What’s a PCR test cycle threshold and why it matters

8 April 2021
What was claimed

The World Health Organisation confirmed that the PCR test is flawed and all tests with a high cycle threshold (Ct) are invalid.

Our verdict

False. It reiterated guidance that tests with a high Ct may need to be retested. There are lots of different reasons for high Ct value results. Interpreting them requires clinical context.

A post on Instagram claims that the World Health Organisation (WHO) has pronounced PCR tests for Covid-19 as flawed, and all positive tests in the past 14 months ‘invalid’ if a cycle threshold of 35 or above has been used.   

A PCR test is performed by repeatedly replicating target viral material in the sample to the point that it becomes detectable. The number of cycles before the virus is detectable is known as the cycle threshold (Ct). We have written previously about how PCR tests work, and how effective they are. 

A positive test with a high Ct value may indicate a test from someone who had a very small amount of detectable viral RNA on their initial swab, and may not be infectious or have ongoing active infection. 

However, there are other clinical scenarios that can result in a positive test with high Ct value in someone who may still be infectious or who may soon become infectious.  The claims in the Instagram post misinterpret clarifications released by the WHO.  

What are cycle thresholds?

During a PCR test for SARS-CoV-2  (the virus that causes Covid-19), viral genetic material on the swab sample is purified, stabilised and concentrated.

If the genetic material of interest is present in the sample, it is then copied again and again by heating and cooling the material in the presence of various substances. Each iteration of this is called a thermal cycle. 

As genetic material is amplified (with real time PCR) fluorescence is produced; how this happens exactly varies by PCR method, but basically involves those substances added to the test releasing fluorescent particles or becoming more fluorescent. 

Eventually the fluorescence is strong enough to be detected. The number of thermal cycles required to reach this point is known as the cycle threshold. 

The fewer cycles required before that fluorescence is observed, the greater the concentration of viral genetic material in the original sample, roughly speaking. Conversely, the more cycles that are required, the smaller the concentration of viral material on the original sample. 

Why the controversy?

During the course of the pandemic, it has been recognised that positive tests with a high cycle threshold may be detecting very small amounts of viral genetic material or “non viable fragments” rather than active virus. 

There is concern that these positive tests may not represent people with an active infection, or who are most infectious. 

Recent posts have suggested that an announcement from the WHO in January 2021 somehow represents a change in approach, or an acceptance that there are flaws in PCR testing, to do with cycle thresholds that were not previously recognised. 

The announcement from the WHO did not retract previous guidance which promotes the use of PCR testing, or suggest that PCR results are ‘invalid’. 

Instead, it re-iterated the advice given in its September guidance and said that: 

“ ...careful interpretation of weak positive results is needed (1). The cycle threshold (Ct) needed to detect virus is inversely proportional to the patient’s viral load. Where test results do not correspond with the clinical presentation, a new specimen should be taken and retested using the same or different [nucleic acid testing] technology.” 

That aside, a high Ct positive test doesn’t always mean that the person the swab was taken from isn’t infectious, or about to become infectious.  

The exact relationship (between Ct value and infectivity) is still being researched, and interpreting these results depends on the clinical context.

Public Health England says although a positive test at the higher cycle thresholds does normally represent someone with a lower concentration of virus at the time that the test was done, there are other factors to consider, all of which depend on the “ clinical history and context”. 

It is important to remember that these tests are a snapshot in time. Therefore, a positive result with high Ct value could represent somebody with lower amounts of virus, and lower infectivity (for example, somebody who has recently recovered from infection). It could also indicate somebody who may have been tested shortly before infection, when viral levels are low, but which may develop “[...] into symptomatic infection with high viral load and infectivity.” 

There are other factors which could also give a high Ct positive result even for someone who is infectious and has a high viral load, for example, if test samples are poorly collected or stored, or people who develop severe lower respiratory tract Covid-19 infection (but are swabbed from an upper respiratory tract site). 

And, while the maximum number of thermal cycles a coronavirus PCR test will do is typically around 35-40, many positive samples will be detectable at much lower levels. 

So the idea that positive tests with high Ct values are “invalid” and should be dismissed is overly simplistic.  

This article is part of our work fact checking potentially false pictures, videos and stories on Facebook. You can read more about this—and find out how to report Facebook content—here. For the purposes of that scheme, we’ve rated this claim as false because the WHO didn’t say PCR tests are flawed and didn’t tacitly admit that all PCR tests conducted at a high cycle threshold are invalid. Sometimes a high CT value may indicate a non-infectious case but, there are many scenarios where they may represent an infectious case.

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