Polymerase chain reaction (PCR) and cycle threshold (Ct) values in COVID-19 testing

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About cycle threshold (Ct) values

Most tests that detect the ribonucleic acid (RNA) or genetic fingerprint of the virus  that causes COVID-19 (e.g., a polymerase chain reaction, or PCR test) use a process where specific bits of the genetic fingerprint are amplified using a temperature cycling reaction that repeats up to 45 times. These are called amplification cycles. The amount of genetic material doubles after each cycle. The number of amplification cycles required to create enough copies of the viral RNA to be detected is called the cycle threshold or Ct value.

The more RNA that is present in the patient sample, the fewer cycles are required for the signal to reach the detection threshold (low Ct value). The less RNA present in the clinical sample, the more cycles are required. So a low Ct value corresponds to a high viral load, while a high Ct value corresponds to a low viral load.

For an example of a real-time amplification curve on a logarithmic scale, see Figure 1 in Public Health Ontario: An Overview of Cycle Threshold Values and their Role in SARS-CoV-2 Real-Time PCR Test Interpretation.

Curves can also be viewed on a linear scale, which will look different but does not change the Ct interpretation. Not all commercial real-time PCR assays provide Ct values or amplification curves for viewing by the user. In addition, some molecular assays are based on other technologies (e.g., flow cytometry), and hence, do not provide Ct values.

How Ct values are used

The Ct value is the cut-off that calls a test positive. This is defined by the manufacturer of the test or the laboratory during the validation process. It ensures that the PCR test is correctly detecting the presence of the virus and not false signals.

In certain circumstances, such as in patients with compromised immune systems, samples may need to be retested following recovery from COVID-19. Ct values can be used to monitor changes in the amount of virus present in a person’s samples over time. This can be complex and typically requires consultation between health care providers and laboratory specialists.

Ct values and infectiousness

A frequent question is whether Ct values can help determine whether an individual is infectious or not. It is not possible to directly translate a Ct value into degree or duration of infectiousness.

A person is deemed infectious if they shed virus particles that are intact and able to go on to infect others. PCR tests cannot distinguish viral genomic material coming from intact viral particles in persons who are infectious or viral particle fragments that are present in individuals who have recovered.

There is good evidence that when more than 35 cycles are required to detect virus, the virus concentration is so low that it is unlikely to grow the virus in the laboratory. However, the cells used in the laboratory to grow the virus are different from the cells in the back of the throat and nose (nasopharynx) or the lungs in people. So just because one can’t grow the virus in a laboratory that does not mean that it won’t transmit.

Many believe that with low viral RNA copy numbers (high Ct value) the virus is not likely to be transmitted. A recent study which followed patients who were symptomatic but did not require hospitalization showed that those with higher viral loads (lower Cts) infected a higher proportion of their immediate contacts. But we do not know how much virus is actually    required to cause an infection in someone and there are other important factors that may influence infectiousness, including the health of the person exposed and the type of exposure that has happened.

Factors to consider when interpreting Ct values

Ct values will depend on the stage of infection

Between exposure to the virus and symptom onset (e.g., incubation or pre-symptomatic period), the amount of virus in a person’s sample can be initially too low to be detectable (negative). A person with an initially negative result may progress to give a test with a high Ct value i.e. >30 (low viral load), then to a lower Ct value (increased viral load) dramatically within a couple of days. Laboratories across the country have seen many cases where the person is tested early during their course of infection and the initial sample had a very high Ct value ~35 (low virus RNA concentration) and the following day the Ct was approximately 14 (high virus RNA concentration).

Ct values are affected by the type of the sample taken from the person

Nasopharyngeal swabs (those that go deep into the nose to swab the back of the upper throat) are the most sensitive specimen type for people who do not need admission to hospital. Throat/nasal swabs, and gargles/saliva may not have as much virus in them (so they would give a positive test with a higher Ct value). In people where COVID- 19 has infected their lungs, these samples from the nose/throat can be negative and a deeper sample like sputum is needed to detect the virus. In addition, the type of swabs used for collecting samples may also influence the Ct value.

Ct values are affected by the quality of the sample taken from the person

The quality of the sample collected is very important. If you don’t get the best possible sample, less virus will be in it and this can lead to a sample with an artificially high Ct value in a person who could have a lot of virus in their system.

Ct values cannot be compared between different PCR tests

There is no standard yet to be able to compare one test to another so the Ct range can greatly differ by the type of test used, that may use different signal detection methods. In fact, even when testing identical samples using different PCR tests,   the results can differ by up to 8 Ct values (e.g., from 22 to 30). This has been observed in the laboratories from different jurisdictions (e.g., Ontario, British Columbia and Saskatchewan).

The genetic fingerprint of the virus can be picked up long after the virus is no longer infectious

PCR can be positive for over 100 days or more after infection, usually with tests that have high Ct values but in most cases are unlikely to transmit to others beyond 10 days post symptom onset. This finding has been considered in the Infection Prevention and Control (IPAC) and public health practice that recommends patient isolation based on symptom onset, disease severity and the presence of any underlying, immunocompromising conditions instead of on PCR results alone both in some healthcare facilities and more so in the community setting.

The impact of new variants on Ct values is not clear

Our current tests can detect the new COVID-19 variants of concern (VOCs) - B.1.1.7 (first detected in the United Kingdom), B.1.351 (first detected in South Africa) and P.1 (first detected in Brazil). It has been documented that B.1.1.7 and B.1.351 variants are more infectious, and patients with B.1.1.7 infections have lower Ct values (higher viral loads) compared with those infected with the originally circulating (non-variant) SARS-CoV-2 virus. B.1.351 and P.1 are undergoing further study. We are closely following the VOC-positive samples in Canada to better understand the impact of these variants on our laboratory tests.

Key points and recommendations

References

  1. An Overview of Cycle Threshold Values and their Role in SARS-CoV-2 Real-Time PCR Test Interpretation. https://www.publichealthontario.ca/-/media/documents/ncov/main/2020/09/cycle- threshold-values-sars-cov2-pcr.pdf?la=en
  2. Basile K, McPhie K, Carter I, et al. Cell-based culture of SARS-CoV-2 informs infectivity and safe de-isolation assessments during COVID-19. Clinical Infectious Diseases, 2020 Oct. DOI: https://doi.org/10.1093/cid/ciaa1579
  3. Ct Values: What They Are and How They Can Be Used. Association of Public Health Laboratories. 2020, November 9. Retrieved from: https://www.aphl.org/programs/preparedness/Crisis-Management/Documents/APHL-COVID19-Ct-Values.pdf#search=What%20They%20Are%20and%20How%20They%20Can%20be%20Used
  4. Frequently Asked Questions about Coronavirus (COVID-19) for Laboratories, CDC. Retrieved from: https://www.cdc.gov/coronavirus/2019-ncov/lab/faqs.html#Interpreting-Results-of-Diagnostic-Tests
  5. Marks A, Millat-Martinez P, Ouchi D, et al. Transmission of COVID-19 in 282 clusters in Catalonia, Spain: a cohort study - The Lancet Infectious Diseases 2021. Published Online February 2, 2021 https://doi.org/10.1016/S1473-3099(20)30985-3
  6. Rhoads D, Peaper DR, She RC, Nolte F, Wojewoda C, Anderson N, Pritt BS, College of American Pathologists (CAP) Microbiology Committee Perspective: Caution Must Be Used in Interpreting the Cycle Threshold (Ct) Value, Clinical Infectious Diseases, 2020, ciaa1199, https://doi.org/10.1093/cid/ciaa1199
  7. Singanayagam A, Patel M, Charlett A, Lopez Bernal J, Saliba V, Ellis J, Ladhani S, Zambon M, Gopal R .Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Euro Surveill. 2020 Aug; 25(32):2001483. DOI: https://doi.org/10.2807/1560-7917.ES.2020.25.32.2001483
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