SARS-CoV-2 Testing

© MMXX V.1.0.0
by Morley Evans

An Introduction to
SARS-CoV-2 Testing

by Michael Hardy, MD

Just a brief reminder that the name of the virus is SARS-CoV-2 and that it causes the disease Covid-19. Think of the virus HIV (Human Immunodeficiency Virus) that results in the disease AIDS (Acquired Immune Deficiency Syndrome).

Before I get into it, I’ll need to explain four terms commonly employed in this area of medicine.

Firstly, a False Positive result (over-diagnosis) is when a test wrongly identifies disease in a perfectly healthy individual. This can be expressed as Specificity, a test’s ability to correctly produce a negative result in people who are not affected (False Positives are few). The consequences of False Positive results are unnecessary further investigations and potentially harmful treatments in people who do not need them.

Secondly, a False Negative result (under-diagnosis) is when a test incorrectly gives a clean bill of health to someone who really does have the disease. This can be expressed as Sensitivity, a test’s ability to correctly produce a positive result in those affected (False Negatives are few). The consequences of False Negative results are withholding additional investigations and prompt treatment in those who really need them.

In the case of SARS-CoV-2, False Positive results might result in essential front-line workers being kept from returning to important work while False Negative results could allow affected individuals to expose others to a potentially deadly infection.

In a perfect world, every medical test would be 100% specific which is to say that absolutely everybody with the disease would test ‘Positive’ and 100% sensitive when everybody who did not have the disease would test ‘Negative’. Unfortunately, this ideal is rarely achieved in practice.

You’ve probably heard that there are two basic tests used in the investigation of SARS-CoV-2. The first looks for genetic information (RNA) in the virus itself collected on a swab taken from the nose or better still from the nasopharynx and sent away to a lab where it can be measured using a technique called RT-PCR (reverse transcriptase-polymerase chain reaction). It’s recently been reported that in general, the specificity of these tests is excellent if conducted properly. That is to say, a positive result can generally be relied upon to accurately identify those with an active infection, are shedding the virus and potentially able to spread the disease to others. Unfortunately, earlier versions of the test and the way it was carried out had an unacceptably low sensitivity of around only around 70%. That is to say, out of every hundred people with the disease 30 were ‘missed’, wrongly sent away with a clean bill of health and a false sense of security. Newer swab tests and a better understanding of how and when to collect samples should eventually begin to improve these figures.

The second kind of test is called an antibody or serology test. After we develop a viral or bacterial disease, our bodies generate antibodies that can help fight the contagion and defend us from future infections with the same micro-organism. Antibodies can remain in our bloodstream for weeks, months and even years. Their principal value in the present context is to let us know whether or not we have already been infected with SARS-CoV-2. It is rather like looking at a video taken over the past few months whereas the swab test is more like a photograph that tells us whether we have Covid-19 at the time the swab was taken. Diabetics will be particularly familiar with this analogy. A blood glucose test tells them what their blood sugar is at the time the blood was collected like a snapshot. The HbA1C level provides an estimate of the average blood sugar control over the previous three months like a video.

Before I go on to describe the SARS-CoV-2 antibody test in more detail, we’ll try to develop one through a series of refinements for a Honda car!

Our first test was positive for ‘things that move’ and negative for ‘things that don’t move’. This was not a very helpful test. It was certainly 100% sensitive because all Honda cars move but unfortunately, the specificity was so low as to make it useless. It couldn’t tell the difference between a Honda, a 747, an elephant and a cruise ship because they all move.

Back to the lab and at our next attempt we were able to modify the test so that it detected only ‘things that move and have wheels’. This test is still 100% sensitive in that all Hondas move and have wheels. It is considerably more specific since it is now negative for all boats and animals but not specific enough to be helpful. In addition to Hondas, the test will still be positive for every other car, van, lorry, skateboard and aeroplane.

Our next refinement was to make the test positive only for ‘things that move and have four wheels’. Sensitivity is still 100% but specificity is improving since now our test is negative for aeroplanes, bicycles, tricycles, heavy lorries etc.

At this stage of development, we’ll amend our test to include only four-wheeled vehicles, with two side mirrors, and a rearview mirror. Sensitivity is still 100% but specificity is still too low because our test is still positive for all cars.

Hopefully, our final improvement will be to include a requirement for that easily recognizable H-shaped badge on the front of the car. In theory, our test should now be 100% sensitive in that it’s positive for all Honda cars (there are no false negatives) and 100% specific in that all other makes of cars will test negative (there are no false positives).

It’s time to confirm the accuracy of our test for Honda cars. Fortunately, there are twenty cars parked on the street outside. Ten of them test ‘positive’ for Honda and ten of them test ‘negative’. Unfortunately, on closer examination one of the cars testing ‘positive’ was found not to be a Honda. This is a one in ten, or 10%, false-positive which means our test is only 90% specific. Another one in ten, also 10%, that tested ‘negative’ is found to be a real Honda. This false-negative reduces our test’s sensitivity to 90%. What could possibly have gone wrong? It seems that a neighbour, embarrassed at driving a Russian-built Lada had stolen the Honda emblem from another neighbour’s car. This dastardly deed caused the Lada to be misclassified as a Honda (a false positive) and the Honda to be misclassified as something else (a false negative). This problem was easily solved by adding a further requirement to our test, an ownership certificate matching the VIN number of the car to the name of the manufacturer. Our test is now both 100% specific and 100% sensitive.

This rarely happens in the real world of laboratory investigations. In a rush to find a serological test for SARS-C0V-2 antibodies, dozens of tests from different manufacturers from all over the world were released prematurely. Many of these tests had both unacceptably low sensitivity and specificity. And as we rapidly learned, an unreliable test giving incorrect results can be worse than no test at all.

I’ll give you a couple of examples, although there were probably many more. SARS-CoV-2 is only one out of seven major strains of coronavirus. The first four are responsible for many cases of the relatively innocuous common cold. The other three were far more serious. SARS-Cov-1 and MERS were responsible for the lethal coronavirus outbreaks that began in 2002 and 2012, respectively. Imagine a test that couldn’t tell the difference between antibodies from SARS-CoV-2 and one of the coronaviruses causing the common cold! We could be advising healthcare workers that they were relatively safe coming into close contact with Covid-19 patients,  not because they had already been infected with SARS-CoV-2 and had developed some degree of immunity but because they had a cold a few weeks earlier. Such false positives and low specificity might prove disastrous.

On the other side of the coin, imagine another test that failed to detect antibodies in people who had been affected and recovered. This might have arisen because the test was not sufficiently sensitive to respond to low levels of antibodies. Such false negatives (low sensitivity) might prevent some key employees from returning to work.

The good news is that Roche has produced an antibody test that appears to have an amazing 99.8 sensitivity and 100% specificity. However, it’s not a test that can be done at home with a finger-prick blood sample applied to a test strip. It does involve a properly collected venous blood sample and analyzed using specific laboratory instrumentation.

At this point, I must stress that we still don’t know the full story of the relationship between antibody levels and immunity. Until more information becomes available we can only assume that prior infection does result in some level of immunity but we don’t know how strong that immunity may be or how long it may last. From our experience of other viral infections, some appear to give only mild to moderate immunity for a few weeks or months while others give almost total immunity that can last a lifetime.

And in conclusion, this is some very new information on the live virus swab test. This is the test designed to establish whether or not an individual is infected with SARS-CoV-2 at the time the swab was taken.

Some worrying reports have begun to emerge. Apparently, patients with typical symptoms of Covid-19 such as cough and fever and a positive swab test have apparently recovered becoming asymptomatic with a negative swab test. Then, some weeks later, the swab test was found to have turned positive again. It was assumed either that the original infection had reemerged or that the patient had developed little or no immunity and had become reinfected.

The WHO has recently announced a third possibility. Dead lung cells containing fragments of SARS-CoV-2 are being expelled and that this is a normal part of the long-term recovery process.  Because swab tests are designed to detect parts of the SARS-CoV-2 genetic material, it can’t distinguish between remnants of a dead (non-infectious) virus and a live virus that could still transmit the disease to others. Let’s hope they’re right.