Test Test Test: How Covid-19 tests work

08 May 2020

BLOOD-TEST-SAMPLES

Blood samples being tested in a laboratory

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“Test, test, test” that was the message from the World Health Organisation (WHO) on how to limit the Covid19 crisis. But which test? And what do they actually measure? Here’s a brief explainer on two of the most commonly talked about tests for coronavirus infection...

Polymerase chain reaction - PCR

This is the standard method being used to confirm Covid19 cases in the UK. The test itself works by measuring the level of viral genetic material from patients using samples collected with a swab from the nose and throat. Apart from being easy sites to access, they are also prime areas for infection. If the virus is present, its genetic code, contained in a molecule called RNA (ribonucleic acid), will be caught on the swab. RNA is very similar to DNA and consists of a string of genetic “letters” – A, C, U and G – that spell out the genetic instructions coding for the coronavirus. In the lab, the RNA is extracted from the swab and separated out from any other debris in the sample.

The purified RNA is mixed with enzymes that can copy RNA sequences, and a fresh supply of genetic letters. The first step is to make a “mirror image” DNA copy of the RNA. This copy is known as cDNA and is then used as the starting point for making millions more DNA copies of one specific part of the viral genetic sequence. In this way, the tiny amounts of viral RNA that were present on the swab can be amplified to measurable levels. Special dye molecules included in the mixture make the DNA copies “glow” when light of a certain colour shines on them. The more copies there are, the brighter the glow. This tells scientists that DNA sequences corresponding to the coronavirus are being copied, and the brightness tells them how much material is there.

PCR tests like this are highly sensitive and commonly used in research. Indeed, a swathe of academic and research labs have offered to support the existing NHS coronavirus testing labs as this is a familiar technique. False positives are rare, the results are very reproducible between labs, and the technique is extremely reliable. However, false negatives can occur if the swabs are collected incorrectly, and PCR is relatively labour-intensive, so there is a reasonable margin for error. PCR also requires specific reagents, which, with worldwide testing going on for coronavirus, are now in high demand. Additionally, the PCR test will only show an active infection, so it won’t reveal if someone has been infected previously and has since recovered. For the UK, PCR testing has been the mainstay, but access to testing has proved challenging as Public Health England has striven to ensure the quality of all testing sites and ensure the correct reagents are available to all. Testing has been limited to those admitted to hospital, and slowly been expanded to symptomatic key works and their households, and care home residents. With this current testing strategy it is impossible to build a full picture of infection across the country, or to know the level of asymptomatic infection.

Antibody testing

Unlike PCR, which reveals only if a person is infected at the moment that the sample is taken, antibody testing can show who has been infected in the past, even if they’ve now recovered. When the immune system encounters a new pathogen, it will begin to generate an antibody response around 7 days later. Antibodies are highly specific proteins which can bind and neutralise pathogens to limit infection. After the initial infection has been resolved, antibodies are still produced at low levels to protect against future infections, forming immune memory. If SARS-CoV-2 specific antibodies are present in a person’s blood, this confirms that they have previously been infected with SARS-CoV-2, even if they had mild or no symptoms. The presence of antibodies also suggests protection against future SARS-CoV-2 infections, although it is not yet clear what level of protection these offer, or for how long. Developing a test that is sensitive and specific is challenging, and it can take years to perfect and needs to be validated against samples from thousands of people. 

Enzyme-Linked Immunosorbent Assays (ELISAs) are one way to test for antibodies. ELISAs are created by identifying which parts of a virus antibodies are most likely to recognise. These targets are known as viral antigens and are usually parts of the viral coat. To create an ELISA, samples of these antigens are stuck down onto a test plate. When blood from a patient is added, any antibodies that recognise the stuck-down viral antigens will attach to them. The plate is then washed to remove any unattached antibodies, leaving behind only the antibodies that recognise the viral antigens. A second antibody is then added that binds onto human antibodies. This second antibody is labelled with a special marker so it can be seen. The plate is again washed to remove any antibody that has not bound. Any residual colour indicates that there are human antibodies present stuck to the antigens. These are the ones that recognise SARS-CoV-2 antigens, proving that the patient has been exposed to the new coronavirus and is probably now also immune.

Currently, this type of test can be carried out only in diagnostics labs, but other tests are also in development such as “lateral flow” antibody tests. These work using a similar principle, with SARS-CoV-2 antigens being bound by specific antibodies in the blood which causes a colour change as the antibody-antigen complex passes over the detection strip. Lateral flow antibody tests are the technology behind home pregnancy tests: they’re simple to use at home and can give a result in a few minutes. Significant efforts are being put into development of an at-home test, but at the time out writing none have performed well enough to be approved by regulators and released to the public.

The development of these tests is made harder because although SARS-CoV-2 is a novel human pathogen, there are other coronaviruses which have been circulating in the populations for generations. They are some of the many respiratory viruses that circulate each winter causing colds. As a result, almost everyone in the population will have antibodies to other coronaviruses, which can confuse the test. Developers have to ensure that their tests are specific just for the SARS-CoV-2 coronavirus and that no related viruses can accidentally trigger the test and produce a false positive result. They also have to make sure that they select antigens which the immune systems of most members of the population will have responded to. Otherwise, people who have had SARS-CoV-2 infections could appear to be negative simply because the antibodies made by their immune systems are specific for antigens different to those used in the test.

We don’t know yet if antibodies are protective against future infection. Most antibody responses are protective for at least a few years, but we don’t know about SARS-CoV-2 yet. We can make some predictions though. The 2003 SARS outbreak infected over 8,000 people, and antibody testing on those that recovered from the infection showed they still had neutralising antibodies 2 years after the infection. Some people still test positive for antibodies even now, 17 years later. The development of antibody testing will provide vital epidemiological evidence, but the WHO has cautioned against using them to create “immunity passports” until we can know for sure that they prevent reinfection...

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