COVID-19 Testing and Detection: How It Works [Transcript of Video]

Dr. Frederick Schaffer Explains COVID-19 Testing and Detection

Hello, I’m Dr. Fred Schaffer, and I’m a board-certified allergist immunologist and a former university associate professor. We’re here today to discuss, in more depth than usual, the detection of COVID-19 infections. As you may know from a lot of press and television discussions, the topic is rather confusing and hopefully I can simplify some of it so it’s more understandable. 

How To Identify COVID-19

There are two common ways in which the infection is identified. The first way deals with a process called RT-PCR which is a molecular technique, and the second way is using an antibody assay technique. 

RT-PCR

The RT-PCR involves getting a sample of the virus on a swab, usually either from the nasopharynx or from the throat. For patients who are hospitalized and on a ventilator, they sometimes will actually get fluid from the trachea which leads to the lungs directly, and as you would imagine, the trachea samples tend to be more accurate. However, it’s extremely uncomfortable and not most commonly done. Then, the nasopharyngeal swab samples are put into media, sent onto a sophisticated lab where the studies are run. 

Usually the studies take about six to eight hours to complete and most patients get their results several days later due to a backlog in most of the commercial labs out there today. The test result tends to be relatively accurate, however false negatives can occur if the technician or the nurse who did the swabbing did not get enough viral material on the swabs. If there’s a mutation in the virus, parts of the sequence of the RNA in the virus are used in the process of RT-PCR, so if a mutation occurs that was not known then the process may not be totally accurate. Once the RT-PCR is done, the real issue there is over what time period is it sensitive and accurate, and how good is it for doctors and patients? 

Well, the incubation period for COVID-19 is usually two to 10 days after exposure to the virus and the RT-PCR has been said to be effective in terms of accuracy within the first five and a half days of disease. Subsequent to that, the accuracy decreases. One study in China showed that during the first week of disease, the accuracy of the methodology they use was approximately 67 percent accurate and correct, but during the second week after day seven it went down to about 45 percent. 

Antibody Assay

In terms of the antibody study, it’s sort of the opposite perspective. During the first week, it’s not that fantastic because the antibody test measures the antibodies produced by your immune system to respond to the viral infection. Initially, an IgM antibody is made and is usually detectable after about five to seven days of infection. Then later, the more important IgG antibody is made and that usually appears to be detectable by antibody assays by approximately ten to fourteen days after infection starts. So, you can see given the windows of detectable opportunity, that for the first five days the better test would be the RT-PCR test, while later on, after five to seven days, the antibody test tends to be better. 

In one study, the antibody test was shown to be superior to the PCR test for detection during the second week of disease, meaning after seven days. Both tests have been hypothesized to be used together, and thus increase the accuracy during the first week. The claim in the paper was that 98.6 percent accuracy would occur, which is higher than either one of the tests independently on their own. 

About The BioHit SARS-2 IgM and IgG Antibody Assay Test

One of the exciting items I’d like to bring to your attention is one of the antibody tests. This is a BioHit SARS-2 IgM IgG antibody assay test kit, and if you want to get further information than what I speak about in this video, check out our FAQs for further information. But in essence, let’s kind of break this box open and see what we have. 

Now, I pre opened one of these to make it a little easier to follow. This contains a cassette that you would do the test on, and you may not be able to see it, but there’s a trough there to put a drop of blood with some diluent, and then there’s a area below that where the solution will diffuse through and then within 15 minutes give you some results. Now the way that this tests works is as follows.

How The Test Works

In the cassette, there are strips of nitrocellulose paper, and in the strips there are certain pieces of protein from the COVID virus. If your blood has antibodies, either IgM or IgG, against the virus, it will stick to that patch of the paper within the cassette that has the particular protein from the virus. Then there are two specific separate areas, and in one area on top of having the virus protein embedded, has an anti-human IgG antibody and the other area has anti-human IgM antibody. 

So in essence, once the patient’s blood solution sticks to the area where the virus particles are, then the anti-human IgM in one line will light up if it’s an IgM antibody. If the blood solution contains a virus-specific IgG antibody, then teh embedded anti-human IgG antibody area of the strip will light up in addition. This all takes place in less than 15 minutes and you’ll quickly get a picture if the patient has either IgM, IgG, or no immune response at that point. And finally, there’s a line in there which checks the validity of the test. It’s a C line or constant line that needs to always light up otherwise it means that the test is invalid. 

Now let me show you a picture of what that may look like. To give you a simulation of that test as performed, one can look at these pictures and you can see on the right, where I’m pointing, that the constant line is showing up, but not the IgG or IgM. That could represent the results of a patient that is disease-free, or during that first five day period where the IgM antibody has not been produced yet. But you can see that the constant is there, suggesting that this is a valid result.

Alternatively, if you look at the panel right next to it, you can see that the IgM and IgG light up, but the constant line is missing. That means that though there is M and G, this is not a valid test, so one cannot interpret these results, so it’s really important to have the constant line in your results in order to say it’s a valid test. In order to know how good, or get an idea of the accuracy of the RT-PCR test, or the BioHit antibody test, or other antibody tests, it’s important to know what’s known as the sensitivity and the selectivity of these tests.

Sensitivity and Specificity

Sensitivity is essentially the measure of a true positive rate, or in essence, the proportion of positive test results that are currently identifying patients that may have disease. Alternatively, the specificity is the true negative rate, or the proportion of negative test results that are correctly identifying patients that do not have disease. So, when we look at those two statistical parameters for the BioHit test, we find the following:

The sensitivity for IgM is very high at 97.5 percent. For IgG it’s the same at 97.5 percent, and for total antibody, meaning IgG plus IgM, it’s also 97.5 percent. When we look at the specificity, we find that for IgM it’s even higher 99.5 percent, it’s 100 percent for IgG, and 100 percent for both IgM and IgG together is total antibody. 

Let’s briefly look at an interpretation of the last segment. So the specificity of the IgG for example is 100 percent, that means that there will be no false negative tests and all the tests that come back negative are truly negative. Alternatively, for the sensitivity for the IgG, and not specificity but sensitivity, which is 97.5 percent, that means at best maybe up to 2.5 percent of patients testing positive might be false positive results. In either case, these are very good results. 

PPV and NPV

Now there’s something called PPV and NPV, which we won’t go into, which has to do with the prevalence of the disease in a particular community. But you could check out our website to get some idea of how that plays a role in these parameters. 

These specific statistical parameters, meaning the specificity, sensitivity, and as alluded to the NPV and the PPV, have all been checked by the manufacturers at a medical university in their own locale. The initial testing involved 40 patients who were COVID-19 positive, 78 patients with other respiratory diseases, and 108 normal individuals. Using the results of testing with the BioHit antibody assay on these populations of patients, those statistics were ascertained. Some of the statistics are reproduced in the FAQs on our website.

Study Conducted by Yale University

I was just informed, not more than an hour ago, that Yale University published a study which is just hitting the press where they use the same BioHit antibody test on 1,800 patients, which is a large study. They found the accuracy and specificity are very similar to what was just stated in the previous sections of this talk. So validation is there, and it should reinforce the use of this assay test kit in the future.

How To Use the BioHit Antibody Test to Interpret Patient Scenarios

I’m going to be showing you figures of test cassette results that come from an excellent paper by Jacofsky and his associates (https://www.arthroplastyjournal.org/action/showPdf?pii=S0883-5403%2820%2930442-3). So let’s go on to some examples and see how we can use the BioHit antibody assay test to interpret different patient scenarios.

Example A

Let’s presume that a patient is asymptomatic, but he thinks he was exposed to a couple of his friends about a week ago and he found that after the fact that they were both diagnosed with COVID-19. And though he’s feeling great, he was concerned that he might be the carrier and unfortunately pass it onto others, or that his own personal situation clinically may get worse. In that particular scenario, a doctor ordered an antibody assay, the BioHit one by the way, and the results are pictured in this set of panels. As you can tell, in this panel, the constant is lit up, which means it is a valid test, and only the IgM is lit up, but not the IgG. 

Let’s see if we can interpret that.

This was done one week after the patient thought he was exposed. If you recall from our antibody discussion earlier, IgM is produced within five to seven days or more after exposure, so the fact that the IgM is positive would theoretically fit the scenario that this antibody was detected within the timeframe that IgM is usually produced in response to a COVID-19 exposure.

The IgG was absent, but you may recall that IgG is not detectable until approximately 10 to 14 days after exposure. So, this would verify the timeframe that the patient may have been exposed in. Now, his physician may want to further validate it with a PCR test which would be able to detect evidence of the virus in the swab

Even though the patient is asymptomatic, I imagine the physician would tell him after the test results are acquired, to isolate himself, to take it easy, enjoy Netflix, and have ongoing conversations about his health status. Now, approximately 2 to 3 weeks later, or a little bit less if need be, the test can be redone. 

The IgG comes up at approximately 10 to 14 days, but the IgM is minimal or disappears closer to 21 days. So, two weeks after the seven day period would put him at around 28 days out. At this point, if he repeated his BioHit antibody assay, it theoretically may show the following:

As you can see, the constant line is showing, verifying it is a valid test. But the only line that’s showing is IgG, and not IgM. The reason that occurs is because most IgM is undetectable after 21 days onset after disease. Alternatively, the IgG would be positive. IgG hits its initial peak at the time of disease recovery, and then continues to increase slightly and has been known to stay present for many months, and even years, in part since the IgG half life is rather long. 

The PCR test, which I wouldn’t recommend and I don’t think his doctor would either at 28 days, would indirectly be helpful but not very because most PCR tests done after 21 days show minimal or no detection. This means that, while not proven, there is less viral shedding at that point. So again, the BioHit test showing the IgG presence, or alternative the negative PCR test, can show along those lines. 

Example B

Let’s look at another possible application. Let’s say we have a patient who has been mildly symptomatic for about 16 days, but never got in touch with his doctor until he heard more about COVID-19 cases in his community on the local news channel. Once he called his doctor, his doctor set up a BioHit antibody assay, and he found the following results:

He has the constant band lit up, showing the test is valid, and both the IgG and IgM. This would mean the following. Since IgM does not disappear in terms of detection until around 21 days, you would expect to see it at 16 days. And you may recall that the IgG starts to be detected at approximately 10 to 14 days and increases in concentration over the next several months for some patients. So at 16 days, you would expect to see some, which is why this cassette result shows that both the IgG and the IgM are positive.

If a RT-PCR test was done this day or the next day, that would also be positive, because, as you would recall, detectability from swab samples does not diminish until around 21 days of disease activity. 

Example C

I want to talk about one more case. As you may have read in the news, the use of transfused blood products from patients who have recovered from COVID-19 would hopefully help those with life-threatening or very severe cases of COVID-19. 

For example, if a patient has a terrible pneumonia and is intubated, and his outcome does not look great, the FDA recently put out new guidelines on May 1, 2020 that the acquisition of convalescent plasma from patients who have already recovered from COVID-19 infections may be used.

What this entails is finding patients who have fully recovered, they have to be at least 14 days free of symptoms, and they have had to either been diagnosed early during their disease course by RT-PCR methods, or if not diagnosed, then at that point and time when they are willing to become donors, an antibody study such as the BioHit IgM IgG study should be done. If that study shows evidence of IgG, then they would be probably recovered, and anti-viral IgG is the important antibody you would transfuse in the convalescent plasma. 

In some cases, the plasma that the volunteer is willing to donate after his or her disease activity is tested for high titers of neutralizing antibody which is perfectly what you need to do to transfuse in patients in critical conditions with COVID-19 in order to improve their status. 

So then again, for those who were not initially diagnosed by RT-PCR, who do become or want to become convalescent plasma donors, using the BioHit antibody study would be helpful in selecting those appropriate patients. There’s a whole lot of other guidelines involved, but to keep it simple and short, this is a pathway in which it can be currently done. These pathways are investigational, but under current guidelines, local doctors with severe cases or hospitals with severe cases can pursue the direction to acquire those samples of convalescent plasma. 

Conclusion

Well, that sort of ends our talk. I hope it was both understandable and somewhat comprehensive. Again the article by Jacofsky would be very helpful. He goes through similar cases in his paper and he goes more in-depth about the immune responses I hope that this has been educational and provides you with further information. 

If you’d like further information about the BioHit IgM IgG antibody assay kit, please check our website and look at the FAQs. There’s a place there to ask questions so if any questions have gone unanswered feel free to throw us a question we’ll be glad to try to respond. 

Thank you. I appreciate your attention.