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1.
J Clin Microbiol ; 60(4): e0228321, 2022 04 20.
Article in English | MEDLINE | ID: covidwho-1759279

ABSTRACT

Tools to detect SARS-CoV-2 variants of concern and track the ongoing evolution of the virus are necessary to support public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. To this aim, an allele-specific probe PCR (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. The comparative advantage for ASP-PCR over NGS was most pronounced in samples with cycle threshold (CT) values between 26 and 30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. ASP-PCR is well suited to augment but not replace NGS. The method can differentiate SARS-CoV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer-target base mismatch through altered oligonucleotide chemistry or chemical additives.


Subject(s)
COVID-19 , SARS-CoV-2 , Alleles , COVID-19/diagnosis , Humans , Polymerase Chain Reaction , SARS-CoV-2/genetics
2.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-307811

ABSTRACT

We describe the innate and adaptive immune system trajectory in Multi-system inflammatory syndrome of childhood (MIS-C), at acute(within 72 hours of hospitalization), resolution (at clinical improvement) and convalescent phase. In our cohort, in the acute phase, 68% of the children were SARS-CoV-2 seropositive, with hypercytokinenemia (high interleukin(IL)-1beta,IL-6,IL-8,IL-10,IL-17, interferon gamma), procoagulant state, myocardial dysfunction, activated neutrophils and monocytes;differential T and B cell subset lymphopenia;activated chemokine receptor type-7 positive and gamma-delta T cell subsets;antigen presenting cells had reduced HLA-DR expression;and B-cell class-switch responses occurred with illness resolution. MIS-C is an immunopathogenic illness associated with SARS-CoV-2 infections in children.

3.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-295804

ABSTRACT

ABSTRACT Background Treatment of COVID-19 patients with convalescent plasma containing neutralising antibody to SARS-CoV-2 is under investigation as a means of reducing viral loads, ameliorating disease outcomes, and reducing mortality. However, its efficacy might be reduced in those infected with the emerging B.1.1.7 SARS-CoV-2 variant. Here, we report the diverse virological characteristics of UK patients enrolled in the Immunoglobulin Domain of the REMAP-CAP randomised controlled trial. Methods SARS-CoV-2 viral RNA was detected and quantified by real-time PCR in nasopharyngeal swabs obtained from study subjects within 48 hours of admission to intensive care unit. Antibody status was determined by spike-protein ELISA. B.1.1.7 strain was differentiated from other SARS-CoV-2 strains by two novel typing methods detecting the B.1.1.7-associated D1118H mutation with allele-specific probes and by restriction site polymorphism (SfcI). Findings Of 1260 subjects, 90% were PCR-positive with viral loads in nasopharyngeal swabs ranging from 72 international units [IUs]/ml to 1.7×10 11 IU/ml. Median viral loads were 45-fold higher in those who were seronegative for IgG antibodies (n=314;28%) compared to seropositives (n=804;72%), reflecting in part the latter group’s possible later disease stage on enrolment. Frequencies of B.1.1.7 infection increased from early November (<1%) to December 2020 (>60%). Anti-SARS-CoV-2 seronegative individuals infected with wild-type SARS-CoV-2 had significantly higher viral loads than seropositives (medians of 1.2×10 6 and 3.4 ×10 4 IU/ml respectively;p=2×10 −9 ). However, viral load distributions were elevated in both seropositive and seronegative subjects infected with B.1.1.7 (13.4×10 6 and 7.6×10 6 IU/ml;p=0.18). Interpretation High viral loads in seropositive B.1.1.7-infected subjects are consistent with increased replication capacity and/or less effective clearance by innate or adaptive immune response of B.1.1.7 strain than wild-type. As viral genotype was associated with diverse virological and immunological phenotypes, metrics of viral load, antibody status and infecting strain should be used to define subgroups for analysis of treatment efficacy.

4.
J Infect Dis ; 224(4): 595-605, 2021 08 16.
Article in English | MEDLINE | ID: covidwho-1367024

ABSTRACT

BACKGROUND: Convalescent plasma containing neutralizing antibody to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is under investigation for coronavirus disease 2019 (COVID-19) treatment. We report diverse virological characteristics of UK intensive care patients enrolled in the Immunoglobulin Domain of the REMAP-CAP randomized controlled trial that potentially influence treatment outcomes. METHODS: SARS-CoV-2 RNA in nasopharyngeal swabs collected pretreatment was quantified by PCR. Antibody status was determined by spike-protein ELISA. B.1.1.7 was differentiated from other SARS-CoV-2 strains using allele-specific probes or restriction site polymorphism (SfcI) targeting D1118H. RESULTS: Of 1274 subjects, 90% were PCR positive with viral loads 118-1.7 × 1011IU/mL. Median viral loads were 40-fold higher in those IgG seronegative (n = 354; 28%) compared to seropositives (n = 939; 72%). Frequencies of B.1.1.7 increased from <1% in November 2020 to 82% of subjects in January 2021. Seronegative individuals with wild-type SARS-CoV-2 had significantly higher viral loads than seropositives (medians 5.8 × 106 and 2.0 × 105 IU/mL, respectively; P = 2 × 10-15). CONCLUSIONS: High viral loads in seropositive B.1.1.7-infected subjects and resistance to seroconversion indicate less effective clearance by innate and adaptive immune responses. SARS-CoV-2 strain, viral loads, and antibody status define subgroups for analysis of treatment efficacy.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/therapy , SARS-CoV-2/immunology , Viral Load/immunology , Aged , Antibodies, Neutralizing/immunology , COVID-19/virology , Critical Illness , Female , Humans , Immunization, Passive , Immunoglobulin G/immunology , Male , Middle Aged , RNA, Viral/immunology , Serologic Tests/methods , Spike Glycoprotein, Coronavirus/immunology , United Kingdom
6.
Cancer Cell ; 39(2): 257-275.e6, 2021 02 08.
Article in English | MEDLINE | ID: covidwho-1009339

ABSTRACT

Given the immune system's importance for cancer surveillance and treatment, we have investigated how it may be affected by SARS-CoV-2 infection of cancer patients. Across some heterogeneity in tumor type, stage, and treatment, virus-exposed solid cancer patients display a dominant impact of SARS-CoV-2, apparent from the resemblance of their immune signatures to those for COVID-19+ non-cancer patients. This is not the case for hematological malignancies, with virus-exposed patients collectively displaying heterogeneous humoral responses, an exhausted T cell phenotype and a high prevalence of prolonged virus shedding. Furthermore, while recovered solid cancer patients' immunophenotypes resemble those of non-virus-exposed cancer patients, recovered hematological cancer patients display distinct, lingering immunological legacies. Thus, while solid cancer patients, including those with advanced disease, seem no more at risk of SARS-CoV-2-associated immune dysregulation than the general population, hematological cancer patients show complex immunological consequences of SARS-CoV-2 exposure that might usefully inform their care.


Subject(s)
COVID-19/immunology , Neoplasms/immunology , Neoplasms/virology , Severe Acute Respiratory Syndrome/immunology , Adult , Aged , Aged, 80 and over , COVID-19/etiology , COVID-19/mortality , Female , Hematologic Neoplasms/immunology , Hematologic Neoplasms/mortality , Hematologic Neoplasms/therapy , Hematologic Neoplasms/virology , Humans , Immunophenotyping , Male , Middle Aged , Nasopharynx/virology , Neoplasms/mortality , Neoplasms/therapy , Severe Acute Respiratory Syndrome/etiology , Severe Acute Respiratory Syndrome/mortality , Severe Acute Respiratory Syndrome/virology , T-Lymphocytes/virology , Virus Shedding , Young Adult
7.
Nat Med ; 26(11): 1701-1707, 2020 11.
Article in English | MEDLINE | ID: covidwho-722216

ABSTRACT

Recent reports highlight a new clinical syndrome in children related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1-multisystem inflammatory syndrome in children (MIS-C)-which comprises multiorgan dysfunction and systemic inflammation2-13. We performed peripheral leukocyte phenotyping in 25 children with MIS-C, in the acute (n = 23; worst illness within 72 h of admission), resolution (n = 14; clinical improvement) and convalescent (n = 10; first outpatient visit) phases of the illness and used samples from seven age-matched healthy controls for comparisons. Among the MIS-C cohort, 17 (68%) children were SARS-CoV-2 seropositive, suggesting previous SARS-CoV-2 infections14,15, and these children had more severe disease. In the acute phase of MIS-C, we observed high levels of interleukin-1ß (IL-1ß), IL-6, IL-8, IL-10, IL-17, interferon-γ and differential T and B cell subset lymphopenia. High CD64 expression on neutrophils and monocytes, and high HLA-DR expression on γδ and CD4+CCR7+ T cells in the acute phase, suggested that these immune cell populations were activated. Antigen-presenting cells had low HLA-DR and CD86 expression, potentially indicative of impaired antigen presentation. These features normalized over the resolution and convalescence phases. Overall, MIS-C presents as an immunopathogenic illness1 and appears distinct from Kawasaki disease.


Subject(s)
COVID-19/blood , COVID-19/immunology , Leukocytes/classification , Leukocytes/pathology , SARS-CoV-2/immunology , Systemic Inflammatory Response Syndrome/blood , Systemic Inflammatory Response Syndrome/immunology , Adolescent , Age of Onset , Blood Coagulation/physiology , COVID-19/complications , COVID-19/epidemiology , Cardiomyopathies/blood , Cardiomyopathies/etiology , Cardiomyopathies/immunology , Case-Control Studies , Child , Child, Preschool , Cohort Studies , Female , Humans , Immunophenotyping , Inflammation/blood , Inflammation/etiology , Inflammation/immunology , Leukocytes/immunology , Male , Systemic Inflammatory Response Syndrome/complications , Systemic Inflammatory Response Syndrome/epidemiology
8.
Nat Med ; 26(10): 1623-1635, 2020 10.
Article in English | MEDLINE | ID: covidwho-717130

ABSTRACT

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.


Subject(s)
Antibodies, Viral/immunology , B-Lymphocytes/immunology , Coronavirus Infections/immunology , Cytokines/immunology , Dendritic Cells/immunology , Pneumonia, Viral/immunology , T-Lymphocytes/immunology , Aged , B-Lymphocyte Subsets/immunology , Basophils/immunology , Betacoronavirus , COVID-19 , Case-Control Studies , Cell Cycle , Chemokine CXCL10/immunology , Chemokines/immunology , Cohort Studies , Coronavirus Infections/blood , Disease Progression , Female , Flow Cytometry , Hospitalization , Humans , Immunologic Memory , Immunophenotyping , Interleukin-10/immunology , Interleukin-6/immunology , Leukocyte Count , Lymphocyte Activation/immunology , Male , Middle Aged , Pandemics , Pneumonia, Viral/blood , Prognosis , SARS-CoV-2 , Severity of Illness Index , T-Lymphocyte Subsets/immunology , Up-Regulation
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