Why Ct value is not important for COVID19 treatment?

Ct value is not important for treatment of COVID19, but why is it so?

Clinicians should avoid treating the patients based on Ct value alone because till today no definitive research has shown that Ct value is affected by SARS-CoV-2 viral load.

What is Ct value?

Ct value is short form Threshold Cycle. In a RT- PCR test, Ct value demonstrates the minimum threshold value that is needed to call a fluorescence curve as “actual amplification curve”. It is located in between the region where the curve changes from lag phase to log phase. A horizontal baseline is set at a height to rule out all background noise. The point on sigmoidal amplification curve where the baseline intersect, is set the Ct value.

Ct value-RT PCR curve

As the Rt-PCR reaction proceeds, the original amount of DNA is increased by a factor of 2n. Here ‘n’ is the number of Cycle. For example, 1 starting copies will become 2 after 1 cycles, 4 after 2 cycles, 8 after 3 cycles etc.  normally there are 40-45 cycles so at the end there are millions of copies of DNA.

PCR Amplification

After each cycle, there is a fluorescence released. This fluorescence is detected by real time PCR instrument on 2D chart which has Y axis has fluorescence intensity and X axis as no. of cycles. After each cycle, the fluorescence is multiplied. Initially the instrument is not able to detect fluorescence and it is known as Lag phase.  After certain number of cycles, the instrument can detect the fluorescence and the curve enter Log phase. Towards the last phase, the reagents start getting limited and no new fluorescence is detected even if DNA is multiplying. This is known as plateau phase or stationary phase. Thus the curve becomes a S shaped or sigmoidal shaped amplification curve.

Why is Ct value important?

Normally, the Ct value is direct indication of virus copies present in sample. If a sample has more virus RNA, the Ct value will be closer to zero. As the virus load is reduced, the Ct value shifts towards 40.

This is because of the fact that more starting copies of DNA/RNA will achieve the Threshold Cycle(Ct) faster. It will have smaller lag phase. Less copies in sample will have long lag phase and its Ct will be higher.

This principal has long been used to detect Viral loads of various pathogens like HCV, HBV etc. Additionally, it can be used to asses non quantitative viral load in patients by analyzing Ct value pattern over time.

2d animation tutorial

Now the question arises?

If Ct value is indeed such an important marker for viral load, why is not useful in COVID19?

There are many reasons to this. Ct value is a direct indication of viral load in extracted sample that is used for analysis, that is true. But the viral load in extracted sample is dependent on many factors. A slight change in anyone can lead to a difference in Ct values.

Let us understand few important reasons.

 

  1. Sample collection:- The biggest and most important factor in affecting the Ct value is sample quality. In this pandemic, it has been observed that too many untrained staff are collecting nasopharyngeal swabs. Only skilled healthcare worker would know how to properly collect a NP Swab. A swab collected in haste or without proper procedure will result in low or no copies of detectable virus. For a patient, two different swabs collected in same day by different individuals at different locations may give different Ct values. For accurate measure of correct viral load representation, consistency is important. Same healthcare worker, collecting swabs with same carefulness is important when other parameters are constant.
  2. RNA extraction:- in this pandemic, many small companies came into existence all providing Viral RNA extraction kits. These companies which earlier did not have any experience in molecular extraction kits, are now selling “high quality Viral extraction kits”. This is a point concern as there is insecurity about consistent performance of these kits which may not go through exhaustive quality checks. To meet demand, QC is compromised. Therefore, a lab which use high quality automatic extraction system may have different RNA copies extracted as compared to a lab which changes manual extraction kits regularly. Both may give different Ct values for same patient.
  3. Laboratory staff:- RT PCR testing is extremely sensitive to handling. Poor lab practices and inexperienced staff lead to the assay not working optimally. This results in changes in assay efficiency which directly affects the Ct value. Also, incorrect manual baseline setting will change Ct values as every technician may set baseline differently.
  4. Sample collection Vial:- Another factor that slightly affects the Ct value indirectly is the Sample collection vial which has poor quality Viral Transport Media.

All these factors work in tandem regularly for each patient. A moderately positive patient may be low positive or high positive at other labs.

Another aspect is that many non-symptomatic individuals may have low Ct (towards high viral load) while fully symptomatic individuals may have high Ct( towards low viral load)

Clinicians should understand that Ct value in case of Covid19 pandemic is not same as Ct they have been using for other diseases. It is still an important factor to monitor many other diseases but only time will tell if Ct value corresponds to viral load in COVID19 patients.

Reference: ICMR CT value Guidelines

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