COVID-19 RT-PCR Ct Values. What Do They Mean?

What is a Ct Value?

The “cycle threshold” or Ct value of an RT-PCR assay refers to the number of cycles needed to amplify viral RNA to reach a detectable level.

Are Ct Values Absolute?

No. Commercially available COVID-19 diagnostic RT-PCR assays are qualitative tests producing positive or negative results. In qualitative RT-PCR assays Ct values are relative, not absolute numbers. Qualitative RT-PCR assays are not designed to provide semi-quantitative or quantitative measurement of the level of viral RNA in a sample.

Are Ct Values Variable?

Yes, they can be. For example, in one study, 26 labs that test for the coronavirus participated in a proficiency test. In this study they saw a variability of Ct values of up to eight cycles between labs when testing the same specimen (Public Health Ontario, 2020).

Do Ct Values Correlate with Viral Load?

There is a relationship between Ct values and amount of virus in a patient specimen, but it is not linear (Livak and Schmittgen, 2001). A high Ct value does not necessarily equate to a low viral load. There are many reasons a sample could have a very high viral load and a high Ct value including:

• The amount of material collected by nasopharyngeal swabs is imprecise (Garcia-Rodriguez et al, 2020)
• Inefficient extraction of viral RNA
• The patient consumed something that inhibited the real-time PCR reaction
• The sample was packaged or stored inappropriately and the viral RNA in the specimen degraded

Are Ct Values from One Test Comparable to another Test?

No. Ct values and cutoffs are assay- and method-specific (APHL, 2020). Ct values can vary up to two to three logs from test to test (Pritt, et al, 2020). Additionally, test sensitivity varies between different assays. The FDA developed a reference panel to determine the relative sensitivity of the COVID-19 RT-PCR tests. The data reported on the FDA website shows that there is a 1000-fold difference in Limits of Detection (LoD) between these assays (FDA, 2021).

Are Samples with High Ct Positives Amplifying Non-specific Targets?

Specificity measures a test’s ability to correctly generate a negative result for samples which does not contain the SARS-CoV-2 virus. RT-PCR assays are tested for specificity before they can be used to diagnose COVID-19 – cross-reactivity of the assay is evaluated using both in silico analysis and wet testing against normal and pathogenic organisms found in the respiratory tract.

Are Results Above a Certain Ct Value Unreliable?

No. Because Ct values are relative and not absolute, a single Ct value cannot be used as a cutoff for reporting a positive sample across different tests. Every RT-PCR assay’s Ct values are individually defined and then validated. NGS is an orthogonal option that can be used to verify RT-PCR results.

Can People with High Ct Positives Infect Others?

Yes, they can. As stated above, there are many reasons a Ct value may not correlate with viral load. A sample with high Ct value may have come from a person with high viral load.

Additionally, the SARS-CoV-2 replication process must also be considered. If a sample is collected shortly after infection, the viral load may still be low as the virus has not had a lot of time to replicate. However, this person may just be reaching the most infectious stage of infection.

A specimen collected many days to weeks after the initial infection may have a low viral load, and viral RNA can be detectable for many weeks after infection in some patients. There is insufficient evidence to rule out infectiousness in such cases.

References:

APHL. (2020) Ct Values: What They Are and How They Can be Used. Version 1.

Dahdouh E., Lázaro-Perona F., Romero Gomez M.P., Mingorance J., and Garcia-Rodriguez J. (2020) Ct values from SARS-CoV-2 diagnostic PCR assays should not be used as direct estimates of viral load. J Infect. Doi:10.1016/j.jinf.2020.10.017.

FDA Reference Panel Comparative Data. Feb. 25, 2021.

Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25(4):402–8. doi:10.1006/meth.2001.1262.

Public Health Ontario, (2020) 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

Rhoads D., Peaper DR, She RC, Nolte FS, Wojewoda CM, Anderson NW, and Pritt BS. (2020) College of American Pathologists microbiology committee perspective: Caution must be used in interpreting the cycle threshold (Ct) value. Clin. Infect Dis., doi:10.1093/cid/ciaa1199.