Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking
Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking
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DOI：
10.1093/clinchem/hvab158
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发表时间：
2021-10-05
影响因子：
9.3
通讯作者：
Wang, Ping
中科院分区：
文献类型：
--
作者：
Chen, Hui;Li, Zhao;Wang, Ping
背景：高灵敏度的严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）抗原检测是减少假阴性结果的理想方法。可用于量化和跟踪不同人群呼吸道样本中SARS-CoV-2抗原负担的数据有限。方法：采用智能手机读出的微鼓泡SARS-CoV-2抗原检测方法（MSAA），在鼻咽拭子中检测出0.5 pg/mL （10.6 fmol/L）核衣壳抗原或4000拷贝/mL灭活SARS-CoV-2病毒。我们开发了一种基于计算机视觉和机器学习的自动微泡图像分类器，用于准确识别阳性和阴性，量化和跟踪重症监护病房新冠肺炎住院患者和免疫功能低下患者的抗原动态。结果：与定性逆转录聚合酶链反应方法相比，MSAA在372份剩余临床NP拭子的临床验证研究中显示97%的阳性百分比一致性（95% CI 92%-99%）和97%的阴性百分比一致性（95% CI 94%-100%）。在免疫正常的个体中，尽管核酸持续呈阳性，但拭子抗原阳性率随着症状出现后天数的增加而下降。在免疫功能低下的血液恶性肿瘤患者中检测抗原的时间更长，时间多变。总微泡体积是抗原负荷的定量标志，与周期阈值和症状后天数呈负相关。在长时间高抗原负荷患者中检测到病毒序列变异。结论：MSAA能够灵敏和特异性地检测急性感染，量化和跟踪抗原负担，可以作为纵向研究中的筛选方法，以确定可能正在经历活跃的复制轮的患者，并保证密切的病毒序列监测。
BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations.METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone read-out, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients.RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptomonset. Viral sequence variations were detected in patients with long duration of high antigen burden.CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.