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Zhejiang Da Xue Xue Bao Yi Xue Ban ; 50(6): 748-754, 2021 Dec 25.
Article in English | MEDLINE | ID: covidwho-1753705


To explore the application value of nanopore sequencing technique in the diagnosis and treatment of secondary infections in patients with severe coronavirus disease 2019 (COVID-19). A total of 77 clinical specimens from 3 patients with severe COVID-19 were collected. After heat inactivation, all samples were subjected to total nucleic acid extraction based on magnetic bead enrichment. The extracted DNA was used for DNA library construction, then nanopore real-time sequencing detection was performed. The sequencing data were subjected to Centrifuge software database species matching and R program differential analysis to obtain potential pathogen identification. Nanopore sequencing results were compared with respiratory pathogen qPCR panel screening and conventional microbiological testing results to verify the effectiveness of nanopore sequencing detection. Nanopore sequencing results showed that positive pathogen were obtained in 44 specimens (57.1%). The potential pathogens identified by nanopore sequencing included , , and , et al. , , were also detected in clinical microbiological culture-based detection; was detected in respiratory pathogen screening qPCR panel; was only detected by the nanopore sequencing technique. Comprehensive considerations with the clinical symptoms, the patient was treated with antibiotics against , and the infection was controlled. Nanopore sequencing may assist the diagnosis and treatment of severe COVID-19 patients through rapid identification of potential pathogens.

COVID-19 , Coinfection , Nanopore Sequencing , Nanopores , COVID-19/diagnosis , Humans , Sequence Analysis, DNA/methods
Biosci Trends ; 15(2): 93-99, 2021 May 11.
Article in English | MEDLINE | ID: covidwho-1154737


As the COVID-19 epidemic is still ongoing, a more rapid detection of SARS-CoV-2 infection such as viral antigen-detection needs to be evaluated for early diagnosis of COVID-19 disease. Here, we report the dynamic changes of SARS-CoV-2 viral antigens in nasopharyngeal swabs of COVID-19 patients and its association with the viral nucleic acid clearance and clinical outcomes. Eighty-five COVID-19 patients were enrolled for detection of SARS-CoV-2 viral antigens, including 57 anti-SARS-CoV-2 antibody negative cases and 28 antibody positive cases. The viral antigen could be detected in 52.63% (30/57) patients with SARS-CoV-2 antibody negative at the early stage of SARS-CoV-2 infection, especially in the first 5 days after disease onset (p = 0.0018) and disappeared in about 8 days after disease onset. Viral antigens were highly detectable in patients with low Ct value (less than 30) of SARS-CoV-2 nucleic acid RT-PCT assay, suggesting the expression of viral antigen was associated with high viral load. Furthermore, positive antigen detection indicated disease progression, nine cases with positive antigen (9/30, 30.0%), in contrast to two cases (2/27, 7.40%) (p = 0.0444) with negative antigen, which progressed into severe disease. Thus, the viral antigens were persistent in early stages of infection when virus was in highly replicating status, and viral antigen detection promises to rapidly screen positive patients in the early stage of SARS-CoV-2 infection.

Antigens, Viral/analysis , COVID-19 Testing/methods , COVID-19/diagnosis , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Antigens, Viral/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Nucleic Acid Testing , COVID-19 Serological Testing , COVID-19 Testing/trends , China/epidemiology , Disease Progression , Early Diagnosis , False Negative Reactions , Female , Humans , Male , Middle Aged , Nasopharynx/immunology , Nasopharynx/virology , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Time Factors , Viral Load , Young Adult
Front Immunol ; 12: 625881, 2021.
Article in English | MEDLINE | ID: covidwho-1133910


T cells play a critical role in coronavirus diseases. How they do so in COVID-19 may be revealed by analyzing the epigenetic chromatin accessibility of cis- and trans-regulatory elements and creating transcriptomic immune profiles. We performed single-cell assay for transposase-accessible chromatin (scATAC) and single-cell RNA (scRNA) sequencing (seq) on the peripheral blood mononuclear cells (PBMCs) of severely ill/critical patients (SCPs) infected with COVID-19, moderate patients (MPs), and healthy volunteer controls (HCs). About 76,570 and 107,862 single cells were used, respectively, for analyzing the characteristics of chromatin accessibility and transcriptomic immune profiles by the application of scATAC-seq (nine cases) and scRNA-seq (15 cases). The scATAC-seq detected 28,535 different peaks in the three groups; among these peaks, 41.6 and 10.7% were located in the promoter and enhancer regions, respectively. Compared to HCs, among the peak-located genes in the total T cells and its subsets, CD4+ T and CD8+ T cells, from SCPs and MPs were enriched with inflammatory pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway and tumor necrosis factor (TNF) signaling pathway. The motifs of TBX21 were less accessible in the CD4+ T cells of SCPs compared with those in MPs. Furthermore, the scRNA-seq showed that the proportion of T cells, especially the CD4+ T cells, was decreased in SCPs and MPs compared with those in HCs. Transcriptomic results revealed that histone-related genes, and inflammatory genes, such as NFKBIA, S100A9, and PIK3R1, were highly expressed in the total T cells, CD4+ T and CD8+ T cells, both in the cases of SCPs and MPs. In the CD4+ T cells, decreased T helper-1 (Th1) cells were observed in SCPs and MPs. In the CD8+T cells, activation markers, such as CD69 and HLA class II genes (HLA-DRA, HLA-DRB1, and HLA-DRB5), were significantly upregulated in SCPs. An integrated analysis of the data from scATAC-seq and scRNA-seq showed some consistency between the approaches. Cumulatively, we have generated a landscape of chromatin epigenetic status and transcriptomic immune profiles of T cells in patients with COVID-19. This has provided a deeper dissection of the characteristics of the T cells involved at a higher resolution than from previously obtained data merely by the scRNA-seq analysis. Our data led us to suggest that the T-cell inflammatory states accompanied with defective functions in the CD4+ T cells of SCPs may be the key factors for determining the pathogenesis of and recovery from COVID-19.

CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/physiology , COVID-19/immunology , Chromatin/metabolism , SARS-CoV-2/physiology , COVID-19/genetics , Calgranulin B/genetics , Chromatin/genetics , Class Ia Phosphatidylinositol 3-Kinase/genetics , Epigenome/immunology , Gene Expression Profiling , Humans , Immunity, Cellular/genetics , Inflammation/genetics , Lymphocyte Activation , NF-KappaB Inhibitor alpha/genetics , Sequence Analysis, RNA , Single-Cell Analysis , Transposases/metabolism , Up-Regulation