COVID-19 Emergency Response, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, USA.
Clin Infect Dis. 2022 Nov 14;75(10):1698-1705. doi: 10.1093/cid/ciac282.
The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of US Food and Drug Administration emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, that is, the presence of replicable virus. As the number of tests conducted increased, persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA positivity by reverse-transcription polymerase chain reaction (RT-PCR) in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike, nucleocapsid, membrane, envelope, and ORF8. Viral RNA abundances calculated from these assays were compared with antigen presence, self-reported symptoms, and culture outcome (virus isolation) using samples from a 14-day longitudinal household transmission study. By characterizing the clinical and molecular dynamics of infection, we show that sgRNA detection has higher predictive value for culture outcome compared to detection of gRNA alone. Our findings suggest that sgRNA presence correlates with active infection and may help identify individuals shedding culturable virus.
新型冠状病毒大流行引发了对检测病毒核酸、病毒蛋白和相应抗体的分析物的前所未有的需求。获得美国食品和药物管理局紧急使用授权的 320 种分子诊断方法主要侧重于病毒检测；然而，目前没有批准的测试可以用于推断传染性，即可复制病毒的存在。随着检测数量的增加，一些个体通过逆转录聚合酶链反应（RT-PCR）持续检测到严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）RNA 呈阳性，这引发了对检疫指南的关注。为此，我们试图设计一种分析物检测方法，减少那些不释放可培养病毒的个体的阳性检测结果的频率。我们描述了用于检测 SARS-CoV-2 的基因组 RNA（gRNA）和亚基因组 RNA（sgRNA）物种的多重定量 RT-PCR 分析物，包括刺突、核衣壳、膜、包膜和 ORF8。使用来自为期 14 天的家庭传播纵向研究的样本，比较了这些分析物计算的病毒 RNA 丰度与抗原存在、自我报告的症状和培养结果（病毒分离）。通过对感染的临床和分子动力学进行特征分析，我们发现 sgRNA 检测比单独检测 gRNA 对培养结果具有更高的预测价值。我们的研究结果表明，sgRNA 的存在与活跃感染相关，并且可能有助于识别可释放可培养病毒的个体。
