Large-scale testing and contact tracing are central to efforts to control the pandemic. This relies on diagnostic tests that can rapidly, accurately and reliably detect SARS-CoV-2.
In December 2019, the novel coronavirus SARS-CoV-2 first emerged in China and it has since rapidly spread around the world. The disease it causes, COVID-19, has resulted in over one million deaths and put extreme pressure on health systems and economies.
“Until a vaccine is developed, our main approach is going to be testing and preventing transmission of the virus,” says Chaz Langelier, associate medical director at UCSF.
Researchers have raced to develop diagnostic tests that can quickly and accurately identify people who are currently infected with SARS-CoV-2, either through the detection of specific viral nucleic acids (molecular testing) or proteins (antigen testing), or if they have had previous exposure to the virus, via the detection of anti-SARS-CoV-2 antibodies (serological testing).
“SARS-CoV-2 has an RNA genome which is surrounded by a lipid membrane containing several different proteins,” says Langelier. “The one that’s probably best known is the “spike” protein, which is important for mediating entry of the virus into host cells.”
Many clinical diagnostics laboratories currently rely on molecular tests based on the polymerase chain reaction (PCR) for detecting SARS-CoV-2 infection in patient samples, such as throat or nose swabs.
“PCR-based tests are probably the most widely used and are considered the “gold standard’” in terms of sensitivity and specificity,” says Langelier.
However, as PCR testing requires specialized equipment and trained personnel, attention is shifting towards the development of low-cost, portable technologies that can provide more rapid results. Antigen tests are already starting to be deployed as a low-cost screening tool – and alternative molecular tests offer huge potential for the future.
PCR tests are the “gold standard”
Before the end of January 2020, the SARS-CoV-2 genome sequence was made available, enabling researchers to design the PCR assays that are now in routine use today.
The technique involves extracting viral RNA from a patient sample, which is usually a swab collected from the upper respiratory tract. After converting it into complementary DNA, specific sections of the viral genome are amplified in a thermal cycler, and the products are then detected.
“PCR tests are designed to detect sections of the viral genome,” says Langelier. “But the exact targets may be different, depending on the assay.”
Researchers are keeping a close eye on viral evolution – as the emergence of new strains could potentially lead to false-negatives.
“SARS-CoV-2 mutates something like once every two weeks or so, which is relatively slow for a virus,” says Emily Crawford, assistant adjunct professor at UCSF and group leader at the Chan Zuckerberg BioHub. “But a single point mutation in a primer or probe binding site can essentially kill the ability of that PCR assay to detect the virus.”
To combat this, most assays are based on at least two different targets, dramatically lowering the chance of spontaneous mutations arising in both places at once.
“The test we use in our laboratory targets both the N gene and the E gene of the virus,” explains Crawford. “So, we’d be quickly alerted if we did see a sample where one gene was detected very robustly and the other at a low level, or not at all.” The N gene contains the instructions for the virus’s nucleocapsid protein and the E gene encodes the envelope protein.
The sensitivity and specificity of PCR assays can be more than 95% when administered under ideal conditions. But in the real world, the performance of swab tests might be lower due to other factors, such as when and how the sample was collected.
“We know that the viral load peaks about a day before a person develops any symptoms – if they’re going to develop them – and then it decreases,” says Langelier. “So if you collect a sample two weeks after their first symptoms, the amount of virus is going to be much lower, which affects the sensitivity of the test.”
The specificity of PCR-testing can become a major issue through the cross-contamination of samples with other viral material. For example, earlier this year the contamination of testing kits assembled in the US Centers for Disease Control and Prevention laboratories rendered them ineffective.
“The viral load in some patient samples is so high that if they are not handled with the utmost care, they could cross-contaminate others,” explains Langelier. “It’s probably more of a common issue than we appreciate.”
Another major challenge is that the threshold value for the viral load that is needed for a person to be infectious is currently unknown.
“We have seen huge differences in the viral load of different samples, from around one hundred viral genome copies up to around a hundred billion copies per milliliter,” says Crawford. “That poses a challenge because we need to have an assay that can accurately detect both extremely high and low titers of the virus – but we have no idea whether someone with low titers is likely to pass it on to others.”
Rapid antigen tests are a potential game-changer
To slow down the spread of SARS-CoV-2, most experts agree that the focus should be on identifying and isolating people who are infectious. In theory, this could be achieved through the use of new rapid antigen tests, the first of which recently received emergency use authorization from the US Food and Drug Administration (FDA).
A typical antigen test also usually starts with a nose or throat swab – although some are being developed that use saliva samples. After adding a solution to break down the virus, the mix is then added to a laminar flow strip containing an antibody that binds to a specific viral antigen.
“These assays can be put into something like a small card that will give you a read-out,” describes Langelier. “So, you might see a band that only appears if the SARS-CoV-2 antigen is present – similar to a home pregnancy test.”
Antigen-based tests are much cheaper and faster than PCR-based assays – and because they do not require any specialist equipment, they could be employed in a point-of-care setting. But the cost is reduced sensitivity.
“Current estimates suggest that the limit of detection of antigen tests might be ten to a hundred-fold higher than PCR-based assays,” says Langelier. “So it might give you a false-negative result when the person has low amounts of SARS-CoV-2 in their body.” A negative result obtained using an antigen test may then need to be confirmed with a molecular test to help inform treatment decisions.
But this may be less important if the goal of testing is to identify people when they are contagious.
“There is an increasing amount of data suggesting that most people with mild or even moderate illness are no longer infectious after 7 to 10 days,” explains Langelier. “So antigen-based tests could be highly effective at identifying people when they are most likely to be able to pass it on – as long as they’re done often enough.”
Antibody-based tests offer another diagnostic strategy. These are blood-based immunoassays that are designed to detect specific antibodies produced by the immune system in response to a SARS-CoV-2 infection.
“As it takes around 7 to 10 days for someone to produce sufficient antibodies to be detected by an assay, they don’t really have a role in diagnosing an acute infection,” says Langelier. “But they might be useful to help confirm cases who are highly suspected to have COVID-19, but have remained negative on PCR tests.” Antibody tests may help in confirming a SARS-CoV-2 infection when it is not possible to get an accurate result with PCR, such as if the sample was collected more than 8 days after the person’s symptoms began.
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New molecular tests in the pipeline
An alternative technique known as Loop-mediated Isothermal Amplification (LAMP) is attracting increasing attention as it has the potential to be cheaper, quicker and easier to carry out than PCR. The technique amplifies the viral genetic material in a targeted way just like PCR, but it doesn’t require thermal cycling.
“In theory, that means it could be used in a point-of-care setting where you don’t have access to a fancy machine,” explains Crawford. “But there are drawbacks as these techniques are generally not as sensitive or specific as PCR.”
CRISPR, which is best known as a gene-editing technology, also has huge potential as a diagnostic tool. The approach involves binding a protein to a specific sequence in the viral genome and then detecting that interaction.
“It’s very specific, but it’s not as sensitive as PCR,” says Crawford. “But what people are doing is combining isothermal amplification with CRISPR, which means you can get really good sensitivity and specificity.”
But many researchers are cautious about the readiness of CRISPR-based tests as a means for helping address the current pandemic.
“I think there’s a temptation to look for a cool new toy that going to solve all our problems,” says Crawford. “But, in reality, there are still a lot of challenges with it.”
Another potential approach involves detecting changes in the host immune response rather than the presence of the virus itself. For example, research from Langelier’s team has shown that the pattern of gene activity in the upper respiratory track is unique to SARS-CoV-2 compared to other respiratory illnesses, which he says, “could be taken advantage of in an assay”.
Diagnostics takes center stage
Large-scale rapid diagnostic testing of people with symptoms of COVID-19 is essential for identifying SARS-CoV-2 infections, containing its spread and preventing deaths.
“Until we have a vaccine, our main approach is going to be testing and preventing transmission,” says Langelier. “Easy and frequent access to diagnostic testing could help overcome a lot of the current challenges in infection control and prevention.”
The long-term goal is to reach a point where widespread testing for SARS-CoV-2 infection is no longer necessary. But this is unlikely to happen anytime soon.
“Even if we have a vaccine, I think that diagnostic testing will continue to play a key role in terms of identifying and preventing infection,” says Langelier.