Testing for COVID-19: Here’s What You Need to Know

Currently two testing methods are being used to detect COVID-19 infections, caused by the SARS-CoV-2 virus (novel coronavirus). The more well-established method, considered the gold standard, is called the real time Reverse Transcription Polymerase Chain Reaction (RT-PCR) test. And  the other testing method being used is the Rapid Antibody Test. Guidelines for carrying out these tests have been laid down by the World Health Organization (WHO).

What is the real fundamental difference between real time RT-PCR and rapid antibody testing methods?

Real time RT-PCR is a diagnostic test for infection of the novel coronavirus. This molecular test detects the presence of genetic material of the virus of an infected person. On the other hand, the rapid antibody test is a serological method (serum is the pale yellow liquid part of the blood) that detects proteins called immunoglobulins, or antibodies, in the blood plasma. Antibodies are produced by white blood cells to fight antigens – the novel coronavirus in this case – and are found in the blood of those who are fighting or have recovered from a COVID-19 infection.

How are the samples collected for these tests?

For the real time RT-PCR test,the sample is collected by the swab technique. A  sterile cotton swab, less than 3 mm in diameter at the tip, is placed at the back of the nasal passage, a pathway for air from the nose to the upper respiratory tract. The sample is a secretion of mucus and saliva, and will contain the virus if it is present in the person being tested. This swab is immediately placed in a liquid-filled tube ready for transport and further testing. The rapid antibody test, however, requires a blood sample, typically drawn from the finger of the person being tested, which is then placed on a kit that has markers to detect the antibodies.
 

 
How do these tests work?

In a real time RT-PCR test, the swab sample is first treated with several chemical solutions to eliminate proteins and fats, thus ensuring that only the virus’s RNA is extracted. Since the coronavirus does not have DNA, the RNA template of the virus is transcribed to synthesise DNA (reverse transcription). The sample, along with reagents and enzymes, is then placed in a PCR machine, where it undergoes a series of temperature changes, a process called thermal cycling. This triggers chemical reactions which allows for exponential amplification of any genetic material that might be present. 

As new copies of the DNA are made, marker labels attach themselves to the DNA strands and release a fluorescent dye. The degree of fluorescence is measured by the machine's computer which presents the results in real time. The test not only indicates the presence of the virus but also reveals the severity of the infection. However, it is important to keep in mind that this method is used only to test for current infection; it cannot tell us whether the person tested was infected earlier and has since recovered.

The rapid antibody test detects two types of immunoglobulins: IgG and IgM. They are produced by the immune system in response to the presence of the virus. IgM is typically produced 10 to 15 days after initial exposure to the virus. About two days later, it is replaced by IgG. Both these antibodies can be detected by the rapid antibody test. On the test strip, their presence is indicated by the appearance of a “C line” (Control) and either an “IgG” or an “IgM” line. But if only the “C line” appears, it means that antibodies were not detected by the sample. The results usually appear in less than 30 minutes.  Though the rapid antibody test cannot detect the virus, it can reveal whether the body fought off an infection earlier because antibodies last in the bloodstream for many months after the infection. Unlike the real time RT-PCR, the rapid antibody test can detect infection only a few days after infection. A positive test indicates exposure to the virus, but a negative test does not rule out its infection. 

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How accurate are these tests?

The answer to the question requires a statistical detour. The accuracy of a test is assessed based on two complementary measures: sensitivity and specificity. Sensitivity is a measure of the true positive rate. For instance, a test with 90% sensitivity means that 90% of the total number of positive cases have been identified correctly (the false negative rate is 10%). On the other hand, specificity is a measure of the true negative rate. For instance, a test with 90% specificity means that 90% of the total number of negative cases have been identified correctly (the false positive rate is 10%). Ideally, one would want a test to have high rates of accuracy, or in other words tests with low error rates (false negatives and false positives).

However, in the case of the real time RT-PCR test, a high false negative rate is a more serious problem because many who will have been given a clean bill of health may not avail the required treatment and are likely to unknowingly spread the infection to others. And in the case of a rapid antibody test, a high false positive rate may be of greater concern because those who test positive could then be assumed to have become immune to the coronavirus. 

Though researchers are still assessing the accuracy and error rates of these tests, PCR tests are traditionally thought to be more reliable.  An organisation called Foundation for Innovative New Diagnostics, or FIND, headquartered in Geneva, Switzerland, is evaluating the accuracy of COVID-19 tests. Three hundred molecular tests have been submitted for evaluation to FIND. Of these, five have been tested for accuracy. All five achieved 100% sensitivity on positive samples, and at least 96% specificity on negative samples. While some antibody tests too achieved impressive accuracy scores in FIND tests, there have been several reports  of faulty test kits from across the world.

Therefore, In India and indeed in the rest of the world, real time RT-PCR is being used as the principal diagnostic test. Both the WHO and the Indian Council for Medical Research (ICMR) do not recommend the use of the rapid antibody test for diagnosing; they are to be used only for screening and surveillance. 

 
Are there any other downsides to these tests?

RT-PCR testing requires ICMR-approved laboratories equipped with expensive PCR machines. The tests themselves are costlier than antibody tests and it takes about a day for the results to come out. In places where labs are not present, the sample needs to be collected, transported, and then processed in the lab, which may take a few additional days. Besides, sample collection and testing for both the tests can only be done by trained experts. 

What is pooled testing?

Given the limited resources at our disposal, one strategy to save time and money is to use what is known as pooled testing (ICMR too has recommended the use of this strategy but in specific contexts: it is to be conducted only for surveillance purposes in areas with low prevalence). As the name suggests, it involves testing samples that are pooled together. A recent article in Scientific American describes its rationale thus: “Let’s say you are examining 100 people, and one of them is positive. Normally you would do 100 diagnostic tests, searching for genetic material from the virus in each individual. But with group testing, you can divide those 100 people into five groups of 20. That gives you five pools with 20 samples, and you use one test per pool. If the first four sample pools test negative, you have eliminated 80 people with four tests. If the last pool tests positive, you retest each sample in that last pool individually to identify the one with the disease. In the end, you did 25 tests instead of 100.”

What are the latest developments on the testing front?

There are many organizations and pharmaceutical companies in the race to develop tests that are quick, inexpensive, and accurate. We discuss three promising tests here. The Swiss pharma giant Roche has come up with an antibody test (Elecsys Anti-SARS-CoV-2 test) for the novel coronavirus that they claim shows 100% sensitivity and 99.81% specificity (14 days after the PCR confirmation test). The test, already approved by the United States Food and Drug Administration (USFDA), yields a result in just 18 minutes. Roche Diagnostics India has obtained an import license for the test kit and is readying to bring it in India. 

Another test, this one developed indigenously by India’s CSIR (Council of Scientific and Industrial Research), is a paper-based antibody test called FNCAS9 Editor Linked Uniform Detection Assay or simply Feluda (named after Satyajit Ray’s famous fictional detective). It is based on CRISPR technology for COVID-19 detection. According to CSIR, the test, which can be done in most pathology labs, has an accuracy of 100% and the results can be obtained in just minutes. ICMR has signed an MoU with Tata Sons for the transfer of know-how so that it can be manufactured for mass testing. 
And finally, a new test that can detect antigens of the novel coronavirus in an active infection within 15 minutes has just been announced. It has been granted emergency use authorization by the USFDA. The antigen test, developed by Qidel, is called 'Sofia 2 SARS Antigen FIA'. It detects the nucleocapsid proteins located on the virus and is conducted on a mucus sample taken with a nasal swab. The company claims that the positive results are highly accurate. However, the rate of false negatives will have to be tested with RT-PCR tests.

Meena Kharatmal is a scientific officer and Ph.D. student at the Homi Bhabha Centre for Science Education (TIFR) in Mumbai