A masked female in a surgical gown holding a COVID-19 sample kit

As the world grapples with the ongoing COVID-19 pandemic, public health organizations are trying to help clinicians grasp how best to treat their patients with the most accurate and useful testing possible. One proposed recommendation, as set out by the World Health Organization, is to use cycle threshold (Ct) values set by quantitative polymerase chain reaction (qPCR) as a performance target for newly developed diagnostic technologies.

In this way, the WHO uses Ct values as a surrogate for the level of viral load. WHO has proposed a Ct value of 25 as the minimum level of virus that should be detected by a point-of-care diagnostic test. Ct also has been proposed by some to be used to tell who has a high viral load and might be at higher risk for severe disease, who might be infectious and likely to spread disease, and who can be released from quarantines.

AACC has come out against using Ct values for managing patients with COVID-19. In a statement published in July 2021, AACC emphasized that SARS-CoV-2 tests are not standardized in any one country, let alone around the world (www.myadlm.org/science-and-research/covid-19-resources). The same Ct value can therefore mean widely different things for different tests, potentially leading to inappropriate patient care.

“I can understand why people would want to compare Ct values, but there is a potential for harm when looking at an assay that is not validated to be quantitative. SARS-CoV-2 tests are validated to be qualitative,” said Deborah Payne, PhD, MT(ASCP), ABMM, DABCC, FADLM, a clinical consultant and a member of the AACC group that wrote the AACC Ct values statement.

No Two Nostrils Are Alike

Payne said that it’s natural to think Ct values can be used to determine severity of COVID-19. That’s because Ct values are routinely used to detect viral burden in infections like HIV and hepatitis C and B viruses. That number is valuable because collection of urine or blood is uniform, she said. However, “collection from a swab is not.”

To start, not every swab, nose, or even each nostril in the same person is the same. “There’s biological variance between the nasal cavities. You can have one nostril with detectable SARS-CoV-2 and the other nostril without, which is why the recommendation is to swab both sides of the nose,” she said.

Clinical laboratory professionals also don’t yet have the data to understand the optimal conditions for taking samples for a PCR test for a respiratory disease. Some urine assays are best taken in the morning, for example, and some blood samples drawn after a patient fasts. “We don’t really know enough about the respiratory system when it comes to this kind of testing and what the optimal time of day is to collect a sample,” Payne said. “We don’t know enough about respiratory virus shedding to know whether there’s a particular time it’s better to take a test, or whether there’s a difference between morning and evening, and how that will affect results.”

On top of not being able to adjust for differences in swabs and nostrils, and not having established optimal sampling conditions, the sheer number of assays given emergency use authorization (EUA) in the U.S. and the equivalent in other countries means that there are no standards across testing platforms and laboratories either. They aren’t calibrated against each other, so a Ct value of 25 on one test could mean high viral load in a patient in California, low viral load in a patient in New Jersey, and somewhere in between in a patient in Australia.

Without calibration, a Ct value is “virtually meaningless,” said Jim Francis Huggett, PhD, of the U.K. National Measurement Laboratory at LGC, and professor in analytical microbiology at the University of Surrey, who set out to find out just how meaningless it is.

Uncalibrated Tests Lead to Wildly Different Results

Huggett revealed the trouble with relying on Ct values by leading a team of researchers to study the differences in SARS-CoV-2 tests from labs around the world. One aim was to determine how WHO’s suggested Ct cutoff of 25 impacts coronavirus test performance, if at all.

In the resulting paper, which was published in AACC’s journal Clinical Chemistry, researchers analyzed the results of more than 6,000 patients who underwent PCR testing at clinical laboratories in the U.K., Belgium, and the Republic of Korea (Clin Chem 2021; doi: 10.1093/clinchem/hvab21). For this particular study, all PCR tests used were considered to have 100%
clinical sensitivity.

When Huggett’s team interpreted the test results using WHO’s cutoff, the tests’ sensitivity dropped, varying from approximately 16% to 90%, depending on the patient cohort. “There were three different groups at three different labs and we got three distinct differences,” Huggett said.

The researchers also conducted analysis of data from 732 additional laboratories and found that an individual Ct value can correspond to widely different viral loads depending on the lab. For example, a Ct value range of 25 to 30 should correspond to 106 copies of SARS-CoV-2/mL. But researchers also found that the Ct range can correspond to as many as 108 copies to as few as 103 copies.

“That’s a range of a hundred thousand fold difference,” he said. “It’s chaos.”

What To Do with Ct Values

AACC isn’t the only organization that has come out against the WHO Ct value recommendation. In a December letter to the editor in Clinical Infectious Diseases, the American College of Pathologists also cautioned against using Ct values for COVID-19, citing many of the same concerns as AACC, including variables in testing collection, no quantitative assay standardization, the wide range of Ct values among laboratories, and that some PCR tests use isothermal amplification methods and therefore don’t produce Ct values at all.

This doesn’t mean that Ct values have no use when it comes to COVID-19. “As long as you control the experiments, you can use a Ct value in your lab as a guide to compare one result to another,” Huggett said. “But as soon as you try to compare a number between laboratories, it means nothing because of the large amounts of variation,” he added. “If you’re going to use the Ct value, you need to work out what is your own scale, and be aware of how it may differ over time and cognizant of the limitations of using such an approach."

Right now, epidemiologists can also use Ct values of COVID tests if they’re “looking at a large number of points. Then if there’s an outlier, that outlier can be accounted for,” said Payne. It can give a bigger picture of the virus in one population without having an impact on individual patient care.

For example, researchers used mathematical modeling to show that capturing the Ct values of positive SARS-CoV-2 PCR results could give a real-time estimate of the growth rate of the virus in a community (Science 2021; doi: 10.1126/science.abh0635). “The Ct value is a measurement with magnitude, which provides information on underlying viral dynamics,” the authors wrote. “Although there are challenges to relying on single Ct values for individual-level decision-making, the aggregation of many such measurements from a population contains substantial information. These results demonstrate how one or a small number of random virologic surveys can be best used for epidemic monitoring.”

For Ct values to be useful for managing patients on a worldwide scale, though, there needs to be a standard calibration and sample standardization, much like what happened in order for laboratories to use Ct scores for HIV and other diseases.

Talking to Clinicians about Ct Values in COVID-19

The AACC statement does acknowledge that laboratories are “in a difficult position,” and that “there may also be pressure from their clinical colleagues to report or, at a minimum, have access to Ct values.” For laboratories that decide to report Ct values, AACC suggests including an interpretative comment with the test results:

The utilization of Ct values to guide patient management is discouraged. Correlation with viral load, viral burden, or infectivity has not been established for qualitative SARS-CoV-2 tests. Numerous factors such as biological variance, adequacy of sample, time of exposure, instrumentation, methodology, lack of certified reference material, and regulatory factors influence the Ct values detected in qualitative SARS-CoV-2 assays. Therefore, AACC discourages reporting or disclosing Ct values to guide patient management.

Meanwhile, as WHO considers making a specific Ct value minimum recommendation, the U.S. Centers for Disease Control and Prevention has not. In their COVID-19 FAQ for laboratories, updated on August 25, 2021, they stated that “Ct values should not be used to determine an individual’s viral load, how infectious an individual person
may be, or when an individual person can be released from isolation or quarantine.”

Despite such a blunt statement, a contrarian view by WHO could still hold sway, especially in countries without their own robust public health organizations, Huggett said. Established use of Ct values in other conditions also can muddy the waters. Huggett knows it can be difficult to explain to clinicians why the logical conclusion that the Ct might be used to guide management of COVID-19 patients is wrong—he himself is married to a medic, he joked. He also doesn’t want to knock Ct values completely as they do have some value now.

“To say it’s rubbish is incorrect. If it’s one laboratory and standardized, that could be fit for purpose,” he said. “What’s not fit for purpose is using the same number in different labs. It has to be very specific to that laboratory.”

Jen A. Miller is a freelance journalist who lives in Audubon, New Jersey. @byJenAMiller