Humoral responses following SARS-CoV-2 breakthrough infection in COVID-19 vaccinated individuals

Numerous studies have shown that a prior SARS-CoV-2 infection can greatly enhance the antibody response to COVID-19 vaccination, with this so called "hybrid immunity" leading to greater neutralization breadth against SARS-CoV-2 variants of concern. However, little is known about how breakthrough infection (BTI) in COVID-19 vaccinated individuals will impact the magnitude and breadth of the neutralizing antibody response. Here, we compared neutralizing antibody responses between unvaccinated and COVID-19 double vaccinated individuals (including both AZD1222 and BNT162b2 vaccinees) who have been infected with the delta (B.1.617.2) variant. Rapid production of Spike-reactive IgG was observed in the vaccinated group providing evidence of effective vaccine priming. Overall, potent cross-neutralizing activity against current SARS-CoV-2 variants of concern was observed in the BTI group compared to the infection group, including neutralization of the omicron (B.1.1.529) variant. This study provides important insights into population immunity where transmission levels remain high and in the context of new or emerging variants of concern.

Antibody longevity and cross-neutralizing activity following SARS-CoV-2 wave 1 and B.1.1.7 infections

As SARS-CoV-2 variants continue to emerge globally, a major challenge for COVID-19 vaccination is the generation of a durable antibody response with cross-neutralizing activity against both current and newly emerging viral variants. Cross-neutralizing activity against major variants of concern (B.1.1.7, P.1 and B.1.351) has been observed following vaccination, albeit at a reduced potency, but whether vaccines based on the Spike glycoprotein of these viral variants will produce a superior cross-neutralizing antibody response has not been fully investigated. Here, we used sera from individuals infected in wave 1 in the UK to study the long-term cross-neutralization up to 10 months post onset of symptoms (POS), as well as sera from individuals infected with the B.1.1.7 variant to compare cross-neutralizing activity profiles. We show that neutralizing antibodies with cross-neutralizing activity can be detected from wave 1 up to 10 months POS. Although neutralization of B.1.1.7 and B.1.351 is lower, the difference in neutralization potency decreases at later timepoints suggesting continued antibody maturation and improved tolerance to Spike mutations. Interestingly, we found that B.1.1.7 infection also generates a cross-neutralizing antibody response, which, although still less potent against B.1.351, can neutralize parental wave 1 virus to a similar degree as B.1.1.7. These findings have implications for the optimization of vaccines that protect against newly emerging viral variants.

Detection of SARS-CoV-2 variants by Abbott molecular, antigen, and serological tests

BackgroundViral diversity presents an ongoing challenge for diagnostic tests, which need to accurately detect all circulating variants. The Abbott Global Surveillance program monitors severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants and their impact on diagnostic test performance. ObjectivesTo evaluate the capacity of Abbott molecular, antigen, and serologic assays to detect the SARS-CoV-2 B.1.1.7, B.1.351 and the P.1 variants. Study designVirus variant culture stock dilutions (B.1.1.7, BEI NR-54011; B.1.351, BEI NR-54008 and 54009; P.1, BEI NR-54982) and clinical samples from patients with confirmed B.1.1.7 variant infection were run on the Abbott ID NOW COVID-19, m2000 RealTime SARS-CoV-2, Alinity m SARS-CoV-2, and Alinity m Resp-4-Plex molecular assays; the BinaxNOW COVID-19 Ag Card and Panbio COVID-19 Ag Rapid Test Device; and the ARCHITECT/Alinity i SARS-CoV-2 IgG and AdviseDx IgM assays, Panbio COVID-19 IgG assay, and ARCHITECT/Alinity i AdviseDx SARS-CoV-2 IgG II assay. ResultsCultured virus stocks and B.1.1.7 clinical samples were detected with molecular, antigen, and serologic assays in the expected ranges, confirming in silico predictions. The ratio between genome equivalents (GE) and calculated median tissue culture infectious dose (TCID50) were 31-to 83-fold higher for B.1.1.7 cultures compared to B.1.351 and P.1 cultures, demonstrating that GE are more consistent units between cultures than TCID50. ConclusionsAbbott molecular and antigen assays effectively detect B.1.1.7, B.1.351, and P.1 variant infections and Abbott serologic assays detect B.1.1.7 antibodies in patient sera. Future studies with SARS-CoV-2 virus cultures should use quantitative viral load values to compare detection of variants. HighlightsO_LIAbbott SARS-CoV-2 molecular and antigen assays detect B.1.1.7, B.1.351, and P.1 variants C_LIO_LIAbbott SARS-CoV-2 antibody assays detect B.1.1.7 antibodies in recovered patient sera C_LIO_LIQuantitation of viral load in genome equivalents allows comparison of assay performance C_LI

First and second SARS-CoV-2 waves in inner London: A comparison of admission characteristics and the effects of the B.1.1.7 variant

IntroductionA second wave of SARS-CoV-2 infection spread across the UK in 2020 linked with emergence of the more transmissible B.1.1.7 variant. The emergence of new variants, particularly during relaxation of social distancing policies and implementation of mass vaccination, highlights the need for real-time integration of detailed patient clinical data alongside pathogen genomic data. We linked clinical data with viral genome sequence data to compare cases admitted during the first and second waves of SARS-CoV-2 infection. MethodsClinical, laboratory and demographic data from five electronic health record (EHR) systems was collected for all cases with a positive SARS-CoV-2 RNA test between March 13th 2020 and February 17th 2021. SARS-CoV-2 viral sequencing was performed using Oxford Nanopore Technology. Descriptive data are presented comparing cases between waves, and between cases of B.1.1.7 and non-B.1.1.7 variants. ResultsThere were 5810 SARS-CoV-2 RNA positive cases comprising inpatients (n=2341), healthcare workers (n=1549), outpatients (n=874), emergency department (ED) attenders not subsequently admitted (n=532), inter-hospital transfers (n=281) and nosocomial cases (n=233). There were two dominant waves of hospital admissions, with wave one starting from March 13th (n=838) and wave two from October 20th (n=1503), both with a temporally aligned rise in nosocomial cases (n=96 in wave one, n=137 in wave two). 1470 SARS-CoV-2 isolates were successfully sequenced, including 216/838 (26%) admitted cases from wave one, 472/1503 (31%) admitted cases in wave two and 121/233 (52%) nosocomial cases. The first B.1.1.7 variant was identified on 15th November 2020 and increased rapidly such that it comprised 400/472 (85%) of sequenced isolates from admitted cases in wave two. Females made up a larger proportion of admitted cases in wave two (47.3% vs 41.8%, p=0.011), and in those infected with the B.1.1.7 variant compared to non-B.1.1.7 variants (48.0% vs 41.8%, p=0.042). A diagnosis of frailty was less common in wave two (11.5% v 22.8%, p<0.001) and in the group infected with B.1.1.7 (14.5% v 22.4%, p=0.001). There was no difference in severity on admission between waves, as measured by hypoxia at admission (wave one: 64.3% vs wave two: 65.5%, p=0.67). However, a higher proportion of cases infected with the B.1.1.7 variant were hypoxic on admission compared to other variants (70.0% vs 62.5%, p=0.029). ConclusionsAutomated EHR data extraction linked with SARS-CoV-2 genome sequence data provides valuable insight into the evolving characteristics of cases admitted to hospital with COVID-19. The proportion of cases with hypoxia on admission was greater in those infected with the B.1.1.7 variant, which supports evidence the B.1.1.7 variant is associated with more severe disease. The number of nosocomial cases was similar in both waves despite introduction of many infection control interventions before wave two, an observation requiring further investigation.

Comparative performance of SARS-CoV-2 lateral flow antigen tests demonstrates their utility for high sensitivity detection of infectious virus in clinical specimens

BackgroundRapid antigen lateral flow devices (LFDs) are set to become a cornerstone of SARS-CoV-2 mass community testing. However, their reduced sensitivity compared to PCR has raised questions of how well they identify infectious cases. Understanding their capabilities and limitations is therefore essential for successful implementation. To address this, we evaluated six commercial LFDs on the same collection of clinical samples and assessed their correlation with infectious virus culture and cycle threshold (Ct) values. MethodsA head-to-head comparison of specificities and sensitivities was performed on six commercial rapid antigen tests using combined nasal/oropharyngeal swabs, and their limits of detection determined using viral plaque forming units (PFU). Three of the LFDs were selected for a further study, correlating antigen test result with RT-PCR Ct values and positive viral culture in Vero-E6 cells. This included sequential swabs and matched serum samples obtained from four infected individuals with varying disease severities. Detection of antibodies was performed using an IgG/IgM Rapid Test Cassette, and neutralising antibodies by infectious virus assay. Finally, the sensitivities of selected rapid antigen LFTs were assessed in swabs with confirmed B.1.1.7 variant, currently the dominant genotype in the UK. FindingsMost of the rapid antigen LFDs showed a high specificity (>98%), and accurately detected 50 PFU/test (equivalent N1 Ct of 23.7 or RNA copy number of 3x106/ml). Sensitivities of the LFDs performed on clinical samples ranged from 65 to 89%. These sensitivities increased in most tests to over 90% for samples with Cts lower than 25. Positive virus culture was achieved for 57 out of 141 samples, with 80% of the positive cultures from swabs with Cts lower than 23. Importantly, sensitivity of the LFDs increased to over 95% when compared with the detection of infectious virus alone, irrespective of Ct. Longitudinal studies of PCR-positive samples showed that most of the tests identified all infectious samples as positive, but differences in test sensitivities can lead to missed cases in the absence of repeated testing. Finally, test performance was not impacted when re-assessed against swabs positive for the dominant UK variant B.1.1.7. InterpretationIn this comprehensive comparison of antigen LFD and virus infectivity, we demonstrate a clear relationship between Ct values, quantitative culture of infectious virus and antigen LFD positivity in clinical samples. Our data support regular testing of target groups using LFDs to supplement the current PCR testing capacity, to rapidly identify infected individuals in situations where they would otherwise go undetected. FundingKings Together Rapid COVID-19, Medical Research Council, Wellcome Trust, Huo Family Foundation.

Application of respiratory metagenomics for COVID-19 patients on the intensive care unit to inform appropriate initial antimicrobial treatment and rapid detection of nosocomial transmission

BackgroundClinical metagenomics (CMg) is being evaluated for translation from a research tool into routine diagnostic service, but its potential to significantly improve management of acutely unwell patients has not been demonstrated. The SARS-CoV-2 pandemic provides impetus to determine that benefit given increased risk of secondary infection and nosocomial transmission by multi-drug resistant (MDR) pathogens linked with expansion of critical care capacity. MethodsProspective evaluation of CMg using nanopore sequencing was performed on 43 respiratory samples over 14 weeks from a cohort of 274 intubated patients across seven COVID-19 intensive care units. ResultsBacteria or fungi were cultured from 200 (73%) patients, with a predominance of Klebsiella spp. (31%) and C. striatum (7%) amongst other common respiratory pathogens. An 8 hour CMg workflow was 93% sensitive and 81% specific for bacterial identification compared to culture, and reported presence or absence of {beta}-lactam resistance genes carried by Enterobacterales that would modify initial guideline-recommended antibiotics in every case. CMg was also 100% concordant with quantitative PCR for detecting Aspergillus fumigatus (4 positive and 39 negative samples). Single nucleotide polymorphism (SNP)-typing using 24 hour sequence data identified an MDR-K. pneumoniae ST307 outbreak involving 4 patients and an MDR-C. striatum outbreak potentially involving 14 patients across three ICUs. ConclusionCMg testing for ICU patients provides same-day pathogen detection and antibiotic resistance prediction that significantly improves initial treatment of nosocomial pneumonia and rapidly detects unsuspected outbreaks of MDR-pathogens.

Combined epidemiological and genomic analysis of nosocomial SARS-CoV-2 transmission identifies community social distancing as the dominant intervention reducing outbreaks

Many healthcare facilities report SARS-CoV-2 outbreaks but transmission analysis is complicated by the high prevalence of infection and limited viral genetic diversity. The contribution of different vectors to nosocomial infection or the effectiveness of interventions is therefore currently unclear. Detailed epidemiological and viral nanopore sequence data were analysed from 574 consecutive patients with a PCR positive SARS-CoV-2 test between March 13th and March 31st, when the pandemic first impacted on a large, multisite healthcare institution in London. During this time the first major preventative interventions were introduced, including progressive community social distancing (CSD) policies leading to mandatory national lockdown, exclusion of hospital visitors, and introduction of universal surgical facemask-use by healthcare-workers (HCW). Incidence of nosocomial cases, community SARS-CoV-2 cases and infection in a cohort of 228 HCWs followed the same dynamic course, decreasing shortly after introduction of CSD measures and prior to the main hospital-based interventions. We investigated clusters involving nosocomial cases based on overlapping ward-stays during the 14-day incubation period and SARS-CoV-2 genome sequence similarity. Our method placed 63 (79%) of 80 sequenced probable and definite nosocomial cases into 14 clusters containing a median of 4 patients (min 2, max 19) No genetic support was found for the majority of epidemiological clusters (31/44, 70%) and genomics revealed multiple contemporaneous outbreaks within single epidemiological clusters. We included a measure of hospital enrichment compared to community cases to increase confidence in our clusters, which were 1-14 fold enriched. Applying genomics, we could provide a robust estimate of the incubation period for nosocomial transmission, with a median lower bound and upper bound of 6 and 9 days respectively. Six (43%) clusters spanned multiple wards, with evidence of cryptic transmission, and community-onset cases could not be identified in more than half the clusters, particularly on the elective hospital site, implicating HCW as vectors of transmission. Taken together these findings suggest that CSD had the dominant impact on reducing nosocomial transmission by reducing HCW infection.

Automating the Generation of Antimicrobial Resistance Surveillance Reports: Proof-of-Concept Study Involving Seven Hospitals in Seven Countries.

BACKGROUND: Reporting cumulative antimicrobial susceptibility testing data on a regular basis is crucial to inform antimicrobial resistance (AMR) action plans at local, national, and global levels. However, analyzing data and generating a report are time consuming and often require trained personnel. OBJECTIVE: This study aimed to develop and test an application that can support a local hospital to analyze routinely collected electronic data independently and generate AMR surveillance reports rapidly. METHODS: An offline application to generate standardized AMR surveillance reports from routinely available microbiology and hospital data files was written in the R programming language (R Project for Statistical Computing). The application can be run by double clicking on the application file without any further user input. The data analysis procedure and report content were developed based on the recommendations of the World Health Organization Global Antimicrobial Resistance Surveillance System (WHO GLASS). The application was tested on Microsoft Windows 10 and 7 using open access example data sets. We then independently tested the application in seven hospitals in Cambodia, Lao People's Democratic Republic, Myanmar, Nepal, Thailand, the United Kingdom, and Vietnam. RESULTS: We developed the AutoMated tool for Antimicrobial resistance Surveillance System (AMASS), which can support clinical microbiology laboratories to analyze their microbiology and hospital data files (in CSV or Excel format) onsite and promptly generate AMR surveillance reports (in PDF and CSV formats). The data files could be those exported from WHONET or other laboratory information systems. The automatically generated reports contain only summary data without patient identifiers. The AMASS application is downloadable from https://www.amass.website/. The participating hospitals tested the application and deposited their AMR surveillance reports in an open access data repository. CONCLUSIONS: The AMASS is a useful tool to support the generation and sharing of AMR surveillance reports.

Clinical utility of targeted SARS-CoV-2 serology testing to aid the diagnosis and management of suspected missed, late or post-COVID-19 infection syndromes: results from a pilot service

OBJECTIVEDetermine indications and clinical utility of SARS-CoV-2 serology testing in adults and children. DESIGNProspective evaluation of initial three weeks of a daily Monday to Friday pilot SARS-CoV-2 serology service for patients. SETTINGEarly post "first-wave" SARS-CoV-2 transmission period at single centre London teaching hospital that provides care to the local community, as well as regional and national referral pathways for specialist services. PARTICIPANTS110 (72 adults, 38 children, age range 0-83 years, 52.7% female (n=58)). INTERVENTIONSPatient serum from vetted referrals tested on CE marked and internally validated lateral flow immunoassay (LFIA) (SureScreen Diagnostics) detecting antibodies to SARS-CoV-2 spike proteins, with result and clinical interpretation provided to the direct care team. MAIN OUTCOME MEASURESPerformance characteristics, source and nature of referrals, feasibility and clinical utility of the service, particularly the benefit for clinical decision-making. RESULTSThe LFIA was deemed suitable for clinical advice and decision making following evaluation with 310 serum samples from SARS-CoV-2 PCR positive patients and 300 pre-pandemic samples, giving a sensitivity and specificity of 96.1% and 99.3% respectively. For the pilot, 115 referrals were received leading to 113 tests performed on 108 participants (sample not available for two participants); paediatrics (n=35), medicine (n=69), surgery (n=2) and general practice (n=2). 43.4% participants (n=49) had detectable antibodies to SARS-CoV-2. There were three main indications for serology; new acute presentations potentially triggered by recent COVID-19 infection e.g. PIMS-TS (n=26) and pulmonary embolism (n=5), potential missed diagnoses in context of a recent compatible illness (n=40), and making infection control and immunosuppression treatment decisions in persistently SARS-CoV-2 RNA PCR positive individuals (n=6). CONCLUSIONSThis study shows acceptable performance characteristics, feasibility and clinical utility of a SARS-CoV-2 serology service using a rapid, inexpensive and portable assay for adults and children presenting with a range of clinical indications. Results correlated closely with a confirmatory in-house ELISA. The study showed the benefit of introducing a serology service where there is a reasonable pre-test probability, and the result can be linked with clinical advice or intervention. Experience thus far is that the volume of requests from hospital referral routes are manageable within existing clinical and laboratory services; however, the demand from community referrals has not yet been assessed. Given recent evidence for a rapid decline in antibodies, particularly following mild infection, there is likely a limited window of opportunity to realise the benefit of serology testing for individuals infected during the "first-wave" before they potentially fall below a measurable threshold. Rapidly expanding availability of serology services for NHS patients will also help understand the long-term implications of serostatus and prior infection in different patient groups, particularly before emergence of any "second-wave" outbreak or introduction of a vaccination programme. SUMMARY BOXO_ST_ABSWHAT IS ALREADY KNOWN ON THIS TOPICC_ST_ABSThe mechanisms and utility of providing a SARS-CoV-2 (COVID-19) serology service is under evaluation. There are different technologies detecting antibodies against different SARS-CoV-2 proteins. Antibodies are known to appear from about 10 days after symptom onset but it is unclear how long they persist. WHAT THIS STUDY ADDSA SARS-CoV-2 serology service using a validated lateral flow immunoassay measuring antibodies against spike protein can be rapidly introduced with clinical benefit demonstrated for a broad range of individuals. Indications include missed diagnoses where COVID-19 infection has been suspected but SARS-COV-2 RNA tests were either negative or not performed, conditions potentially triggered by COVID-19 such as pulmonary embolism, and predicting infectivity or immunity in patients with persistently detectable SARS-CoV-2 RNA. Testing is quick, simple to perform and inexpensive, however emerging evidence that antibodies fall rapidly particularly in mild disease, and the observed breadth of emerging indications highlight the urgent need for targeted testing with clinical interpretation provided on a case-by-case basis.

Longitudinal evaluation and decline of antibody responses in SARS-CoV-2 infection

Antibody (Ab) responses to SARS-CoV-2 can be detected in most infected individuals 10-15 days following the onset of COVID-19 symptoms. However, due to the recent emergence of this virus in the human population it is not yet known how long these Ab responses will be maintained or whether they will provide protection from re-infection. Using sequential serum samples collected up to 94 days post onset of symptoms (POS) from 65 RT-qPCR confirmed SARS-CoV-2-infected individuals, we show seroconversion in >95% of cases and neutralizing antibody (nAb) responses when sampled beyond 8 days POS. We demonstrate that the magnitude of the nAb response is dependent upon the disease severity, but this does not affect the kinetics of the nAb response. Declining nAb titres were observed during the follow up period. Whilst some individuals with high peak ID50 (>10,000) maintained titres >1,000 at >60 days POS, some with lower peak ID50 had titres approaching baseline within the follow up period. A similar decline in nAb titres was also observed in a cohort of seropositive healthcare workers from Guys and St Thomas Hospitals. We suggest that this transient nAb response is a feature shared by both a SARS-CoV-2 infection that causes low disease severity and the circulating seasonal coronaviruses that are associated with common colds. This study has important implications when considering widespread serological testing, Ab protection against re-infection with SARS-CoV-2 and the durability of vaccine protection.

Comparative assessment of multiple COVID-19 serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings

There is a clear requirement for an accurate SARS-CoV-2 antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. We therefore evaluated the performance of a variety of antibody testing technologies and their potential as diagnostic tools. A highly specific in-house ELISA was developed for the detection of anti-spike (S), -receptor binding domain (RBD) and -nucleocapsid (N) antibodies and used for the cross-comparison of ten commercial serological assays - a chemiluminescence-based platform, two ELISAs and seven colloidal gold lateral flow immunoassays (LFIAs) - on an identical panel of 110 SARS-CoV-2-positive samples and 50 pre-pandemic negatives. There was a wide variation in the performance of the different platforms, with specificity ranging from 82% to 100%, and overall sensitivity from 60.9% to 87.3%. However, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over 95% seen in several tests when assessing samples from more than 20 days post onset of symptoms. Furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. Rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of SARS-CoV-2 infections.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna(R) Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP (PHE guidelines). All RNA extraction methods provided similar results. FastVirus and Luna proved most sensitive. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrate that treatment of nasopharyngeal swabs with 70 degrees for 10 or 30 min, or 90 degrees for 10 or 30 min (both original variant and B 1.1.7) inactivates SARS-CoV-2 employing plaque assays, and that it has minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable to settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.