Best practices for influenza testing: Methodologies and implementation

Oct. 24, 2016


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1. Identify the viruses and strains that cause different types of influenza.
2. Discuss current epidemiology of influenza and how the surveillance of yearly outbreaks is used.
3. Describe the different testing methodologies that are available for influenza identification and limitations of each method.
4. Discuss factors that are needed and must be implemented to provide fast, accurate, and cost-effective diagnoses of influenza.


Influenza is a contagious, seasonal respiratory illness that results in significant mortality, morbidity, and economic burden as reflected in medical costs, lost productivity, and lost earnings.1 It is caused by human influenza A and B (flu A and flu B) viruses that infect the nose, throat, and lungs. Currently circulating flu A viruses include H1N1, a new variant of which caused an epidemic in the spring of 2009, and H3N2. In this nomenclature, H stands for hemagglutinin and N for neuraminidase, both surface proteins of the virus. Flu B viruses are categorized by lineage (e.g., flu B/Yamagata and flu B/Victoria). Both flu A viruses and one or two flu B viruses are generally included in the seasonal flu vaccine each year.

Flu symptoms include fever, cough, sore throat, runny or stuffy nose, body aches, headache, chills, and fatigue and, less commonly, vomiting and diarrhea. Antiviral drugs are effective in reducing symptoms, shortening the course of the disease, and, importantly, preventing serious complications such as pneumonia in vulnerable populations: young children, the elderly, immunocompromised patients, and those with chronic conditions such as asthma, diabetes, or heart disease.

Diagnosis and the role of testing

Flu testing is important for diagnosis or confirmation of diagnosis based on clinical presentation to guide treatment decisions. Symptoms often associated with flu are not unique to flu A and flu B, and testing can identify the cause and help the clinician to focus treatment. The choice between antivirals and antibiotics, for example, has important implications in patient outcome as well as antimicrobial stewardship.

Despite the importance of testing, empiric treatment is a common practice, since antiviral flu drugs work best when they are started within two days of illness2 and, until recently, testing methodologies that can deliver accurate results in a timely manner at the point of care have been lacking. Efficacious treatment is especially important for pregnant women, individuals with a weakened immune system, young children, the elderly, and those with chronic health conditions. In such cases, treatment is adjusted as appropriate once test results are available, but at a cost to the clinical workflow, since patients will need to be re-contacted.

Testing and surveillance

Testing is an important surveillance tool. State health departments collect and publish influenza surveillance data to track outbreaks and monitor flu activity.3 In institutional settings such as hospitals, nursing homes, or chronic care facilities, flu testing is useful in guiding decisions such as precautionary cohorting of infected patients. In addition to identifying flu outbreaks and their cause, it is a public health priority to maintain vigilance for emerging flu subtypes and strains. These novel subtypes, a result of genetic changes in the virus, can cause more severe disease and pandemics. Information about emerging subtypes is also needed for the design of vaccines for the coming year.

Current testing methodologies

Viral culture has been the conventional standard in flu testing. Only culture can provide specific information regarding circulating viral strains and subtypes, for comparing current circulating flu strains with vaccine strains and formulating vaccines for the coming year. Viral culture can also help identify causes of flu-like outbreaks or potential novel viral agents that may pose a pandemic threat. The emergence of antiviral resistance can be identified. In clinical research, viral culture can play an important role in providing isolates for epidemiology studies and evaluation of mechanisms of antiviral resistance for new drugs.4

As a diagnostic tool, however, viral culture takes one to three days, a turnaround time that is not optimal for treatment decisions. Also, viral culture requires specialized skills that may not be accessible in smaller labs, especially those in the community setting. And a combination of specialized skills and facilities is critical to ensure accurate testing and avoid culture contamination and cross-contamination.

A number of rapid influenza diagnostic tests (RIDTs), also called rapid antigen direct tests (RADTs), are commercially available for the qualitative detection of flu A and flu B viral nucleoprotein antigens in respiratory specimens. They provide results in less than 30 minutes, and many have been CLIA-waived and so can be used in laboratories licensed as CLIA waived point-of-care settings. However, their utility is limited by their modest sensitivities, and both the U.S. Centers for Disease Control and Prevention (CDC) and the U.S. Food and Drug Administration (FDA) have published guidance urging caution in their use.4-6

The CDC notes that RIDTs have suboptimal test sensitivity, and false-negative results are common when flu activity is high. Conversely, RIDTs can have relatively high false-positive rates, especially during times when flu activity is low. Thus confirmatory testing using PCR or culture is necessary. The FDA notes that none of the FDA-cleared RIDTs could differentiate among flu A subtypes or differentiate those that commonly infect humans (H3N2 and H1N1) from those that typically infect birds and other animals. It is also not clear whether similar sensitivity and specificity of RIDTs would be experienced when a new subtype emerges as a predominant circulating flu A type.

The FDA further notes that RIDTs “may have lower sensitivity for adults than for children because children tend to shed virus more abundantly and for longer periods of time” and that “any rapid test using frozen samples may likely show greater sensitivity than with freshly collected samples.” In sum, “rapid tests may be useful in [strategies for influenza diagnostic testing for inpatients and outpatients] by providing preliminary tools for guiding treatment and patient management in a clinically relevant time frame (less than 30 minutes).…When interpreting results from any rapid influenza test, laboratories and clinicians must use clinical experience, further laboratory testing, [and] surveillance information about circulating influenza strains and the current level of influenza activity, along with an understanding of the limitation of these rapid tests.”6 Clinicians share this cautionary view. For clinicians, the inability to rule out a flu diagnosis based on RIDT results and the potential for misdiagnosis and overtreatment across a range of disease prevalence are reasons not to base treatment on RIDT results.7

Molecular assays are increasingly used in flu testing, offering superior sensitivity and specificity compared with other methods such as RIDTs. There are now two FDA-cleared molecular assays with time to results of 20 minutes, making rapid molecular tests for flu diagnosis a reality. The availability of the assays on CLIA-waived systems makes rapid molecular testing a viable option for point-of-care. The two systems use different nucleic amplification technology. One is based on reverse transcription-polymerase chain reaction (RT-PCR) and the other on isothermal amplification.

While molecular tests offer several advantages, such as faster time to results (especially with rapid molecular assays), improved sensitivity and specificity, and the ability to distinguish among specific flu A subtypes, they are generally more expensive when compared with viral culture and RIDTs. In considering molecular assays for flu testing, it is important to analyze the costs and benefits in the context of overall patient care. These include improving patient management by limiting unnecessary antibiotic or antiviral use and reducing hospital stay or time in the ER. And while policies vary by payer and by location (state), molecular tests generally receive higher reimbursement compared with non-molecular tests. Also to be considered is the value in preventing or limiting community spread of flu by timely identification of an outbreak and the ability to characterize the epidemiology of flu virus infections.8 Importantly, judicious use of point-of-care testing can be driven by guidelines put in place by multidisciplinary teams for determining when point-of-care flu testing is appropriate and when testing can be done in the lab, often more economically.

Planning and implementation

The primary goal of flu testing is to enhance patient care through timely and accurate diagnosis for guiding treatment and precautionary measures in the institutional environment. Flu testing is also necessary to support public health reporting requirements. Thus flu testing must be an integral part of an overall program to prevent and manage respiratory viral infections—one that will consider the clinical workflow in its entirety from diagnosis to treatment, and one that will benefit significantly from multidisciplinary participation including the lab, infectious disease, administration, emergency medicine, nursing, public health laboratories, infection control, pharmacy, and antimicrobial stewardship. The clinical workflow will guide planning on interrelated issues such as which patients to test, when they should be tested, what tests should be used, whether tests should be done at the point of care or in the lab, and when test results need to be confirmed, with the added context of the time frame within the flu season. Each member of the team must be aware of his or her role in this initiative and be fully engaged in it.9 Each institution is unique in its patient population, rural vs. urban setting, payer mix and financial considerations, and the flu testing program must be customized accordingly.

From the point of view of the lab, two key points in flu test implementation are noteworthy. First is the value of standardization in processes and test methodologies. When it is optimal to provide flu testing both in the central lab and at the point of care, choosing a methodology that is available in multiple formats, optimized for each setting, can make it easier to compare results and streamline workflow. Second, a well-thought-out plan for specimen collection and transportation will reduce the likelihood of poor results due to sample degradation or inappropriate samples for the test methodology.

When waived testing is modified by changing the approved test method or changing the sample type tested, the classification of the test will be modified to a more complex test. Often this will move that waived test to a complexity beyond that allowed by the point-of-care laboratory.9 Toward this end, it is important to follow manufacturer’s directions in the package insert unless validations can be performed and the test is completed in a highly complex laboratory.

Making a difference

As molecular flu testing at the point of care becomes reality, lab professionals have the opportunity to update clinician colleagues about the power of having test results in hand to support treatment decisions—improving patient outcome, streamlining clinical workflow, reducing costly unnecessary treatment, and embracing antimicrobial stewardship. Lab professionals also have the opportunity to participate in designing and implementing a test strategy, with the goal of bringing best practices in flu testing to their institution.


  1. Molinari NA, Ortega-Sanchez IR, Messonnier ML, et al. Vaccine. 2007;25:5086-5096.
  2. U.S. Centers for Disease Control and Prevention. Influenza antiviral medications: summary for clinicians.
  3. North Carolina Department of Health and Human Services. 2015–16 influenza season: update for NC clinicians.
  4. Ginocchio CC, McAdam AJ. Current best practices for respiratory virus testing.
    J Clin Microbiol .2011;49(9):S44-S48.
  5. U.S Centers for Disease Control and Prevention. Guidance for clinicians on the use of rapid influenza diagnostic tests.
  6. U.S Food and Drug Administration. Cautions in using rapid tests for detecting influenza A viruses.
  7. DeLaney M. Four reasons not to order a rapid flu test.
  8. Mahony JB, Blackhouse G, Babwah J, et al. Cost analysis of multiplex PCR testing for diagnosing respiratory virus infections. J Clin Microbiol. 2009;47(9):2812-2817.
  9. Sautter RL, Lipford EH. Point-of-care testing: guidelines and challenges. NC Med J.2007;68(2):132-135.



Robert L. Sautter, PhD, HCLD (ABB), CC, is principal of RL Sautter Consulting LLC, where he is director of Microbiology and Point of Care. He has more than 120 published abstracts and more than 40 publications in peer reviewed journals, and is a member of several professional societies, including the American Society for Microbiology and the American Board of Bioanalysis. Based on his extensive studies, he has given numerous presentations nationally.