The RT-PCR test
"Making something out of nothing": PCR tests, CT values and false positives
Niels Harrit PhD
27 May 2021
If vaccination can be used as verification of the Corman-Drosten RT-PCR test for Covid-19, about 50% of the positive results reported should be considered false when no more than 35 cycles are used. If only 25 cycles are used, the proportion of false positives drops to 20%.
The effectiveness of the RT-PCR test used to identify infection with the SARS-CoV-2 virus and "cases" of Covid-19 disease is widely disputed. In these discussions, it is often claimed that the test produces 97% false positives. This claim references a study by a group from Marseille, who published their findings in a letter to the Oxford Academic (https://academic.oup.com/cid/article/72/11/e921/5912603) on September 28, 2020.
The first author is R. Jaafar, so it is referred to as the Jaafar paper. It represents an extended dataset compared to an earlier study led by B. La Scola. This publication is referred to as the La Scola paper.
Overall, the results presented in the Jaafar paper do not independently prove that the test produces 97% false positives. This comment attempts to extract the essential conclusions from their data.
There has also been confusion about the abbreviation "RT-PCR," which is sometimes referred to as "Reverse Transcriptase Polymerase Chain Reaction," while in other cases it is explained as "Real-Time PCR."
It is both. It is Real-Time RT-PCR.
The enzyme reverse transcriptase addresses single-stranded RNA in the swab and converts it into double-stranded DNA in several steps. Then the polymerase enzyme begins making copies of selected DNA. The selection is determined by a pair of so-called primers necessary to start the process.
Replication occurs in cycles. Each cycle begins by heating the sample to separate the DNA double helix into two free DNA strands. These serve as templates for the polymerase to produce complementary strands from the building blocks present in the mix.
Upon cooling, the strands recombine. The cycle is complete. The result is a doubling of the number of DNA molecules present before the cycle.
During production, the DNA is labeled with a probe molecule that fluoresces only when incorporated into the DNA. So, the sample emits visible light when illuminated with a small laser. Fluorescence intensity is recorded for each PCR cycle as a measure of the amount of DNA produced. This is where real-time comes in. When a predetermined level is reached, the multiplication process is stopped, and the test is called "positive."
The number of cycles required to produce the critical level of fluorescence is called the cycle threshold, Ct, which is a characteristic of each sample. Clearly, if the process starts with a large number of RNA fragments, the threshold fluorescence intensity is reached early, and Ct is low. If the initial load is only a few RNA molecules, or perhaps even a single one, it may take many cycles to get the critical fluorescence signal.
This means that the Ct value has the potential to provide a quantitative measure of the viral load in a person. It can be practical if you want a quantitative measure of your temporary condition.
A future conversation could be like this:
"How are you feeling?"
"I went to the test center. They told me I'm fine. I have a Ct of 42 today."
As will become evident from the following, this exchange could mean that person #2 had a cold last fall but no clinical symptoms today. All samples are positive if 60 cycles are used because "PCR makes something out of nothing," as Kary Mullis - Nobel Prize winner and inventor of PCR technology - once said.
When it is only a matter of cycles before the test is positive, we must all have DNA and/or RNA fragments - foreign or domestic - in our bodies that are targeted by the current primers. At high Ct values, you end up amplifying the "background molecular noise" of harmless genetic fragments.
WE ALL HAVE A CT ALL THE TIME!
The Jaafar article is a contribution to the important discussion about the therapeutic utility of PCR methodology. More specifically: "What is the tipping point for Ct below which a PCR provides a meaningful test for Covid-19 and above which it is meaningless"?
From the beginning of the Covid-19 outbreak until the research was completed in this publication, the institute in Marseille performed 250,566 SARS-CoV-2 tests for 179,151 patients.
Of these, 13,161 tested positive within 35 cycles of RT-PCR. It is 7.3%.
Of these positive samples, 3790 were inoculated and processed for culture. The process of inoculation is more or less described in the La Scola article.
They write that "0.5 ml of swab fluid was centrifuged and inoculated onto VERA cells (monkey kidney cell line) and observed for cytopathic effect" - for an unspecified number of days.
That is, did the cells die and disintegrate? This observation must be made under an optical microscope. If they observed cell death, a fluid sample was taken from the vial and then processed for observation in a scanning electron microscope.
The authors call this "Presumptive detection of virus in supernatant showing cytopathogenic effect." In Danish it means: "If we see the monkey cells die and disintegrate in the optical microscope, we take the sample to the electron microscope and think that whatever we see there must be virus."
However, no images from the electron microscope were presented.
The presence of the virus is further "confirmed" with RT-PCR on this fluid. Very importantly, the Ct values of these RT-PCRs run on the samples subjected to electron microscopy are not given. If the DNA/RNA selected by the pre- and post-inoculation primers targeted the same virus, the final Cts should be significantly lower than the Cts obtained from the crude swabs. If they weren't, we really don't know why the cells died.
But let's assume for now that cell death is a criterion for successful inoculation and that the deaths are due to the same virus quantified in the RT-PCR test.
Thus, Jaafar et al. report that inoculation was successful in 1941 cases out of the 3790 PCR positives processed for culture. This leads us to the immediate assessment that 49% of the positive PCR tests may have been false, in the sense that the patient's viral load must have been negligible.
Whether the 51% successful inoculated samples truly represent a positive diagnosis for the disease called Covid-19 depends on whether SARS-CoV-2 is truly a unique entity and whether it has been isolated and proven to be pathogenic.
In this assessment, we therefore reserve the term "positive" for a sample that reaches the critical fluorescence limit. The "positives" include the inoculable and the non-inoculable samples (which are false positives).
The data from the Jaafar article is reproduced in Figure 1. It shows the distribution between inoculable and non-inoculable samples for each group of Cts ranging from 11 (Ct11) to 37 (Ct37) cycles.
Yes, inoculables make up only 3% of the Ct35 group. However, since only 74 samples needed to be taken so far, this does not mean that the RT-PCR test generally produces 97% false positives. The picture is more diverse.
The same data is shown in a more traditional way in Figure 2.
We seek an answer to the question: How many cycles should be standard if we want 80% of the positives to represent an inoculable sample?
In Figure 3 (left), the number of inoculatable or non-inoculable samples up to the Ct value. That is, the curves in Figure 2 are integrated.
In Figure 3 (right), the same data are shown as a percentage of the total number of cultured samples up to the Ct.
It is seen that at Ct25, 80% of the samples called "positive" by the RT-PCR test will be inoculable. However, 20% of the "positives" will be false if inoculation is a benchmark for the efficiency of RT-PCR. Some may find it acceptable.
So if 1) something like a unique SARS-CoV-2 virus exists, if 2) this virus causes severe respiratory symptoms, if 3) the virus is inoculable into VERA cells, if 4) VERA cells are a valid representation of humans, and if 5) the Corman-Drosten test really detects SARS-CoV-2 specifically, it may have some advantages for a physician, where the clinical setting is a patient with severe respiratory symptoms, to run an RT-PCR until Ct = 25 as a supplementary test.
That's a bit far-fetched though, isn't it?
It really boils down to the primers, their specificity and utility at low concentrations. How can they target a deadly SARS-CoV-2 virus when its existence has yet to be proven? In addition, the sequences of the various primers used are found not only in ca. 100 bacteria, but they are also abundant in the human genome.
Pairing of two strands of DNA - hybridization - does not have to be perfect to occur. If the two strands are, say, only 80% complementary, the binding constant will be reduced. But hybridization happens anyway. When a grossly exaggerated concentration of primers is used in the standard Corman-Drosten test, it is deliberately bound to pick up other DNA floating around, whether produced by the reverse transcriptase or not.
AN RT-PCR TEST RUN AT CT25
What result can be expected IF the Corman-Drosten test detected something called SARS-CoV-2 and was run at a maximum of 25 cycles?
Consider in Figure 4 (left) how the total number of cultured samples increases partially linearly with the number of cycles. This is in accordance with the view that the number of cultures per Ct group reaches a plateau beyond Ct ~ 20 (Figure 2), but only noticeable if the selection of samples subjected to culture was random.
Of this subset (3790) submitted to culture - selected from those called positive by the RT-PCR test within 35 cycles - only 1813 would have been recorded as positive if only 25 cycles had been used (Figure 4, left) , corresponding to 48% of the subgroup (Figure 4, right).
Since 7.3% tested positive by RT-PCR within 35 cycles, it can be expected that 7.3 x 0.48 = 3.5% will be returned as positive if the number of cycles is limited to 25.
Of these, 2.8% may be credible (80% according to inoculation), while 0.7% may be false positives - IF inoculation is a valid confirmation method and all other conditions are met!
FINAL REMARK
To be sick is to have symptoms. If you are not sick, you are not a carrier of the infection. It used to be common sense that you are healthy unless proven otherwise.
Common sense is no longer common during the alleged Covid-19 pandemic. Now you are sick until proven otherwise - and basically you are contagious. The vehicle for this scam is the RT-PCR test run at >35 cycles and above. Stop testing and survive.