1. No PCR test has been validated against the gold standard of a purified/isolated “SARS-COV-2.”
“RT-PCR assays in the UK have analytical sensitivity and specificity of greater than 95%, but no single gold standard assay exists 1.“
Diverging for a moment to look at the citation:
“No test gives a 100% accurate result; tests need to be evaluated to determine their sensitivity and specificity, ideally by comparison with a “gold standard.” The lack of such a clear-cut “gold-standard” for Covid-19 testing makes evaluation of test accuracy challenging.”
Why is the lack of a gold standard important?
“In any case, those tests were not built on a “gold standard” which means purification of an actual virus. Purification means the pathogen has been separated from all else.”
2. No “SARS-COV-2” isolate was used in the creation of the CDC and Drosten tests, the two most widely used around the world.
This is from the FDA emergency use authorization of the CDC’s PCR test used in the USA:
“Since no quantified virus isolates of the 2019-nCoV were available for CDC use at the time the test was developed and this study conducted, assays designed for detection of the 2019-nCoV RNA were tested with characterized stocks of in vitro transcribed full length RNA (N gene; GenBank accession: MN908947.2) of known titer (RNA copies/µL) spiked into a diluent consisting of a suspension of human A549 cells and viral transport medium (VTM) to mimic clinical specimen.”
From the Drosten PCR Test used around the world:
“The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur.”
“We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.”
“Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology.”
“In the present case of 2019-nCoV, virus isolates or samples from infected patients have so far not become available to the international public health community. We report here on the establishment and validation of a diagnostic workflow for 2019-nCoV screening and specific confirmation, designed in absence of available virus isolates or original patient specimens. Design and validation were enabled by the close genetic relatedness to the 2003 SARS-CoV, and aided by the use of synthetic nucleic acid technology.”
3. Both the CDC and Drosten PCR tests determined WATER was positive for “SARS-COV-2.”
“The E Charité and N2 US CDC assays were positive for all specimens, including negative samples and negative controls (water). These false-positive results were explored (details below), but the sensitivity of these assays was not further assessed.”
“No false-positive results were obtained on clinical samples that tested negative for SARS-CoV-2 and/or positive for other viruses than SARS-CoV-2, except for E Charité and N2 US CDC, which were positive for all specimens.”
4. Detection of “viral” RNA does not mean infectious “virus” is present nor that what is detected is the cause of symptoms.
From the CDC PCR Test:
“Detection of viral RNA may not indicate the presence of infectious virus or that 2019-nCoV is the causative agent for clinical symptoms.
• The performance of this test has not been established for monitoring treatment of 2019-nCoV infection.
• The performance of this test has not been established for screening of blood or blood products for the presence of 2019-nCoV.
• This test cannot rule out diseases caused by other bacterial or viral pathogens.”
5. None of the PCR tests are FDA-approved and are only out on EUA which forgoes many quality controls:
“The FDA granted the first Emergency Use Authorization (EUA) for a SARS-CoV-2 rRT-PCR diagnostic test on February 4, 2020. Consequently, hundreds of tests for SARS-CoV-2, among them rRT-PCRs, other types of nucleic acid amplification tests (NAATs), and automated and/or multiplex methods based on proprietary platforms, obtained FDA Emergency Use Authorization (EUA). As of August 4th, 2020, the FDA has granted EUAs to 203 diagnostic tests, including 166 molecular tests, 35 antibody assays, and 2 antigen tests. Although the FDA began requiring the submission of validation methods and results as part of EUA application for SARS-CoV-2 diagnostic tests, these tests were not initially required to undergo the rigorous assessment that would normally be part of the FDA approval process.”
“So the agency uses an alternative evaluation process that’s designed to vet things more quickly than the usual FDA approval regimen. If a drug or vaccine passes muster, it’s granted an emergency use authorization, or EUA.”
6. The Cycle Threshold (Ct) Values used to determine one “infected” or not are completely arbitrary (i.e. RANDOM).
This is a huge problem as explained by PCR expert Professor Stephen Bustin:
“Bustin, in the podcast, described reliance on an arbitrary Ct number as “absolute nonsense, it makes no sense whatsoever”. It certainly cannot be assumed that the same Ct number on tests done at different laboratories indicates the same original quantity of RNA.”
“Professor Bustin stated that cycling more than 35 times was unwise, but it appears that nobody is limiting cycles to 35 or less (the MIQE guidelines recommend less than 40). Cycling too much could result in false positives as background fluorescence builds up in the PCR reaction.
7. According to the CDC, Ct Values related to “infectiousness” are UNKNOWN.
“The exact RT-PCR Ct values associated with the presence of infectious SARS-CoV-2 is unknown”
8. The CDC states Ct Values can not be used to determine if one is not “infectious.”
According to the CDC:
Can cycle threshold (Ct) values be used to access when a person is no longer infectious?
“No. Although attempts to culture virus from upper respiratory specimens have been largely unsuccessful when Ct values are in high but detectable ranges, Ct values are not a quantitative measure of viral burden. In addition, Ct values are not standardized by RT-PCR platform nor have they been approved by FDA for use in clinical management. CDC does not endorse or recommend use of Ct values to assess when a person is no longer infectious.”
9. Fauci has stated any Ct Value over 35 is just “dead nucleotides.”
“Dr. Anthony Fauci had the following to say on Jul 16, 2020,
“…What is now sort of evolving into a bit of a standard that if you get [perform the PCR test at] a cycle threshold of 35 or more…the chances of it being replication-competent [aka accurate] are minuscule… you almost never can culture virus [detect a true positive result] from a 37 threshold cycle… even 36… it’s just dead nucleotides, period.” Dr. Anthony Fauci
10. Most PCR tests are run at 40 or more cycles.
“In a list of test kits approved by the US FDA one manufacturer each recommended 30 cycles, 31, 35, 36, 37, 38 and 39. 40 cycles was most popular, chosen by 12 manufacturers, and one each recommended 43 and 45.”
“Bustin’s advice in my interview with him was that not more than 35 cycles be used. With either 35 or less than 40, the majority of COVID-19 RT-PCR tests approved by the FDA may be pushing RT-PCR to its limits or beyond.”
“One solution would be to adjust the cycle threshold used now to decide that a patient is infected. Most tests set the 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.
Tests with thresholds so high may detect not just live virus but also genetic fragments, leftovers from infection that pose no particular risk — akin to finding a hair in a room long after a person has left, Dr. Mina said.
“Any test with a cycle threshold above 35 is too sensitive, agreed Juliet Morrison, a virologist at the University of California, Riverside. “I’m shocked that people would think that 40 could represent a positive,” she said.”
11. The Infectious Disease Society of America and the Association for Molecular Pathology released a joint statement about the PCR Test Ct Values warning healthcare providers not to rely on them.
“Although there is a relative relationship between Ct values and the amount of virus in a clinical specimen, Ct values generated by qualitative PCR tests should not be considered quantitative measures of viral load. Due to the myriad of analytical and clinical factors known to impact Ct values, caution is advised when applying published correlations of Ct values with disease severity or as a predictor of active infection and hence transmissibility. At the current time, routine use of Ct values to inform clinical decision making is not advised. Development of a quantitative SARS-CoV-2 real-time PCR assay may overcome some of the limitations outlined above. The development of such tests may eventually occur if clinical utility for measurement of
SARS-CoV-2 viral load is shown.”
“In a blog post on Wednesday, the Association for Molecular Pathology cautioned healthcare providers against basing clinical decisions on cycle threshold values from PCR tests.”
12. PCR tests are NOT STANDARDIZED across the world and what Ct Value equals positivity is determined by the manufacturers of the test.
“The Food and Drug Administration said in an emailed statement that it does not specify the cycle threshold ranges used to determine who is positive, and that “commercial manufacturers and laboratories set their own.”
“The Centers for Disease Control and Prevention said it is examining the use of cycle threshold measures “for policy decisions.” The agency said it would need to collaborate with the F.D.A. and with device manufacturers to ensure the measures “can be used properly and with assurance that we know what they mean.”
“Accuracy — The accuracy and predictive values of SARS-CoV-2 NAATs have not been systematically evaluated. They are highly specific tests [50,51]. Although NAATs have high analytic sensitivity in ideal settings (ie, they are able to accurately detect low levels of viral RNA in test samples known to contain viral RNA), clinical performance is more variable.”
“However, the clinical application of the Ct is uncertain . Ct values are not standardized across RT-PCR platforms, so results cannot be compared across different tests. Furthermore, no clinical studies have validated use of Ct to guide management.
Who determines the Ct cutoff?
- The Ct cutoff is determined by the manufacturer of the test, not the state or laboratory performing the test. The cutoffs are reviewed during the submission process for the FDA’s Emergency Use Authorization (EUA). Once a test receives the EUA, the Ct cutoffs are set and cannot be changed by laboratories.
- Not all test manufacturers use the same Ct cutoffs, each test differs based on how it is designed and what part of the SARS-CoV-2 genetic material it targets for detection. Test manufacturers establish the cutoffs based on evaluation of their test with known positive and negative samples.
Why isn’t the Ct value reported on SARS-CoV-2 tests?
- The FDA EUA limits molecular diagnostic tests to report qualitative (positive/negative) of SARS-CoV-2 results and not quantitative (Ct value) results.
Ct values and cutoffs differ by test and thus cannot be compared from one test to another. A specimen with a Ct=36 may be considered positive by one test but produce a different Ct value and be considered negative or indeterminate on another.
13. PCR tests are prone to CONTAMINATION which leads to FALSE-POSITIVE results.
This is the CDC’s guidelines on false-positive PCR test results for the original SARS:
“The most common cause of false-positive results is contamination with previously amplified DNA. The use of real-time RT-PCR helps mitigate this problem by operating as a contained system. A more difficult problem is the cross-contamination that can occur between specimens during collection, shipping, and aliquoting in the laboratory. Liberal use of negative control samples in each assay and a well-designed plan for confirmatory testing can help ensure that laboratory contamination is detected and that specimens are not inappropriately labeled as SARS-CoV positive.
In the absence of SARS-CoV transmission worldwide, the probability that a positive test result will be a “false positive” is high. To decrease the possibility of a false-positive result, testing should be limited to patients with a high index of suspicion for having SARS-CoV disease.
In addition, any positive specimen should be retested in a reference laboratory to confirm that the specimen is positive. To be confident that a positive PCR specimen indicates that the patient is infected with SARS-CoV, a second specimen should also be confirmed positive. Finally, all laboratory results should be interpreted in the context of the clinical and epidemiologic information available for the patient.”
“The most important source of contamination is from the repeated amplification of the same target sequence, which leads to accumulation of amplification products in the laboratory environment. Even minute amounts of carryover can lead to false-positive results. A typical PCR generates theoretically as many as 108 copies of target sequence . If uncontrolled, amplification products will contaminate laboratory reagents, equipment, and ventilation systems. Carryover of previously accumulated amplified DNA is considered the major source of contamination.”
“Although PCR is a valuable technique, it does have limitations. Because PCR is a highly sensitive technique, any form of contamination of the sample by even trace amounts of DNA can produce misleading results (Bolognia et al, 2008; Smith & Osborn, 2009). In addition, in order to design primers for PCR, some prior sequence data is needed. Therefore, PCR can only be used to identify the presence or absence of a known pathogen or gene. Another limitation is that the primers used for PCR can anneal non-specifically to sequences that are similar, but not completely identical to target DNA. In addition, incorrect nucleotides can be incorporated into the PCR sequence by the DNA polymerase, albeit at a very low rate.”
- The DNA polymerase used in the PCR reaction is prone to errors and can lead to mutations in the fragment that is generated
- The specificity of the generated PCR product may be altered by nonspecific binding of the primers to other similar sequences on the template DNA
- In order to design primers to generate a PCR product, some prior sequence information is usually necessary.
14. PCR accuracy depends upon disease prevalence which can only be determined by clinical diagnosis. Diagnosis of “Covid-19” by clinical symptoms alone is impossible.
“WHO reminds IVD users that disease prevalence alters the predictive value of test results; as disease prevalence decreases, the risk of false positive increases (2). This means that the probability that a person who has a positive result (SARS-CoV-2 detected) is truly infected with SARS-CoV-2 decreases as prevalence decreases, irrespective of the claimed specificity.”
How Disease Prevalence is calculated:
15. “Viral” load estimates are useless and meaningless.
“Viral load information is in the PCR tests done to confirm SARS-CoV-2 infection but the tests aren’t approved by the FDA for that quantitative information, Satlin says.”
“Qualitative real-time PCR tests, however, are not designed to provide a quantitative or semi-quantitative measurement of nucleic acid in a sample. This is because qualitative test Ct values are not normalized to standardized controls of known concentration. Qualitative assays are also not optimized to have a linear relationship between the Ct value and the concertation of target nucleic acid in the specimen, which may disproportionally impact the reliability of Ct values obtained from samples containing either high or low viral loads. Truly quantitative viral load tests are typically performed using blood specimens. Phlebotomy is an easily standardized and reproducible procedure. In contrast, respiratory specimen types are less amendable to quantitative PCR testing due to the variability inherent to collection process as well as difficulties associated with testing complex sample types like saliva or sputum.”
“Little effort has been made to track viral loads in Covid-19 patients. This month, however, the Food and Drug Administration said clinical labs might report not just whether a person was infected with the coronavirus, but an estimate of how much virus was carried in their body.”
“But the use of Ct values to estimate viral load is a fraught practice. Viral load measurements for H.I.V. are highly precise, because they are based on blood samples. Tests for the coronavirus rely on swabbing the nose or throat — a procedure that is subject to user error and whose results are less consistent.”
“The exact relationship between a Ct value and the corresponding viral load can vary between tests. Rather than validate this quantitative relationship for each machine, the F.D.A. authorized the tests to deliver diagnoses based on a cutoff for the cycle threshold.
Most manufacturers conservatively set their machine’s thresholds for diagnosis from 35 to 40, values that generally correspond to an extremely low viral load. But the exact threshold for a positive result, or for a specific Ct to indicate infectiousness, will depend on the instrument used.”
16. PCR is prone to false-positives.
“Wang Chen, president of the Chinese Academy of Medical Sciences, admitted that “PCR tests are only 30 to 50% accurate”
“Gina Kolata’s story in the New York Times about what happened in 2006 at Dartmouth-Hitchcock Medical Center makes for astonishing reading. I can’t recount it better than she did — you should really just go and read it right now. But if you’d rather not, I’ll quote some of the most important parts (emphasis mine):
Not a single case of whooping cough was confirmed with the definitive test, growing the bacterium, Bordetella pertussis, in the laboratory. Instead, it appears the health care workers probably were afflicted with ordinary respiratory diseases like the common cold.
… specialists say the problem was that they placed too much faith in a quick and highly sensitive molecular test that led them astray … At Dartmouth the decision was to use a test, P.C.R., for polymerase chain reaction … their sensitivity makes false positives likely, and when hundreds or thousands of people are tested, as occurred at Dartmouth, false positives can make it seem like there is an epidemic.
There are no national data on pseudo-epidemics caused by an overreliance on such molecular tests, said Dr. Trish M. Perl … but, she said, pseudo-epidemics happen all the time. The Dartmouth case may have been one the largest, but it was by no means an exception, she said. There was a similar whooping cough scare at Children’s Hospital in Boston last fall that involved 36 adults and 2 children. Definitive tests, though, did not find pertussis.
“Because we had cases we thought were pertussis and because we had vulnerable patients at the hospital, we lowered our threshold,” she said.
“If we had stopped there, I think we all would have agreed that we had had an outbreak of whooping cough and that we had controlled it,” Dr. Kirkland said.
“It’s a problem; we know it’s a problem,” Dr. Perl said. “My guess is that what happened at Dartmouth is going to become more common.”https://blog.plan99.net/pseudo-epidemics-7603b2da839
From Dr. Andrew Cohen:
“Contrary to the practice during previous epidemics, with COVID-19 health authorities have treated a single positive result from a PCR-based test as confirmation of infection, irrespective of signs, symptoms and exposure. This is based on a widespread belief that positive results in these tests are highly reliable. However, evidence from external quality assessments and real-world data indicate enough a high enough false positive rate to make positive results highly unreliable over a broad range of scenarios. This has clinical and case management implications, and affects an array of epidemiological statistics, including the asymptomatic ratio, prevalence, and hospitalization and death rates, as well as epidemiologic models. Steps should be taken to raise awareness of false positives and reduce their frequency. The most important immediate action is to check positive results with additional tests, at least when prevalence is low.”
More from Dr. Cohen:
17. CDC admits that PCR testing of the asymptomatic leads to high ffalse-positives.
“However, only patients with signs and symptoms consistent with pertussis should be tested by PCR to confirm the diagnosis. Testing asymptomatic persons should be avoided as it increases the likelihood of obtaining falsely-positive results. Asymptomatic close contacts of confirmed cases should not be tested and testing of contacts should not be used for post-exposure prophylaxis decisions.”
18. A review of many PCR tests found the studies behind them flawed/lacking and raised questions about their accuracy.
The estimation of diagnostic accuracy of tests for COVID-19: A scoping reviewThe estimation of diagnostic accuracy of tests for COVID-19: A scoping review
“In our scoping review of 49 articles concerning test performance characteristics of rRT-PCR and other NAAT used for the diagnosis of COVID-19, we were able to observe several overarching themes. Clinical diagnosis by the case definition for COVID-19 used in the early period of the pandemic does not correlate well with positive rates of COVID-19 rRT-PCR (Table 1). The result of the initial rRT-PCR performed on a patient, if negative, may not be reflective of the result after multiple repeated rRT-PCRs for that patient (Table 2).”
“These findings should be viewed cautiously as the SARS-CoV-2 tests in these studies have not undergone rigorous evaluation necessary for FDA approval due to the emergency state generated by the COVID-19 pandemic. In addition, during our scoping review, we found substantial heterogeneity among available studies in terms of test types, reference standards, metrics, and details of study design and methodology.”
“Evaluation of the performance characteristics of SARS-CoV-2 diagnostic tests is vital to control of the ongoing COVID-19 pandemic. While more than 200 SARS-CoV-2 molecular diagnostic tests have received FDA EUAs, we have described in this scoping review that the performance of few of these tests has been assessed appropriately. The lack of robust test performance that we noted in many studies published to date is undoubtably due in part to the critical need for tests, which resulted in accelerated test development. However, our scoping review also uncovered imperfect methods for estimating diagnostic test performance in the absence of a gold standard and demonstrate that the accuracy of these tests should be interpreted with caution.”
19. 22 respected scientists reviewed the Drosten PCR test, which is the standard protocol all other tests have been built upon, and found numerous fatal scientific errors.
“The detailed explanation of these scientific errors exceeds the remit of this editorial, which is why they are only listed below.
- Extremely high concentrations of primers, DNA polymerase and magnesium sulfate. This leads to an increase in nonspecific binding, amplification, and lack of specificity to identify the SARS-CoV-2 virus.
- Non-specific primers (oscillating letters) that could give rise to various sequences of forward primers and as many inverse ones that are not related at all to SARS-CoV-2, so the test is not a specific tool for its diagnosis
- The test cannot discriminate between the complete virus (infectious) and the viral fragments.
- A difference of 10 °C with respect to the annealing temperature of the first pair of RdRp primers (direct and reverse) when it should be 2 °C.
- The genes chosen were wrong because:
- They did not represent the entire length of the virus.
- The E gene is nonspecific and is present in all coronaviruses.
- The N gene that at least ensured that it was a SARS-1 or SARs-CoV2 was removed by the WHO from the protocol due to lack of sensitivity.
- The RdPd gene proposed by Corman, et al.  contains too many oscillating letters so that 2 forward primers, 4 different reverse primers and 8 different probes could be synthesized, which provides excessive variability from the point of view of commercial tests to ensure its specificity.
- The PCR products have not been validated at the molecular level.
- The PCR test does not contain a single positive control to evaluate its specificity for SARS-CoV-2 or a negative control to exclude the presence of other coronaviruses, which makes the test unsuitable as a specific diagnostic tool to identify SARS- CoV-2 Virus
- The number of cycles is not specified for a test to be positive. Later, the WHO recommended between 40 and 45 cycles, which is totally wrong from a scientific point of view.
Recently, Bruno, et al. . conducted another critical review of the article by Corman, et al.  focused on the “Non-specificity of the Real Time RT-PCR test to detect COVID-19” in which to show the inability of the RT-PCR test to discriminate between different coronavirus strains and to confirm the molecular diagnosis of infection with the novel SARS-CoV-2 virus.”
20. Kary Mullis, inventor of the PCR test, regularly criticized it’s misuse as a diagnostic test and was very critical of his scientific fraternity.
“Quantitative PCR is an oxymoron.”
“I think misuse PCR is not quite… I don’t think you can misuse PCR. The results, the interpretation of it, if they could find this virus in you at all, and with PCR, if you do it well, you can find almost anything in anybody. It starts making you believe in the sort of Buddhist notion that everything is contained in everything else. Right, I mean, because if you can amplify one single molecule up to something which you can really measure, which PCR can do, then there’s just very few molecules that you don’t have at least one single one of them in your body, okay. So that could be thought of as a misuse of it, just to claim that it’s meaningful.”
“It’s [PCR] just a process that’s used to make a whole lot of something out of something. That’s what it is. It doesn’t tell you that you’re sick and it doesn’t tell you that the thing you ended up with really was going to hurt you or anything like that.”
These are UNDENIABLE FACTS. They are all sourced. No opinion here.