Why do some recovered COVID-19 patients test positive again after testing negative? Do these patients really become “re-infected”? (Spoiler alert: probably not). A thread. (1/n)

The answer to this question has to do with the test that is used to diagnose infection. But before I get to that, I would like to briefly clarify that coronaviruses do not “lay dormant” or “reactivate.” (2/n)

The dormancy phenomenon is kind of unique to viruses like herpesviruses, papillomaviruses and HIV. Coronaviruses don’t do that! They enter a cell, replicate and get out to infect others. (3/n)

All they leave behind is a “memory” of the infection, in the form of memory B cells and T cells. These cells can recognize the virus upon a second infection and clear it before the virus has a chance to cause any disease at all, or at least before it can cause severe disease. 4/n

Now about the test: The predominant test used to diagnose SARS-CoV-2 infection is called a quantitative real-time polymerase chain reaction (qRT-PCR) test. I know this is a mouthful, but I will explain below what that means and how it works. (5/n)

A nasopharyngeal swab is taken from the patient. This swab contains mucus, which possibly contains virus. The sample is mixed with reagents which first of all inactivate the virus. All that is left behind is the viral nucleic acid – which is RNA. (6/n)

However, RNA is unstable in the test tube, so it is first turned into DNA by a process called reverse transcription. That DNA then serves as a template in polymerase chain reaction (PCR), which is done in a PCR machine. (7/n)

During PCR, probes attach to the DNA and amplify it with the help of an enzyme called polymerase (the “polymerase” part of PCR). (8/n)

These probes are small pieces of DNA that are complementary to specific sequences of the viral genome and contain fluorescent markers that are detected by the PCR machine. There are typically 40 cycles of amplification. (9/n)

The PCR machine provides a read-out which quantifies the amount of fluorescence that is detected (this is the “quantitative” part of the test). This read-out is a number called the “Ct number” (stands for “cycle threshold”). (10/n)

The Ct number is analyzed relative to a gene that is usually present in cells at relatively stable amounts – called a “housekeeping gene”. (11/n)

The Ct is defined as the number of cycles required for the fluorescent signal to cross a threshold, meaning that it exceeds the signal of the housekeeping gene detected. The Ct number is inversely related to the amount of viral DNA detected in a sample. (12/n)

That means that the lower the Ct number, the more viral DNA is detected (kind of like a golf score – big number means less viral DNA, small number means more viral DNA). (13/n)

The “real-time” part of the reaction means that the increase in fluorescence is being monitored in real time, rather than just detecting an end product. (14/n)

To determine what a positive test is, you have to determine what the cutoff value is for that Ct value, and this can vary from lab to lab. The cutoff Ct value for most labs for a positive test is 37. (15/n)

This means if the amount of viral DNA detected at cycle 37 is greater than the background level of the housekeeping gene, the test is considered positive. So as you can see, a positive test is not a black and white scenario. (16/n)

The viral RNA in some of those patients who tested negative and then tested positive again, was probably fluctuating right around the cutoff line. (17/n)

So the positive/negative/positive test results might be more about what cutoff threshold was used to determine what a positive test result is. As opposed to the patient really having zero virus and then boom… having virus again. (18/n)

To put this another way, the people who tested positive, then negative, then positive again probably still had the original infection going on, because we do know that some patients can shed viral RNA in their mucus, saliva, feces, urine, etc. for weeks, and weeks, and weeks. 19/

That middle negative test was probably just a false negative (because of the blurry Ct cutoff line). The specific length of viral RNA shedding is unclear right now and it is unclear why some patients shed longer than others. This is currently a topic of intense research. (end)