Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses (preprint)

Neurological complications occur in a significant proportion of COVID-19 cases. In order to identify key biomarkers, we measured brain injury markers, inflammatory mediators, and autoantibodies in 203 participants admitted to hospital for management of COVID-19; 111 provided acute sera (1-11 days post admission) and 56 with COVID-19-associated neurological diagnoses provided convalescent sera (up to76 weeks post admission). Compared to 60 controls, brain injury biomarkers (total-Tau, GFAP, NfL, UCH-L1) were increased in acute sera, significantly more so for NfL and UCH-L1, in participants with altered consciousness. Total-Tau (tTau) and NfL remained elevated in convalescent sera, particularly following cerebrovascular and neuroinflammatory disorders. Acutely, inflammatory mediators (including IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) were higher in participants with altered consciousness and correlated with brain injury biomarker levels. Inflammatory mediators were lower in convalescent sera than acute sera. Levels of CCL2, CCL7, IL-1RA, IL-2Rα, M-CSF, SCF, IL-16 and IL-18 in individual participants correlated with tTau levels even at later time points. When compared to acute COVID-19 patients with a normal Glasgow Coma Scale score (GCS), network analysis showed significantly altered immune responses in patients with acute alteration of consciousness, and in convalescent patients who had suffered an acute neurological complication. The frequency and range of autoantibodies did not associate with neurological disorders. However, autoantibodies against specific antigens were more frequent in patients with altered consciousness in the acute phase (including MYL7, UCH-L1, GRIN3B, and DDR2), and in patients with neurological complications in the convalescent phase (including MYL7, GNRHR, and HLA antigens). In a novel low-inoculum mouse model of SARS-CoV-2, while viral replication was only consistently seen in mouse lungs, inflammatory responses were seen in both brain and lungs, with significant increases in CCL4, IFNγ, IL-17A, and microglial reactivity in the brain. Neurological injury is common in the acute phase of COVID-19 and we found brain injury markers persist during convalescence and may be driven by a para-infectious process involving a dysregulated host response.

The contribution of hospital-acquired infections to the COVID-19 epidemic in England in the first half of 2020 (preprint)

Background: SARS-CoV-2 is known to transmit in hospital settings, but the contribution of infections acquired in hospitals to the epidemic at a national scale is unknown. Methods: We used comprehensive national English datasets to determine the number of COVID-19 patients with identified hospital-acquired infections (with symptom onset >7 days after admission and before discharge) in acute English hospitals up to August 2020. As patients may leave the hospital prior to detection of infection or have rapid symptom onset, we combined measures of the length of stay and the incubation period distribution to estimate how many hospital-acquired infections may have been missed. We used simulations to estimate the total number (identified and unidentified) of symptomatic hospital-acquired infections, as well as infections due to onward community transmission from missed hospital-acquired infections, to 31 st July 2020. Results: In our dataset of hospitalised COVID-19 patients in acute English hospitals with a recorded symptom onset date (n = 65,028), 7% were classified as hospital-acquired. We estimated that only 30% (range across weeks and 200 simulations: 20-41%) of symptomatic hospital-acquired infections would be identified, with up to 15% (mean, 95% range over 200 simulations: 14.1%-15.8%) of cases currently classified as community-acquired COVID-19 potentially linked to hospital transmission. We estimated that 26,600 (25,900 to 27,700) individuals acquired a symptomatic SARS-CoV-2 infection in an acute Trust in England before 31st July 2020, resulting in 15,900 (15,200-16,400) or 20.1% (19.2%-20.7%) of all identified hospitalised COVID-19 cases. Conclusions: Transmission of SARS-CoV-2 to hospitalised patients likely caused approximately a fifth of identified cases of hospitalised COVID-19 in the “first wave” in England, but less than 1% of all infections in England. Using time to symptom onset from admission for inpatients as a detection method likely misses a substantial proportion (>60%) of hospital-acquired infections.

Safety, tolerability and viral kinetics during SARS-CoV-2 human challenge (preprint)

To establish a novel SARS-CoV-2 human challenge model, 36 volunteers aged 18-29 years without evidence of previous infection or vaccination were inoculated with 10 TCID50 of a wild-type virus (SARS-CoV-2/human/GBR/484861/2020) intranasally. Two participants were excluded from per protocol analysis due to seroconversion between screening and inoculation. Eighteen (~53%) became infected, with viral load (VL) rising steeply and peaking at ~5 days post-inoculation. Virus was first detected in the throat but rose to significantly higher levels in the nose, peaking at ~8.87 log10 copies/ml (median, 95% CI [8.41,9.53). Viable virus was recoverable from the nose up to ~10 days post-inoculation, on average. There were no serious adverse events. Mild-to-moderate symptoms were reported by 16 (89%) infected individuals, beginning 2-4 days post-inoculation. Anosmia/dysosmia developed more gradually in 12 (67%) participants. No quantitative correlation was noted between VL and symptoms, with high VLs even in asymptomatic infection, followed by the development of serum spike-specific and neutralising antibodies. However, lateral flow results were strongly associated with viable virus and modelling showed that twice-weekly rapid tests could diagnose infection before 70-80% of viable virus had been generated. Thus, in this first SARS-CoV-2 human challenge study, no serious safety signals were detected and the detailed characteristics of early infection and their public health implications were shown. ClinicalTrials.gov identifier: NCT04865237.

Pandemic, epidemic, endemic: B cell repertoire analysis reveals unique anti-viral responses to SARS-CoV-2, Ebola and Respiratory Syncytial Virus (preprint)

Immunoglobulin gene heterogeneity reflects the diversity and focus of the humoral immune response towards different infections, enabling inference of B cell development processes. Detailed compositional and lineage analysis of long read IGH repertoire sequencing, combining examples of pandemic, epidemic and endemic viral infections with control and vaccination samples, demonstrates general responses including increased use of IGHV4-39 in both EBOV and COVID-19 infection cohorts. We also show unique characteristics absent in RSV infection or yellow fever vaccine samples: EBOV survivors show unprecedented high levels of class switching events while COVID-19 repertoires from acute disease appear underdeveloped. Despite the high levels of clonal expansion in COVID-19 IgG1 repertoires there is a striking lack of evidence of germinal centre mutation and selection. Given the differences in COVID-19 morbidity and mortality with age, it is also pertinent that we find significant differences in repertoire characteristics between young and old patients. Our data supports the hypothesis that a primary viral challenge can result in a strong but immature humoral response where failures in selection of the repertoire risks off-target effects.

Performance of the Innova SARS-CoV-2 Antigen Rapid Lateral Flow Test in the Liverpool Asymptomatic Testing Pilot (preprint)

Background: The Innova SARS-CoV-2 antigen rapid lateral flow test (LFT) offers fast detection of COVID-19 cases. This study assesses the performance of LFT in the general population attending asymptomatic testing centres.Methods: Observational cohort study to compare LFT with reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) results based on two self-administrated swab samples per participant taken within minutes among individuals declaring no classic symptoms, attending asymptomatic testing sites in Liverpool, UK, between 6th and 29th of November 2020.Findings: 5869 individuals attended 48 testing sites in Liverpool. The relative sensitivity of LFT versus RT-qPCR, excluding void tests, was 40∙0% (95% CI: 28∙5 to 52∙4; 28/70). The specificity was 99∙9% (99∙8 to 99∙99; 5431/5434), positive predictive value was 90∙3% (74∙2 to 98∙0; 28/31) and negative predictive value was 99∙2% (99∙0 to 99∙4; 5431/5473). For cases with RT-qPCR cycle threshold CT <18∙3 (approximate viral loads > 10 6 RNA copies/ml), sensitivity was 90∙9% (58∙7 to 99∙8; 10/11), for CT <24∙4 (viral load > 10 4 RNA copies/ml), sensitivity was 69∙4% (51∙9 to 83∙7; 25/36), and for CT >24∙4 (viral load <10 4 RNA copies/ml), sensitivity was 9∙7% (1∙9 to 23∙7; 3/34).Interpretation: Innova LFT is a helpful tool for identifying infections among people who declare no symptoms of COVID-19, particularly those with high viral load and so more likely to infect others. The number of cases with lower CT (indicating higher viral load) missed by LFT, although small, should be considered, with due caution over using single LFT in high-consequence settings. Clear and accurate communication with the public about how to interpret test results is important. Further research is needed to understand how infectiousness is reflected in the viral antigen shedding detected by LFT versus the viral loads approximated by RT-qPCR.Funding: This evaluation was commissioned by the UK Department of Health and Social Care.Declaration of Interests: This evaluation was commissioned by the UK Department of Health and Social Care. IEB, MGF, MGS, DMH, GB and CPC received grant funding from the UK Department of Health and Social Care to evaluate LFT in the Liverpool pilot discussed in this article. IEB reports fees from AstraZeneca as chief data scientist adviser via Liverpool University and a senior investigator grant from the National Institute for Health Research (NIHR) outside the submitted work. MGS is Chair of the Infectious Disease Scientific Advisory Board and a minority shareholder in Integrum Scientific LLC, Greensboro, NC, USA, a company that has interests in COVID-19 testing but not with lateral flow technology, and reports grants from the NIHR, the Medical Research Council, and the Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool.Ethics Approval Statement: The University of Liverpool provided secondary data analysis as part of a UK national service evaluation with data collection by the UK Department of Health and Social Care (DHSC; Sponsor) As per the National Health Service (NHS) Research Authority guidelines, this work did not require ethical approval.

Detection and Quantification of Antibody to SARS-CoV-2 Receptor Binding Domain Provides Enhanced Sensitivity, Specificity and Utility (preprint)

Background: Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity.Methods: The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA) provides the most sensitive format. It has been exploited in a novel hybrid manner employing an S1 solid-phase preferentially presenting RBD once solid-phase bound, coupled with a labelled RBD conjugate, used in a two-step sequential assay.Findings: This assay showed a specificity of 100% on 825 pre COVID-19 samples and a potential sensitivity of 99.6% on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralisation and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine. The early response at presentation with illness, elevated responsiveness with disease severity, detection of asymptomatic seroconversion and persistence after the loss of antibody to the nucleoprotein (anti-NP) are all documented.Trial Registration: The ISARIC WHO CCP-UK study was registered at https://www.isrctn.com/ISRCTN66726260 and designated an Urgent Public Health Research Study by NIHR.Interpretation: The hybrid DABA displays the attributes necessary for an antibody test to be used in both clinical and reference serology. It allows the neutralising antibody response to be inferred early in infection and potentially in vaccine recipients. It is also of sufficient sensitivity to be used to provide serological confirmation of prior infection and provides a more secure measure for seroprevalence studies in the population generally than does anti-NP based on the Architect platform.Funding: This work is variously supported by grants from: the National Institute for Health Research (NIHR; award CO-CIN-01), the Medical Research Council (MRC; grant MC_PC_19059 and MC_PC_19078), MRC NIHR (grant CV220-111) and by the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford (award 200907), NIHR HPRU in Respiratory Infections at Imperial College London with PHE (award 200927), Wellcome Trust and Department for International Development (DID; 215091/Z/18/Z), the Bill and Melinda Gates Foundation (OPP1209135), Liverpool Experimental Cancer Medicine Centre (grant reference C18616/A25153), NIHR Biomedical Research Centre at Imperial College London (IS-BRC-1215-20013), EU Platform for European Preparedness Against (Re-)emerging Epidemics (PREPARE; FP7 project 602525), and NIHR Clinical Research Network for providing infrastructure support for this research.Declaration of Interests: RST, MOM and PC report patent pending (Patent Application No. 2011047.4 for “SARS-CoV-2 antibody detection assay). All other authors declare no competing interests.Ethics Approval Statement: The use of tissues was approved by the CDRTB Steering Committee in accordance with the responsibility delegated by the National Research Ethics Service (South Central Ethics Committee – C, NRES reference 15/SC/0089).Written informed consent was obtained from all patients. Ethical approval was given by the South Central–Oxford C Research Ethics Committee in England (reference: 13/SC/0149), Scotland A Research Ethics Committee (reference: 20/SS/0028) and World Health Organization Ethics Review Committee (RPC571 and RPC572l; 25 April 2013)

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2 (preprint)

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, detection of seroconversion after vaccination, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests have a long history in blood typing, and general serology through linkage of reporter molecules to the red cell surface. They do not require special equipment, are read by eye, have short development times, low cost and can be applied as a Point of Care Test (POCT). We describe a red cell agglutination test for the detection of antibodies to the SARS-CoV-2 receptor binding domain (RBD). We show that the Haemagglutination Test (HAT) has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. The HAT can be titrated, detects rising titres in the first five days of hospital admission, correlates well with a commercial test that detects antibodies to the RBD, and can be applied as a point of care test. The developing reagent is composed of a previously described nanobody to a conserved glycophorin A epitope on red cells, linked to the RBD from SARS-CoV-2. It can be lyophilised for ease of shipping. We have scaled up production of this reagent to one gram, which is sufficient for ten million tests, at a cost of ~0.27 UK pence per test well. Aliquots of this reagent are ready to be supplied to qualified groups anywhere in the world that need to detect antibodies to SARS-CoV-2, but do not have the facilities for high throughput commercial tests.

Ethnicity and Outcomes from COVID-19: The ISARIC CCP-UK Prospective Observational Cohort Study of Hospitalised Patients (preprint)

Background: Reports of ethnic inequalities in COVID-19 outcomes are conflicting and the reasons for any differences in outcomes are unclear. We investigated ethnic inequalities in critical care admission patterns, the need for invasive mechanical ventilation (IMV), and in-hospital mortality, among hospitalised patients with COVID-19. Methods: We undertook a prospective cohort study in which dedicated research staff recruited hospitalised patients with suspected/confirmed COVID-19 from 260 hospitals across England, Scotland and Wales, collecting data directly and from records between 6th February and 8th May 2020 with follow-up until 22nd May 2020. Analysis used hierarchical regression models accounting for confounding, competing risks, and clustering of patients in hospitals. Potential mediators for death were explored with a three-way decomposition mediation analysis. Findings: Of 34,986 patients enrolled, 30,693 (88%) had ethnicity recorded: South Asian (1,388, 5%), East Asian (266, 1%), Black (1,094, 4%), Other Ethnic Minority (2,398, 8%) (collectively Ethnic Minorities), and White groups (25,547, 83%). Ethnic Minorities were younger and more likely to have diabetes (type 1/type 2) but had fewer other comorbidities such as chronic heart disease or dementia than the White group. No difference was seen between ethnic groups in the time from symptom onset to hospital admission, nor in illness severity at admission. Critical care admission was more common in South Asian (odds ratio 1.28, 95% confidence interval 1.09 to 1.52), Black (1.36, 1.14 to 1.62), and Other Ethnic Minority (1.29, 1.13 to 1.47) groups compared to the White group, after adjusting for age, sex and location. This was broadly unchanged after adjustment for deprivation and comorbidities. Patterns were similar for IMV. Higher adjusted mortality was seen in the South Asian (hazard ratio 1.19, 1.05 to 1.36), but not East Asian (1.00, 0.74 to 1.35), Black (1.05, 0.91 to 1.26) or Other Ethnic Minority (0.99, 0.89 to 1.10) groups, compared to the White group. 18% (95% CI, 9% to 56%) of the excess mortality in South Asians was mediated by pre-existing diabetes. Interpretation: Ethnic Minorities in hospital with COVID-19 were more likely to be admitted to critical care and receive IMV than Whites, despite similar disease severity on admission, similar duration of symptoms, and being younger with fewer comorbidities. South Asians are at greater risk of dying, due at least in part to a higher prevalence of pre-existing diabetes. Trial Registration: The study was registered at https://www.isrctn.com/ISRCTN66726260. Funding Statement: This work is supported by grants from: the National Institute for Health Research [award CO-CIN-01], the Medical Research Council [grant MC_PC_19059] and by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford [NIHR award 200907], Wellcome Trust and Department for International Development [215091/Z/18/Z], and the Bill and Melinda Gates Foundation [OPP1209135], and Liverpool Experimental Cancer Medicine Centre for providing infrastructure support for this research (Grant Reference: C18616/A25153). JSN-V-T is seconded to the Department of Health and Social Care, England (DHSC).Declaration of Interests: All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: AB Docherty reports grants from Department of Health and Social Care, during the conduct of the study; grants from Wellcome Trust, outside the submitted work; CA Green reports grants from DHSC National Institute of Health Research UK, during the conduct of the study; PW Horby reports grants from Wellcome Trust / Department for International Development / Bill and Melinda Gates Foundation, grants from NIHR , during the conduct of the study; JS Nguyen-Van-Tam reports grants from Department of Health and Social Care, England, during the conduct of the study; and is seconded to the Department of Health and Social Care, England (DHSC); PJM Openshaw reports personal fees from consultancies and from European Respiratory Society; grants from MRC, MRC Global Challenge Research Fund, EU, NIHR Biomedical Research Centre, MRC/GSK, Wellcome Trust, NIHR (HPRU in Respiratory Infection), and NIHR Senior Investigator outside the submitted work. His role as President of the British Society for Immunology was unpaid but travel and accommodation at some meetings was provided by the Society. JK Baillie reports grants from Medical Research Council UK; MG Semple reports grants from DHSC National Institute of Health Research UK, grants from Medical Research Council UK, grants from Health Protection Research Unit in Emerging & Zoonotic Infections, University of Liverpool, during the conduct of the study; other from Integrum Scientific LLC, Greensboro, NC, USA, outside the submitted work. EM Harrison, H Ardwick, J Dunning, R Pius, L Norman, KA Holden, JM Read, G Carson, L Merson, J Lee, D Plotkin, L Sigfrid, S Halpin, C Jackson, and C Gamble, all declare: no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; and no other relationships or activities that could appear to have influenced the submitted work.Ethics Approval Statement: Ethical approval was given by the South Central – Oxford C Research Ethics Committee in England (Ref: 13/SC/0149), and by the Scotland A Research Ethics Committee (Ref: 20/SS/0028).