Basic Diagnosis
Testing for COVID-19
Two kinds of tests are available for COVID-19:
- Viral tests (Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
- Antibody tests (IgM & IgG)
1. Viral Testing: Reverse-Transcription Polymerase Chain Reaction (RT-PCR):
Technology overview
PCR is a very common scientific technique that has been widely used in research and medicine for around 20-30 years to detect genetic information. RT-PCR is a special version used when RNA is being detected and it is now being used as a test to detect SARS-CoV-2, the virus causing COVID-19. This type of test has frequently been used as a frontline test for COVID-19 as it directly tests for the presence of the virus RNA.
RT-PCR tests are fairly quick, sensitive and reliable, capable of producing results in 3-4 hours, although this usually takes longer if samples must first be sent to specialized external laboratories (6-8 hours on average).
Many diagnostic and research companies produce RT-PCR products, tests and machines so the technology is widely available. Some RT-PCR tests are developed as an `all in one’ kit, reducing laboratory handling and potential for contamination.
How it works
Sample collection: These samples are taken from the nose or throat using either long or short swabs, but samples can be collected in other ways too. Collecting samples from where the virus is shedding or multiplying, improves the accuracy of the test.
Once a sample has been collected, chemicals are used to remove any proteins, fats and other molecules, leaving only RNA behind. This will be a mixture of a person’s genetic material as well as any viral RNA that might be present.
What does the test detect?
RT-PCR detects whether or not viral RNA is present in samples from a patient. It does this by capturing and amplifying regions of the virus’ genetic material, usually the Spike protein, N protein or Envelope (see picture).
To measure the viral RNA, it is converted to DNA, copied many times using repeated temperature cycles in a PCR machine and then fluorescent markers are used to detect the virus. If the amount of fluorescence goes above a certain level, this confirms that the virus is present. The number of temperature cycles the machine performs to reach this threshold is recorded to estimate how much virus was present in the patient sample. The lower the number of cycles, the more virus was present.
Advantages and limitations
Advantages:
- RT-PCR is accepted by scientists and medical staff as a robust and well documented technique.
- With RT-PCR being so common in research and medicine, the technology is already in place to test for COVID-19.
- RT-PCR can detect current infections of disease, allowing medical staff to determine who is currently infected and who is not.
Disadvantages:
- RT-PCR relies on capturing and detecting the virus and so it is possible to miss patients who have cleared virus and recovered from disease.
- The distribution of virus across the respiratory tract varies between patients, so even if a person is infected, the virus may only be detectable in sputum or nasopharyngeal swab but not necessarily at both locations at the same time.
- RT-PCR for COVID-19 can only tell if a person is currently infected with this particular coronavirus. It can’t provide information on other diseases or symptoms
2. Antibody testing: Lateral Flow / Colloidal Gold Immunochromatography
Technology overview
Lateral flow assays have commonly been referred to as ‘Antibody tests’ in the media as they are currently used to detect antibodies to disease in a patient’s blood. The technology is also being tested for antigen use too.
Antibody lateral flow tests for SARS-CoV-2 are produced as test kits used by a specialist or clinician rather than by patients themselves. They require a drop of patient blood, either from a vein or from a small finger prick, similar to a finger prick test used for blood sugar monitoring in certain types of diabetes.
These types of tests work very differently to RT-PCR and detect the patient’s immune antibody response to the virus rather than detecting the virus itself.
A major advantage for this type of test is also the ability to see if patients are currently infected or have recovered from COVID-19 even if they have fully recovered and cleared the virus months ago. However, it cannot distinguish between an active and a previous infection.
How it works
Lateral flow immunoassays for COVID-19 are simple devices that can detect antibodies in the blood.
A small sample of patient blood is taken from a vein or from a finger-prick by a clinician and dropped onto a spongey pad within the test device. A few drops of a diluting liquid called a ‘buffer’ are added to help the blood sample flow across the device.
As the sample moves through the device, antibodies against SARS-CoV-2 that are present in the sample will attach to chemicals in the device, capturing the antibodies on the test and control lines. This capturing and binding process results in a colour change along the test and control lines which can be seen by eye, producing one, two or three lines depending on the type of antibodies are present (IgM or IgG).
What does the test detect?
Lateral flow immunoassays for SARS-CoV-2 detect two types of protective antibodies that are produced by the body when the immune system recognizes a foreign structure, in this case SARS-CoV-2, the virus causing COVID-19.
Antibody lateral flow immunoassays can be designed to detect IgM or IgG alone or both together.
These antibodies help fight the disease and remain in the blood for months after the virus and disease is cleared. The presence of antibodies in the body is often referred to as immunity or that a person is immune to a virus, as these antibodies protect against re-infection and return of the same disease.
When we are infected by virus, our immune system produces early `prototype’ antibodies (IgM) with intermediate strength binding to virus, that are able to start working to clear virus about 5 days after a new infection. Typically at 8 to 10 days after infection, IgG antibodies with high binding strength, can work to help more rapid virus clearance. Antibodies act by developing a matching contoured surface to stick to foreign antigens, using a sophisticated selection process to amplify antibodies with the best surface match and strongest binding.
What does the result mean?
Antibody lateral flow immunoassays detect antibodies to the virus in the blood. They don’t detect the virus itself. Using the antibody response alone does not allow distinction between individuals who are currently infected and those who have cleared the virus infection.
Antibody tests provide a hugely important ability to detect past infection with virus to identify people who were asymptomatic, people who have cleared the virus and so no longer risk being infected or spreading the virus to others. In addition, antibody tests are critical for assessing population spread of the virus and the level of ‘herd’ immunity in the population.
How to read the test?
The antibody IgG and IgM lateral flow immunoassay tests are very simple to read:
A control line must appear to show that the assay has worked correctly. Then, test lines will appear if either of the antibody types are found in the sample. The appearance of lines for IgG or IgM, or both indicate a positive test – showing that the patient has been infected with the COVID-19 coronavirus.
Advantages and limitations
Advantages:
- Lateral flow assays are extremely quick per patient, giving results in just 15 minutes.
- Testing levels of antibody in blood allows a single patient sample from one accessible part of the body where sampling is non-invasive to be tested for presence of virus.
- These tests require very little training to perform and don’t rely on specialist laboratories or scientists to analyse.
Disadvantages:
- The technology is new and the evidence for its accuracy in coronavirus diagnosis is still being evaluated.
- So far, available lateral flow tests can only determine if a patient has at some point been infected with COVID-19. Further testing would be needed to check if a patient is currently infected. Future versions of this technology might allow clinicians to detect current infections.
- Lateral flow tests are more expensive and time consuming for large batch testing than specialist laboratory based antibody tests such as ELISA.