Department of Internal Medicine, College of Medicine, Chosun Universitygrid.254187.dgrid.464555.3, Gwangju, Republic of Korea.
Department of Premedical Science, College of Medicine, Chosun Universitygrid.254187.dgrid.464555.3, Gwangju, Republic of Korea.
Microbiol Spectr. 2022 Feb 23;10(1):e0059121. doi: 10.1128/spectrum.00591-21. Epub 2022 Feb 16.
Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (C) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (0.0001) or CDC (N1: 0.0012, N2: 0.0013, N3: 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.
新型冠状病毒病（COVID-19）是一种由严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）引起的轻度至重度呼吸道疾病。疾病预防控制中心（CDC）或世界卫生组织（WHO）推荐的实时聚合酶链反应（RT-qPCR）引物在临床实践中的诊断准确性尚未得到证实。我们使用针对核衣壳蛋白的内部设计的引物集（iNP）进行了一项针对 RT-qPCR 准确性的前瞻性研究，以及各种推荐和商业引物。准确性通过培养或血清转换进行评估。我们共招募了 12 名确诊的 COVID-19 患者，共采集了 590 份临床样本。当将循环阈值（C）的截止值设定为 35 时，使用 WHO RdRp 引物和 CDC N1、N2 和 N3 引物的 RT-qPCR 在痰中的灵敏度为 42.1%至 63.2%，特异性为 90.5%至 100%，在鼻咽样本中的灵敏度为 65.2%至 69.6%，特异性为 65.2%至 69.6%。iNP RT-qPCR 在痰和鼻咽样本中的灵敏度和特异性分别为 94.8%/100%和 69.6%/100%。痰检测的灵敏度最高，其次是鼻咽检测（0.0193）；自我采集的唾液样本比口咽样本具有更好的特征（0.0032）。我们的结果表明，iNP RT-qPCR 比使用 WHO（0.0001）或 CDC（N1：0.0012，N2：0.0013，N3：0.0012）引物的 RT-PCR 具有更好的灵敏度和特异性。痰 RT-qPCR 分析的灵敏度最高，其次是鼻咽、唾液和口咽检测。我们的研究表明，需要对用于检测 SARS-CoV-2 的 RT-qPCR WHO 和 CDC 引物进行相当大的改进。许多研究活动已经解决了 SARS-CoV2 病毒检测的各种引物的绝大多数临床和诊断特异性和敏感性。尽管在解决大流行方面取得了令人瞩目的进展，但在临床实践中，仍需要不断积极改进用于诊断的引物。我们的研究在比较不同标本和不同引物的特异性和灵敏度方面，其规模明显超过了先前发表的研究，并且是迄今为止对当前使用的引物集进行持续监测最全面的研究。因此，我们的结果表明，痰样本的灵敏度最高，其次是鼻咽、唾液和口咽样本。CDC 建议使用口咽标本，这导致了 CDC 和 IDSA 制定的指南之间存在一定差异。我们证明了口咽样本对 SARS-CoV-2 的检测灵敏度最低。
