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Published November 22, 2021 | Version v1
Journal article Open

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

  • 1. National Measurement Laboratory, LGC, Teddington, Middlesex, UK; Department of Microbial Sciences, School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey, Guildford, UK;
  • 2. National Measurement Laboratory, LGC, Teddington, Middlesex, UK
  • 3. Gesellschaft zur Foerderung der Qualitaetssicherung in Medizinischen Laboratorien e. V. (INSTAND), Dusseldorf, Germany; IQVD GmbH, Institut fuer Qualitaetssicherung in der Virusdiagnostik, Berlin, Germany
  • 4. Department of Microbial Sciences, School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey, Guildford, UK
  • 5. GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, Germany
  • 6. Health Policy and Management, School of Public Health, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel
  • 7. Department of Biomolecular Medicine, Ghent University, Ghent, Belgium
  • 8. Seoul Medical Center, Seoul, Republic of Korea; iBiogazelle, Zwijnaarde, Belgium;
  • 9. Department of Virology, NHS East and South East London Pathology Partnership, Royal London Hospital, Barts Health NHS Trust
  • 10. Department of Laboratory Medicine, Yonsei University College of Medicine, Severance Hospital, Seoul, Republic of Korea
  • 11. Department of Microbiology, Virology and Infection Prevention and Control, Level 4 Camelia Botnar Laboratories, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK;
  • 12. Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea
  • 13. Gesellschaft zur Foerderung der Qualitaetssicherung in Medizinischen Laboratorien e. V. (INSTAND), Dusseldorf, Germany; IQVD GmbH, Institut fuer Qualitaetssicherung in der Virusdiagnostik, Berlin, Germany; GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, Germany;

Description

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.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.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 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI).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.

Notes

This project (18HLT03 SEPTIMET) has received funding from the EMPIR programme co-financed by the Participating States and from the European Union's Horizon 2020 research and innovation programme

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18HLT03 Septimet Supplementary Funding Acknowledgement CLIN CHEM.pdf

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