Category: Matrix

What are Ct values?  

In quantitative PCR (qPCR) and reverse transcriptase quantitative PCR (RT-qPCR), the cycle threshold (Ct) value is the number of nucleic acid amplification cycles required for the signal generated by the amplification of a specific target DNA to cross a set threshold. Labelling the DNA primers with a fluorescent tag is what makes this PCR ‘real time’; as the target genetic material increases with each amplification cycle so does the fluorescence signal.

The lower the amplification cycles required to meet the threshold, the higher the level of target nucleic acid (and thus virus in the case of SARS-CoV-2) in the patient sample. Therefore Ct values are inversely related to viral load. Within a specific test the Ct value result is compared to a value often known as a ‘cut-off’. A value lower than the cut-off is a positive result (for COVID-19 meaning the patient most likely has the infection), whereas a value higher than the cut-off is a negative result.

Many of Logical Biological’s COVID-19 swab samples are tested using Thermo-Fisher Taqpath, which has a positive cut-off of Ct ≤37. In that test, a sample with a Ct of 36 would be a low positive. At the other end of the range, Ct values below 20 would be considered a strong positive, and we have infrequently seen values below 15.

Ct values and COVID-19 

The course of SARS-CoV-2 infection shows viral levels vary throughout the duration of the disease, peaking a few days after symptom onset. Correspondingly RT-qPCR Ct values should alter depending on the timeline of an individuals infection. The lower the Ct values the increased probability the patient is near to peak-infection. However there are multiple practical factors unrelated to the actual infection that may also affect the Ct value achieved.

Correspondence between development of viral load during SARS-CoV-2 infection, clinical course and positivity of quantitative rRT-PCR assays (r = real-time). (Lippi et al, 2020).

qPCR/RT-qPCR Ct value variables include but are not limited to:

1. Swabbing protocol
2. Swabbing technique
3. Swab type
4. Swab brand
5. Sample storage time
6. Sample storage conditions
7. UTM/VTM brand
8. RNA extraction method
9. PCR reagents used
10. PCR test kit used
11. Patient days since infection
12. Patient viral load
13. Environmental contamination
14. Standard quantification curves

With so many variables any comparison of Ct values between individuals has obvious limitations.

Clinical Use of Covid-19 Ct data

Cycle threshold values are beginning to be used for COVID-19 patients as there is a suggested relationship between viral load and prognosis. Lower Ct values have been linked to a worse course of illness and poorer outcomes. Clinicians have been  encouraged to interpret the SARS-CoV-2 RT-qPCR test results and consider the Ct value where appropriate.

A positive PCR test alone does not correlate with infectivity (Tom and Mina, 2020); a positive test with high Ct value may indicate the patient is not infectious. If this information could be used to inform patient isolation decisions it could transform quarantine restrictions and take significant steps in reducing the already extensive economic consequences of the pandemic, not to mention the mental health implications of isolation. Conversely, low Ct values may indicate a high level of infectiousness, acting as a signal to healthcare professionals to ensure the patient remains isolated and avoid releasing them into the general population until their Ct value increases sufficiently. The challenge here is reliably using Ct values to determine infectiousness.

Contradictory research regarding viral load and shedding in asymptomatic cases require further investigation. Measuring asymptomatic Ct values provided evidence of little difference when compared to symptomatic cases, implying similar viral load. On the other hand, other researchers found asymptomatic cases tested had no viable SARS-CoV-2 virus and no evidence of viral transmission for these carriers. This particular study should be viewed in the context of the virus persisting undetected in a population (Wuhan) where non-pharmaceutical interventions had been used to almost eradicate the virus from the population over a relatively long period of time; selection for a weakened strain may have occurred.

Ct values and disease diagnosis test development

While the value of Ct values to clinicians is debatable, they definitely have utility in in vitro diagnostic (IVD) assay development. Assay sensitivity can be defined as the proportion of true positive samples that actually test positive. SARS-CoV-2 test makers are motivated to produce sensitive tests able to even detect virus in patient samples where the viral load is low. Lateral Flow SARS-CoV-2 antigen test manufacturers, for example, may use patient samples with known RT-qPCR Ct values to determine how the samples test in their assay. Samples with very low Ct counts (high viral loads) would be expected to test positive, but by testing samples with increasing Ct counts (lower viral loads) assay developers can assess the performance of their test, understand what viral loads correspond with a failure of their test to detect, and work to optimise assay performance where necessary.

Conclusions

Ct values have limited use in clinical settings. A multitude of variables impact on the viral load and a separate large set of variables impact on patient outcomes (e.g. age). Ultimately Ct values are not easily comparable between assays or patients. It is possible they can be used to assist patient prognosis in some circumstances where a full clinical history is available. 

However, in vitro diagnostic assay developers find Ct values useful as a way of quickly determining the lower limit of detection of the tests they are developing. In this way, Ct data has a great value in contributing to the development of sensitive tests. 

Available from Logical Biological

At Logical Biological we supply nasopharyngeal/oropharyngeal/nasal SARS-CoV-2, FluA and Flu B swabs available in Universal Transport Medium (UTM), Inactivating Transport Medium (ITM), saline or in dry frozen format. Negative swabs and COVID-19 / pre-COVID saliva are also available, as are serum and plasma. Typically, our swabs are provided together a Ct value measured from a ‘companion swab’ taken simultaneously. For UTM & ITM we can provide remnant samples at your preferred Ct values.

Table: Products available from Logical Biological

This article covers swab type and selection, transport products and medium enabling a smooth transfer of patient samples to the laboratory for analysis. Additionally, we discuss the implications of cycle thresholds and whether they should be assessed in patient prognosis.

Swabs

Swabs come in all shapes and sizes and some favourites have become well known in recent months:

• Nasal Swab: needs to be inserted at least 1cm into the naris (nostril), directed upwards. May cause sneezing.
• Nasopharyngeal Swab: one method is to measure the distance from the corner of the nose to the front of the ear, then half it. This is the approximate distance the nasopharyngeal swab should be entering the nasal cavity. Use the medial side of the septum as a guide and gently rotate to facilitate insertion. The swab should run parallel to the palate and needs to be left in place for ‘several seconds’. Taking a sample is a skill in itself. The complexity and invasive nature of this test paired with lack of discrepancy compared to nasal swab results has lead to a shift in favour of nasal swabs. May cause sneezing and watering eyes.
• Oropharyngeal Swab: enters through the oral cavity (mouth). This swab aims to take material from the tonsils and the oropharyngeal wall (back of the throat), and avoids touching the tongue, teeth and gums. May cause retching.  

Generally the shape of these swabs are incredibly similar, essentially long shafted cotton buds. The tips can be selected to best accommodate the chosen antigen or marker of interest and the shafts chosen for access purposes. Options include long-armed pharmaceutical grade cotton buds, or perhaps Rayon or Dacron swabs are better suited if considering a dry swab. Rayon swabs are made from synthetic fibres spun from wood pulp, whereas Dacron is made from polystyrene. Swabs destined for transport medium require different properties such as polyester or foam, they can be made less or more absorbent, have different porosities and have good ‘release’ factors. Canadian researchers investigated how specific these swabs need to be especially in the current pandemic with supplies at a premium. They determined swab type had limited impact on test results.

Transport Medium

Having collected samples with wisely chosen swabs, samples must be secured in transport medium to make their way to the lab. Again there are many choices. Generally used, Universal Transport Medium (aka UTM) will protect and conserve samples. This is a well know method and low risk regarding the preservation of the sample. Examples are the BD universal viral transport system, which can accommodate storage at room temperature or at +4°C, and Copan UTM, which contains proteins for virus stablisation, plus buffer, antibiotics and antimycotics. 

Other media can provide more specific care depending on sample consistency, for example Viral Transport Medium (VTM) has specific ingredients suited to the transport of viral content. However, UTMs are also suitable for virus sample maintenance and the ingredients list of VTMs is similar. Generon outline a mix including heat inactivated fetal bovine serum, gentamicin and amphotericin B, an antifungal agent. Some recipes suggest including additional calcium and magnesium. If you are making your own sterility can be ensured by filtration or using sterile ingredients and aseptic technique upon mixing. A newer alternative is the Inactivated Transport Medium (ITM), or Molecular Transport Medium.

Inactivation – Health & Safety

It is not a large leap to identify the advantages of using ITM, and in fact there has been a call for this to take place with SARS-CoV-2 from back in April. From a UK perspective, SARS-CoV-2 is classified as Hazard Group 3 (HG3) by the UK Advisory Committee for Dangerous Pathogens. The Containment Level 3 list of requirements for low-risk handling of active HG3 infectious organisms are long and require specialist facilities, including:

• Maintenance of a negative air pressure
• HEPA systemic exhaust air extraction and filtration 
• Sealable laboratory to enable fumigation

For SARS-CoV-2 research and assay development, the ability to transport and analyse inactivated viral samples potentially negates the need for such stringent containment measures. This would assist COVID-19 test developers because they would no longer need to access Containment Level 3 / BSL 3 facilities as part of their test development. Such facilities are few and far between and potentially extremely expensive to access. The CDC advises transport of inactivated COVID-19 samples in a nucleic acid extraction buffer. The nucleic acids remain intact and identifiable, the medium inactivates the protein cap (using guanidine thiocyanate and N-Lauroylsarcosine (sodium) in ethanol) and renders the virus unable to reinfect. Of course, each user will need to abide by their local risk assessments and satisfy themselves that their choice of ITM truly inactivates the virus. Inactivation may denature the antigen of interest so users would need to be sure that the ITM would be compatible with their project objectives. Users should also seek specialist advice on the containment measures required when using any ITM. Logical Biological offers SARS-CoV-2 remnant swabs inactivated in NEST Scientific ITM.

Cycle Threshold Values

With an inactivated sample safely in the lab, the molecular components are ready to be amplified and analysed. Cycle Threshold (Ct) values are important as the quantitative unit of measurement in PCR methodologies and using them in diagnostic testing can be invaluable for patient prognosis. Essentially the lower the number of amplification cycles required to reach a threshold level for the target gene is, the higher the level of target nucleic acid (and thus virus in the case of SARS-CoV-2) in the patient sample; Ct values are inversely related to viral load. Generally speaking a very high Ct value indicates a low amount of target nucleic acid in the sample, and the possibility of environmental contamination should be borne in mind. Often a Ct value of around 34 is the cut-off point for a patient to be considered positive for the infection. Many of Logical Biological’s COVID-19 swab samples are tested using Thermo-Fisher Taqpath, which has a positive cut-off of Ct ≤37. At the other end of the range we have infrequently observed Ct values as low as 9, with Ct values below 15 considered an extremely strong positive. Some Logical Biological clients wish to access a selection of swabs with a range of different Ct values, to ensure that their assay is able to detect the various different ‘strengths’ of infection. This is something we have been able to accommodate readily

SARS-COV-2 PCR Ct values have been analysed and the scientific and clinical communities are assessing if indeed Ct values, that are not often reported in clinical results, should be included and used as a marker for future prognosis. As an example please refer to Rao et al, 2020. 

Available from Logical Biological

At Logical Biological we supply nasopharyngeal/oropharyngeal/nasal SARS-CoV-2, FluA and Flu B swabs available in UTM, Inactivating TM, saline or dry. Negatives, and COVID-19 / pre-COVID saliva is also available.