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

Daniel Evans, Simon Cowen, Martin Kammel, Denise M O’Sullivan, Graham Stewart, Hans-Peter Grunert, Jacob Moran-Gilad, Jasper Verwilt, Jiwon In, Jo Vandesompele (UGent) , et al.
(2022) CLINICAL CHEMISTRY. 68(1). p.153-162
Author
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Abstract
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (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 cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing. METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment 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 cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 10(6) copies/mL was applied. The interlaboratory 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 (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory 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. 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.
Keywords
Biochemistry (medical), Clinical Biochemistry

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MLA
Evans, Daniel, et al. “The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples : A Lesson from COVID-19.” CLINICAL CHEMISTRY, vol. 68, no. 1, 2022, pp. 153–62, doi:10.1093/clinchem/hvab219.
APA
Evans, D., Cowen, S., Kammel, M., O’Sullivan, D. M., Stewart, G., Grunert, H.-P., … Huggett, J. F. (2022). The dangers of using Cq to quantify nucleic acid in biological samples : a lesson from COVID-19. CLINICAL CHEMISTRY, 68(1), 153–162. https://doi.org/10.1093/clinchem/hvab219
Chicago author-date
Evans, Daniel, Simon Cowen, Martin Kammel, Denise M O’Sullivan, Graham Stewart, Hans-Peter Grunert, Jacob Moran-Gilad, et al. 2022. “The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples : A Lesson from COVID-19.” CLINICAL CHEMISTRY 68 (1): 153–62. https://doi.org/10.1093/clinchem/hvab219.
Chicago author-date (all authors)
Evans, Daniel, 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, Nathaniel Storey, Suzie Hingley-Wilson, Ulf Dühring, Young-Kyung Bae, Carole A Foy, Julian Braybrook, Heinz Zeichhardt, and Jim F Huggett. 2022. “The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples : A Lesson from COVID-19.” CLINICAL CHEMISTRY 68 (1): 153–162. doi:10.1093/clinchem/hvab219.
Vancouver
1.
Evans D, Cowen S, Kammel M, O’Sullivan DM, Stewart G, Grunert H-P, et al. The dangers of using Cq to quantify nucleic acid in biological samples : a lesson from COVID-19. CLINICAL CHEMISTRY. 2022;68(1):153–62.
IEEE
[1]
D. Evans et al., “The dangers of using Cq to quantify nucleic acid in biological samples : a lesson from COVID-19,” CLINICAL CHEMISTRY, vol. 68, no. 1, pp. 153–162, 2022.
@article{8763142,
  abstract     = {{BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (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 cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing.

METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment 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 cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 10(6) copies/mL was applied. The interlaboratory 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 (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory 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. 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.}},
  author       = {{Evans, Daniel and Cowen, Simon and Kammel, Martin and O’Sullivan, Denise M and Stewart, Graham and Grunert, Hans-Peter and Moran-Gilad, Jacob and Verwilt, Jasper and In, Jiwon and Vandesompele, Jo and Harris, Kathryn and Hong, Ki Ho and Storey, Nathaniel and Hingley-Wilson, Suzie and Dühring, Ulf and Bae, Young-Kyung and Foy, Carole A and Braybrook, Julian and Zeichhardt, Heinz and Huggett, Jim F}},
  issn         = {{0009-9147}},
  journal      = {{CLINICAL CHEMISTRY}},
  keywords     = {{Biochemistry (medical),Clinical Biochemistry}},
  language     = {{eng}},
  number       = {{1}},
  pages        = {{153--162}},
  title        = {{The dangers of using Cq to quantify nucleic acid in biological samples : a lesson from COVID-19}},
  url          = {{http://doi.org/10.1093/clinchem/hvab219}},
  volume       = {{68}},
  year         = {{2022}},
}
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