In this post we’ll cover the different kinds of tests available, some background on the testing principles, what the tests tell us, and a few dos and don’ts.

PCR (polymerase chain reaction) tests

The PCR method was invented in 1985 by Kary Mullis. You will sometimes see people on Internet posts say that PCR tests are not valid for SARS-Cov2 because PCR as originally invented was not meant to be used as a diagnostic test. Rubbish! Science has moved on hugely since 1985 and is still exploring novel ways to apply PCR techniques.

Basically, one takes a sample containing the DNA of interest, heats it up to denature it (it separates into two pieces), and an enzyme called Taq polymerase (“Taq”) is added so that two new DNA strands can be made. The process is repeated many times and is automated. The idea is to amplify the tiny amount of DNA in the sample so one ends up with enough DNA to analyze.

PCR testing for viruses was one of the first applications of the technique. For SARS-Cov2 testing the test detects viral RNA and employs the reverse transcriptase polymerase chain reaction, or RT-PCR, which is a version of the basic PCR approach, to produce complementary DNA.

RNA contains the sugar ribose, while DNA contains the slightly different sugar known as deoxyribose and RNA has the nucleobase uracil while DNA contains thymine. Nucleotides (the sugar plus the nucleobase) are attracted to each other through electrostatic forces known as hydrogen bonds. DNA that is complimentary to the viral RNA has the opposite sequence; so if the RNA strand has an adenine-based nucleotide, it’s paired DNA nucleotide will be thymine based (i.e., A-T pairing).

Regions of the DNA coding for particular viral gene fragments are detected by primers, which are small pieces of DNA designed to only connect to a genetic sequence that is specific to the viral DNA, ensuring that only viral DNA is duplicated. After the primers attach, new complementary strands of DNA extend along the template strand. As this occurs, fluorescent dyes attach to the DNA, providing a marker of successful duplication. At the end of the process, two identical copies of viral DNA are created. The cycle is then repeated some 25-40 times to create enough DNA to be detected through fluorescence (Figure 1).

To ensure really good sensitivity, meaning few false negative results, and reasonable specificity, meaning few false positive results, there has to be a balance between over-amplifying and under amplifying the DNA. The cycle threshold (Ct) value is the actual number of cycles it takes for the PCR test to detect the virus. This number estimates how much virus was likely in the sample to start with, although not the actual amount. If the virus is found in a low number of cycles (Ct value under 30), it means that the virus was easier to find in sample and that the sample started out with a large amount of the virus.  A good analogy is the zoom viewing feature  on your computer. If you only have to zoom in a bit(like 110%), it means that item was large to start with but if you have to zoom a lot (like 200%), it means that the item was small to start with. If you want to know more about the mechanics of SARS-Cov2 PCR testing, the Public health Department in Ontario, Canada, has a nice web page on the subject.

Separation of the DNA template strands, attachment of the primers, and duplication of DNA strands containing the primers and attached fluorescent strands. Figure courtesy of the National Human Genome Research Institute.

Fig 1. Separation of the DNA template strands, attachment of the primers, and duplication of DNA strands containing the primers and attached fluorescent strands. Figure courtesy of the National Human Genome Research Institute.

The primers in PCR tests target several conserved viral epitopes, meaning parts of the viral DNA that are common to all variants or strains, so although in theory there could be some interference in some evolved strains, in practice it has been found that this is not a problem. There are also tests that target multiple viruses, such as the multiplex PCT test which tests for flu and SARS-Cov2 simultaneously.

Generally speaking, if you become infected, a PCR test will pick it up in 1-3 days, so there is a short time in which testing won’t find the virus. PCR testing is currently the gold standard and good tests have a sensitivity and specificity of about 99.9% (some are considerably worse). However, it is expensive (like $100, though some tests have come down a lot in price) and can take 1-2 days to get the results. Moreover, after infection one can test positive for several weeks because the test is so sensitive. Because of the expense and delays, and need to see when a person is recovering (repeated tests) and can no longer infect someone else, more rapid tests were devised.

Antigen Tests

Antigen tests work by detecting the presence of viral proteins using simple immunochromatography methods, commonly referred to as lateral flow assays. Rapid antigen tests target the SARS-CoV-2 nucleocapsid protein, the most abundant protein expressed by the virus. They directly assess the presence of viral proteins, making them different from serology tests, which look for antibodies produced by the host in response to the infection. Thus, rapid antigen tests assess for acute infection only, not prior infection or response to vaccination.

Rapid antigen tests offer a number of advantages over molecular assays. Testing can be deployed outside of hospital laboratories, and many can be performed by members of the general public. They are relatively inexpensive relative to RT-PCR (like $5 per test plus shipping/handling, etc.) and simple to interpret (think: pregnancy test in which you either have a colored line or you don’t; see Figure 2). They have a turnaround time as fast as 15 minutes. They are also compatible with samples taken from the anterior nasal area rather than the nasopharynx, making them more comfortable!

The primary disadvantage of the antigen test is a risk of false negative results in people with low viral loads who may be early in their infection, and who go on to spread it to others in subsequent days. In practice, this has not been found to be a big deal. There is also a slightly elevated rate of false positives relative to PCR tests, though the rate is dependent on the prevalence of disease and the proportion of people who are symptomatic. For many commonly used rapid antigen tests, the negative predictive value (e.g., the likelihood someone with a negative test is truly negative for infection) is greater than 98%, which is still a very good number.

Recommendations

For every day testing I favor antigen testing, but for travel to other countries in which one needs one or more negative tests to enter I would want to have a PCR test if I was non-symptomatic and an antigen test if I had recovered from an infection a few weeks ago, assuming one has a choice.

Abbott’s BinaxNOW™ COVID-19 SARS-Cov2 lateral flow test device

Fig 2. Abbott’s BinaxNOW™ COVID-19 SARS-Cov2 lateral flow test device

Antibody Tests

Antibody tests, sometimes known as serology tests, check for antibodies as a result of SARS-Cov2 infection. Antibodies generally show up 1-3 weeks post-infection. They are useful to check if someone really had SARS-Cov2 as opposed to something like flu. If you had a vaccination for SARS-Cov2, you would also test positive. For research studies, these tests can help gauge the likely herd immunity in a geographic area or given population.

The most common tests are point-of-care (POC), which are diagnostic tests performed at or near the place where a specimen is collected and can provide results within minutes rather than hours. In general, these tests are like lateral flow antigen device tests, and detect IgG, IgM, or total antibody in fingerstick whole blood. Laboratory tests include lateral flow, ELISA, or chemiluminescent immunoassay (CIA) methods for antibody detection in serum, plasma, or whole blood. Based on the test, total antibody (Ig) can be detected, or IgG and IgM can be detected separately. Most tests are designed to detect antibodies against either multiple forms of the S (spike) protein (full-length (S1+S2) or partial (S1 domain or receptor binding domain or RBD)) or the N (nucleocapsid) protein. The choice of antigenic targets can help address different aspects of immune response; for example, antibody detection against the RBD is considered to have higher correlation toward functional aspects like ability to neutralize virus. If one tests positive for antibodies against the S protein but not the N protein, this means you were vaccinated but not infected with SARS-Cov2. A positive test for both types of antibodies means that you were infected by SARs-Cov2 but the results won’t tell you if you were vaccinated.

These days, antibody tests are really only useful for individuals if you are unvaccinated and are trying to decide whether you should be vaccinated based on your infection status (maybe you had minimal symptoms or were asymptomatic) and thus have some immunity. That said, those tests won’t tell you when you were infected and what strain you were infected with. I should also say that while natural immunity is good, immunity will wane 6-12 months post-infection, and a booster shot, depending on how healthy you are, is a good idea a year or more after infection.