It’s Flu Season Somewhere: How Clinical Labs Can Approach Influenza Diagnostic Testing

Nov. 11, 2019

It is almost influenza (flu) season in North America, but anytime is a good time to talk about flu because it is always flu season somewhere! As you can see in the GIF of World Health Organization (WHO) biweekly updates, there’s always flu going around somewhere in the world. Flu seasonality is real, but the factors that influence the seasonality of  viruses are poorly understood.

Biweekly flu activity summaries from WHO 2017-2019. Country/region colors indicate % tests positive; pie chart grey/blue/green (flu A subtypes) and orange (flu B) indicate flu types detected by percent of positive tests reported. via GIPHY

Influenza viruses are enveloped RNA viruses with segmented genomes; influenza A and influenza B have 8 genome segments each. Genome segmentation plays a key role in the emergence of new subtypes of flu: cells co-infected with 2 types of influenza virus will produce virions with an assortment of genome segment combinations (an example of genetic shift). For example, an influenza subtype that primarily infects birds or pigs but isn’t able to efficiently infect humans may co-infect a host with a human-adapted influenza strain, leading to the production of virions that are able to infect humans but are antigenically different than strains encountered by humans previously. Influenza A virus subtypes are defined by the hemagglutinin (HA or H) and neuraminidase (NA or N) surface glycoproteins (for example H1N1).

Smaller genetic mutations also occur over time, and the accumulation of these mutations leads to genetic drift. Influenza strain identification names can be quite long: you may see a longer name that includes the type, host from which the virus was isolated, country of origin, strain number, and year isolated (example: A/quail/Vietnam/36/2004 H5N1). The genetic diversifications due to shift and drift have an impact on the pathogenicity of influenza strains and can also influence our ability to detect them with diagnostic tests–more on that later.

Before considering how to test for influenza infection, one must consider whether testing is needed for a particular patient situation. The CDC has posted a guide to aid in deciding which patients to test, and whether to initiate antiviral therapy. Naturally the decision tree starts with ascertaining whether the patient has signs or symptoms of an influenza-like illness, and further decision points include whether the patient will be admitted to a hospital and whether the test result would influence therapy decisions. Some people at higher risk for severe flu-related outcomes (elderly, infants, immunocompromised), or those who co-habit with members of these demographics, may require special consideration. Testing may not be required in many outpatient or emergency care situations. If the time at presentation is not within 72 hours of symptom onset, the sensitivity of diagnostic tests may be significantly reduced. Additionally, antiviral treatment is less effective if started >48 hours after illness offset.

Saying that testing for flu is not always necessary in symptomatic patients should not diminish the importance of testing for certain patient populations or the pathogenic potential of the virus. During the last flu season alone (2018-2019), more people died in the United States from flu (~61,000) than the number of American soldiers who died in the entire Vietnam War (~57,000), the 4th deadliest war in the history of our country. During my clinical microbiology fellowship, I was asked by a technologist to look at an unusual cerebrospinal fluid Gram stain with cells they did not recognize. It turned out there was brain matter in the CSF, and the child died of influenza with cerebral hemorrhage. The child had not been vaccinated – vaccination against influenza A and B is an important way to reduce flu-related morbidity and mortality. Testing for suspected influenza infection, especially in vulnerable populations, can lead to earlier treatment and better patient outcomes.

Testing is primarily performed by detecting influenza viral antigens by lateral flow assay or viral nucleic acid in clinical specimens (usually nasopharyngeal swabs). There are also culture and flu antigen targeting immunofluorescence assays but these are not commonly used. Antigen tests are cheaper, more rapid, and generally simpler to use than other testing methodologies. Unfortunately, antigen tests also tend to be less sensitive than molecular testing methods, which led to the FDA in 2017 reclassifying antigen-based rapid influenza diagnostic tests (RIDTs)–different classes of medical devices require different levels of regulatory control based on potential risk to patients–and after reclassification, flu RIDTs were moved from class I to class II. The FDA also established minimum performance requirements, which includes a minimum of 80% sensitivity relative to approved molecular methods. These new standards cut a fair number of RIDTs out of the market because they could not demonstrate this level of sensitivity. That said, there have been improvements recently in antigen test development. RIDT digital antigen immunoassays, which use  automated readers, have demonstrated higher sensitivities relative to manually read RIDTs. A recent publication of an immunochromatographic RIDT demonstrated results that compared favorably to molecular testing, with approximately 95% sensitivity. Despite the lower cost per test, RIDTs may not be the most cost-effective testing strategy.

Molecular tests have clearly demonstrated improved sensitivity relative to antigen based tests. Molecular tests from several manufacturers are making strides in 2 areas where RIDTs used to have the advantage: ease of use and turn-around-time. The molecular test my lab uses takes less than 5 minutes to set up, and has a run time of about 25 minutes. Several competitors have similar rapidity and ease of use. A number of molecular tests are now also CLIA waived and can be utilized in point-of-care settings. Molecular tests are still more expensive than antigen tests (antigen tests are typically $8-12 each, molecular tests $25-50). Many molecular test manufacturers offer combined flu and respiratory syncytial virus (RSV) tests, which is convenient for labs that want to offer both up front. This is useful for patient room management, where infection control practices isolate patients with either virus. Genetic shift or drift may impact either primary type of testing (antigen or molecular), and assays that include multiple targets may have a significant advantage in years where atypical strains predominate.  Currently I’m happy with our molecular test, but will keep an open mind about newer RIDTs if sensitivity can be reliably improved relative to molecular tests.

Here’s hoping for a lighter flu season than last year!
The above represent the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology.

Further Information

Author: Matthew Pettengill

Matthew Pettengill
Matthew Pettengill is the Scientific Director of Clinical Microbiology at Thomas Jefferson University Hospital in Philadelphia, Pennsylvania, and is a Diplomate of the American Board of Medical Microbiology.