COVID-19: what are the different types of tests available?
Tuesday, August 27, 2019. Author FitnessGenes
Tuesday, August 27, 2019. Author FitnessGenes
FitnessGenes are proud to announce we’re one of the approved providers on the UK government’s Test to Release scheme.
From December 15th, travellers to England
Must be a PCR test.
What is a PCR test and what are the different tests available?
Broadly speaking, there are two main categories of COVID-19 tests:
The first category includes tests that detect the presence of genetic material (called RNA) inside viral particles. PCR (or RT-PCR) tests, such as the one offered by FitnessGenes, are an example of this. Antigen tests, which detect proteins on the surface of viral particles (rather than looking at genetic material), also fall into the first category.
A variety of techniques can be used to detect and measure viral proteins or RNA, including: RT-PCR, LAMP, and lateral flow assays.
We’ll talk about how these different testing techniques work, and their relative merits, later in this article.
The second category of tests, also known as serological or antibody testing, is used to detect the presence of antibodies in blood serum. Again, various testing techniques can be employed in antibody tests, including ELISA and lateral flow assays.
For the sake of brevity, this article will focus on the first category: tests that tell you whether you are currently carrying the SARS-CoV-2 virus.
Different tests all have their respective advantages and disadvantages.
RT-PCR tests, for example, are highly sensitive to small amounts of the SARS-CoV-2 virus, but it may take several hours for results to be ready.
By contrast, lateral flow rapid antigen tests can give you a result in just 15-30 minutes, but they’re not as sensitive as RT-PCR tests, and may incorrectly give you a negative result if you’re carrying low amounts of the virus.
Given these differences between tests, how should governments, institutions and individuals decide which type/s of test to use? Below are some key factors to consider:
- Cost and availability of resources
Some tests cost more than others.
This largely stems from differences in the technology and human capital required for the various testing techniques.
For example, the technology and reagents used in LAMP (Loop-mediated Isothermal Amplification) tests are newer, more sophisticated, and less widely available than those used in RT-PCR testing. Consequently, as the graph below shows, the cost per test is slightly higher.
One of the most important features of a COVID-19 test is its sensitivity: the ability to give a positive result when someone does truly have COVID-19.
Someone who does truly have the disease and is given a positive a result is known as a true positive case.
However, tests can sometimes miss people who have COVID-19, and incorrectly give them a negative result. Such cases are known as false negatives.
If we know the number of true positive and false negative cases generated by a test, we can calculate its sensitivity as follows:
A test with a 95% sensitivity, for example, will correctly return a positive result for 95% of people who have COVID-19. It will also, however, incorrectly return a negative result for 5% of people who do in fact have the disease.
Perhaps it may seem counterintuitive, but tests with high sensitivity are better at ‘ruling out’ people with a negative COVID-19 test result. As high sensitivity tests generate a low number of false negatives, we can be more confident that someone with a negative result truly does not have COVID-19.
In this respect, a negative result from an RT-PCR test, which have a higher sensitivity (around 98% according to one study), would carry more weight than a negative result from a lateral flow rapid antigen test, which have a lower sensitivity (76.8% according to one study).
As well as flagging up people with COVID-19, a good test will also correctly identify people without COVID-19. This is reflected in a test’s specificity: the ability of a test to give a negative result when someone truly does not have COVID-19.
Someone who truly does not have the disease and is given a negative a result is known as a true negative case.
Occasionally, however, a test can mistakenly return a positive result, even though someone does not have COVID-19. These cases are known as false positives.
The number of true negative and false positive cases can be used to calculate specificity as follows:
A test with a 95% specificity, for example, will correctly return a negative result for 95% of people who do not have COVID-19. It will also, however, incorrectly return a positive result for 5% of people who, in reality, do not have the disease.
Again, it may seem counterintuitive, but tests with high specificity are better at ‘ruling in’ people with a positive COVID-19 test result. As high specificity tests generate a low number of false positives, someone with a positive test result is likely to truly have COVID-19.
On this note, positive results carry more weight in tests where the specificity is higher than the sensitivity. Lateral flow rapid antigen tests are one such example: with studies suggesting a 99.68% specificity and 76.8% sensitivity for the Innova SARS-CoV-2 Antigen Rapid Qualitative Test.
An important related point to make is that test results and sensitivity/specificity figures should be used in conjunction with clinical judgement. A negative test result is less likely to be correct if someone is showing overt symptoms of COVID-19 (such as a cough, fever, loss of smell/taste).
Certain tests, such as lateral flow tests and some LAMP tests, are known as ‘point-of-care’ tests. This means they use portable equipment and samples do not have to been sent to specialist laboratories in order to get results. This makes it easier to conduct testing and deliver results on site, which is clearly useful for settings such as outpatient clinics, care homes, airports, workplaces etc.
- Speed of testing
The time from providing a sample to getting a test result varies considerably from test to test.
On an individual basis, tests that require processing in a laboratory using specialist equipment and expertise, such as RT-PCR tests, typically take longer than ‘point-of-care’ tests, which can be delivered on site.
By contrast, specialist lab equipment such as PCR machines can often be used to test large batches of samples at the same time. This may make them faster than point-of-care tests for large scale testing and more suited to high through-put testing.
It’s worth bearing in mind that the perfect test doesn’t exist and that different test types involve trade-offs between the above criteria. For example, rapid lateral flow antigen testing sacrifices sensitivity for quicker results.
Similarly, some tests may have a high sensitivity (thereby picking up more people with COVID-19 and producing more positive results), but at the expense of specificity (producing more false positive results).
On a related note, when looking at sensitivity and specificity, the associated ‘costs’ of false positive and false negative results need to be weighed up. For example, someone who has COVID-19 but is incorrectly given a negative result (i.e. a false negative case) may inadvertently spread the virus to others. This is arguably worse than someone without the disease but with a positive result (i.e. false positive case) self-isolating needlessly.
RT-PCR stands for Reverse Transcription Polymerase Chain Reaction. This type of test analyses nasal swab, sputum, stool, or saliva samples for the presence of genetic material - called RNA – that is specific to the SARS-CoV-2 virus.
As it is a widely established technique and shown to be highly sensitive to viral RNA, RT-PCR is considered to be the ‘gold-standard’ of COVID-19 testing.
How do RT-PCR tests work?
We’ve outlined the RT-PCR process in more detail in this article. Readers are encouraged to visit that article for an in-depth overview, but we’ll briefly describe the process here too.
In order to enter human cells, hijack human cell machinery, and make copies of itself, the SARS-CoV-2 virus needs to produce various proteins. The instructions or genetic code for making these proteins is contained within a molecule called RNA.
The aim of RT-PCR is to isolate and amplify specific portions of this RNA code. If your saliva/sputum/swab sample contains these pieces of viral RNA code, then this will show up on RT-PCR testing and you will receive a positive result.
Some types of RT-PCR test, known as quantitative RT-PCR, can also tell you your viral load: the amount of virus someone is carrying in a given volume of bodily fluid (e.g. sputum, saliva, or blood).
Stages of RT-PCR
We can break down RT-PCR into 4 main steps:
Step 1: Sample collection
RT-PCR can be used to test for viral RNA in nasopharyngeal swab, saliva, sputum, blood, and stool samples.
Step 2: Extraction and purification of viral RNA
Specialised chemicals are used to break open or ‘lyse’ viral particles to release RNA. Other chemicals are also used to remove other cell material and molecules, leaving only purified RNA.
Step 3: Amplification
Samples will only contain a miniscule amount of RNA, so we next have to amplify and create millions of copies in order to get a detectable amount of genetic material.
Using an enzyme called reverse transcriptase, viral RNA is converted into a complementary copy of viral DNA. This reverse transcription step is required because only DNA can be copied and amplified.
Specially designed molecules, called primers, are then added, which ensure that only the DNA copies of specific sections of the SARS-CoV-2 RNA code are amplified. Fluorescent markers are also added: these are later used to detect the amplified DNA copies of the viral RNA.
The entire mixture of viral DNA, primers and fluorescent markers is then put in a PCR machine. Using a process, called thermocycling, the PCR machine repeatedly heats and cools the mixture to catalyse chemical reactions that create more and more copies of the viral DNA. Each heating and cooling cycle doubles the number of DNA copies.
Typically, the PCR machine will go through 35 cycles, meaning that 2^35 or about 35 billion DNA copies of the original viral RNA will be produced.
Step 4: Detection
During the amplification stage, fluorescent markers are attached to viral DNA copies. These produce a light signal, which is read by the PCR machine. If the light signal exceeds a certain threshold, it indicates that SARS-CoV-2 RNA was present in your sample. In this case, you will receive a positive result.
The number of heating and cooling cycles required for this light threshold to be met can be used estimate how much viral RNA was in your sample. If fewer cycles are required, then this indicates your sample contained more virus particles.
If no SARS-CoV-2 RNA was found in your sample, then no complementary viral DNA will be created and amplified during the RT-PCR process, leading to no light signal. In this case, you will receive a negative result.
RT-PCR tests have a high sensitivity, meaning they have an excellent ability to pick up people with COVID-19. Moreover, they are less likely to return a false negative result, meaning that a negative RT-PCR test result (alongside clinical judgement) can be reliably used to rule someone out from having COVID-19.
A meta-analysis of COVID-19 tests found that RT-PCR had a sensitivity of 98% when using nasopharyngeal swab samples, and 91% for saliva samples.
Studies suggest that RT-PCR tests have a specificity of greater than 95%. Due to the lower chance of false positive results, a high specificity means that a positive RT-PCR test result can be reliably used to rule someone in as having COVID-19.
Sensitive to small amounts of virus
One of the reasons that RT-PCR is considered the gold standard of COVID-19 testing is that it can detect extremely small amounts of the SARS-CoV-2 virus.
This is captured in a measure known as limit of detection: the lowest concentration of SARS-CoV-2 RNA that is detectable.
As the graph below illustrates, studies have found that RT-PCR tests can detect just a single molecule of viral RNA.
Slower than other tests
RT-PCR can deliver a COVID-19 test result in 3 hours, although laboratories typically take between 6 - 8 hours for results to be ready.
Longer testing times using RT-PCR partly result from the heating and cooling cycles needed to amplify DNA.
Require a laboratory
RT-PCR requires a laboratory, specialised reagents, and a PCR machine to process samples. Accordingly, RT-PCR cannot be performed on site (e.g. in a care home, workplace), and swabs/saliva samples must be sent to a laboratory for testing.
That said, point-of-care of RT-PCR tests, which use portable devices and do not require a laboratory, are currently being trialled. One of these is the CovidNudge test, which involves placing a nasopharyngeal swab directly into a cartridge that then performs RT-PCR and can yield results in 90 minutes.
LAMP (or RT-LAMP) stands for Loop-Mediated Isothermal Amplification.
It is a very similar process to RT-PCR, in that it also tests for SARS-CoV-2 RNA by creating and then amplifying viral DNA copies. The key difference, however, is that the amplification process in LAMP is isothermal: - it uses a constant temperature rather than using heating and cooling cycles.
A similar process to LAMP is called RPA (recombinase polymerase amplification). This uses a different enzyme for amplification, which can also be conducted at a constant temperature with no need for heating and cooling cycles. RPA and LAMP can also be combined into a protocol known as RAMP.
Another feature of LAMP (as well as RPA and RAMP) is that the presence of amplified copies of viral DNA causes the sample to become cloudy or change colour. These changes are visible to the naked eye, meaning that a scientist or clinician can quickly confirm a positive or negative result.
As with RT-PCR, LAMP tests can be used on a variety of samples, including nasopharyngeal swabs, sputum and saliva.
Quicker than RT-PCR
By obviating the need for heating and cooling cycles (thermocycling) used in RT-PCR, LAMP tests can cut the time needed for amplification of DNA (as shown in the green bars in the diagram below).
As such, LAMP can return results in a much quicker time than RT-PCR, often in 1-2 hours.
High sensitivity and specificity
Studies suggest that LAMP testing for COVID-19 has a high specificity and sensitivity comparable to (albeit slightly lower than) that of RT-PCR.
As it is a fairly new procedure, however, there are a lack of thorough meta-analyses on LAMP testing.
Portable / point-of-care testing
Although not yet widely available, portable instruments can be used for the amplification and detection steps of LAMP. This means that samples do not need to be sent to a laboratory and can be tested on site. In fact, LAMP testing is currently being offered in Heathrow airport.
The downside of portable, point-of-care LAMP testing, however, is that large numbers of samples cannot be tested at the same time.
Less widely available than RT-PCR
LAMP testing is a much newer technology than RT-PCR and is therefore less widely established across hospitals and laboratories. The testing protocols may still need to be validated in some areas.
May require a laboratory
High through-put LAMP testing still requires much of the same technology as RT-PCR, including access to a PCR machine. As such, samples will need to be sent to a laboratory.
Lateral flow assays use the same technology found in pregnancy tests.
They are small testing strips that can be used on small droplets of blood to detect the presence of antibodies to the SARS-CoV-2 virus. More recently, lateral flow tests have been developed to test for viral antigens- proteins on the surface of virus particles.
Lateral flow antigen tests are therefore different from RT-PCR and LAMP tests in that they do not test for genetic material (RNA), but test for surface proteins expressed by the SARS-CoV-2 virus.
Currently, lateral flow rapid antigen tests, such as the one created by Innova, are in the process of being trialled across the UK.
Lateral flow antigen tests are similar to pregnancy tests in that they are immunochromatographic assays – absorbent test strips that contain antibodies which are targeted to a specific protein (in this case, proteins on the surface of the SARS-CoV-2 virus). If these proteins are present in a sample, they will bind to the antibodies, producing a coloured line on the test strip that is visible to the naked eye.
We can break down the process of lateral flow rapid antigen tests into 4 key steps:
Step 1: Sample collection
The Innova lateral flow rapid antigen test is currently designed to be used with nasopharyngeal swabs. Other lateral flow tests are being developed to use saliva. Once collected, samples are applied to the sample application pad of the test strip.
Step 2: Sample binds to detection antibodies
On one side of the test strip is an area called the conjugate release pad. This area contains detection antibodies that are specifically targeted to surface proteins of the SARS-CoV-2 virus.
If your sample contains virus particles, then the detection antibodies will bind to the viral surface proteins forming a complex (viral antigen- detection antibody complex). The detection antibodies also have coloured labels which are designed to show up further along the test strip.
Step 3: Sample diffuses across the test strip to the test line.
Once bound to the detection antibodies, the sample complex moves across the test strip membrane. At some point on the test strip, there is a test line. This is a line of ‘capture antibodies’ that are also targeted to the SARS-CoV-2 antigen proteins.
As their name suggests, the capture antibodies bind to and capture the sample complex (viral antigen- detection antibody complex). This triggers a colour change in the detection antibody, causing the test line to become visible.
The appearance of the test line indicates a positive result, suggesting your sample contained the SARS-CoV-2 virus.
If your sample did not contain any virus particles, then no sample (viral antigen-detection antibody) complexes will be formed or captured by the test line. Consequently, the test line will not appear and remain invisible. This indicates a negative result.
Step 4: Sample diffuses across to the control line
Located further along the test strip is another line of capture antibodies, known as the control line. This line contains antibodies that are targeted to the detection antibody. Any excess detection antibody is captured by this line, again triggering a colour change.
The appearance of a coloured control line is mainly to ensure the test has worked properly and the sample has properly diffused across the test strip.
Quicker results than RT-PCR and LAMP
The main advantage of lateral flow antigen tests is that they can produce results in just 30 minutes.
Point of care testing
As a laboratory is not required, lateral flow tests can easily be distributed to and conducted in a variety of settings e.g. care homes, airports, workplaces.
Test strips are fairly inexpensive to mass produce and the lateral flow testing technique does not require any specialist laboratory equipment or reagents. Furthermore, the tests are easy to use, so no specialist training is needed to conduct and interpret the tests.
Lateral flow rapid antigen tests have been found to have very high specificity (99.68% in one study). This suggests that a positive test result can be used to ‘rule in’ someone as being likely to have COVID-19.
Lower sensitivity than RT-PCR and LAMP
The largest drawback of lateral flow antigen tests is their poorer sensitivity. Preliminary analysis of the Innova SARS-CoV-2 Antigen Rapid Qualitative Test, for example, has found the test to have an overall sensitivity of 76.8%.
This means that a substantial portion of people tested may incorrectly receive a negative result while actually having COVID-19. This is more likely to be the case for people with low viral loads i.e. people infected with SARS-CoV-2 but carrying lower amounts of virus in their respiratory tract.
Low sensitivity is likely to be particularly problematic if using tests to control local outbreaks, as people given a false negative result can continue to spread the virus unwittingly.
Limited use in isolation
Given the lower sensitivity of lateral flow antigen tests, people receiving a negative result may require another more sensitive test (e.g. RT-PCR) to confirm they do not indeed have COVID-19.
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