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 106 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.

中文翻译：

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使用 Cq 量化生物样品中核酸的危险：COVID-19 的教训

背景 已经提出通过逆转录定量 PCR (RT-qPCR) 测量的严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) RNA 量用于对临床风险进行分层或确定分析性能目标。我们调查了可重复性以及设置诊断临界值如何改变 2019 年冠状病毒病 (COVID-19) 检测的临床敏感性。方法分析了来自英国、比利时和大韩民国 3 个临床实验室的 6000 多名患者的 SARS-CoV-2 RNA 定量分布[定量周期 (Cq) 和拷贝/mL]。评估了 Cq 截止值对临床敏感性的影响。2020 年 6 月/7 月 INSTAND 外部质量评估计划 SARS-CoV-2 材料用于估计实验室报告的拷贝数/mL，并估计给定 Cq 的拷贝数/mL 的变化。结果当应用 WHO 建议的 Cq 截止值 25 时，临床敏感性下降到约 16%。当应用 106 拷贝/mL 的模拟检测限时，临床敏感性也下降到约 27%。给定 Cq 值的实验室间变异大于 1000 倍拷贝/mL (99% CI)。结论 虽然 RT-qPCR 有助于应对 COVID-19，但由于实验室间重现性差，我们建议不要使用 Cq（循环阈值或交叉点）值来设定临床临界值或诊断性能目标；校准的基于拷贝的单位（在病毒学的其他地方使用）提供了更多可重复的替代方案。我们还报告了一种现象，即诊断性能可能会相对于有效再生数发生变化。我们的研究结果表明，在评估或部署诊断测试时，患者群体之间的时间差异是一个重要的考虑因素。这与不断演变的流行病的紧急情况尤其相关。