How to read a Covid-19 test report?

I hope everyone reading this post is safe, happy (Hard in Covid times, but we can try! 😛), and healthy. This post is my attempt at helping those who themselves or whose loved-ones have unfortunately been infected by Covid-19, in better understanding their RT-PCR Covid report. 

I have written it in such a manner that it should be accessible to those of us who do not have an idea about molecular biology. Consequently, I have not gone into any details and have discussed various concepts quite superficially. Some of my relatives who got infected with Covid-19 (and all have recovered, thanks to God and the doctors!) were kind enough to share their reports with me. This allowed me to have a look at how an actual report looks like and also motivated me to write this post.

What you need to know to understand this post (basic concepts):

(Skip this section if you have ever done even a single molecular biology course)

 1. COVID-19 is caused by a virus (called SARS-CoV2)

 2. Virus is a very small (sub-microscopic) infectious agent. One virus particle is made up of a chain of certain molecules (called nucleic acids) capsuled in a protein covering. The aforementioned chain is called RNA or Ribonucleic acid.

3. Multiple virus particles are required to cause the infection. A single virus particle is generally not capable of causing the infection. 

4. What are Genes? 

The functioning of our body, like muscular movement, digestion, etc is carried out by proteins present in the cells of our body. Proteins are chains of certain molecules called amino acids. The sequence of amino acids in a protein is critical for its optimal functioning. The sequence that works the best for a protein differs between different types of protein. Now, what determines or dictates, or provides information about this sequence are the genes. 

Our body is made of a number of cells (~30 trillion) where each cell contains DNA or Deoxyribonucleic acid. DNA, like RNA, is also a chain of nucleic acids. Only certain parts of DNA guide the production of proteins and these parts are called genes.  

A particular gene may correspond to a particular protein in two ways: 

  A. By dictating the sequence in which the amino acids will be chained together to form that protein. The sequence of amino acids is dictated by the sequence of nucleic acids in the gene in a 2 step process of transcription and translation.

  B. By dictating the amount of the protein that would be produced in the body.

💡 Note that genes, which dictate protein production and virus particles, both are made of nucleic acids. So, if a virus enters our cells, would it interfere with protein production in our body? Yes! (and this is what exactly happens)

5. Viral particles enter the cells of our body and use the cell's resources to manufacture their proteins. This interferes with the cell's normal functioning.

How do Covid-19 tests work? 

Think of the virus as a criminal, your body as the site of the crime or the victim, and the Covid-19 test as the detective trying to solve the case. The detective wants to know if it is indeed the virus (SARS-CoV2) that's been troubling you. Following are the ways detective can determine that:

1. Check if the virus is present at the site of crime (your body) - RT PCR test, antigen-tests

2. Check what kind of defense (immune response) is the victim (your body) using to fight the virus, the 'type' and 'magnitude' of defense can inform about the attacker - Immunoassays

Reading the Covid-19 test report: RT-PCR

Let us discuss the RT-PCR test report here:

RT-PCR amplifies the viral load (see the red circled part of report) by making copies of it over a certain number of cycles (see the blue circled part of the report). Let's decode this statement:

Short story: More the number of cycles required (Ct value), the less is the viral load and the better it is for you. But, if you are interested in a longer story, please read on.

What is RT-PCR?

Generally, the DNA content isolated from cells of organisms is in quantities that are not detectable. To detect and further study this DNA like finding its sequence, we need to amplify it in quantity. PCR is a tool or a method to amplify the quantity of DNA in a sample. PCR stands for Polymerase Chain Reaction and works by making copies of the DNA strands over certain cycles.

Wait! the virus is made of RNA, but the PCR only amplifies DNA, right? Right!. So, to detect and study RNA, RNA strands are first converted to DNA using a process called reverse transcription. Then this DNA, also referred to as complementary DNA or cDNA is amplified using PCR. The above process of converting RNA to DNA and then amplifying is called RT-PCR or reverse-transcription PCR.

What is it that the RT-PCR amplifies exactly?

In a COVID test, RT-PCR is used to amplify the viral RNA. Viral RNA consists of several parts (genes) that dictate the production of various proteins that help the virus survive and spread in the body. The test amplifies one or more of these genes. Following are the names of some of these genes - E, N, RdRp, ORF1a, ORF1b, etc

What are these 'cycles' or the Ct number reported against the name of virus genes?

It is the number of cycles that were required to get a certain quantity of a particular gene. Let's consider an example:

Instead of biscuits, there are DNA strands in RT-PCR. Recall, that these DNA strands are gotten from viral RNA strands in, say a nose swab from the patient. As one can understand from this example, when the initial viral load (the viral load in the nose swab) is more, the number of cycles (Ct) required to reach a certain specific quantity by doubling that viral load would be less. So, a smaller Ct value represents a greater viral load and a larger Ct value represents a lower viral load. As one recovers from Covid-19, the reports would show a higher Ct value than what was shown early in the infection. 

References 

1. Sheikhzadeh, E., Eissa, S., Ismail, A., & Zourob, M. (2020). Diagnostic techniques for COVID-19 and new developments. Talanta, 121392.

2. Molecular biology courses that I followed as part of my undergraduate career at the Indian Institute of Technology (IIT) Madras, India