiric antiviral treatment would have been completed.

Which test and when?

From the CDC’s 2004-5 influenza interim guidelines :

During the current flu season, the diminished supply of influenza vaccine could increase the demand for influenza testing; thus, this document was developed to help clinicians determine when they should order testing. Diagnostic testing should be considered when an institutional outbreak of influenza is suspected or if test results would influence clinical decision making.

Influenza Tests

A variety of tests are available to diagnose influenza. Rapid diagnostic tests have been increasingly used because they can yield results in a clinically relevant time frame, i.e., approximately 30 minutes; however, the reference standard for diagnosis of influenza remains virus culture. For a comparison of the various available tests see Lab Diagnosis.

The above CDC statement was meant to apply to a typical influenza season, not a pandemic flu situation. However, even rapid tests can quickly become in short supply during a typical flu season (and they cost 20–25 US dollars each… Connecticut, for example, is projected to have 800,000 cases in a moderate pandemic and testing everyone would cost 20 million dollars - just for rapid flu testing!). When tests are not available, influenza is diagnosed on clinical grounds and on the basis of prevalence in the community.

In a pandemic flu setting, triage of patients to designated ‘flu centers’ or (as in Tasmania) ‘fever centres’ to sort out who does or does not have flu may be needed. This ‘reverse triage’ strategy is designed to keep flu patients from overwhelming hospitals and Emergency Departments. Some sort of rapid test would be important to have in this setting, although as noted, test kits may not be available in plentiful supply.

What does CLIA and CLIA waived mean?

CLIA is the Clinical Laboratory Improvement Amendments which are designed to regulate quality standards for laboratory testing in the United States regardless of where the test was performed. When a lab test is considered simple and safe enough it can be designated as ‘waived’ or ‘CLIA waived’, meaning the lab test does not have to meet strict standards of documentation, training, etc. Tests that must meet CLIA standards are generally run by commercial, hospital and research labs. Only one of the 5 rapid tests is approved for use in CLIA-waived labs (QuickVue Influenza A/B Test).

More on this topic undoubtedly to come.