Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, New Orleans, Louisiana, USA.
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China.
J Clin Invest. 2021 Apr 1;131(7). doi: 10.1172/JCI146031.
BACKGROUNDCirculating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA may represent a more reliable indicator of infection than nasal RNA, but quantitative reverse transcription PCR (RT-qPCR) lacks diagnostic sensitivity for blood samples.METHODSA CRISPR-augmented RT-PCR assay that sensitively detects SARS-CoV-2 RNA was employed to analyze viral RNA kinetics in longitudinal plasma samples from nonhuman primates (NHPs) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for coronavirus disease 2019 (COVID-19) diagnosis in adults cases confirmed by nasal/nasopharyngeal swab RT-PCR results; and to identify suspected COVID-19 cases in pediatric and at-risk adult populations with negative nasal swab RT-qPCR results. All blood samples were analyzed by RT-qPCR to allow direct comparisons.RESULTSCRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days after infection, and these increases preceded and correlated with rectal swab viral RNA increases. In a patient cohort (n = 159), this blood-based assay demonstrated 91.2% diagnostic sensitivity and 99.2% diagnostic specificity versus a comparator RT-qPCR nasal/nasopharyngeal test, whereas RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified patients with COVID-19 using one or more negative nasal swab RT-qPCR results.CONCLUSIONResults of this study indicate that sensitive detection of SARS-CoV-2 RNA in blood by CRISPR-augmented RT-PCR permits accurate COVID-19 diagnosis, and can detect COVID-19 cases with transient or negative nasal swab RT-qPCR results, suggesting that this approach could improve COVID-19 diagnosis and the evaluation of SARS-CoV-2 infection clearance, and predict the severity of infection.TRIAL REGISTRATIONClinicalTrials.gov. NCT04358211.FUNDINGDepartment of Defense, National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the National Center for Research Resources.
背景：循环严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) RNA 可能比鼻腔 RNA 更能可靠地反映感染情况，但定量逆转录聚合酶链反应 (RT-qPCR) 对血液样本的诊断灵敏度不足。
方法：采用一种 CRISPR 增强的 RT-PCR 检测方法，对暴露于病毒后非人类灵长类动物 (NHPs) 的纵向血浆样本中的病毒 RNA 动力学进行分析；评估血液 SARS-CoV-2 RNA 检测对经鼻/鼻咽拭子 RT-PCR 结果确诊的成人 2019 年冠状病毒病 (COVID-19) 诊断的实用性；并识别儿科和高危成人人群中经鼻拭子 RT-qPCR 结果为阴性但疑似 COVID-19 的病例。所有血液样本均通过 RT-qPCR 进行分析，以便直接比较。
结果：CRISPR 增强的 RT-PCR 从感染后 1 至 28 天，始终能在实验感染的 NHPs 血浆中检测到 SARS-CoV-2 RNA，这些增加先于并与直肠拭子病毒 RNA 增加相关。在患者队列中（n=159），与比较 RT-qPCR 鼻/鼻咽拭子检测相比，该血液检测法的诊断灵敏度为 91.2%，特异性为 99.2%，而 RT-qPCR 对相同血液样本的诊断灵敏度为 44.1%，特异性为 100%。该 CRISPR 增强的 RT-PCR 检测还能准确识别具有一个或多个阴性鼻拭子 RT-qPCR 结果的 COVID-19 患者。
结论：本研究结果表明，CRISPR 增强的 RT-PCR 对血液中 SARS-CoV-2 RNA 的灵敏检测可准确诊断 COVID-19，且能检测到具有一过性或阴性鼻拭子 RT-qPCR 结果的 COVID-19 病例，这提示该方法可能会改善 COVID-19 诊断以及对 SARS-CoV-2 感染清除的评估，并预测感染的严重程度。
试验注册：ClinicalTrials.gov，NCT04358211。
资助：美国国防部、美国国立过敏和传染病研究所、美国国立儿童健康与人类发育研究所以及美国国家研究资源中心。
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