Before 2019, most people probably wouldn’t have heard of polymerase chain reaction (PCR) tests unless their doctor suggested one. But, as the coronavirus pandemic took hold, PCR became part of every day parlance. Suddenly, millions of people needed Covid-19 testing – and they needed it fast. By 18 June 2022, over 912 million tests had been done in the US alone; across the pond in the UK, that figure was around 503 million by 19 May of the same year, according to Our World in Data.
That’s over one billion samples collected and processed. Time was of the essence, too: the quicker a positive result was returned, the sooner that person could isolate themselves to prevent further spread. Pulling off this behemoth of a task stretched global health systems and supply chains beyond imagination. But, as a result, respiratory virus testing has advanced considerably.
“Covid has pushed the market remarkably in many aspects,” says associate professor of pathology and director of molecular virology at John Hopkins Medicine, Heba Mostafa. It progressed research and spurred development of different testing options: for instance, a wider variety of sample types can now be used. This means that there’s new information for healthcare professionals to weigh up when deciding how to go about diagnosis. With all these options, how can they select the right test, at the right time, for the right patient?
Detecting the genome
Post-pandemic, PCR is still seen as the gold standard for detecting respiratory viral pathogens. It’s a type of nucleic acid amplification test (NAAT), which work by making copies of the virus’ genetic material so it can be analysed by the lab. Yet other NAATs have emerged which are quicker and easier to use.
Though PCR is considered the most sensitive method, meaning that it’s less likely to miss any cases of disease, “these tests can take upwards of several hours to run and usually require sophisticated lab machinery,” says the president of the Association for Diagnostics and Laboratory Medicine (ADLM) and associate professor in pathology, critical care medicine and clinical and translational science at the University of Pittsburgh’s School of Medicine, Octavia Peck Palmer.
Loop-mediated isothermal amplification (LAMP) is an alternative that gained steam during the pandemic. Unlike PCR, isothermal methods like LAMP can be done at a consistent temperature, so tests can be run using compact equipment. Another isothermal amplification method that was used for Covid-19 detection is transcription-mediated amplification (TMA).
Isothermal amplification was routinely used for STI testing before the pandemic, Mostafa explains. But, thanks to a surge in research and interest, it evolved significantly during that time and more tests became available. “There was a lot of literature showing that it can be sensitive and showing that it can facilitate [Covid-19] testing,” she says. For instance, in 2020, the UK’s NHS found that the OptiGene RT-LAMP test was sensitive enough to be used for Covid-19 testing. Though LAMP and TMA can be faster and cheaper than PCR, they’re typically less accurate, says Palmer.
Some NAATs can be done at home and with a variety of sample types, although your choice of specimen may be limited by what’s approved for use with particular assays. In guidance written for healthcare professionals in navigating respiratory virus testing post-pandemic, the ADLM suggests that clinicians consider how quickly a result is needed, whether collection of a quality specimen is feasible and how sensitive the testing method is for that specimen when deciding which sort of sample to use. Prior to the pandemic, nasopharyngeal swabs (NPS) were the standard for upper respiratory tract infection testing. But they require a skilled healthcare professional to collect. The need to facilitate self-testing at scale and reduce strain on supply chains not only saw greater uptake of nasal swabs, but the collection of saliva, too. “It was previously assumed that saliva shouldn’t be used for respiratory testing,” says Palmer.
According to research done by Mostafa and her team at Johns Hopkins, nasal swabs and NPS have comparable sensitivity when testing for influenza, respiratory synthetic virus (RSV) and Covid-19. Though saliva is less sensitive, it could be used when it isn’t practical to collect other sample types, per the ADLM. For instance, patients aged over 65 might have some nasal dryness. The ADLM notes that there’s currently no clear consensus on which sample type should be routinely used when detecting different viruses, though NPS is preferred for upper respiratory tract infections.
“There was a lot of literature showing that [isothermal amplication] can be sensitive and showing that it can facilitate [Covid-19] testing.”
Heba Mostafa
Rapid antigen testing
The rise of rapid antigen testing is perhaps a key hallmark of the pandemic. These tests work by detecting specific proteins (antigens) on the surface of the virus. And, before Covid-19, they’d never been used for respiratory tract infections, says professor of virology at the University of Nottingham, Will Irving.
“They ended up being very successful because they could be operated by anyone. You didn’t need a laboratory. They’re not as sensitive as PCR, but they serve a major function in allowing people to self-test.”
Antigen testing can be done using a simple nasal swab and delivers results within 15 minutes. People can also do it at home, which isn’t the case for all NAATs. While they’re less accurate, antigen tests may be more useful in stopping the spread of disease as they give people actionable information faster that was critical during the pandemic. Outside of a public health emergency, speed can still be important if knowing a patient’s result sooner would change how you manage their treatment. For instance, the influenza medication Tamiflu is only licensed for use in the first 48 hours after symptoms start.
Currently, antigen tests are only approved or have emergency use authorisation for influenza A, influenza B, SARS-CoV-2 (the virus that causes Covid-19) and RSV. The ADLM writes that NAATs are the preferred method of diagnosis for respiratory viruses, though antigen tests could be used if a NAAT isn’t available.
While it’s not as quick as an antigen test and can’t be done via self-testing, rapid PCR testing is a possible alternative here. This is another breakthrough pushed by the pandemic, says Irving, and can provide results in up to an hour. You need a rapid PCR testing machine, which can be operated at the point of care. Simply register the sample, put it into the machine and wait for the results, Irving explains. “These machines just need an electricity supply and someone who’s trained to know which buttons to press. So you can move the diagnosis away from the hospital, and that’s been accelerated by our experience in the pandemic.”
Which test is best?
What’s most appropriate for each case is unique to that patient. But there are certain things that should factor into the decision to use a particular test, or whether to go down the testing route at all. “Tests should be performed only if there is a high pretest probability of respiratory viral infection based on clinical presentation and local prevalence, and in cases where results will change clinical management,” says Palmer.
If, from there, it makes sense to move forward, the ADLM recommends evaluating tests based on their accuracy, whether they’re feasible for the lab, their cost versus their value, how regularly the lab runs that test (testing volume) and how the results will impact the clinical outcome. Diagnostic algorithms, which are step-by-step methods outlining what to do in different scenarios, can be particularly helpful here. The ADLM guidance, on which Mostafa was a coauthor, includes a suggested testing algorithm for respiratory viruses.
“Who you test and when to test and why to test should actually be thought of when you decide which direction in the algorithm to take,” Mostafa explains. She gives an example: if you had a patient come to the hospital with symptoms but who wasn’t immunocompromised, you might consider testing for influenza and Covid-19. If the results came back negative, you might choose not to do further tests for viruses where no treatment options are available.
Though if that same patient was immunocompromised or had an underlying condition, you’d also be thinking about infection control, monitoring, and whether to isolate them. As there’s a risk their infection could lead to severe disease, it would be important to know which virus is present. Perhaps you’d then take a different direction in the algorithm and test for a larger range of viruses.
Looking forward
The coronavirus pandemic has progressed research into respiratory virus diagnostics and our capabilities in detecting infection. This is true even if some of the advances made are yet to have an application in clinics.
While next-generation sequencing and CRISPRbased diagnostics, which identify a virus’ DNA or RNA, were granted emergency use authorisation during the pandemic, there are currently no approved tests available for respiratory infections. But work is underway in labs around the world to change that. These tests can be simpler to run than PCR while having a comparable sensitivity, says Mostafa. Whatever happens, the progress made during this pandemic stands us in good stead, says Irving. “If and when the next pandemic comes, we’ll be better prepared for it.”
