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Study Finds 42% False Discovery Rate for SARS-CoV-2 PCR Tests

by Nov 9, 2022Health Freedom, Special Reports0 comments

A molecular biologist prepares an RT-PCR reaction (Photo: IAEA, licensed under CC BY 2.0)
A new study estimates a 42% false discovery rate for SARS-CoV-2 PCR tests, suggesting invalidation of data from COVID-19 vaccine clinical trials.

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The Problem of False Positive PCR Test Results

Since the outbreak of COVID‑19 and declaration of a global pandemic in March 2020, PCR tests have been widely used to identify “COVID‑19 cases”. However, it is known that the tests frequently give a positive result for people who do not have an active infection with SARS‑CoV‑2, the coronavirus that causes the disease. The tests are designed to detect viral RNA fragments and do not prove the presence of whole viable virus, so the tests can return positive for people who do not have an active infection.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays, more commonly known simply as PCR tests, work by reverse transcribing viral RNA into DNA and then cyclically amplifying the genetic material to a detectable level. The number of cycles required to reach the threshold for positivity is a proxy measure of “viral load”: a fewer number of cycles indicates a larger amount of viral RNA in the sample, whereas a larger number of cycles indicates a smaller amount of viral RNA.

The fact that many people were being counted as “COVID‑19 cases” despite not having the disease or even an infection was acknowledged during an interview in July 2020 by Dr. Anthony Fauci, the infamous director of the National Institute for Allergy and Infectious Diseases (NIAID) under the National Institutes of Health (NIH), Chief Medical Advisor to the President, and member of the White House Coronavirus Task Force.

One way to verify the presence of viable virus is to use cell culture to observe cytopathic effects and viral replication. If you get a cycle threshold of 35 or more with a PCR test, Fauci explained, “the chances of it being replication competent are miniscule”. With such high cycle threshold (Ct) values, he said, “you almost never can culture” the virus. 

As similarly explained in an article published in Nature Medicine on June 18, 2020, the viral load inferred by the number of cycles required to reach the threshold for positivity “does not equate with viral infectivity, and further evaluation is needed to determine the respiratory SARS‑CoV‑2 viral load that is correlated with culturable virus.”

But that was never done. PCR tests were being used to diagnose “COVID‑19” even in the absence of symptoms and without any confirmation that viable SARS‑CoV‑2 was present in the patient sample. The systematic misuse of PCR tests to count people who had no infection and were not contagious as “COVID‑19 cases” was admitted in a New York Times article on August 29, 2020, titled “Your Coronavirus Test Is Positive. Maybe It Shouldn’t Be.

As the Times reported, the tests were being used with such a high threshold that “up to 90 percent of people testing positive” were “not likely to be contagious”.

The media’s faux “fact checkers” nevertheless maintained the false claim that a positive PCR test necessarily meant that the tested individual was a “COVID‑19 case”. On social media, people were censored for telling the truth that the systematic misuse of PCR tests was biasing case counts upwards.

Apart from detecting non-viable SARS‑CoV‑2 RNA fragments, PCR tests can give false positive results due to “background noise”, as explained by the World Health Organization (WHO) in a medical product alert on December 14, 2020. While it was removed from the WHO website in late January 2021, that document acknowledged the problem of “an elevated risk for false SARS‑CoV‑2 results” from PCR tests.

If the prevalence of SARS‑CoV‑2 infection is low, “the positive predictive value” of the tests “also decreases”, which means that “the probability that a person who has a positive result . . . is truly infected with SARS‑CoV‑2 decreases as positivity rate decreases, irrespective of the assay specificity.” Health care providers were therefore “encouraged to take into consideration testing results along with clinical signs and symptoms”.

In other words, a positive PCR test should not be used as the sole basis for diagnosing “COVID‑19”. Routine testing including asymptomatic individuals was certain to bias COVID‑19 case counts upwards.

The WHO further warned of the need “to determine if manual adjustment of the PCR positivity threshold is necessary to account for any background noise which may lead to a specimen with a high cycle threshold (Ct) value result being interpreted as a positive result.” In some circumstances, “the distinction between background noise and actual presence of the target virus is difficult to ascertain.” Therefore, in some cases, “the cut-off should be manually adjusted to ensure that specimens with high Ct values are not incorrectly assigned SARS‑CoV‑2 detected due to background noise.”

In an updated version of its medical product alert published on January 13, 2021, the WHO reminded of the need for “careful interpretation of weak positive results”, especially in light of how “disease prevalence alters the predictive value of test results; as disease prevalence decreases, the risk of false positive increases.” PCR tests should be used as “an aid for diagnosis”, not as the sole basis for labeling a person a COVID‑19 case.

The medical product alert referenced an interim guidance document on diagnostic testing for SARS‑CoV‑2 published by the WHO on September 11, 2020. The WHO in that document similarly emphasized that the decision to test “should be based on both clinical and epidemiological factors”, noting that there was “a risk of false positive or false negative SARS‑CoV‑2 results”. According to a flowchart presented in the guidance document, only patients who present with clinical disease should be tested, and test results should be carefully interpreted because PCR tests could “produce false signals at high Ct values.”

The WHO further advised that positive PCR test results can be supported by secondary testing with an alternative PCR test, or the positive result can be confirmed “via virus sequencing”. Genomic sequencing is a more practical alternative than viral isolation in cell culture, which requires trained staff and biosecurity level three (BSL-3) laboratories.

An even earlier WHO interim guidance document on laboratory testing for SARS‑CoV‑2, published on March 19, 2020 (eight days after having declared COVID‑19 a pandemic), had similarly emphasized that the decision to test “should be based on clinical and epidemiological factors and linked to an assessment of the likelihood of infection.” The only situation in which the WHO advised testing of asymptomatic individuals was if they had had a clear exposure to the virus from someone presenting with the clinical disease.

PCR tests should be used for “confirmation”, not as the sole basis for diagnosing COVID‑19, and test results should be further confirmed “by nucleic acid sequencing when necessary.” The WHO advised that, in areas of low prevalence, a positive test could be confirmed “by sequencing partial or whole genome of the virus”.

To understand why the proportion of false positive results will increase as prevalence decreases, here are two helpful videos, the first a general overview of “Bayes’ Theorem”, and the second applying the theorem specifically to COVID‑19 tests:

Gold Standard Validation of PCR Tests Hasn’t Been Done

The US regulatory system had previously established the use of sequencing as the gold standard method of validation for PCR test results, as noted in a new study in the journal Science, Public Health Policy, and the Law, a publication of the Institute for Pure and Applied Knowledge (IPAK), founded by Dr. James Lyons-Weiler. The author of the study is Dr. Sin Hang Lee, a pathologist and director of the Milford Molecular Diagnostics Laboratory in Connecticut. Published on October 31, the paper is titled “Evidence-Based Evaluation of PCR Diagnostics for SARS‑CoV‑2 and the Omicron Variants by Gold Standard Sanger Sequencing”.

After the outbreak of severe acute respiratory syndrome (SARS) in 2002, the US Food and Drug Administration (FDA) and US Centers for Disease Control and Prevention (CDC) established a protocol for validating PCR test results to determine whether patients presenting with symptoms were infected with the novel coronavirus that caused the disease. As Dr. Lee explains, the CDC recommended sequencing “to verify the authenticity of the amplified product”. The FDA reaffirmed this gold-standard approach in a guidance document from January 2009 on the use of PCR tests to detect enterovirus RNA.

As Lee explains:

Contrary to the previously established protocol and guideline set by the CDC and the FDA for the diagnosis of SARS‑CoV‑2 and for RNA viruses, the SARS‑CoV‑2 commercial RT‑qPCR assay kits are generating a Ct number, an unproven surrogate for nucleotide sequence, for “the presumptive qualitative detection of nucleic acid from the 2019‑nCoV [later renamed SARS‑CoV‑2]” under emergency use authorization.

In a previous study, published in the International Journal of Geriatrics and Rehabilitation in July 2020, Lee utilized the recommended approach of verifying PCR test results with sequencing to retest twenty reference samples provided by the Connecticut State Department of Public Health. He found that 20 percent of samples classified as negative by the CDC’s PCR test were in fact positive for SARS‑CoV‑2, whereas 30 percent of samples classified as positive were in fact negative.

As Lee commented in the paper:

Using nucleotide sequencing to validate every positive PCR amplicon practically eliminates all false-positive results. For timely accurate molecular diagnosis and characterization of infectious agents, concerned scientists have been urging to bring microbial sequencing to the hospital laboratories to improve individual and population health. . . .

RT-qPCR tests are known to generate both false-negative and false-positive results. Highly sensitive and accurate laboratory tests for SARS‑CoV‑2 are needed for certain populations, especially for the people of advanced age living in long-term care facilities and their care takers. Patients with false-negative test results may transmit the virus to family, friends, or caregivers. Uninfected residents in long-term care facilities with false-positive results may be isolated in rooms with Covid‑19 patients, which puts them at risk of becoming true positives. Convalescent hospitalized patients must be tested by an extremely sensitive, no-false positive nucleic acid test prior to being discharged into the communities.

In another study, published in June 2021 in COVID‑19 Pandemic: Case Studies & Opinions, Dr. Lee explained (emphasis added):

RT-qPCR for molecular detection of SARS‑CoV‑2 in human respiratory tract specimens is inherently flawed. . . . PCR is a tool invented to provide templates for DNA sequencing analysis. The fluorescence signal generated in RT-qPCR is not a reliable surrogate marker for the DNA sequence of a target template. . . . It is well known that a distinction between background noise and actual presence of the target virus is sometimes very difficult to ascertain by the RT-qPCR assay alone. Routine partial SARS‑CoV‑2 gene sequencing should be used to improve diagnostic accuracy, as the WHO advised.

The Finding of a 42% False Discovery Rate for PCR Tests

In his new study, Dr. Lee used Sanger sequencing to reexamine fifty reference samples taken from patients during January 2022 that had tested positive for SARS‑CoV‑2 by RT-qPCR assay. He found that SARS‑CoV‑2 was detected by PCR and Sanger sequencing in only twenty-nine of the fifty samples—a false positive rate of 42 percent for the provided reference samples.

One reason for background noise, Lee explains, is that “PCR primer/template hybridization is not fully sequence-specific because PCR primers may attach to non-target DNAs and amplify unwanted DNAs if these DNAs are present and partially match the primers in nucleotide sequence. As a result, relying on PCR, especially the qPCR technology using Ct numbers as the surrogate for actual PCR product analysis, for disease diagnosis is bound to generate false positives.”

There has been a “general assumption” that a PCR primer “extends a matched, but not mismatched, nucleotide”, Lee remarks, but this is “incorrect.”

As an example, he states that “the SARS‑CoV‑2 N gene reverse nested PCR primer has been shown to initiate a PCR amplification of a segment of human chromosome 1 gene due to a 6‑base match in its 3’ terminus with a human genomic sequence, a mechanism that may contribute to the 21 RT‑qPCR false-positive reference specimens.”

He further comments:

Non-target DNA amplification by PCR was clearly demonstrated in Figures 18-21, in which a set of PCR primers was found to amplify a shorter DNA segment instead of the fully matched longer target template when the shorter DNA segment offered a 9‑base sequence matching the 3’ terminal sequence of a PCR primer. PCR always prefers amplification of shorter templates when there is such an option.

His findings showed that “RT-qPCR is generating a significant number of false-positive test results at the current stage of the COVID‑19 pandemic”, which problem could be overcome with routine monitoring of PCR products by DNA sequencing.

He summarized:

The flaw of the RT-qPCR as a diagnostic assay is that it depends on a number, which may vary from laboratory to laboratory and from test run to test run, to distinguish between the positives and the negatives of a test result. The analyte of PCR is a segment of target DNA, the presence of which can only be verified by demonstrating its nucleotide sequence.

Lee notes the curiosity that for diagnosis using PCR tests of other infectious diseases, including Monkeypox, the CDC employs a more stringent standard (emphasis added):

Using qPCR for the diagnosis of infectious diseases, such as Monkeypox virus infections, the CDC requires the testing laboratories to establish their own positive control Ct cut-off value or to prepare a standard curve in order to identify the samples that are truly positive for Monkeypox virus DNAs. However, no such requirement is set for the SARS‑CoV‑2 RT-qPCR assays. As a result, the diagnostic laboratories do not have a validated quantitative standard curve or a verified Ct cut-off value for SARS‑CoV‑2 RT‑qPCR tests; cut-off values differ from laboratory to laboratory. In some circumstances, the distinction between background noise and actual presence of the target virus is difficult to ascertain in these RT‑qPCR assays; a 42% false discovery rate in SARS‑CoV‑2 RT-qPCR assays is not unexpected. The need for a confirmatory test with 100% specificity was already recognized by the current CDC director 2 years ago.

Lee proceeds to explain that one implication of this result is that the estimates of vaccine efficacy from the clinical trials for COVID‑19 vaccines are unreliable. The trials were designed to determine vaccine efficacy against symptomatic infection, with a case being defined by having one or more symptoms plus a positive PCR test.

As Lee remarks, “Using RT‑qPCR tests with false-positive results to evaluate the endpoint in COVID‑19 vaccine development might have artificially inflated the vaccine effectiveness. . . . Without confirmatory Sanger sequencing of the RT‑qPCR products, the claim of the BNT162b2 [Pfizer-BioNTech COVID‑19 vaccine] being 95% effective against COVID‑19 becomes questionable.”

The implications of Dr. Lee’s study are further elucidated in a Substack article by Dr. James Lyons-Weiler, the CEO of IPAK and Editor-in-Chief of the journal Science, Public Health Policy & the Law.

Assuming a 5 percent prevalence rate for SARS‑CoV‑2 infections in a population, the 42 percent false discovery rate of PCR tests means that for every 50 true positives, there will be 36 false positive results. The number of “COVID‑19 cases” reported based on PCR testing will be overstated by a factor of 72 percent.

Given such unreliability of PCR tests, “There are no credible COVID‑19 vaccine trial data. . . . The published efficacy of SARS‑CoV‑2 vaccines, the published breakthrough rate, the published re-infection rate estimates—all of it is potentially rubbish given the 42% false discovery rate. No credible data exist on the efficacy or real-world effectiveness of SARS‑CoV‑2 targeting vaccines.”

Lyons-Weiler also comments on how the CDC and FDA had failed to follow their own guidelines establishing Sanger sequencing as the gold-standard method of verifying PCR test results:

The reasons why this was abandoned must be examined by a Senate hearing. FDA has designated sequencing a “complex” procedure, and yet Dr. Lee reminds us that any hospital can conduct Sanger sequencing. . . . In fact, in his study, he advises that hospitals develop their own Sanger sequencing testing capacity and not wait for FDA approval. Dr. Lee has developed a set of primers every hospital can use, and has published them in the report.

In a follow-up article further discussing the implications of such a high false discovery rate in situations of low prevalence, Lyons-Weiler remarks:

When CDC launched and FDA approved of PCR testing with no way to estimate the baseline cycle threshold (Ct; for every patient, every time), they set us up for a future with unacceptably high false discovery rates. . . . At any false discovery rate that is not close to zero, at low prevalence mass testing will be a disaster because false positives will greatly outnumber true positives.

The clinical trials were conducted in populations “that had very low prevalence”, and therefore “the outcomes of the trials in terms of number of cases in the vaccinated groups and number of cases in the unvaccinated groups are bogus.”

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