Polymerase Chain Reaction and SARS-CoV-2 RT-PCR

Polymerase Chain Reaction (PCR) was developed in the early 1990s by Karry Banks Mullis from the USA who together with Michael Smith (Canada) was awarded the Nobel Prize in Chemistry (1993) for his efforts in method development in DNA-based chemistry for the discovery of PCR. method.
Also called “molecular photocopying” – PCR is a fast and inexpensive technique used to “amplify” – copy – small segments of DNA. Because significant amounts of a sample of DNA are required for molecular and genetic analyzes, studies of isolated DNA fragments are almost impossible without so-called PCR amplification.

To amplify a DNA segment by PCR, the sample is first heated so that DNA denatures, or is separated into two pieces of single-stranded DNA. Then an enzyme called “Taq polymerase” synthesizes two new strands of DNA using the original strands that grind. This process results in duplication of the original DNA, with each of the new molecules containing an old and a new DNA strand. Then each of these strings can be used to make two new copies, and so on and so forth. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, resulting in more than one billion exact copies of the original DNA segment. The whole process is automated and can be completed in just a few hours. It is controlled by a machine called a thermocycler, which is programmed to change the reaction temperature every two minutes which leads to DNA denaturation and allows synthesis.

Are you infectious if you have a positive PCR test?

PCR-positive (SARS-CoV-2 RT-PCR) that have no symptoms of Covid19 infection are registered as “infection” cases and reported together with those who have symptoms of Covid-19. What is the probability that these positive tests are sign of a infectious SARS-Cov2.

The key to being able to develop a successful PCR test is to have access to the original so-called -master copy- of the entire genetic sequence based on a pure virus isolate. If the master copy is contaminated in any way, the test is considered unreliable. The master copy of SARS-CoV-2 used for PCR testing was derived from a synthetic RNA strand from viral fragments produced by Chinese researchers that could not be confirmed to come from a pure virus isolate but only an assumption based on the study of a partial RNA gene sequeuncing from lung fluid of a patient with symptom picture of Covid-19 in Wuhan.

With this as a starting point, an article entitled “Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR” was published on 25 January 2020, in the journal Eurosurveillance. This publication by Corman and Drosten was used as a basis for laboratories around the world to develop a PCR test for Covid-19 and apply for the authorisation for the emergency use of these (EUA).

Corman-Drosten’s publication eventually came under considerable criticism where it was claimed that due to lack of isolation of virus and without for the PCR test is not linked to a complete sequencing of a virus isolate, the test does not have the ability to accurately distinguish between SARS CoV-2 and other of the many Corona cold viruses.

The authors also emphasize that when using 35 cycles or more, signals are captured that cannot be said to represent true positives and (as is the case in most laboratories in Europe and the USA), the probability that the person is actually infected is less than 3%. that is, the probability that PCR is false positive is 97%.

The cycle threshold level for detecting live virus will vary by setting (hospital vs. community); depending on the symptom severity and the duration of symptoms, as well as the quality of the testing. Cycle thresholds are the times that the amplifying test has to be repeated to get a positive result. The higher the viral concentration the lower amplification cycles are necessary.

Why does the cycle threshold cut-off matter?

RT-PCR uses an enzyme called reverse transcriptase to change a specific piece of RNA into a matching piece of DNA. The PCR then amplifies the DNA exponentially, by doubling the number of molecules time and again. A fluorescent signal can be attached to the copies of the DNA, and a test is considered positive when the fluorescent signal is amplified sufficiently to be detectable.

The cycle threshold (referred to as the Ct value) is the number of amplification cycles required for the fluorescent signal to cross a certain threshold. This allows very small samples of RNA to be amplified and detected.

The lower the cycle threshold level the greater the amount of RNA (genetic material) there is in the sample. The higher the cycle number, the less RNA there is in the sample.

What does this mean?

This detection problem is ubiquitous for RNA viruses detection. SARS-CoV, MERS, Influenza Ebola and Zika viral RNA can be detected long after the disappearance of the infectious virus.

The immune system works to neutralise the virus and prevent further infection. Whilst an infectious stage may last a week or so, because inactivated RNA degrades slowly over time it may still be detected many weeks after infectiousness has dissipated.

It is also known that fragments of an infectious virus can remain in the body for up to 31 days. One person cannot infect another person with viral fragments and therefore a positive PCR test, regardless of the number of cycles used, can not readily conclude that the person is infected with SARS-CoV-2.

The figure below shows how the probability of SARS-CoV-2 infectious virus is greater (the red bars) when the cycle threshold is lower (the blue line) and when symptoms to test time is shorter – beyond 8 days, no live virus was detected.

Shedding of infectious virus in hospitalized patients with coronavirus disease-2019 (COVID-19): duration and key determinants medRxiv 2020.06.08.20125310

PCR manufacturers state that the PCR tests should not be used as the sole evidence for clinical diagnosis and treatment. Symptoms and signs of Covid-19 are necessary to indicate that a person is or may be contagious.

A “PCR-true” positive test cannot say how long the person has had the virus in their body or whether it is “active”. For example, if 20% of the population is PCR-positive, the number of PCR-positive will depend on the amount of samples taken. This means that the more PCR tests are performed, the greater the proportion of the population that is said to be infected, but where in reality parts of this population may have been infected long before these tests were performed.

PCR Positiv test and virulens 

The shaded area shows the number of days the virus is contagious. However, the virus is still present for many days after this. This may sound like a PCR-positive test, but it does not mean that the virus is virulent or contagious, where residues of “inactive” viral RNA are still detected by PCR.

A comprehensive systematic review of the medical literature from the University of Oxford, Center of Evidence Based Medicine concludes with the following:

«For a proper use of PCR, it is necessary to test a reference with the use of cell culture samples in each country that uses SARS-CoV-2 RT-PCR. This is to determine the usefulness and reliability of PCR for Covid-19 and its relationship to the concept of infection. The term “infection” must be weighed against the date of onset of symptoms and the cycle threshold level of the test. A binary Yes / No approach to the interpretation of RT-PCR regardless of threshold level that is not validated against viral culture will result in false positives with possible segregation of a large number of people who are no longer contagious and thus not a threat to public health.

A positive PCR test does not mean that the person necessarily presents any danger to society. The virus cannot be transmitted when cell culture shows that the virus is not contagious. The conclusion is that PCR tests are useful as long as the limitations are understood; but because the test detects RNA in very small amounts – caution must be exercised. This is not done now.

A Portuguese Court of Appeal refers to this basic criticism and rejected the PCR technology now used (SARS-CoV-2 R T-PCR) as a basis for imposing isolation or quarantine. They stated: “Based on the available scientific evidence, this RT-PCR test is not in itself able to establish that positivity actually corresponds to infection with the SARS-CoV-2 virus.” Courts in Austria and Belgium have taken similar stands

Lab Alert: Changes to CDC RT-PCR for SARS-CoV-2 Testing

Division of Laboratory Systems (DLS), CDC

Lateral Flow Test (Rapid Test) and SARS-Cov2

The Lateral Flow test is an established technology, adapted to detect the presence of a particular protein target, and is routinely used in healthcare settings because it is affordable, easy to use, delivers fast results and has a high level of accuracy. The best-known example of a lateral flow test is the home pregnancy test kit. 

In the case of COVID-19, the target of the lateral flow test is proteins, or antigens supposedly found in the COVID-19 virus. 

The COVID-19 test kit is a hand-held device with an absorbent pad at one end and a reading window at the other. Inside the device is a strip of test paper that changes colour in the presence of COVID-19 antigens. Using one swab, a sample is taken first from the tonsils and then from the nostril. Alternatively, a saliva sample may be used.  

“A Cochrane Systematic Review of the evidence for use of LFT i for testing in asymptomatic cohorts conclude that: .

Test accuracy studies cannot adequately assess the ability of antigen tests to differentiate those who are infectious and require isolation from those who pose no risk, as there is no reference standard for infectiousness.”

Rapid, point‐of‐care antigen and molecular‐based tests for diagnosis of SARS‐CoV‐2 infection, Cochrane Systematic Review.