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Here we describe a retrospective clinical evaluation of the QIAGEN artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay that detects SARS-CoV-2 RNA without the need for a nucleic acid eluate extraction procedure. Using Roche SARS-CoV-2 RT-PCR on the cobas® 8800 platform as a reference standard, a total of 225 confirmed SARS-CoV-2 positive and 320 negative nasopharyngeal swabs in viral transport media, were used to evaluate the artus® assay. Using the RT-PCR cycle threshold as a semi-quantitative marker of viral load, an assessment of over 370,000 SARS-CoV-2 RT-PCR positive results was used in the design of the reference positive specimen cohort. The viral load of all reference positive specimens used in the evaluation was a unique and accurate representation of the range and levels of SARS-CoV-2 positivity observed over a 13-month period of the COVID-19 pandemic. The artus® RT-PCR detects the presence of SARS-CoV-2 RNA, an internal control, and the human RNase P gene to ensure specimen quality. The diagnostic sensitivity of artus® was 92.89% with a specificity of 100%. To assess the analytical sensitivity, a limit of detection was performed using the 1st WHO NIBSC SARS-CoV-2 international standard, recording a 95% LOD of 1.1 × 103 IU/ml. The total invalid rate of specimens was 7.34% due to a lack of detectable RNase P (Ct >35). The artus® SARS-CoV-2 Prep&Amp UM RT-PCR assay is a new rapid RT-PCR assay, which may be considered to produce acceptable levels of diagnostic sensitivity and specificity whilst potentially halving the laboratory processing time.
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BackgroundMolnupiravir was licensed for treating high-risk patients with COVID-19 based on data from unvaccinated adults. AGILE CST-2 (NCT04746183) Phase II reports safety and virological efficacy of molnupiravir in vaccinated and unvaccinated individuals. MethodsAdult out-patients with PCR-confirmed SARS-CoV-2 infection within five days of symptom onset were randomly assigned 1:1 to receive molnupiravir (800mg twice daily for five days) or placebo. The primary outcome was time to swab PCR-negativity, compared using a Bayesian model for estimating the probability of a superior virological response (Hazard Ratio>1) for molnupiravir over placebo. Secondary outcomes included change in viral titre at day 5, safety and tolerability, clinical progression and patient reported outcome measures. We analysed outcomes after the last participant reached day 29. FindingsOf 180 participants randomised (90 molnupiravir, 90 placebo), 50% were vaccinated. Infections with SARS-CoV-2 variants Delta (40%), Alpha (21%), Omicron (21%) and EU1 (16%) were represented. The median time to negative-PCR was 8 versus 11 days for molnupiravir and placebo (HR=1{middle dot}30, 95% CrI 0{middle dot}92-1{middle dot}71, p=0{middle dot}07 by Logrank and p=0{middle dot}03 by Breslow-Gehan tests). Although small numbers precluded subgroup analysis, no obvious differences were observed between vaccinated and unvaccinated participants. Using a two-point prior the probability of molnupiravir being superior to placebo (HR>1) was 75{middle dot}4%, which was just below our defined threshold of 80% for establishing superiority. Using an uninformative continuous prior, the probability of HR>1 was 94{middle dot}7%. As an exploratory analysis, the change in viral titre on day 5 (end of treatment) was significantly greater with molnupiravir compared with placebo. A total of 4 participants reported severe adverse events (grade 3+), 3 of whom were in the placebo arm. InterpretationWe found molnupiravir to be well-tolerated, with evidence for high probability of antiviral efficacy in a population of vaccinated and unvaccinated individuals infected with a broad range of viral variants. FundingFunded by Ridgeback Biotherapeutics and UK National Institute for Health and Care Research infrastructure funding. The AGILE platform infrastructure is supported by the Medical Research Council (grant number MR/V028391/1) and the Wellcome Trust (grant number 221590/Z/20/Z).
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The Omicron variant of SARS-CoV-2 is now globally dominant but despite high prevalence little is known regarding the immune response in children. We determined the antibody and cellular immune response following Omicron infection in children aged 6-14 years and related this to prior SARS-CoV-2 infection and vaccination status. Primary Omicron infection elicited a weak antibody response and only 53% of children developed detectable neutralising antibodies. In contrast, children with secondary Omicron infection following prior infection with a pre-Omicron variant developed 24-fold higher antibody titres and neutralisation of Omicron. Vaccination elicited the highest levels of antibody response and was also strongly immunogenic following prior natural infection with Omicron. Cellular responses against Omicron were robust and broadly equivalent in all study groups. These data reveal that primary Omicron infection elicits a weak humoral immune response in children and may presage a clinical profile of recurrent infection as seen with antecedent seasonal coronaviruses. Vaccination may represent the most effective approach to control infection whilst cellular immunity should offer strong clinical protection.
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BackgroundThe role of educational settings on SARS-CoV-2 infection and transmission remains controversial. We investigated SARS-CoV-2 infection, seroprevalence and seroconversions rates in secondary schools during the 2020/21 academic year, which included the emergence of the more transmissible Alpha and Delta variants, in England. MethodsThe UK Health Security Agency (UKHSA) initiated prospective surveillance in 18 urban English secondary schools. Participants had nasal swabs for SARS-CoV-2 RT-PCR and blood sampling for SARS-CoV-2 Nucleoprotein and Spike protein antibodies at the start (Round 1: September-October 2020) and end (Round 2: December 2021) of the autumn term, when schools reopened after national lockdown was imposed in January 2021 (Round 3: March-April) and end of the academic year (Round 4: May-July). FindingsWe enrolled 2,314 participants (1277 students, 1037 staff). In-school testing identified 31 PCR-positive participants (20 students, 11 staff). Another 247 confirmed cases (112 students, 135 staff) were identified after linkage with national surveillance data, giving an overall positivity rate of 12.0% (278/2313; staff [14.1%, 146/1037] vs students [10.3%, 132/1276; p=0.006). Nucleoprotein-antibody seroprevalence increased for students and staff between Rounds 1-3 but changed little in Round 4, when the Delta variant was the dominant circulating strain. Overall, Nucleoprotein-antibody seroconversion was 18.4% (137/744) in staff and 18.8% (146/778) in students, while Spike-antibody seroconversion was higher in staff (72.8% (525/721) than students (21.3%, 163/764) because of vaccination. InterpretationSARS-CoV-2 infection and transmission in secondary schools remained low when community infection rates were low because of national lockdown, even after the emergence of the Delta variant FundingDHSC
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BackgroundIn England, the rapid spread of the SARS-Cov-2 Alpha (B.1.1.7) variant from November 2020 led to national lockdown, including school closures in January 2021. We assessed SARS-CoV-2 infection, seroprevalence and seroconversion in students and staff when secondary schools reopened in March 2021. MethodsPublic Health England initiated SARS-CoV-2 surveillance in 18 secondary schools across six regions in September 2020. Participants provided nasal swabs for RT-PCR and blood samples for SARS-CoV-2 antibodies at the beginning (September 2020) and end (December 2020) of the autumn term and at the start of the spring term (March 2021). FindingsIn March 2021, 1895 participants (1100 students, 795 staff) were tested; 5.6% (61/1094) students and 4.4% (35/792) staff had laboratory-confirmed SARS-CoV-2 infection between December 2020 and March 2021. Nucleoprotein antibody seroprevalence was 36.3% (370/1018) in students and 31.9% (245/769) in staff, while spike protein antibody prevalence was 39.5% (402/1018) and 59.8% (459/769), respectively, similar to regional community seroprevalence. Between December 2020 and March 2021 (median 15.9 weeks), 14.8% (97/656; 95% CI: 12.2-17.7) students and 10.0% (59/590; 95% CI: 7.7-12.7) staff seroconverted. Weekly seroconversion rates were similar from September to December 2020 (8.0/1000) and from December 2020 to March 2021 (7.9/1000; students: 9.3/1,000; staff: 6.3/1,000). InterpretationBy March 2021, a third of secondary school students and staff had serological evidence of prior infection based on N-antibody seropositivity, and an additional third of staff had evidence of vaccine-induced immunity based on S-antibody seropositivity. Further studies are needed to assess the impact of the Delta variant. Research in ContextO_ST_ABSEvidence Before this studyC_ST_ABSThe Alpha variant is 30-70% more transmissible than previously circulating SARS-CoV-2 strains in adults and children. One outbreak investigation in childcare settings estimated similar secondary attack rates with the Alpha variant in children and adults. There are limited data on the impact of the Alpha variant in educational settings. In England, cases in primary and secondary school aged children increased rapidly from late November 2020 and peaked at the end of December 2020, leading to national lockdown including school closures. Added Value of This StudySeroconversion rates in staff and students during December 2020 to March 2021, when the Alpha variant was the primary circulating strain in England, were similar to the period between September 2020 and December 2020 when schools were fully open for in-person teaching. By March 2021, a third of students overall and more than half the students in some regions were seropositive for SARS-CoV-2 antibodies. Among staff, too, around a third had evidence of prior infection on serological testing and a further third had vaccine-induced immunity. Implications of all the Available EvidenceSARS-CoV-2 antibody seroprevalence was high among secondary school students in March 2021 and is likely to be higher following the emergence of an even more transmissible Delta variant in May 2021. Education staff are increasingly being protected by the national COVID-19 immunisation programme. These findings have important implications for countries that are considering vaccination of children to control the pandemic
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Seroepidemiological studies to monitor antibody kinetics are important for assessing the extent and spread of SARS-CoV-2 in a population. Non-invasive sampling methods are advantageous to reduce the need for venepuncture, which may be a barrier to investigations particularly in paediatric populations. Oral Fluids are obtained by gingiva-crevicular sampling from children and adults and are very well accepted. ELISA based on these samples have acceptable sensitivity and specificity compared to conventional serum-based antibody ELISAs and are suitable for population-based surveillance. We describe the development and evaluation of SARS-COV-2 IgG ELISAs using SARS-CoV-2 viral nucleoprotein (NP) and spike (S) proteins in IgG isotype capture format and an indirect receptor-binding-domain (RBD) IgG ELISA, intended for use in children. All three assays were assessed using a panel of 1999 paired serum and oral fluids from children and adults participating in national primary school SARS-CoV-2 surveillance studies during and after the first and second pandemic wave in the UK. The anti NP IgG capture assay was the best candidate, with an overall sensitivity of 75% (95% CI: 71-79%) specificity of 99% (95% CI: 78-99%) when compared with paired serum antibodies measured using a commercial assay SARS-CoV-2 nucleoprotein IgG assay (Abbott, Chicago, IL, USA). Higher sensitivity was observed in children (80%, 95% CI: 71-88%) compared to adults (67%, CI: 60%-74%). Oral fluid assays using spike protein and RBD antigens were also 99% specific and achieved reasonable but lower sensitivity in the target population (78%, 95% CI (68%-86%) and 53%, 95% CI (43%-64%), respectively). Conclusion statementOral Fluid assays based on the detection of SARS-CoV-2 antibodies are a suitable tool for population based seroepidemiology studies in children.
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SARS-CoV-2 infection is generally mild or asymptomatic in children but the biological basis for this is unclear. We studied the profile of antibody and cellular immunity in children aged 3-11 years in comparison with adults. Antibody responses against spike and receptor binding domain (RBD) were high in children and seroconversion boosted antibody responses against seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Seroneutralisation assays against alpha, beta and delta SARS-CoV-2 variants demonstrated comparable neutralising activity between children and adults. T cell responses against spike were >2-fold higher in children compared to adults and displayed a TH1 cytokine profile. SARS-CoV-2 spike-specific T cells were also detected in many seronegative children, revealing pre-existing responses that were cross-reactive with seasonal Alpha and Beta-coronaviruses. Importantly, all children retained high antibody titres and cellular responses at 6 months after infection whilst relative antibody waning was seen in adults. Spike-specific responses in children also remained broadly stable beyond 12 months. Children thus distinctly generate robust, cross-reactive and sustained immune responses after SARS-CoV-2 infection with focussed specificity against spike protein. These observations demonstrate novel features of SARS-CoV-2-specific immune responses in children and may provide insight into their relative clinical protection. Furthermore, this information will help to guide the introduction of vaccination regimens in the paediatric population.
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BackgroundMost individuals with COVID-19 will recover without sequelae, but some will develop long-term multi-system impairments. The definition, duration, prevalence and symptoms associated with long COVID, however, have not been established. MethodsPublic Health England (PHE) initiated longitudinal surveillance of clinical and non-clinical healthcare workers for monthly assessment and blood sampling for SARS-CoV-2 antibodies in March 2020. Eight months after enrolment, participants completed an online questionnaire including 72 symptoms in the preceding month. Symptomatic mild-to-moderate cases with confirmed COVID-19 were compared with asymptomatic, seronegative controls. Multivariable logistic regression was used to identify independent symptoms associated with long COVID. FindingsAll 2,147 participants were contacted and 1,671 (77.8%) completed the questionnaire, including 140 (8.4%) cases and 1,160 controls. At a median of 7.5 (IQR 7.1-7.8) months after infection, 20 cases (14.3%) had ongoing (4/140, 2.9%) or episodic (16/140, 11.4%) symptoms. We identified three clusters of symptoms associated with long COVID, those affecting the sensory (ageusia, anosmia, loss of appetite and blurred vision), neurological (forgetfulness, short-term memory loss and confusion/brain fog) and cardiorespiratory (chest tightness/pain, unusual fatigue, breathlessness after minimal exertion/at rest, palpitations) systems. The sensory cluster had the highest association with being a case (aOR 5.25, 95% CI 3.45-8.01). Dermatological, gynaecological, gastrointestinal or mental health symptoms were not significantly different between cases and controls. InterpretationMost persistent symptoms reported following mild COVID-19 were equally common in cases and controls. While all three clusters identified had a strong association with cases, the sensory cluster had the highest specificity and strength of association, and therefore, most likely to be characteristic of long COVID. FundingPHE. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed using search terms "long covid*" OR "post COVID*" in adults for studies including cohort, case reports, randomised control trials, cross-sectional and systematic reviews published up to 12 March 2020 without any language restrictions. Most reports included a small number of cases. Larger studies included very specific cohorts, including hospitalised cases and self-selected participants with COVID-19. A systematic review identified 15 studies and, using a broad case definition, concluded that 80% (95% CI 65-92) of patients with SARS-CoV-2 developed one or more long-term symptoms. The five most common symptoms were fatigue (58%), headache (44%), attention disorder (27%), hair loss (25%), and dyspnea (24%), but no assessment was made of these symptoms in uninfected adults. Added value of this studyIn a prospective, longitudinal cohort of clinical and non-clinical healthcare workers recruited at the start of the pandemic, we found that most self-reported symptoms were as common in 140 adults who developed mild-to-moderate COVID-19 more than 6 months previously compared to 1,160 controls who were asymptomatic and SARS-CoV-2 antibody negative throughout the surveillance period. Compared to controls, we identified three clusters of symptoms affecting the sensory, neurological and cardiorespiratory systems that were more prevalent among cases. Notably, symptoms affecting other organ systems were as prevalent among cases as controls. The high proportion of cases and controls reporting mental health symptoms highlights the toll that the pandemic has had on healthcare workers Implications of all the available evidenceOur findings highlight the importance of including a representative cohort of cases to assess long-term outcomes of COVID-19 as well as appropriate controls to estimate the relative prevalence of self-reported symptoms to accurately define this new syndrome. Our study adds to the evidence-base for long COVID in adults with mild-to-moderate COVID-19 who contribute to the vast majority of 120+ million infections worldwide. This information is not only important for clinicians, patients and the public, but also for policy makers and healthcare providers who are investing heavily in long-term provisions for COVID-19 survivors.
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BackgroundThe safety of COVID-19 vaccines has been demonstrated in selected populations in recent studies, but more data in specific groups is needed to inform vaccine choice and health policy. ObjectivesAn international, online survey was conducted to compare the safety, tolerability and reactogenicity of available COVID-19 vaccines in different recipient groups. MethodsThis survey was launched in February 2021, for 11 days. Recipients of a first COVID-19 vaccine dose [≥]7 days prior to survey completion were eligible. The incidence and severity of vaccination side effects were assessed. ResultsSurvey was completed by 2,002 respondents, of whom 26.6% had prior COVID-19 infection (68.8% laboratory confirmed). Prior COVID-19 infection was associated with increased risk of any side effect (risk ratio 1.08, 95% confidence intervals [1.05-1.11]), fever (2.24 [1.86-2.70]), breathlessness (2.05 [1.28-3.29]), flu-like illness (1.78 [1.51-2.10]), fatigue (1.34 [1.20-1.49]) and local reactions (1.10 [1.06-1.15]). It was also associated with increased risk of severe side effects, leading to hospital care (1.56 [1.14-2.12]). While mRNA vaccines were associated with a higher incidence of any side effect (1.06 [1.01-1.11]) compared to viral vector-based vaccines, these were generally milder (p<0.001), mostly local reactions. Importantly, mRNA vaccine-recipients reported considerably lower incidence of systemic reactions (RR<0.6) including anaphylaxis, swelling, flu-like illness, breathlessness and fatigue, and of side effects requiring hospital care (0.42 [0.31-0.58]). ConclusionFor the first time, our study links prior COVID-19 illness with increased incidence of vaccination side effects and demonstrates that mRNA vaccines cause milder, less frequent systemic side effects, but more local reactions. Key messages- People with prior COVID-19 illness appear to experience significantly increased incidence and severity of side effects after receiving the COVID-19 vaccine. - In this first head-to-head comparison of the safety and reactogenicity of different types of vaccines, it was demonstrated that mRNA vaccines cause milder, less frequent systemic side effects, compared to viral vector vaccines, but more local reactions. Tweetable SummaryA survey of >2000 COVID-19 vaccine-recipients links prior COVID-19 illness with increased incidence of vaccination side effects; mRNA vaccines cause milder, less frequent systemic side effects, but more local reactions.
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The immune response to SARS-CoV-2 is critical in both controlling primary infection and preventing re-infection. However, there is concern that immune responses following natural infection may not be sustained and that this may predispose to recurrent infection. We analysed the magnitude and phenotype of the SARS-CoV-2 cellular immune response in 100 donors at six months following primary infection and related this to the profile of antibody level against spike, nucleoprotein and RBD over the previous six months. T-cell immune responses to SARS-CoV-2 were present by ELISPOT and/or ICS analysis in all donors and are characterised by predominant CD4+ T cell responses with strong IL-2 cytokine expression. Median T-cell responses were 50% higher in donors who had experienced an initial symptomatic infection indicating that the severity of primary infection establishes a setpoint for cellular immunity that lasts for at least 6 months. The T-cell responses to both spike and nucleoprotein/membrane proteins were strongly correlated with the peak antibody level against each protein. The rate of decline in antibody level varied between individuals and higher levels of nucleoprotein-specific T cells were associated with preservation of NP-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T-cell responses are retained at six months following infection although the magnitude of this response is related to the clinical features of primary infection.
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BackgroundAntibody waning after SARS-CoV-2 infection may result in reduction in long-term immunity following natural infection and vaccination, and is therefore a major public health issue. We undertook prospective serosurveillance in a large cohort of healthy adults from the start of the epidemic in England. MethodsClinical and non-clinical healthcare workers were recruited across three English regions and tested monthly from March to November 2020 for SARS-CoV-2 spike (S) protein and nucleoprotein (N) antibodies using five different immunoassays. In positive individuals, antibody responses and long-term trends were modelled using mixed effects regression. FindingsIn total, 2246 individuals attended 12,247 visits and 264 were seropositive in [≥]2 assays. Most seroconversions occurred between March and April 2020. The assays showed >85% agreement for ever-positivity, although this changed markedly over time. Antibodies were detected earlier with Abbott (N) but declined rapidly thereafter. With the EuroImmun (S) and receptor-binding domain (RBD) assays, responses increased for 4 weeks then fell until week 12-16 before stabilising. For Roche (N), responses increased until 8 weeks, stabilised, then declined, but most remained above the positive threshold. For Roche (S), responses continued to climb over the full 24 weeks, with no sero-reversions. Predicted proportions sero-reverting after 52 weeks were 100% for Abbott, 59% (95% credible interval 50-68%) Euroimmun, 41% (30-52%) RBD, 10% (8-14%) Roche (N) <2% Roche (S). InterpretationTrends in SARS-CoV-2 antibodies following infection are highly dependent on the assay used. Ongoing serosurveillance using multiple assays is critical for monitoring the course and long-term progression of SARS-CoV-2 antibodies.
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BackgroundStudies based on molecular testing of oral/nasal swabs underestimate SARS-CoV-2 infection due to issues with test sensitivity and timing of testing. The objective of this study was to report the presence of SARS-CoV-2 antibodies, consistent with previous infection, and to report the symptomatology of infection in children. DesignThis multicentre observational cohort study, conducted between 16th April - 3rd July 2020 at 5 UK sites, aimed to recruit 900 children aged 2 to 15 years of age. Participants provided blood samples for SARS-CoV-2 antibody testing and data were gathered regarding unwell contacts and symptoms. Results1007 participants were enrolled, and 992 were included in the final analysis. The median age of participants was 10{middle dot}1 years. There were 68 (6.9%) participants with positive SARS-CoV-2 antibody tests indicative of previous SARS-CoV-2 infection. Of these, 34/68 (50%) reported no symptoms. The presence of antibodies and the mean antibody titre was not influenced by age. Following multivariate analysis 4 independent variables were identified as significantly associated with SARS-CoV-2 infection. These were: known infected household contact; fatigue; gastrointestinal symptoms; and changes in sense of smell or taste. DiscussionIn this study children demonstrated similar antibody titres in response to SARS-CoV-2 irrespective of age. The symptoms of SARS-CoV-2 infection in children were subtle but of those reported, fatigue, gastrointestinal symptoms and changes in sense of smell or taste were most strongly associated with antibody positivity. RegistrationThis study was registered at https://www.clinicaltrials.gov (trial registration: NCT04347408) on the 15/04/2020.
