Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China in December 2019. The virus rapidly spread globally, resulting in a public-health crisis including more than 3.1 million cases and 224,000 deaths as of May 1, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (FFPE) cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross detection of the respective SARS-CoV-2 proteins by immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.
Jun Liu, April M. Babka, Brian J. Kearney, Sheli R. Radoshitzky, Jens H. Kuhn, Xiankun Zeng
BACKGROUND. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a novel viral pneumonia (COVID-19), which is rapidly spreading in the world. The positive result of nucleic acid test is a golden criterion to confirm SARS-CoV-2 infection, but the detection features remain unclear. METHODS. We performed a retrospective analysis in 5,630 high-risk individuals receiving SARS-CoV-2 nucleic acid tests in Wuhan, China, and investigated their characteristics and diagnosis rates. RESULTS. The overall diagnosis rate was 34.7% (1,952/5,630). Male (P = 0.025) and older age (P = 2.525 × 10–39) were two significant risk factors of SARS-CoV-2 infection. People were generally susceptible, and most cases concentrated in people of 30- to 69-years-old. Besides, we investigated the association between diagnosis rate and the number of testing in 501 subjects. Results revealed a 1.27-fold improvement (35.5%/27.9%) of diagnosis rate from testing once to twice (P = 5.847 × 10–9), and a 1.43-fold improvement (39.9%/27.9%) from testing once to three times (P = 7.797 × 10–14). More than three testing times was not helpful for further improvement. However, this improvement was not observed in subjects with pneumonia (P = 0.097). CONCLUSION. All populations are susceptible to SARS-Cov-2 infection, and male and older age are two significant risk factors. Increasing the number of testing could significantly improve diagnosis rates, except for subjects with pneumonia. It is recommended to test twice in those high-risk individuals whose results are negative for the first time, and to perform three testing times is better if available.
Na Shen, Yaowu Zhu, Xiong Wang, Jing Peng, Weiyong Liu, Feng Wang, Yanjun Lu, Liming Cheng, Ziyong Sun
In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have potential to propagate inflammation and microvascular thrombosis — including in the lungs of patients with acute respiratory distress syndrome. While elevated levels of blood neutrophils predict worse outcomes in COVID-19, the role of NETs has not been investigated. We now report that sera from patients with COVID-19 (n = 50 patients, n = 84 samples) have elevated levels of cell-free DNA, myeloperoxidase(MPO)-DNA, and citrullinated histone H3 (Cit-H3); the latter two are highly specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute phase reactants including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. In summary, these data reveal high levels of NETs in many patients with COVID-19, where they may contribute to cytokine release and respiratory failure. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts, and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.
Yu Zuo, Srilakshmi Yalavarthi, Hui Shi, Kelsey Gockman, Melanie Zuo, Jacqueline A. Madison, Christopher N. Blair, Andrew Weber, Betsy J. Barnes, Mikala Egeblad, Robert J. Woods, Yogendra Kanthi, Jason S. Knight
Background: The Coronavirus Disease-2019 (COVID-19), infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a severe outbreak in China. The host immunity of COVID-19 patients is unknown. Methods: The routine laboratory tests and host immunity in COVID-19 patients with different severity of illness were compared after patient admission. Results: A total of 65 SARS-CoV-2-positive patients were classified as mild (n=30), severe (n=20), and extremely severe (n=15) illness. Many routine laboratory tests such as ferritin, lactate dehydrogenase and D-dimer were increased in severe and extremely severe patients. The absolute numbers of CD4+ T cells, CD8+ T cells and B cells were all gradually decreased with increased severity of illness. The activation markers such as HLA-DR and CD45RO expressed on CD4+ and CD8+ T cells were increased in severe and extremely severe patients compared with mild patients. The co-stimulatory molecule CD28 had opposite results. The percentage of natural regulatory T cells was decreased in extremely severe patients. The percentage of IFN-γ producing CD8+ T cells was increased in both severe and extremely severe patients compared with mild patients. The percentage of IFN-γ producing CD4+ T cells was increased in extremely severe patients. The IL-2R, IL-6, and IL-10 were all increased in extremely severe patients. The activation of DC and B cells was decreased in extremely severe patients. Conclusions: The number and function of T cells are inconsistent in COVID-19 patients. The hyperfunction of CD4+ and CD8+ T cells is associated with the pathogenesis of extremely severe SARS-CoV-2 infection.
Feng Wang, Hongyan Hou, Ying Luo, Guoxing Tang, Shiji Wu, Min Huang, Weiyong Liu, Yaowu Zhu, Qun Lin, Liyan Mao, Minghao Fang, Huilan Zhang, Ziyong Sun
Complications of COVID-19 have been particularly severe among older adults, who are the focus of this article. Public policy goals should prioritize pandemic preparedness in nursing homes, as well as civic and local government-based support programs for community-dwelling older adults, to ensure that risk of infection is mitigated while promoting wellness during a period of stress and uncertainty.
John P. Mills, Keith S. Kaye, Lona Mody
Lessons from history underline the importance of having direct lines of communication to and from public health officials, who must remain free from policital bias in times of crisis.
Kathleen L. Collins, Howard Markel, Andrew P. Lieberman
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