The dangers of using Cq to quantify nucleic acid in biological samples; a lesson from COVID-19 release_s7jbizkfjzc23mrrzlwfqm3g2e

by Daniel Evans, Simon Cowen, Martin Kammel, Denise M O'Sullivan, Graham Stewart, Hans-Peter Grunert, Jacob Moran-Gilad, Jasper Verwilt, Jiwon In, Jo Vandesompele, Kathryn Harris, Ki Ho Hong (+8 others)

Published in Clinical Chemistry by Oxford University Press (OUP).

2021  

Abstract

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> 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. </jats:sec> <jats:sec> <jats:title>Methods</jats:title> 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. </jats:sec> <jats:sec> <jats:title>Results</jats:title> 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 &amp;gt;1000 fold in copies/mL (99% CI). </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> 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. </jats:sec>
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