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Several media reports have come up with scary headlines that the new SARS-CoV-2 variants are fooling the RT-PCR test, which is considered the gold standard test for detecting the SARS-CoV-2 infection. Anecdotal reports say PCR tests are not able to detect the virus due to the new mutations. Is it true? Let us explore science and find out the truth.
Are mutations frequent in coronaviruses?
NO. Coronaviruses make far fewer replication errors than other RNA viruses, which means fewer mutations and less genetic variability over time.
Targets used by RT PCR to detect SARS-CoV-2:
The ORF1ab/ RdRp, E, N, and S genes are the most frequently used targets for SARS-CoV-2 detection by RT-PCR.
Different protein targets of SARS-CoV-2:
- The nucleocapsid (N) protein has 90% amino acid similarity with SARS-CoV (the 2003 pandemic of Asia), which means that this gene/ sequence has been conserved over time. This stability makes the N gene one of the most promising targets for SARS-CoV-2 detection. Therefore PCR tests designed for the N gene will easily detect SARS-CoV-2.
- Point mutations in the RdRp gene have given the virus the ability to interfere with diagnostic assays and anti-viral treatments (e.g., Remdesivir).
- S gene (for the Spike glycoprotein): has multiple mutations that confer a genetic advantage to the virus (greater transmissibility)
Can a PCR be designed to detect a single viral target?
A PCR test can have a single target, provided it is a "stable" target, such as the N protein.
What does the World Health Organization (WHO) recommend?
WHO recommends at least two targets for SARS CoV-2 detection by RT PCR:
- one sarbecovirus specific E-gene
- one SARS CoV-2 specific gene (N, RdRp or ORF1b, etc.)
Why use multiple targets?
The advantage of using multiple targets in a PCR is that the test remains accurate even if a target sequence mutates. For the same reason, manufacturers often include secondary and tertiary sites in their assays. If one of the targets produces a false negative due to mutations, the other sites can generate PCR signals to detect SARS-CoV-2 correctly.
RT PCR tests in India:
The new variants and their mutations can not escape detection and can not give negative results as the RT-PCR tests used in India target more than two genes. Both sensitivity and specificity of the RT-PCR tests remain the same as earlier.
Then why are people testing negative?
- Testing too early after exposure: The best time to test is one week after exposure. It can be a range of 5 to 7 days. Earlier than that, PCR may test negative.
- Testing too late after infection: 8 to 9 days after infection. At this point, only BronchoAlveolar Lavage Fluid (BALF) samples may test positive and not the nasal/ throat swabs.
- No test is perfect. It is well known that all PCR tests have some false negatives.
Conclusion: False-negative PCR tests in the second wave could be mainly due to any of the three reasons mentioned above, but not due to the new variants and their mutations. Labs may have been overwhelmed by the work overload impacting the quality of test results, but no one should worry about the variants missing the PCR tests purely because of the mutations.
Our recommendations for people who have a suspected exposure to an infected person:
Isolate yourself immediately and prevent the spread to other family members. It is an airborne infection that spreads rapidly indoors. Hence, take maximum precautions at home. Keep windows open. Improve ventilation and air circulation. Get tested after 5 to 7 days from the day of suspected exposure. During isolation too, keep monitoring SpO2 levels (with a pulse oximeter) and body temperature.
The best time to get tested is the first day of symptoms.
- DNA Explainer: How the new COVID-19 variant is fooling RT-PCR tests - all you need to know. https://www.dnaindia.com/explainer/report-dna-explainer-how-the-new-covid-19-variant-is-fooling-rt-pcr-tests-all-you-need-to-know-2886515
- Kalita D, Deka S (2020) Effectiveness of Different Gene-Target Strategies for SARS-CoV-2 Screening by RT-PCR and Other Modalities: A Scoping Review. J Med Diagn Meth 9:298. doi: 10.35248/2168-9784.2020.9.298
- Kocemba-Pilarczyk KA, Bentke-Imiolek A, Dudzik P. Does the absence of SARS-CoV-2 specific genes always exclude the infection? How to interpret RT-PCR results?-The scenario of interactive online workshop. Biochem Mol Biol Educ. 2021 Mar 27. doi: 10.1002/bmb.21506. Epub ahead of print. PMID: 33773033.
- SARS-C0V-2 Tests & Their Targets. https://blog.microbiologics.com/sars-c0v-2-tests-their-targets/
- Viral targets: What makes a good Covid-19 RT- PCR test? https://www.zymoresearch.com/blogs/blog/what-makes-a-good-covid-19-rt-pcr-test
- Variants not leading to false negative test results, says Centre https://www.hindustantimes.com/india-news/variants-not-leading-to-false-negative-test-results-says-centre-101618610884979.html