CT Value in RT PCR: What it Means?

What are Ct Values?

Before we explain what a Ct (Cycle Threshold) value is, let’s go back to the basics of PCR for the use of diagnosis.

PCR stands for Polymerase Chain Reaction. There is cyclic amplification of DNA through the use of specific enzymes. This amplification follows an exponential or chain reaction pattern. In the case of RNA viruses, the RNA in the sample is converted to complementary DNA using a reverse transcriptase enzyme (hence the name RT-PCR: Reverse Transcriptase Polymerase Chain Reaction).

Without going into too much detail, what happens is the following:

• There will be a particular DNA sequence that you are looking for. This sequence is selected so as to ensure that ONLY the organism you are testing for has that particular sequence.
• To target the desired DNA sequence, there are primer-probesets designed to attach to the target sequence. These probes are nothing but short fragments of DNA that are complementary to the target sequence. The probes are ‘tagged’ with a substance that generates a fluorescent signal during the PCR reaction.
• So, the required sample from the patient is introduced into the PCR reaction along with the probes. What follows is an exponential sequence of DNA amplification via replication (eg: 50 strands of DNA become 100, then 200, then 400 etc.). The DNA unwinds and replicates during each cycle of the reaction. Each time it does so, the probe attaches to the specific target sequence on the sample DNA – if it is present. If the target sequence is not present, the probes remain free and do not attach anywhere and amplification does not happen.
• So in a positive sample, every time the DNA replicates, probes attach and generate a fluorescent signal. With each cycle, the amount of DNA, and consequently the probe, increases exponentially. As a result, the fluorescent signal also increases/accumulates with each cycle.
• In a negative sample, the target sequence is not present and so the probes do not get attached. Therefore, there is no amplification of the sequences and no fluorescent signal is generated.
• We are now in a position to define what a Ct value is. The Ct (cycle threshold) is defined as the number of cycles (of amplification) required for the fluorescent signal to cross the threshold (i.e. exceeds background level) in a positive sample. So, in a negative sample, no Ct value is generated.(1)

Does the Ct value correlate with Viral Load in SARS-COV-2?

Theoretically, Ct levels are inversely proportional to the amount of target nucleic acid in the sample.(1)

Presently, qualitative kits are being used for detection of the SARS-CoV-2 virus. These kits are not validated for quantitation of the viral load. That will only be possible with kits specifically developed and validated for measuring the viral load in a given sample.

So, should I use Ct values as a guide of Viral Load and thereby, disease severity?

Not really. Even though the Ct value may theoretically correspond with viral load, there are too many pitfalls associated with using Ct value to guide treatment.

The pitfalls that may occur in using Ct value to interpret viral load belong to four major categories:

1. Host factors: Shedding of the virus in the respiratory tract will be influenced by comorbidities (either diagnosed or undiagnosed), or genetic factors (which may or may not predispose to a more severe disease course). It is unknown at this point whether the amount of virus in an individual or the amount of virus shed by the individual plays any additional role in this context.(2)
2. Viral factors: Viral factors may also influence the interpretation of Ct values.(2) The SARS-CoV-2 virus has shown great variations in its shedding pattern in an infected individual. Cases have been reported where there was prolonged virus shedding even after the clinical symptoms were resolved and the individual developed antibodies.(3) We still require more long-term studies and data to be able to interpret the shedding pattern and duration of the shedding of this virus.
3. Sample collection: Ct values can vary due to a variety of factors including the site of swab collection, type of local immunity & how the sample is collected and transported. This is why the Ct value can vary from sample to sample even in the same individual. One paper on acute respiratory illness stated that, in an adult patient, they found a 1000-fold difference in human DNA concentration between two broncho-alveolar lavage samples, taken by the same pulmonologist, potentially reflecting a similar difference in viral sample size.(2)
4. Laboratory techniques: The Ct value is determined by using a qualitative assay and does not correlate directly with the viral load as none of the kits quantify the same. Ct values for a specimen vary between different kits and techniques. The target genes differ from kit to kit. Primers for the same gene may also differ between kits. Also, to be noted is that Ct values are valid for the kit used and that test-run only. A re-run of the same specimen on the same kit may return a different Ct value based on the assay dynamics. The efficiency of the first step of the RT-PCR procedure, nucleic acid extraction, may also vary between samples with different compositions and nucleic acid concentrations. The efficiency of both steps may vary considerably between different target sequences. A given RNA or DNA concentration may result in different CT values depending on the target sequence, on the reverse-transcription and polymerase enzymes, and on the reaction conditions used.(2)

Conclusion
Only use viral load values from validated quantitative assays (when they become available) to guide treatments. Though Ct values may mirror the viral load in a particular sample, its limitations and inherent variability need to be kept in mind before making clinical decisions based on it.

References
The University of Wisconsin-Madison. Real-time PCR CT Values [Internet]. 2013 [cited 2020 Jul 14]. Available from: https://www.wvdl.wisc.edu/wp-content/uploads/2013/01/WVDL.Info_.PCR_Ct_Values1.pdf
Wishaupt JO, Ploeg T van der, Smeets LC, Groot R de, Versteegh FGA, Hartwig NG. Pitfalls in interpretation of CT-values of RT-PCR in children with acute respiratory tract infections. J Clin Virol. 2017 May;90:1–6.
Liu W-D, Chang S-Y, Wang J-T, Tsai M-J, Hung C-C, Hsu C-L, et al. Prolonged virus shedding even after seroconversion in a patient with COVID-19. J Infect. 2020 Apr;S0163445320301900.