COVID-19 – What is the CT value and what does it convey about infectivity?

by Antonino Napoleone, Giuseppe Scarlata | March 24, 2022.
edited by Ivy Rose Sebastian.

Although testing for COVID-19 has become a part of our daily routine, the diagnostic tests hide technical peculiarities that are not common knowledge. As described in previous articles (Read also: Covid-19 tests current status), there are different types of tests, but molecular tests are the gold standard in diagnosing SARS-CoV-2 infections. In contrast to rapid tests, where the result can be seen within the comfort of your home in just 15 minutes, molecular tests require a longer waiting period and a team of specialised scientific personnel. Here, it is crucial to know how to use laboratory techniques, analyse samples correctly and accurately interpret the final results. How can test results be interpreted for SARS-CoV-2 diagnosis and how is the viral load measured? Above all, can the viral load be calculated, and the severity of the infection determined? Let’s find out how molecular test results are analysed in the laboratory!

Molecular testing

Molecular testing is based on an advanced technology called RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction), which can identify the presence of viral genetic material in the samples collected by a nasopharyngeal swab, with high precision and sensitivity. When we talk about genetic material, we are referring to the genome or nucleic acid of the organism, which can be made up of DNA or RNA, and as we have learned in recent years, the coronavirus has RNA as its genetic material.
This technology uses a biochemical process by which a specific part of the viral genome is targeted and identified through nucleic acid ‘triggers’ called primers, which are highly specific and unique to the viral sequence. In fact, the greatest advantage of this technique lies in its ability to detect even the slightest presence of the pathogenic RNA, within a time frame of one to three hours. This is why the discovery and use of RT-PCR in biomedical laboratories have revolutionised the world of research and diagnostics. (Figure 1).

So, to summarise, the biggest advantages of this technology are:

• Specificity, i.e., the high ability to detect only a specific target, with virtually no risk of cross-contamination (i.e., RNA or DNA from other microorganisms).
• Sensitivity, i.e., the ability to detect even minute amounts of DNA or RNA within a biological sample.

How is RT-PCR performed?

In a previous article, we had covered the journey of the biological sample for SARS-CoV-2 detection, from nasopharyngeal swab collection to the validation of the results (Read also: From the swab to the result). But how is a molecular test carried out? After swabbing, the biological sample is pre-treated and purified and then prepared for the biochemical reaction that takes place in a high-tech instrument (Figure 1).

The reaction involves a mix of reagents prepared in the laboratory:

– Primers, as mentioned earlier, serve as the triggers for the reaction.
– Enzymes, called polymerases, recognise the primers and carry out the key biochemical reaction of the molecular test.
– Nucleotide bases, which are the DNA building blocks, are used by the enzymes during the biochemical reaction. 
– Fluorescent probes or dyes, emit a light signal when they detect and bind to the product of the biochemical reaction synthesised by the enzymes.

The principle of the reaction is based on four steps:

1. Primers act by recognising and specifically attaching to a target sequence in the viral genome.
2. Enzymes recognise the primers and initiate a biochemical reaction using the nucleotide bases to initiate the synthesis of a final product of the biochemical reaction.
3. If the biochemical reaction has taken place, fluorescent probes bind to the final product and emit a light signal that can be detected by the fluorescence detector in the instrument.
4. The biochemical reaction takes place under cyclical conditions of varying temperatures and consists of a synthesis and amplification step for the final product (Figure 2).

If the viral target is present, the reaction takes place and for each cycle, the product is amplified exponentially until a plateau is reached, where the reaction becomes saturated and does not proceed further (Figure 3). Conversely, if the virus is not present, the reaction will not take place and the test result will be negative. 

What is the Ct value in molecular tests?

At the end of the molecular test, the results are indicated by the Ct value. The cycle threshold (Ct) value corresponds exactly to the number of amplification cycles required for the biochemical reaction of the RT-PCR to produce a positive signal above the minimum threshold value (cycle threshold).  As described earlier, a high concentration of virus genetic material in the sample will be detected with a low Ct value (<26), whereas low viral concentrations will require more cycles to be detected, resulting in a higher Ct value (>32). In general, a molecular RT-PCR test has a minimum threshold of 20-22 cycles and a maximum of 40 cycles.

Therefore, Ct value is an indirect indicator of viral load during the course of an infection (Figure 3):

• A lower Ct value indicates a high viral load, which correlates with higher infectivity.
• A higher Ct value indicates a low viral load, which correletes with lower infectivity.

Conclusions

How do these values translate during a SARS-CoV-2 infection? Low Ct values (high viral load) may indicate an increased likelihood of developing marked symptoms and transmitting the virus. High Ct values can be attributed to different clinical scenarios, such as an asymptomatic case, the protective effect of the vaccination (Read also: Vaccine effect on reducing viral load) or a convalescent phase, where the risk of infectivity is lower. Therefore, this value is of great significance, especially from a clinical and diagnostic point of view, as it offers considerable technical and scientific support in the management and monitoring of patients who are positive for SARS-CoV-2 infection. Again, this goes to show how important it is to consider and trust scientific evidence within the standpoint of a cautious and simultaneously, decisive pandemic management process. In other words, the message to entrust medical sciences, rather than media, must be even stronger!

References

• https://www.iaea.org/bulletin/how-is-the-covid-19-virus-detected-using-real-time-rt-pcr
• Understanding cycle threshold (Ct) in SARS-CoV-2 RT-PCR (publishing.service.gov.uk)
• Wikramaratna PS, Paton RS, Ghafari M, Lourenço J. Estimating the false-negative test probability of SARS-CoV-2 by RT-PCR. Euro Surveill. 2020;25(50):2000568. doi:10.2807/1560-7917.ES.2020.25.50.2000568.
• Lan L, Xu D, Ye G, et al. Positive RT-PCR Test Results in Patients Recovered From COVID-19. JAMA. 2020;323(15):1502–1503. doi:10.1001/jama.2020.2783.