Each year in the United States, seasonal flu is responsible for dozens of deaths and thousands of hospitalizations among children, especially those with underlying medical conditions. Rapid diagnosis identifies those children who would be candidates for antiviral therapy that can shorten the course of illness and lessen symptom severity. Unfortunately and until recently, rapid office tests for influenza have been very unreliable. With the introduction of point-of-care (POC) molecular assays, pediatricians can diagnosis influenza A and B during the office visit with sensitivity and specificity comparable to reference assays.
This article will review influenza—its presentation, symptoms and treatment—and review the POC influenza tests currently available to help expedite diagnosis.
The 2 influenza viruses that cause human epidemics are Influenza A and B. Influenza A is categorized into subtypes based on hemagglutinin and neuraminidase protein antigens. Hemagglutinin is responsible for the entry of the virus into cells, while the neuraminidase facilitates the release of virus from host cells. Influenza B is not categorized into subtypes and is separated into 2 genetic lineages: Yamagata and Victoria.
The US Food and Drug Administration (FDA) has recommended that the 2016-2017 influenza trivalent vaccines used in the United States contain an A/California/7/2009 (H1N1) pdm09-like virus, an A/Hong Kong/4801/2014 (H3N2)-like virus, and a B/Brisbane/60/2008-like virus (B/Victoria lineage). It also recommends that quadrivalent vaccines, which have 2 influenza B viruses, contain the viruses recommended for the trivalent vaccines, as well as a B/Phuket/3073/2013-like virus (B/Yamagata lineage). This represents a change in the influenza A(H3N2) component and a change in the influenza B lineage included in the trivalent vaccine compared with the composition of the 2015-2016 influenza vaccines. As recently reported in the medical and popular press, the live attenuated influenza vaccine (LAIV) is not recommended for prophylaxis this season because of its lack of efficacy.
According to the American Academy of Pediatrics Committee on Infectious Disease (aka, the Red Book), influenza viruses are spread through coughing and sneezing as well as with contact with respiratory droplets on surfaces. The incubation period for the disease is 1 to 4 days, and patients shed virus and are considered contagious for 1 day before symptoms begin and during the course of the illness.
Typically, influenza begins in November and lasts until May, with peak season usually from January to March of each year. Symptoms include chills, fever, sore throat, headache, and cough, with myalgia being a very predominant symptom. It is estimated that 10% to 40% of children are infected in any given year.1 Children aged younger than 2 years and patients with an underlying medical condition are at particular risk of contracting severe disease. High-risk patients include those having:1
· Asthma or other chronic pulmonary diseases, such as cystic fibrosis;
· Hemodynamically significant cardiac disease; â¨
· Immunosuppressive disorders or therapy (see Special Considerations, Red Book, page 488); â¨
· Human immunodeficiency virus (HIV) infection;
· Sickle cell anemia and other hemoglobinopathies;â¨
· Diseases that necessitate long-term aspirin therapy, including juvenile idiopathic arthritis or Kawasaki disease; â¨
· Chronic renal dysfunction;â¨
· Chronic metabolic disease, including diabetes mellitus; and â¨
· Any condition that can compromise respiratory function (eg, neurodevelopmental disorders, seizure disorders).
The Centers for Disease Control and Prevention (CDC) recommends antiviral treatment for high-risk patients, as well as for other patients “who in the view of their physician would benefit from treatment.” Antiviral therapy is most effective if started within 48 hours of symptom onset. Available antivirals include oral oseltamivir (Tamiflu) for patients as young as newborns; inhaled zanamivir (Relenza) for patients aged 7 years and older; and intravenous peramivir (Rapivab) for hospitalized patients aged 18 years and older who cannot take other antivirals (Table 12). These are neuraminidase inhibitors that prevent the release of virus from infected cells and reduce further infection. Neuraminidase inhibitors are effective against both influenza A and influenza B virus, and can be used for prophylaxis for patients who are high risk if they are exposed to individuals with influenza.3
Diagnosing influenza: Then and now
Lateral flow immunoassays for influenza first became available in 1998, with the first waived rapid flu test available in 2000. Over 21 million POC rapid tests for influenza are performed in the United States each year. This contrasts with about 50 million strep tests performed each year. These tests involve the extraction of viral nucleoprotein antigen from a swab, the placement of the extracted specimen into a test cartridge or onto a test strip, and the capturing of antigen by an antibody conjugated with a colored nanoparticle. As the antibody antigen complex migrates through the test system, it is captured by an antibody fixed onto the cartridge or strip, producing a colored line. Colored lines indicate the presence of influenza virus A or B and also indicate whether the test was performed correctly. Specimens that can be used with these tests include swabs obtained from the anterior nares, the nasopharynx, as well as from nasal aspirate or wash. Like all similar assays, positive and negative controls need to be run per the manufacturers’ recommendations, usually when an office receives a new shipment or a new lot number of test kits.
The CDC has cautioned physicians regarding the reliability of rapid antigen influenza tests, based on lateral flow immunoassay technology, because their sensitivities have been poor.4 A meta-analysis of over 159 studies demonstrated a sensitivity of 64.6% for Influenza A and 52.2% for influenza B compared with reference assays.5 It should be recognized that physicians fare poorly in distinguishing patients with flu from those who do not, based on history and examination. A recent study showed that physicians working in an emergency department could only diagnosis influenza in adult patients with a sensitivity of 36% when diagnosis was established with PCR testing.6
Helpful when positive, false negative tests are common with lateral flow immunoassay tests, thus negative tests do not exclude the disease. Reference assays for influenza include viral culture, which takes over a week to return, and reverse transcriptase polymerase chain reaction assay (RT-PCR), which can produce results within hours but are only available at reference labs, so specimens need to be sent out for testing, delaying diagnosis. Traditional lateral flow influenza A and B kits cost about $18 to $22 per test with reimbursements running approximately $16 per virus tested. Note that these are billed as 87804 each for both influenza A and influenza B testing, so 2 units of service are charged, bringing the reimbursement into the $32 range.
Newest rapid flu tests
Two new rapid influenza tests with improved sensitivities have appeared on the market over the past few years. These include the Veritor system from BD Diagnostics (Franklin Lakes, New Jersey) and the Sofia Influenza A+B FIA from Quidel (San Diego, California).
The Veritor system, combines a lateral flow immunoassay with an optical reader to detect “weak” positive rapid influenza A and B tests that would otherwise go undetected visually. This was introduced as a Clinical Laboratory Improvement Amendments (CLIA)-waived test for influenza in 2008. The reader costs $300, and the test sells for $16 per cartridge, which tests for both influenza A and B. The test is charged under the CPT code 87804, as mentioned previously. The specimen swab is placed in a tube containing extraction solution and then 3 drops of the solution are placed in the sample well in the cartridge. After 10 minutes, the cartridge is inserted into the optical reader, which then displays the results.
Another advantage of this test for pediatric patients is that the same specimen can be tested using another Veritor assay cartridge for respiratory syncytial virus (RSV). In young patients, RSV can produce pneumonia-like illnesses. Identifying patients with RSV can prevent the inappropriate use of antibiotics to treat a viral infection, whereas identifying influenza within 48 hours of symptom onset would merit consideration of antiviral medication to shorten the course of the illness.
Studies have shown that the Veritor Influenza assay has a sensitivity of 90.2% for influenza A and 87.5% for influenza B.7 In addition, it can detect antigen with fewer viral particles present compared with lateral flow immunoassays. The Veritor system has a level of detection of 105 TCID50/ml (TCID50/ml=tissue culture infectious dose; ie, dose of virus that will produce pathological change in 50% of cell cultures inoculated), better than those of lateral flow assays that usually are of the order of 106 TCID50/ml.8
The Sofia Influenza A+B FIA was introduced in 2011 and received CLIA-waived status in 2012. The test is a fluorescent immunoassay that involves an extraction and testing phase. Following a 1-minute extraction from a nasal swab, nasopharyngeal swab, or nasopharyngeal wash or aspirate, an aliquot of the specimen is dispensed into a testing cassette sample well. During the 15-minute test, the specimen migrates through a test strip containing various “chemical environments.” If influenza A or B antigens are present, they will be bound by antibodies coupled to fluorescent microparticles that migrate through the test membrane. The fluorescent microparticles containing bound antigen will be captured by antibodies on the test membrane where they are detected by the Sofia device.
The test has been shown to have sensitivities of 94% for influenza A and 89% for influenza B.9 The reader costs $4500 and each test costs about $16. As with the Veritor system, the same specimen can be used to test for both influenza and RSV with the Sophia device, with cartridges run simultaneously before insertion in the device.
Waived ‘molecular testing’ is available
Two POC tests are presently available from Alere (Waltham, Massachusetts) and Roche (Pleasanton, California) that utilize nucleic acid amplification technology to detect influenza with sensitivities comparable to RT-PCR assays. In the near future, Cepheid (Sunnyvale, California) will release its own POC influenza waived test for its GeneXpert Xpress System. As noted in my previous Peds v2.0 article on disruptive technologies (Contemporary Pediatrics, July 2016), these devices are “game changers,” bringing diagnostic capability into the office that is on par with those in reference labs.
The Alere i Influenza device detects influenza A or B nucleic acid from nasal specimens using the company’s proprietary isothermal nucleic acid amplification technology (iNAT). Results are available within 15 minutes. A color video displayed on the device’s screen walks users through the testing process. A sample receiver is placed in the machine and warmed for 2 minutes. The specimen swab is placed in the cartridge for just 10 seconds, and then a pipette cartridge is used to transfer the sample into a test base. The lid is closed and the testing cycle begins. You can walk away at this point and return in 15 minutes to see the result on the device’s LCD screen.
The overall sensitivity and specificity of the Alere i is extremely high and on par with results produced by lab-based PCR tests (Table 2).10 In addition, the device requires just hundreds of viral particles versus the thousands required by traditional immunoassays. Because the test is a “molecular test,” it is billed under a unique CPT code—87502—that is reimbursed at a higher rate compared with nonmolecular strep tests that are billed under the code 87804. The cost of the Alere i flu test is about $50, with national payments of about $100 per test. Alere provides the testing device if a minimum number of tests are performed each year.
Roche is marketing its own version of the PCR POC test called the cobas Liat (“lab in a tube”) system. A nasopharyngeal specimen is placed into a tube of transport media, which is then transferred into a test cartridge that is inserted into the system, with results available in just 20 minutes. The Liat features a touch screen that directs the user to scan the cartridge and input or scan identifying patient information. Note that the transport media/swab kit needed to perform a test is not included with the Liat cartridges and must be purchased separately (these cost $2 to $5 per kit). The prices of the Roche system and testing cartridges are not yet available. The Roche test is billed under the CPT code 87502, which is reimbursed at $100 per test. As with the Alere device, the results produced by the Liat are comparable to lab-based PCR systems, with a level of detection superior to all the tests discussed here.11 As of this writing, Roche has just been granted approval for its new test that enables users to test a nasopharyngeal sample for influenza A and B as well as RSV simultaneously.
Whenever a new technology is introduced (such as molecular POC tests), insurance reimbursement can be uncertain. Because the cost of molecular tests is higher than lateral flow assays, it is prudent to get preauthorizations from insurance companies prior to adopting this new technology.
In anticipation of flu season, you now are informed regarding what technologies will enable you to accurately diagnose influenza and make prudent use of antiviral therapy. As always, I appreciate your thoughts and opinions regarding office technology.
1. American Academy of Pediatrics. Influenza. In: Kimberlin DW, Brady MT, Jackson MA, Long SS, eds. Red Book®: 2015 REPORT OF THE COMMITTEE ON INFECTIOUS DISEASES. American Academy of Pediatrics; 2015: 476-493.
2. Centers for Disease Control and Prevention. FluView interactive, 2014-2015. Available at: http://www.cdc.gov/flu/weekly/fluviewinteractive.htm. Published August 13, 2015. Accessed August 10, 2016.
3. Committee on Infectious Diseases, American Academy of Pediatrics. Recommendations for prevention and control of Influenza in children, 2015-2016. Pediatrics. 2015;136(4):792-808.
4. Centers for Disease Control and Prevention (CDC). Evaluation of rapid influenza diagnostic tests for detection of novel influenza A (H1N1) virus—United States, 2009. MMWR Morb Mortal Wkly Rep. 2009;58(30):826-829.
5. Chartrand C, Leeflang MM, Minion J, Brewer T, Pai M. Accuracy of rapid influenza diagnostic tests: a meta-analysis. Ann Intern Med. 2012;156(7):500-511.
6. Dugas AF, Valsamakis A, Atreya MR, et al. Clinical diagnosis of influenza in the ED. Am J Emerg Med. 2015;33(6):770-775.
7. Hassan F, Nguyen A , Formanek A, Bell JJ, Selvarangan R. Comparison of the BD Veritor System for flu A+B with the Alere BinaxNOW influenza A&B card for detection of influenza A and B viruses in respiratory specimens from pediatric patients. J Clin Microbiol. 2014;52(3):906-910.
8. Peters TR, Blakeney E, Vannoy L, Poehling KA. Evaluation of the limit of detection of the BD Veritor system flu A+B test and two rapid influenza detection tests for influenza virus. Diagn Microbiol Infect Dis. 2013;75(2):200-202.
9. Lewandrowski K, Tamerius J, Menegus M, Olivo PD, Lollar R, Lee-Lewandrowski E. Detection of influenza A and B viruses with the Sofia analyzer: a novel, rapid Immunofluorescence-based in vitro diagnostic device. Am J Clin Pathol. 2013;139(5):684-689.
10. Bell J, Bonner A, Cohen DM, et al. Multicenter clinical evaluation of the novel Alere i influenza A&B isothermal nucleic acid amplification test. J Clin Virol. 2014;61(1):81-86.
11. Chen L, Tian Y, Chen S, Liesenfeld O. Performance of the Cobas influenza A/B assay for rapid PCR-based detection of influenza compared to Prodesse ProFlu+ and viral culture. Eur J Microbiol Immunol (Bp). 2015;5(4):236-245.
Dr Schuman, section editor for Peds v2.0, is clinical assistant professor of Pediatrics, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire, and editorial advisory board member of Contemporary Pediatrics. He discloses that he has given lectures sponsored by Alere, mentioned in this article.