The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19

Abstract

Background SARS-CoV-2 RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cut-offs altered the clinical sensitivity of COVID-19 testing. Methods Quantitative SARS-CoV-2 RNA distributions (Cq and copies/mL) from more than 6000 patients from three clinical laboratories in UK, Belgium and the Republic of Korea were analyzed. Impact of Cq cut-offs on clinical sensitivity was assessed. The June/July 2020 INSTAND EQA scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. Results When the WHO suggested Cq cut-off of 25 was applied, the clinical sensitivity dropped to as little as about 16%. Clinical sensitivity also dropped to as little as about 27% when a simulated LOD of 106 copies/mL was applied. The inter-laboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). Conclusion While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (Ct or Cp) values not be used to set clinical cut-offs, or diagnostic performance targets, due to poor inter-laboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number (R). Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.

A roadmap to better COVID-19 testing from the Coronavirus Standards Working Group

“…Testing can also involve a quantitative measurement to determine the abundance of an analyte in a sample. However, viral load is rarely considered during SARS-CoV-2 testing, and quantitative measurements may be reduced to a simple presence or absence result when viral abundance exceeds a threshold 3 .…”

“…Whilst molecular tests have the potential to measure viral abundance, they must be rst calibrated to reference materials to realize this quantitative potential. For example, although the RT-qPCR cycling threshold (Ct, also known as Cq or Cp) score can be used to estimate viral abundance, this abundance can vary up ~1000-fold for a given Ct value between instruments and laboratories 3 . Accordingly, calibration to reference materials is needed for quantitative comparisons of RT-qPCR results 7 , and viral load cannot be considered in clinical strati cation and the assessment of analytical sensitivity due to the current lack of harmonized reporting of RT-qPCR results 26,27 .…”

Add mass spectrometry to the pandemic toolbox

“…Additionally, while RT-PCR tests can assess whether someone is carrying the virus, they cannot reveal how many viral particles someone is releasing into the environment, how infectious a person is, or how a patient will fare. The pandemic ‘readiness toolbox’ must therefore be extended to include methods that can detect other types of biomolecules beyond mRNA, such as viral proteins and peptides ( Evans et al, 2021 ).…”

“…This multiplies the sensitivity many-fold and reduces measurement time, interferences and instrument contamination while also increasing robustness (as also proposed in Van Puyvelde et al, 2021 ). Additionally, without the noise, mass spectrometry can determine the amount of a given target protein extremely accurately, something that is not possible using RT-PCR ( Evans et al, 2021 ).…”

“…Currently, precisely assessing the amount of viral proteins may not help to decide whether a person should be quarantined. However, this information may help to decide when an individual should be released into the community as viral protein might more closely track infectivity in SARS-CoV-2 than viral mRNA, which remains detectable after a patient ceases to be infectious ( Evans et al, 2021 ). Irrespectively, starting to measure viral protein loads in thousands of COVID-19 patients using mass spectrometry could help the technique to mature into a reliable approach to add to the pandemic readiness toolbox; in this effort, the work by Hober et al will help revolutionize the way mass spectrometry is approached in the clinic.…”