It’s ‘scientifically meaningless’ and all false positives, some have claimed about the current polymerase chain reaction (PCR) test for SARS-CoV-2 infection or its disease, Covid-19. False-positive means that the positive result of a test is false, so it’s picking up a signal when there’s none.
There are at least 5 reasons why some doubt its scientific validity:
- The inventor of PCR said it could not detect infectious viruses.
- It has not been compared to a gold-standard, definitive test.
- It’s a qualitative test, not quantitative.
- The cycle threshold (Ct) value used is too high, so it’s detecting coronavirus genes when there are no real viruses.
- There’s no proof that the detected genes belong to SARS-CoV-2 since this coronavirus has never been purified.
This article will look at each of these points — evaluating if they are correct or not. Actually, however, most of the above points are in the grey area that is not outright wrong, which is why they can be misleading if not interpreted properly.
In brief, PCR works by using specific primers to attach to both ends of a gene segment. An enzyme then amplifies that primers-marked gene until its detectable by genetic dye or marker. The type of PCR used to detect SARS-CoV-2 is real-time reverse transcription PCR (RT-PCR). Specifically, the RT-CR test detects the E and RdRp genes coding for the viral envelope and RNA polymerase enzyme, respectively, unique to SARS-CoV-2.
1. It can’t detect infectious viruses?
PCR tests “cannot detect free infectious viruses at all.” a quote attributed to Kary B. Mulis, Ph.D., a biochemist who invented the PCR test worthy of the Chemistry Nobel Laureate award in 1993. He died on 7 August 2019.
Dr. Mulis did not say that quote, however. Instead, it came from a 1996 article by John Lauritsen, an award-winning American reporter.
Anyhow, that quote is not entirely false either. Lauritsen intended to clarify that the PCR test does not detect the whole virion but genetic pieces unique to the virion.
So, it’s true that the PCR test “cannot detect free infectious viruses.” It can be likened to the analogy that finding dog fur in a location doesn’t mean the dog is still there. Similarly, detecting genetic pieces of SARS-CoV-2 doesn’t mean that the complete SARS-CoV-2 virion is still there. Maybe SARS-CoV-2 was already destroyed by the immune system, leaving dead viral fragments. This is undoubtedly one major flaw of the PCR test.
“It is important to note that detecting viral material by PCR does not indicate that the virus is fully intact and infectious, i.e., able to cause infection in other people,” the Public Health England spokesperson told Reuters on November 2020. “The isolation of infectious virus from positive individuals requires virus culture methods. These methods can only be conducted in laboratories with specialist containment facilities and are time-consuming and complex.”
Indeed, there’s a discrepancy between the positive PCR (specifically, RT-PCR) test and culturable SARS-CoV-2. In virology, a culturable virus means that the virus can replicate in cells in a lab dish — indicating that the virus is infectious. After all, viruses can’t survive without a cell host.
An editorial in the International Journal of Infectious Diseases reviewed various studies and stated that SARS-CoV-2 is often not culturable after the 8–10th day of illness, despite on-going positive RT-PCR test results. This is because SARS-CoV-2 may have disseminated deep into the lungs or elsewhere in the body to be sampled from the nose.
Therefore, persons who tested positive for SARS-CoV-2 by RT-PCR may not harbor infectious viruses. The RT-PCR test can’t conclude but can only tell that there might be active or infectious SARS-CoV-2 in the body.
2. It has not been compared to a definitive test?
The accuracy of a particular pregnancy test, for example, is determined by comparing its results to a gold-standard, definitive test. In this case, the definitive test is the visible pregnancy itself. By comparing a particular test to a definitive test, we can discern how accurate it is, such as its false positive and false negative rates.
Admittedly, this comparison has not been made with the current PCR test. But that’s because there’s actually no definitive test for Covid-19 or SARS-CoV-2. Why?
Covid-19 or SARS-CoV-2 can be detected in at least two other ways, although they are not definitive as well:
- A chest CT (computerized tomography) scan showing lung abnormalities typical of Covid-19. But infected people can still spread the virus to others even before symptoms develop and the lungs start showing abnormalities observable with chest scans. About 59% of SARS-CoV-2 transmissions are caused by infected persons who had no symptoms — asymptomatic.
- An attempt to culture viruses from the respiratory samples. But the absence of culturable SARS-CoV-2 doesn’t indicate that the patient is no longer sick. As mentioned, SARS-CoV-2 is not typically culturable after the 8–10th day of illness from nasal swabs, but Covid-19 lasts for a few weeks. This is because the virus may have reached deeper parts of the body, such as the lungs or air sacs, to be culturable from nasal swabs.
Thus, infected persons might not develop an illness, yet they can transmit the virus to others. And sick patients might not harbor culturable viruses. These reasons are why there’s no definitive test for Covid-19 or SARS-CoV-2.
The PCR test may be the best cost-effective option we have to detect possible SARS-CoV-2 infection. It’s only ‘possible’ because the detected SARS-CoV-2 genetic material might be dead, and not culturable or capable of causing Covid-19.
3. It’s qualitative, not quantitative?
Yes, the PCR test is qualitative as it only indicates whether the gene of interest is present or absent. Thus, “they don’t show how many viral particles are in the body,” journalists wrote elsewhere.
But the type of PCR test used to detect SARS-CoV-2 is RT-PCR, which is semi-quantitative. One added functionality of RT-PCR is the cycle threshold (CT) value that indicates the number of amplification that occurred from the PCR.
Therefore, the higher the CT value, the more amplification was needed to detect the gene of interest — indicating that this gene is found in low amounts in the clinical sample. Likewise, the lower the CT value, the less amplification was necessary since the gene of interest is abundant in the sample.
While the RT-PCR test for SARS-CoV-2 or Covid-19 doesn’t indicate the exact number of viruses present, it tells us its abundance. So it's semi-quantitative that offers value in semi-quantifying viral load. Indeed, the Ct value can predict Covid-19 duration, severity, or death, but such clinical utility remains debated since the Ct value can vary between RT-PCR protocols.
4. The Ct threshold value is too high?
As follows, too high of a Ct value indicates minuscule SARS-CoV-2 genetic material in the sample, which might mean that it’s clinically meaningless. Yet, it’s still a positive result.
Anthony Fauci, MD, the director of the National Institute of Allergy and Infectious Diseases and medical advisor of the White House, admitted that the Ct value of >35 most likely indicates “dead nucleotides,” which means harmless virus fragments. Some even consider that the Ct value of over 30 is too high. Indeed, a review of multiple studies concluded that SARS-CoV-2 is usually not culturable at a Ct value of >30 or >33.
“Most [RT-PCR] tests set the [Ct value] limit at 40, a few at 37. This means that you are positive for the coronavirus if the test process required up to 40 cycles, or 37, to detect the virus,” the New York Times reported. Apparently, the FDA did not set the Ct value limit for a positive SARS-CoV-2 test, and that “commercial manufacturers and laboratories set their own.”
Hence, setting the Ct value up to 40 may have overestimated the true number of SARS-CoV-2 cases. If the New York Times is correct in that most RT-PCR tests did that, then the majority of tests might have detected some false positives.
To give an idea of how frequently is the ‘some false positives,’ the Wadsworth Center in New York has kept records of their Ct values. In July 2020, Wadsworth Center detected 872 positive RT-PCR tests for SARS-CoV-2 with Ct value set at 40. However, about 43% and 63% of those positives were no longer positive if the Ct value cutoff is set at 35 and 30, respectively. This means that if the Ct value was set more appropriately at 30–35, we might have recorded about 43–63% fewer SARS-CoV-2 cases today. This is in line with the estimation that 49% of SARS-CoV-2 cases are asymptomatic.
But, newly infected people might show a high Ct value of 37, for example, which may reach 30 or less in the following days as the virus replicates. Recall that higher Ct values mean lesser viral load. Alternatively, the nasal swab may not reach deep enough to sample sufficient viruses. So, the RT-PCR test can pick up every single ‘potentially’ infectious or contagious case.
It’s indeed a dilemmatic situation — to minimize risk by counting every single ‘potentially’ infectious case as true cases or to take some risk by setting the Ct value lower and miss out on some true infectious cases.
Some experts have suggested that persons with high Ct values upon RT-PCR test be tested again soon. Even a rapid test — such as the rapid antigen test — with lower sensitivity can be useful in identifying the most infectious cases, rather than every single potentially infectious case. Indeed, the rapid antigen test works best in high viral load cases with a Ct value of <25.
5. Detected genes don’t belong to SARS-CoV-2?
Opponents of the RT-PCR test have claimed that there’s no proof that the detected gene belongs to SARS-CoV-2. Because nobody has ever purified SARS-CoV-2, to begin with. They went on to argue that SARS-CoV-2, the causative agent of Covid-19, does not exist because it doesn’t fulfill Koch’s postulates for a pathogen.
However, viruses exist following the revised Koch postulates since the original postulates were formed when researchers didn't know viruses exist. As pathogen-host interactions are complex, not even every bacteria can fulfill Koch’s postulates.
Also, unlike bacteria or fungi, viruses can only survive and replicate inside a living cell. Viruses can’t survive in the environment outside cells, except for a brief moment when viruses propagate from cell to cell. In this sense, viruses can’t be purified purely alive like bacteria or fungi, but they can be cultured in a set of living cells.
Therefore, SARS-CoV-2 exists as proven via culturing it in living cells in a lab dish. Electron microscopy can then reveal viral-like particles in SARS-CoV-2 infected cells. Alternatively, SARS-CoV-2 can be separated from the cells before visualization. Genetic material inside infected cells or viruses can then be sequenced to derive the full genome of SARS-CoV-2. SARS-CoV-2 replication inside cells can also be inhibited with drugs or reacted with antibodies. Plus, SARS-CoV-2 can be transmitted among susceptible animal species, causing Covid-like diseases. So, yes, SARS-CoV-2 is real.
Since the full genome of SARS-CoV-2 has been sequenced, we can compare it to the other sequenced viruses in genomic databases. The PCR or RT-PCR test can then be designed to amplify a gene unique to SARS-CoV-2.
Given the topic's complexity, it’s expected that there are misunderstandings. People can be misled into believing that the RT-PCR test for SARS-CoV-2 is a fraud. Indeed, reasons for believing the fraud are not entirely wrong either:
- It’s true that the RT-PCR test can’t detect infectious SARS-CoV-2, but it can detect genes specific to SARS-CoV-2.
- It’s true that the RT-PCR test has not been compared to a gold-standard, definitive test. But that’s because there’s no such definitive test.
- It’s true that the RT-PCR test is qualitative, but it’s also semi-quantitative with a cycle threshold (Ct) value.
- It’s true that the Ct value set is high, making the test very sensitive that it might pick up signals from dead viral fragments. This may have led to SARS-CoV-2 overdiagnoses. But setting the Ct value lower also comes at the risk of overlooking probable infectious cases.
- It’s not true that SARS-CoV-2 has never been ‘purified’; however, that term may mean. SARS-CoV-2 exists, and there are genes specific to SARS-CoV-2 that can be targeted with RT-PCR.
Overall, the RT-PCR test is reliable, although it’s not perfect and may have overestimated the true SARS-CoV-2 case numbers to some extent. Rather than fixating on its flaws, however, proper use and interpretation of RT-PCR tests should be the focus. Public education and correcting misinformation, while easier said than done, also matters.
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