Why the PCR method is used to diagnose SARS-CoV-2?

The responses are generated based on views that are jointly expressed by Estonian Society of Laboratory Medicine (ELMÜ), Estonian Society for Infectious Diseases (EIS) and Estonian Health Board.

Why is PCR diagnostics important?

Detection of SARS-CoV-2 RNA from respiratory tract material using the real-time polymerase chain reaction (PCR) method is recommended by the World Health Organisation (WHO) as well as other international organisations and clinical practices as the co-called golden standard for coronavirus testing.

The PCR method has been regularly used by doctors for many years to detect other viral infections of the respiratory tract (e.g., influenza).

Can the PCR test differentiate between infectious and non-infectious patients?

The PCR test allows detecting only the presence of the genetic material of the virus, not its ability to infect other people. The presence of a viable virus can only be detected by growing the virus in the cell culture; however, this method is unsuitable for regular diagnostics due to its high cost and time-consuming nature (the process takes several weeks).

The SARS-CoV-2 PCR test does not allow exact detection of the amount of virus particles, but their amount can be measured indirectly in the patient’s sample material through the Ct value.

What is the Ct value and how is it related to PCR tests?

  • Ct (cycle threshold) is the number of RNA replication cycles, starting from which the specific test result is considered as positive. The Ct value is often confused with the number of cycles; however, these two are different values. A low Ct value indicates high viral load, which is associated with an increased risk of infecting others.
  • A high Ct value indicates low viral load in the analysed material, which may be due to the following:
    • Beginning of the infectious phase where the viral load is not yet clearly detectable and thus a person is already potentially infectious;
    • Convalescent phase (end of infection), where the person has recently been infected and the risk of him/her infecting others may already be lower.

 

The Ct value is not specified in the response; it is part of the technical data not the response, and it is not validated for clinical use.

Ct values are not standardised and different tests used in laboratories may yield different values from the same sample material. Therefore, comparing these values and drawing conclusions solely based on these values is not clinically justified.

With a view to avoiding false interpretations, laboratories do not issue Ct values. This decision is based on a consensual agreement between the Estonian Society of Laboratory Medicine, Estonian Society for Infectious Diseases and Estonian Health Board.

How are SARS-CoV-2 results interpreted in the SYNLAB Eesti laboratory?

The Ct value used in the test is determined by the test manufacturer and is based on research results.

In the SYNLAB Eesti lab in Tallinn, SARS-CoV-2 is determined with the RT-PCR method, using the ThermoFisher TaqPath COVID-19 CE-IVD RT-PCR reagents. These reagents are designed to detect three SARS-CoV-2 genes, N, S and ORF1ab. According to the rules set forth by the manufacturer, sample material is considered positive if the presence of at least two of these genes is confirmed. In addition to Ct values, the results are also evaluated based on the RFU value (relative fluorescence unit) and rise of the amplification curve.

More detailed information by the manufacturer can be found here.

In addition, SYNLAB Eesti has successfully performed comparative tests with a WHO reference laboratory and participates regularly (several times a year) in international comparative tests.

The methodology has been verified in the lab and is accredited by the Estonian Accreditation Centre. The laboratory has a permission issued by Estonian Health Board for provision of health care services in the field of laboratory medicine.

How would you comment on the claims that most PCR positive patients are patients with residual positive results and are no longer infectious?

A patient’s infectiousness depends on a number of factors: the amount of virus particles in the respiratory tract, the amount of virus particles spread (normal breathing versus singing/sneezing), precautions taken (masks, keeping distance), etc. Laboratory tests analyse the presence of the virus in the respiratory tract. As explained above, PCR does not directly determine the level of infectiousness. The level of infectiousness has been indirectly evaluated by correlating the Ct values with cultivating the virus in the cell culture.

The claim that a large number of PCR positive patients have only residual positive results and thus are no longer infectious may only be valid in certain limited patient groups (e.g., in case of surveillance testing of random people). The majority (ca 90%) of tests sent to the laboratory for diagnostics have extremely high viral loads, indicating infectiousness. Only in fewer than 5% of cases a low infectiousness level can be assumed (based on research data from Jaafar et al. 2020 and SYNLAB Eesti sample statistics).

The possibility that a person is PCR positive but with a certain degree of probability he or she is not infectious cannot be an argument in refusing to wear personal protective equipment or disregarding other rules.

What do doctors of infectious diseases and lab doctors think of Ct values?

As with any other test, the test result, including the Ct result, should be evaluated in the clinical context (presence of symptoms, time from the onset of symptoms and, if necessary, results of additional analyses, e.g., antibodies), and should not be used to automatically determine the patient’s level of infectiousness. The final interpretation falls upon the treating physician, who takes all this into account and decides whether the patient should be isolated or may already have recovered from the infection.

It is important to note that Ct values are not different in symptomatic and asymptomatic patients. With asymptomatic patients, it is difficult to determine precisely the beginning of the infectious period.