Last updated by The POOG on March 27, 2021.

The RT-PCR test is the predominant SARS-CoV-2 testing protocol for many countries. Controversy around it is growing. I’ve decided that it is worth an article of its own. I’ll begin with a short description of the test. Then I will look at issues with the test and the meaning of test results.

The PCR and RT-PCR Tests

PCR stands for ‘polymerase chain reaction’. It wont be on the exam so don’t worry. The RT stands for ‘reverse transcriptase’, hence, RT-PCR (also rRT-PCR and RT-qPCR variant). I’ve cited articles that may offer additional explanation[1][9].

Every cell of every living creature contains a molecule called DNA which is a very long chain of pairs of chemicals called ‘nucleotides’ of which there are 4 distinct varieties. This sequence is like a coded instruction that tells each kind of cell in your body how to build a copy of itself. These different cells make up your hair, your toes, your kidneys – all the parts of your body. Each cell has a small “engine” inside that knows how to read the appropriate part of your DNA code to build a new copy of itself. In the process, something called RNA which is like your DNA, is involved.

Some viruses have their own DNA and some, like SARS-CoV-2, the virus behind the COVID-19 pandemic, has only RNA. Viruses lack the engine to replicate themselves. What they do is to enter some cell in your body and hijack the cell’s engine to make viral copies, killing the cell in the process and releasing the new virus copies to repeat the process in the infection. The PCR test is looking for particular fragments of viral DNA while the RT-PCR test is looking for viral RNA fragments.

To start a test we need a sample from a person to be tested. A nasal swab is the preferred protocol for SARS-CoV-2. In the sample, the presence of viral RNA fragment is usually so small that a direct identification can’t be made. The PCR test solves this problem by an amplification process that doubles the presence of all DNA fragments, viral or not. When testing for RNA, the RNA is first converted to DNA in a precursor step with an enzyme called ‘reverse transcriptase’, hence the RT in front of PCR[1].

This doubling cycle is repeated until there is enough of the viral DNA created that a second stage process can reliably identify. Identification is achieved by comparing the test sample to the know genetic sequences of the virus. The number of cycles performed until enough viral material can be identifies is called the cycle threshold (Ct).

Labs will have a cutoff Ct value, usually 35 – 40, at which point they stop the test and declare a negative result. If a positive result can be determined at a lower Ct value, it has implications for interpreting results.

There are variants of the test such as a ‘Real Time PCR Test’[5] or rRT-PCR. These tests allow for almost immediate determination of the presence or absence of the virus.

Interpreting Test Results Using the Ct

The following video has a good discussion of the complexity of interpreting test results.

The test itself does not necessarily confirm if a viable virus is present or, if one is, if the person is infectious. It is understood that most people are infectious for a period of 7 to 10 days after symptom onset. Beyond that period, they may still have viable virus present but are not transmissive. They may also continue shedding non-viable virus and virus particles for up to 3 months after infection as was a case in China.

For a positive test result, confirmation of infection is made by culturing the amplified test sample. This is a laborious and time consuming process and is usually avoided.

The Cycle threshold (Ct)

If only a small amount of the virus is present, or possibly no virus, the number of cycles required before identification or test cutoff is reached limit increases.

If a person has a high viral load (severe infection), or if the test is done early after symptom onset, the swab is more likely to pick up a higher number of viable virus particles. This results in fewer cycles that have to be run before a positive result is registered. Culturing these low Ct samples is also more successful, confirming infection.

As the Ct increases, the amplification process may introduce spurious DNA other than the viral DNA – background noise. Studies show that beyond a certain Ct, the results become meaningless and if positive is considered a false positive meaning insufficient or no viable virus could be cultured[15].

In an interview, Dr. Anthony Fauci of the CDC stated beginning at the 4:30 minute mark of this video:

… if you get a cycle threshold of 35 or more, that the chances of it being replication competent are minuscule.

… you almost never can culture virus of a 37 threshold cycle … even 36 you gotta say, you know, it’s dead nucleotides, period.

Source: TWiV

A Ct of 35 is a widely recommended cutoff after which a cultivatable sample is unlikely[2]. Gupta cites studies that give a Ct cutoff after which “[N]no culture was obtained from samples with Ct value of > 34.”[6]. The implication then is that “patients with coronavirus disease 2019 (COVID-19) with Ct above 33 to 34 are not contagious“.

Singanayagam et al found that the probability of finding culturable virus dropped with increasing Ct value:

The estimated probability of recovery of virus from samples with Ct > 35 was 8.3% (95% CI: 2.8%–18.4%).

Singanayagam et al (2020)[2]

A paper by Corman, Land, Kaiser, et al, (2020)[7] proposed a protocol with a Ct of 45. The paper has been criticized by Borger, Malhotra, Yeadon, et al. (2020)[8] who noted:

The maximum reasonably reliable Ct value is 30 cycles. Above a Ct of 35 cycles, rapidly increasing numbers of false positives must be expected .

Citing Jaafar et al. 2020 [3]: “At Ct = 35, the value we used to report a positive result for PCR, <3% of cultures are positive.”

Borger, Malhotra, Yeadon, et al. (2020)[8]

Jaafar et al (2020)[12] reviewing more than 100 studies, reported that a Ct maximunm of 30 and possibly as low as 25 should be used.

Several recent publications, based on more than 100 studies, have attempted to propose a cutoff Ct value and duration of eviction, with a consensus at approximately Ct >30 and at least 10 days, respectively [2–5]. However, in an article published in Clinical Infectious Diseases, Bullard et al reported that patients could not be contagious with Ct >25 as the virus is not detected in culture above this value

Jaafar et al (2020)[12]

In a letter to the editors of the New England Journal of Medicine, Min-Chul Kim et al observed that no culturable virus was found above a Ct of 28.4[13].

Interpretation

If the percent of culturable virus is a measure of positive infection, as Ct increases, the likelihood of a false positive result increases because you may be detecting not RNA from viable viruses but genetic fragments from viruses that have become nonviable.

If no result has been determined by the Ct cutoff, the test is declared a negative result. It can still be a false negative if for some reason, the swab did not collect active virus present in the patient. This can happen if the swabbing is done improperly such as insufficient depth of insertion or insufficient time/rotations of the swab[11].

You may be surprised to know that the accuracy of the test itself – not a specific test result – depends on the prevalence of the disease in the population. As the prevalence drops the number of real positives decreases[3][4][10] and false positives increase. This can be explained mathematically using the statistical tool known as Bayes Theorem. Dr. Trefor Bazet explains how[3] the technique works in a video that has other useful information about COVID-19. He gives an example of how if as much as 1% of the population has the disease, the test, with a 95% sensitivity and a 99% specificity, will generate 49% false positives.

Harrit[14] provides an overview of the test and an analysis of false positives and their causes.

Finally, here is the late Kary Mullis, inventor of and Nobel Laureate for the PCR test in what is somewhat a stand-up discussion of the test:

YouTube video deleted by YouTube censors as it contravenes their preferred narrative.

As he says:

PCR is just a process that allows you to make a whole lot of something out of something. It doesn’t tell you that you are sick, or that the thing that you ended up with was going to hurt you or anything like that.”

Source: Kary Mullis, YouTube.

The RT-rPCR Test in Ontario Canada

Public Health Ontario has a document[10] describing their use of the RT-rPCR test.

Tests are performed in more than 40 labs across the province. This requires that careful attention paid to protocols to achieve standardized test results. Each site will calibrate its test using a standard commercial assay of test materials including a control sample.

In the test itself, there is a “background signal (a low level signal that is present in the assay regardless of whether target is present)“. If virus is present, the test is declared to be positive if the detection threshold is reached before the cutoff Ct is reached. Ontario uses a cutoff Ct of38 with values in the 38.1 to 39.9 range as an indeterminate zone.

One test done in Canada found that “specimens with Ct values > 24 were viral culture negative“, and the “US CDC has reported that they were able to culture virus from specimens with Ct values up to the low 30s“. In Ontario, “Variability of Ct values of up to 8 cycles were observed for the same specimen material tested across the participating laboratories.

I interpret their following statement as meaning that the ideal laboratory cutoff for their assay tests is Ct = 38 but that the realistic cutoff for testing should be Ct = 35.

In general, specimens with Ct values well below the assay cut-off for positivity (e.g. Ct < 35 with the laboratory positivity cut-off for that assay set at Ct = 38) are less likely to be false positive.

Public Health Ontario[10]

They note that “test results that are close to the limit of detection (LOD) of an assay have a greater likelihood of being false positives“.

Summary

A negative result does not tell you with certainty that you do not have the disease. It simply tells you that it could not detect the presence of the virus. A false negative can be caused by a number of errors that can occur somewhere in the testing procedure.

A positive result does not tell you with certainty that you do have the disease, and can tell nothing about whether you are infectious or not. The likelihood of a false positive increases with the increase in Ct number. After a certain Ct, the literature says results have to be considered indeterminate. Positive results can register after an infection is over and you no longer have it.

There is widespread agreement as outlined by Jaafar et al (2020)[12] that testing should be stopped at a Ct of 30.

Recommendation

If you are to undergo an RT-PCR test, insist that the testing authority provide the Ct along with the results and their interpretation. If you have read this far, you may know more about the test than the testing agent.

If you want to perform your own Bayesian analysis on the test to determine the percent of false positives expected, ask for:

  • The sensitivity of the test;
  • the specificity of the test; and
  • the percentage prevalence of infection (active cases) in the population.

References

  1. Davis, C.P. Polymerase Chain Reaction (PCR) Test. eMedicineHealth, reviewed on 6/22/2020.
  2. Singanayagam A, Patel M, Charlett A, et al. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Euro Surveill. 2020;25(32):2001483. doi:10.2807/1560-7917.ES.2020.25.32.2001483.
  3. Bazet T. False Positives & Negatives for COVID-19 tests | Using Bayes’ Theorem to Estimate Probabilities. YouTube. 21 May 2020.
  4. Nucleic acid testing (NAT) technologies that use real-time polymerase chain reaction (RT-PCR) for detection of SARS-CoV-2. WHO. 14 December 2020.
  5. Jawerth N. How is the COVID-19 Virus Detected using Real Time RT-PCR? IAEA. 27 March, 2020.
  6. Gupta S. Strong Inverse Correlation Between SARS-CoV-2 Infectivity and Cycle Threshold Value. Infectious Disease Advisor, May 14, 2020.
  7. Corman VM, Land O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance, Volume 25, Issue 3, 23/Jan/2020.
  8. Borger P, Malhotra RB, Yeadon , et al. External peer review of the RTPCR test to detect SARS-CoV-2 reveals 10 major scientific flaws at the molecular and methodological level: consequences for false positive results. Corman-Drosten Review Report, November 27, 2020.
  9. Brueck H and Lee S. Here’s how coronavirus lab tests really work, and why they don’t always give satisfying results. Business Insider. October 1, 2020.
  10. An Overview of Cycle Threshold Values and their Role in SARS-CoV-2 Real-Time PCR Test Interpretation. Public Health Ontario. September 17, 2020.
  11. Staff. Risk of False Results with the Curative SARS-Cov-2 Test for COVID-19: FDA Safety Communication. CDC. January 4, 2021.
  12. Jaafar R, Aherfi S, Wurtz N, et al. Correlation Between 3790 Quantitative Polymerase Chain Reaction–Positives Samples and Positive Cell Cultures, Including 1941 Severe Acute Respiratory Syndrome Coronavirus 2 Isolates. Clinical Infectious Diseases. 28 September 2020. ciaa1491, https://doi.org/10.1093/cid/ciaa1491.
  13. Min-Chul Kim, Chunguang Cui, Kyeong-Ryeol Shin, et al. Duration of Culturable SARS-CoV-2 in Hospitalized Patients with Covid-19.NEJM. January 27, 2021. DOI: 10.1056/NEJMc2027040.
  14. Harrit N. “Making something out of nothing”: PCR tests, CT values and false positives. Off Guardian. March 27, 2021.
  15. Diagnostic testing for SARS-CoV-2. WHO. September 11, 2020.