Global Health Science Education

Coronavirus Testing 1….2…..3…….

By Mrinalini Watsa

Disease testing sounds complicated. Reading about it in the news in the context of the ongoing COVID-19 pandemic is even worse. If you are pretty sure that you’ve read contradictory news articles — you are not wrong!

Most of them don’t slow down to explain that there are actually multiple ‘coronavirus tests’ , which are usually conflated willy-nilly , producing justifiable confusion in a reader.

The goal of this article is simple: clarity. And I will continually work on it until this goal is achieved, so feel free to point out inconsistencies or obscured meanings — never, ever be afraid to admit that you cannot understand this biology. After all, understanding it could be the difference between endless quarantine and a return to normalcy, a diagnosis of COVID-19 or a mild cold.

It’s critical to understand. Thankfully, it’s also relatively simple.

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A Little Biology:

  1. SARS-CoV-2 is the virus causing the disease we call COVID-19. If you want to call it ‘coronavirus’ you can, but it would be like me calling you a primate, instead of at least identifying you by your species — a human. There are many strains or species of coronaviruses, and SARS-CoV-2 is the one we are fighting today.
  2. The coronaviruses are like us in that they have a genome. This means, they have genetic material, or a set of molecules that serve as instructions for our cells to produce the proteins that our bodies need to survive. Every genome contains genes interspaced with other genetic material. Most cells in your body have an entire genome in them.
  3. Viruses are NOT like us in that they do not have cells. A virus is just a shell of proteins with genetic material inside it. People are still arguing about whether we can even deem them to be alive. Creepy, I know.
  4. Viral genomes can contain DNA or RNA: the only difference between them is a single letter in the code. DNA’s code is made up of four molecules we call A, T, C, and G. In RNA, the T is replaced with a U molecule. Further, viral genomes can be double or single stranded and each kind behaves differently inside a host cell to multiply itself.
  5. Viruses are incredibly simple organisms that don’t have a lot of machinery to for self-replication, so they typically hijack a host’s cell’s machinery to make more copies of themselves. First, viruses bind to a host cell through specific receptors on its surface. They then either merge their membranes with the host cell, depositing their contents into the cell, or are eaten by a host cell in a process called endocytosis. Once inside, they strip their protein coats off, and release their genetic material, which form messenger RNA or mRNA. Diabolically, these mRNA are then converted by the host’s own ribosomes (organelles in the cells) into viral proteins!

The illustration below shows how the virus is pulled into a host’s cell, shooting out its messenger RNA (the orange strands) which eventually are the basis for the creation of more viral proteins that are used to construct new viruses.

Test 1: The Genetic or PCR test

Sample: a swab of the infected area, in this case, a nasal passage.

Equipment: Several machines and chemicals that are common to most labs, but hard to acquire as a private citizen, are required for this test.

  • A sterile workspace.
  • A PCR machine: a device that controls temperature precisely to mimic the conditions in a cell in which DNA strands separate, bind to enzymes, and make copies of themselves.
  • Reagents (or chemicals) that specifically target one or more genes in the virus. A reagent is simply a chemical or ingredient in a test.
  • Positive and negative controls. These are key! If you want to know your test is accurate, you need to run (at the same time), a blank that must show up negative (the negative control), and a sample you know must show up positive (the positive control). If your negative control (usually just water) tests positive, then a reagent you used in the test is likely contaminated with viral DNA. Any tests run with those reagents can then become positive due to contamination. If your positive control shows up negative, then your test is not working at all to amplify the virus SARS-CoV-2.

Detection: This test searches for one or more specific genes unique to the genome of the virus.

Implication: A positive test implies that you are infected with the virus SARS-CoV-2. So long as your negative control is negative, and your positive control is positive.

When to take it: When you are feeling sick (a cough and high fever, difficulty breathing) and think you might be infected with the virus

Rapid Tests Currently Available: Abbot (5–13 mins), Cepheid (45 mins), Mesa Biotech (30 min test). Downside to these is most can be run only 1 at a time. The standard PCR test shipped everywhere by the CDC can be run ~96 samples at a time, but takes longer per sample.

Variations on The Genetic Test:

  1. PCR + electrophoresis: Run a PCR test. Then take the PCR product (the thousands of copies of the gene of interest) and put it in a simple device that separates pieces of DNA by size. Since you know the size of your gene target, if you see the right sized fragment, you know you have a positive. This is cheap and easy, but time consuming to scale up and requires visual inspection, which is subject to error.
  2. Quantitative PCR: Run the PCR but with a probe that is tagged with a fluorescent dye. As the test proceeds, the higher the fluorescence detected, the more of the gene is present. The machine continuously monitors this from start to finish of the test, creating a curve that eventually flattens out. If you began with many more copies of the viral genome, such as with a badly infected person, then the curve shoots up at a faster rate, which you can see. Therefore, you can actually quantify the amount of starting material and distinguish a highly infected person from one who is mildly infected.
  3. Isothermal LAMP: Although described in the early 2000s, and much easier to run (only room temperature is needed, with no PCR machine), this is the least used method because of how complicated it can be to design the probes that search for the right gene. If you get it right though, this method can let you create almost any way to signal a positive test. Two common ones are increased fluorescence or turbidity of the sample — both good visual cues. However, you could also hijack a pregnancy test and make it read coronavirus samples by linking the compound the pregnancy test typically reads (human chorionic gonadotrophin, or HCG) to the probe searching for coronavirus. Then, when it is a positive, the HCG test could signal a positive too. The possibilities are endless and rather wonderful.

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Test 2: The Test for Immunity

This test is based on an ELISA — or an Enzyme-Linked ImmunoSorbent Assay. It is designed to target a specific antibody, which is a type of molecule (a protein) that your blood releases in response to an antigen (also a protein) produced by a foreign object, like the virus, in your body.

There are a few different types of ELISAs out there, and here is a brilliant video on how they function. But, there is one important caveat. In the test video below, the antigen = the antibody your body has made in response to the virus. ‘Antigen’ is used here as a generic term for the thing you are trying to detect (the blue, funny shaped molecule). Just remember that the antigen the test is trying to detect is the antibody your body has made in response to the antigen that the virus presents to it during an infection.

Sorry — but sometimes language is used in funny and confusing ways in science. The video below will make it all clear.

Sample: A little blood.

Equipment: These are quite different than the PCR test.

  • A sterile workspace (common to both tests).
  • An enzyme immunoassay reader, which is a machine that can measure qualities of light (i.e. the intensity of absorbance, fluorescence or luminescence, see video above) which can be indicators of the presence of a particular target molecule.
  • Reagents specific to one or more antibody proteins for CoV
  • Positive and negative controls.

Detection: Finds antibodies to SARS-CoV-2 in your blood.

Implication: A positive test implies that you were previously infected with SARS-CoV-2 and currently have some level of immunity to it.

When to take it: If you want to know if you are immune to the disease after a confirmed or suspected bout of illness with SARS-CoV-2

Rapid Tests Currently Available: SureScreen diagnostics (fingerprick only), RayBiotech (ditto).


Final Takeaways:

  1. If you suspect you have SARS-CoV-2, try to get tested with a PCR-based test right away. This can save you a lot of worry later, even if your symptoms are mild. If you are positive and you make it through safely, you should have antibodies to it in your blood.
  2. If you don’t get tested, or receive a negative test result with a PCR test, it doesn’t mean you have not had COVID-19. Many people can be entirely asymptomatic (lucky for them, but unlucky for those whom they come into contact with) and there is a small percent of false negatives possible on the tests, by chance. For these people, the only way to know if they have immunity to the virus is to take the second type of test.
  3. We do not know currently how long the antibodies to the virus will remain in anyone’s blood stream. Anti-rabies vaccines can confer immunity for ~10 years, for example, but SARS-COV-2 antibodies are expected to last for around 1 year.
  4. There are options by which a person with many antibodies could donate plasma for infusion into a sick patient, to potentially relieve their symptoms, but this is not yet standard protocol for this virus.
  5. Immune people can still get SARS-CoV-2. Let me repeat that. Just because you have antibodies to the virus does NOT mean you cannot be infected again, or that you will never feel sick or transmit the disease. ALL of those things are possible. What’s the whole point then, you might wonder? Well, the antibodies should reduce the severity of your next infection, making you get better faster, and therefore, spread the virus to many fewer people than you would have without the antibodies. Why can’t we say for sure? Because all of that research takes time — and we cannot fast forward that process too much.
  6. The single way to counter this pandemic is with an effective vaccine. A vaccine lets you develop antibodies without enduring the full onslaught of a disease. Never before has it been more clear why vaccinations are not a life choice, but a privilege and a GREAT gift to your community.

Cover photo: An illustration of SARS-CoV-2, the virus behind the COVID-19 epidemic. Image credit: Covert