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Multiplex testing helps provide critical insights as respiratory viruses surge

Respiratory viruses, including SARS-CoV-2, can present similar symptoms but vary widely in the treatments they require.Credit: nobeastsofierce Science/Alamy

In typical years, flu season begins to escalate in early autumn and proceeds through winter. In 2021, the pattern for respiratory illnesses appears unsettlingly different.

“We’re seeing all these respiratory viruses right now, like SARS-CoV-2, of course, but also other endemic corona viruses, parainfluenza 2 and 3, and RSV [respiratory syncytial virus],” said Melissa Miller, Ph.D., director of the University of North Carolina’s Clinical Molecular Biology laboratory, on a balmy August day. “This is the most RSV I've seen in my entire career, and it’s still the summer.”

Though no-one can predict the intensity of a particular flu season, the early increase in respiratory illnesses does not bode well. SARS-CoV-2, the virus responsible for COVID-19, has inundated hospitals with symptomatic patients who may or may not be infected with the virus. For the health of patients, healthcare workers, and the public, those showing signs of a respiratory illness require accurate and rapid testing, since therapies and public health protocols vary widely by illness. That is easier said than done.

Symptomatic COVID-19 can present like many other respiratory infections, at least at the outset. An effective diagnostic must be able to differentiate between respiratory illnesses, which will strain labs like Miller’s. To keep pace, these labs will need access to multiplex testing.

The case for multiplexing

SARS-CoV-2 testing has largely consisted of two modalities: Antigen-based and PCR-based testing. Both tests are highly specific, meaning that the risk of a false-positive is low, and physicians can be confident that a positive result is indicative of SARS-CoV-2 infection.

But how should physicians interpret a negative SARS-CoV-2 test when their patient is symptomatic?

While relatively rare, false-negatives in SARS-CoV-2 testing can occur, regardless of whether tests are antigen- or PCR-based; most viral diagnostics have an associated false-negative rate.

It’s also possible that a patient, however symptomatic of COVID-19, is instead infected with one of the many other circulating respiratory viruses, such as influenza A or influenza B. With current testing so narrowly focused on SARS-CoV-2, physicians presented with a negative COVID-19 test lack the diagnostics needed to identify the cause of the patient’s symptoms.

Multiplex tests simultaneously analyse samples for multiple pathogens. Currently available multiplex tests, including those from the life sciences company Thermo Fisher Scientific, leverage PCR techniques to selectively amplify genes specific to each virus of interest. For SARS-CoV-2, the test might amplify genes that code for the virus’ envelope protein (E gene) or the nucleocapsid protein (N gene). For Influenza A, B, and RSV, PCR targets may include virus specific matrix proteins as well as nucleocapsid proteins.

Miller’s team in North Carolina has been increasingly leveraging multiplexed testing for SARS-CoV-2 and other respiratory viruses. ”There are two flavours of multiplexes,” she explained, “there's the mini multiplexes that might just have influenza A, B, RSV, and SARS-CoV-2. Then you have these big multiplexes where there's like 20 or 22 targets. Order volumes for both of those have gone up lately.”

According to Miller, the move to multiplexed testing has streamlined her lab’s workflow, reducing the time, costs, and efforts needed to diagnose most samples.

Patient and public health implications

Multiplex testing offers a number of benefits. Hospitals can better triage patients and allocate resources, but patients stand to gain the most.

While respiratory illnesses can present similarly, their treatment can vary dramatically. Influenza infections, for example, can be treated with antivirals, such as oseltamivir, to reduce the period of infection. But that same medication is markedly less effective against other respiratory pathogens. Case in point, remdesivir, not oseltamivir, is approved as a therapy for more severe COVID-19 cases.1

Likewise, people with severe COVID-19 cases can be prescribed dexamethosone, a steroid, or Interleukin-6 inhibitors to arrest runaway inflammatory responses in the lungs, a precursor to the acute respiratory distress syndrome. But those medications are well known to predispose influenza patients to secondary infections, due to their immuno-suppressive qualities.

For patients, the consequences of an inaccurate diagnosis can be grave. Stephen S. Morse, Professor of Epidemiology at Columbia University, said in an interview, “COVID-19 can go from just feeling sluggish to essentially having to be on a ventilator in a matter of days. If you give dexamethasone or monoclonal antibodies soon enough, it can really help the patient. So the sooner you can diagnose and separate these patients, the faster you can actually start the appropriate treatment.”

Likewise, public health responses could benefit from improved molecular testing of respiratory illness. The infectivity of a virus is often reported as an R0 value, which represents the number of people, on average, that an infected individual will spread the virus to. “One of the most infectious viruses is the measles virus, with an R0 of 15,” Dr. Morse explained. “When we talk about the flu, R0 is usually somewhere between 1 and 2. Now with the Delta variant of SARS-CoV-2, it's probably closer to 6 or 8, which is about the same as the virus responsible for chickenpox.”2

Because SARS-CoV-2 and the now-dominant delta variant are so infectious, multiple necessary events cascade from a positive SARS-CoV-2 diagnosis: Healthcare providers must wear R0 value the necessary personal protective equipment; the patient must be quarantined; and contact tracers need to backtrack the patient’s movements to identify any additional infected individuals.

A time to act

In July of 2021, the CDC announced the imminent phase-out of their singularly focused SARS-CoV-2 RT-PCR test, emphasizing that labs should now consider adoption of multiplexed testing in its place.3 As flu season gets underway, more labs are likely to follow suit.

Many multiplex tests of varying sizes are already available on the market, and many more are currently in development. Both Miller and Morse believe multiplex testing will come to be the norm in the near future as demand rises and advancements expand the diagnostic potential of such tests.

To learn more about diagnostics for respiratory illness, explore this page from Thermo Fisher Scientific.


  1. Flerlage, T., Boyd, D.F., Meliopoulos, V. et al. Influenza virus and SARS-CoV-2: pathogenesis and host responses in the respiratory tract. Nat Rev Microbiol 19, 425–441 (2021).

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  2. McMorrow, M. Improving communication around vaccine breakthrough and vaccine effectiveness. Centers for Disease Control & Prevention (CDC) slide deck (July 2021)

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  3. Centers for Disease Control & Prevention. Lab Alert: Changes to CDC RT-PCR for SARS-CoV-2 testing. CDC Laboratory Outreach Communication System (July 2021)

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