Real-time whole genome sequencing to guide patient-tailored therapy of SARS-CoV-2 infection.

The management of COVID-19 has become more complex due to the expansion of available therapies. The presence of SARS-CoV-2 variants and mutations further complicate treatment due to their differing susceptibilities to therapies. Here we outline the use of real-time whole genome sequencing to characterise infections and guide treatment decisions.

Broad Neutralization of SARS-CoV-2 Variants, Including Omicron, following Breakthrough Infection with Delta 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. IMPORTANCE COVID-19 vaccines have been vital in controlling SARS-CoV-2 infections and reducing hospitalizations. However, breakthrough SARS-CoV-2 infections (BTI) occur in some vaccinated individuals. Here, we study how BTI impacts on the potency and the breadth of the neutralizing antibody response. We show that a Delta infection in COVID-19-vaccinated individuals provides potent neutralization against the current SARS-CoV-2 variants of concern, including the Omicron variant.

Descriptive comparison of admission characteristics between pandemic waves and multivariable analysis of the association of the Alpha variant (B.1.1.7 lineage) of SARS-CoV-2 with disease severity in inner London.

BACKGROUND: The Alpha variant (B.1.1.7 lineage) of SARS-CoV-2 emerged and became the dominant circulating variant in the UK in late 2020. Current literature is unclear on whether the Alpha variant is associated with increased severity. We linked clinical data with viral genome sequence data to compare admitted cases between SARS-CoV-2 waves in London and to investigate the association between the Alpha variant and the severity of disease. METHODS: Clinical, demographic, laboratory and viral sequence data from electronic health record systems were collected for all cases with a positive SARS-CoV-2 RNA test between 13 March 2020 and 17 February 2021 in a multisite London healthcare institution. Multivariate analysis using logistic regression assessed risk factors for severity as defined by hypoxia at admission. RESULTS: There were 5810 SARS-CoV-2 RNA-positive cases of which 2341 were admitted (838 in wave 1 and 1503 in wave 2). Both waves had a temporally aligned rise in nosocomial cases (96 in wave 1 and 137 in wave 2). The Alpha variant was first identified on 15 November 2020 and increased rapidly to comprise 400/472 (85%) of sequenced isolates from admitted cases in wave 2. A multivariate analysis identified risk factors for severity on admission, such as age (OR 1.02, 95% CI 1.01 to 1.03, for every year older; p<0.001), obesity (OR 1.70, 95% CI 1.28 to 2.26; p<0.001) and infection with the Alpha variant (OR 1.68, 95% CI 1.26 to 2.24; p<0.001). CONCLUSIONS: Our analysis is the first in hospitalised cohorts to show increased severity of disease associated with the Alpha variant. The number of nosocomial cases was similar in both waves despite the introduction of many infection control interventions before wave 2.

Evaluating the potential for respiratory metagenomics to improve treatment of secondary infection and detection of nosocomial transmission on expanded COVID-19 intensive care units.

BACKGROUND: Clinical metagenomics (CMg) has the potential to be translated from a research tool into routine service to improve antimicrobial treatment and infection control decisions. The SARS-CoV-2 pandemic provides added impetus to realise these benefits, given the increased risk of secondary infection and nosocomial transmission of multi-drug-resistant (MDR) pathogens linked with the expansion of critical care capacity. METHODS: CMg using nanopore sequencing was evaluated in a proof-of-concept study on 43 respiratory samples from 34 intubated patients across seven intensive care units (ICUs) over a 9-week period during the first COVID-19 pandemic wave. RESULTS: An 8-h CMg workflow was 92% sensitive (95% CI, 75-99%) and 82% specific (95% CI, 57-96%) for bacterial identification based on culture-positive and culture-negative samples, respectively. CMg sequencing reported the presence or absence of ß-lactam-resistant genes carried by Enterobacterales that would modify the initial guideline-recommended antibiotics in every case. CMg was also 100% concordant with quantitative PCR for detecting Aspergillus fumigatus from 4 positive and 39 negative samples. Molecular typing using 24-h sequencing data identified an MDR-K. pneumoniae ST307 outbreak involving 4 patients and an MDR-C. striatum outbreak involving 14 patients across three ICUs. CONCLUSION: CMg testing provides accurate pathogen detection and antibiotic resistance prediction in a same-day laboratory workflow, with assembled genomes available the next day for genomic surveillance. The provision of this technology in a service setting could fundamentally change the multi-disciplinary team approach to managing ICU infections. The potential to improve the initial targeted treatment and rapidly detect unsuspected outbreaks of MDR-pathogens justifies further expedited clinical assessment of CMg.

Neutralizing antibody activity in convalescent sera from infection in humans with SARS-CoV-2 and variants of concern.

COVID-19 vaccine design and vaccination rollout need to take into account a detailed understanding of antibody durability and cross-neutralizing potential against SARS-CoV-2 and emerging variants of concern (VOCs). Analyses of convalescent sera provide unique insights into antibody longevity and cross-neutralizing activity induced by variant spike proteins, which are putative vaccine candidates. Using sera from 38 individuals infected in wave 1, we show that cross-neutralizing activity can be detected up to 305 days pos onset of symptoms, although sera were less potent against B.1.1.7 (Alpha) and B1.351 (Beta). Over time, despite a reduction in overall neutralization activity, differences in sera neutralization potency against SARS-CoV-2 and the Alpha and Beta variants decreased, which suggests that continued antibody maturation improves tolerance to spike mutations. We also compared the cross-neutralizing activity of wave 1 sera with sera from individuals infected with the Alpha, the Beta or the B.1.617.2 (Delta) variants up to 79 days post onset of symptoms. While these sera neutralize the infecting VOC and parental virus to similar levels, cross-neutralization of different SARS-CoV-2 VOC lineages is reduced. These findings will inform the optimization of vaccines to protect against SARS-CoV-2 variants.

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® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in 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/.

Combined epidemiological and genomic analysis of nosocomial SARS-CoV-2 infection early in the pandemic and the role of unidentified cases in transmission.

OBJECTIVES: To analyse nosocomial transmission in the early stages of the coronavirus 2019 (COVID-19) pandemic at a large multisite healthcare institution. Nosocomial incidence is linked with infection control interventions. METHODS: Viral genome sequence and epidemiological data were analysed for 574 consecutive patients, including 86 nosocomial cases, with a positive PCR test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the first 19 days of the pandemic. RESULTS: Forty-four putative transmission clusters were found through epidemiological analysis; these included 234 cases and all 86 nosocomial cases. SARS-CoV-2 genome sequences were obtained from 168/234 (72%) of these cases in epidemiological clusters, including 77/86 nosocomial cases (90%). Only 75/168 (45%) of epidemiologically linked, sequenced cases were not refuted by applying genomic data, creating 14 final clusters accounting for 59/77 sequenced nosocomial cases (77%). Viral haplotypes from these clusters were enriched 1-14x (median 4x) compared to the community. Three factors implicated unidentified cases in transmission: (a) community-onset or indeterminate cases were absent in 7/14 clusters (50%), (b) four clusters (29%) had additional evidence of cryptic transmission, and (c) in three clusters (21%) diagnosis of the earliest case was delayed, which may have facilitated transmission. Nosocomial cases decreased to low levels (0-2 per day) despite continuing high numbers of admissions of community-onset SARS-CoV-2 cases (40-50 per day) and before the impact of introducing universal face masks and banning hospital visitors. CONCLUSION: Genomics was necessary to accurately resolve transmission clusters. Our data support unidentified cases-such as healthcare workers or asymptomatic patients-as important vectors of transmission. Evidence is needed to ascertain whether routine screening increases case ascertainment and limits nosocomial transmission.

Comparative performance of SARS-CoV-2 lateral flow antigen tests and association with detection of infectious virus in clinical specimens: a single-centre laboratory evaluation study.

BACKGROUND: Lateral flow devices (LFDs) for rapid antigen testing are set to become a cornerstone of SARS-CoV-2 mass community testing, although their reduced sensitivity compared with PCR has raised questions of how well they identify infectious cases. Understanding their capabilities and limitations is, therefore, essential for successful implementation. We evaluated six commercial LFDs and assessed their correlation with infectious virus culture and PCR cycle threshold (Ct) values. METHODS: In a single-centre, laboratory evaluation study, we did a head-to-head comparison of six LFDs commercially available in the UK: Innova Rapid SARS-CoV-2 Antigen Test, Spring Healthcare SARS-CoV-2 Antigen Rapid Test Cassette, E25Bio Rapid Diagnostic Test, Encode SARS-CoV-2 Antigen Rapid Test Device, SureScreen COVID-19 Rapid Antigen Test Cassette, and SureScreen COVID-19 Rapid Fluorescence Antigen Test. We estimated the specificities and sensitivities of the LFDs using stored naso-oropharyngeal swabs collected at St Thomas' Hospital (London, UK) for routine diagnostic SARS-CoV-2 testing by real-time RT-PCR (RT-rtPCR). Swabs were from inpatients and outpatients from all departments of St Thomas' Hospital, and from health-care staff (all departments) and their household contacts. SARS-CoV-2-negative swabs from the same population (confirmed by RT-rtPCR) were used for comparative specificity determinations. All samples were collected between March 23 and Oct 27, 2020. We determined the limit of detection (LOD) for each test using viral plaque-forming units (PFUs) and viral RNA copy numbers of laboratory-grown SARS-CoV-2. Additionally, LFDs were selected to assess the correlation of antigen test result with RT-rtPCR Ct values and positive viral culture in Vero E6 cells. This analysis included longitudinal swabs from five infected inpatients with varying disease severities. Furthermore, the sensitivities of available LFDs were assessed in swabs (n=23; collected from Dec 4, 2020, to Jan 12, 2021) confirmed to be positive (RT-rtPCR and whole-genome sequencing) for the B.1.1.7 variant, which was the dominant genotype in the UK at the time of study completion. FINDINGS: All LFDs showed high specificity (≥98·0%), except for the E25Bio test (86·0% [95% CI 77·9-99·9]), and most tests reliably detected 50 PFU/test (equivalent SARS-CoV-2 N gene Ct value of 23·7, or RNA copy number of 3 × 106/mL). Sensitivities of the LFDs on clinical samples ranged from 65·0% (55·2-73·6) to 89·0% (81·4-93·8). These sensitivities increased to greater than 90% for samples with Ct values of lower than 25 for all tests except the SureScreen fluorescence (SureScreen-F) test. Positive virus culture was identified in 57 (40·4%) of 141 samples; 54 (94·7%) of the positive cultures were from swabs with Ct values lower than 25. Among the three LFDs selected for detailed comparisons (the tests with highest sensitivity [Innova], highest specificity [Encode], and alternative technology [SureScreen-F]), sensitivity of the LFDs increased to at least 94·7% when only including samples with detected viral growth. Longitudinal studies of RT-rtPCR-positive samples (tested with Innova, Encode, and both SureScreen-F and the SureScreen visual [SureScreen-V] test) showed that most of the tests identified all infectious samples as positive. Test performance (assessed for Innova and SureScreen-V) was not affected when reassessed on swabs positive for the UK variant B.1.1.7. INTERPRETATION: In this comprehensive comparison of antigen LFDs and virus infectivity, we found 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 with LFDs to supplement the current PCR testing capacity, which would help to rapidly identify infected individuals in situations in which they would otherwise go undetected. FUNDING: King's Together Rapid COVID-19, Medical Research Council, Wellcome Trust, Huo Family Foundation, UK Department of Health, National Institute for Health Research Comprehensive Biomedical Research Centre.

Development and evaluation of a nanopore 16S rRNA gene sequencing service for same day targeted treatment of bacterial respiratory infection in the intensive care unit.

OBJECTIVES: Assess the feasibility and impact of nanopore-based 16S rRNA gene sequencing (Np16S) service on antibiotic treatment for acute severe pneumonia on the intensive care unit (ICU). METHODS: Speciation and sequencing accuracy of Np16S on isolates with bioinformatics pipeline optimisation, followed by technical evaluation including quality checks and clinical-reporting criteria analysing stored respiratory samples using single-sample flow cells. Pilot service comparing Np16S results with all routine respiratory tests and impact on same-day antimicrobial prescribing. RESULTS: Np16S correctly identified 140/167 (84%) isolates after 1h sequencing and passed quality control criteria including reproducibility and limit-of-detection. Sequencing of 108 stored respiratory samples showed concordance with routine culture in 80.5% of cases and established technical and clinical reporting criteria. A 10-week same-day pilot Np16S service analysed 45 samples from 37 patients with suspected community (n=15) or hospital acquired (n=30) pneumonia. Np16S showed concordance compared with all routine culture or molecular tests for 27 (82%) of 33 positive samples. It identified the causative pathogen in 32/33 (97%) samples and contributed to antimicrobial treatment changes for 30 patients (67%). CONCLUSIONS: This study demonstrates feasibility of providing a routine same-day nanopore sequencing service that makes a significant contribution to early antibiotic prescribing for bacterial pneumonia in the ICU.

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 implemented during the first pandemic wave.

During the first wave of the global COVID-19 pandemic the clinical utility and indications for SARS-CoV-2 serological testing were not clearly defined. The urgency to deploy serological assays required rapid evaluation of their performance characteristics. We undertook an internal validation of a CE marked lateral flow immunoassay (LFIA) (SureScreen Diagnostics) using serum from SARS-CoV-2 RNA positive individuals and pre-pandemic samples. This was followed by the delivery of a same-day named patient SARS-CoV-2 serology service using LFIA on vetted referrals at central London teaching hospital with clinical interpretation of result provided to the direct care team. Assay performance, source and nature of referrals, feasibility and clinical utility of the service, particularly benefit in clinical decision-making, were recorded. Sensitivity and specificity of LFIA were 96.1% and 99.3% respectively. 113 tests were performed on 108 participants during three-week pilot. 44% participants (n = 48) had detectable antibodies. Three main indications were identified for serological testing; new acute presentations potentially triggered by recent COVID-19 e.g. pulmonary embolism (n = 5), potential missed diagnoses in context of a recent COVID-19 compatible illness (n = 40), and making infection control or immunosuppression management decisions in persistently SARS-CoV-2 RNA PCR positive individuals (n = 6). We demonstrate acceptable performance characteristics, feasibility and clinical utility of using a LFIA that detects anti-spike antibodies to deliver SARS-CoV-2 serology service in adults and children. Greatest benefit was seen where there is reasonable pre-test probability and results can be linked with clinical advice or intervention. Experience from this pilot can help inform practicalities and benefits of rapidly implementing new tests such as LFIAs into clinical service as the pandemic evolves.

Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans.

Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10-15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting 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 use as diagnostic tools. Highly specific in-house ELISAs were 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.

A comparative evaluation between the Abbott Panbio™ COVID-19 IgG/IgM rapid test device and Abbott Architect™ SARS CoV-2 IgG assay.

INTRODUCTION: Antibodies to SARS-CoV-2 serve as critical diagnostic markers for determining how broadly the COVID-19 pandemic has spread, confirming patient recovery, monitoring potential long-term effects of infection, and evaluating potential protection from reinfection. As new antibody tests become available, it is important to evaluate their performance and utility. The aim of this study was to compare the performance of the Abbott PanbioTM COVID-19 IgG/IgM Rapid Test Device against the Abbott ArchitectTM SARS CoV-2 IgG Assay for the detection of the COVID-19 IgG antibody. METHODS: Two panels of specimens were utilized to challenge both antibody tests: (1) a set of 150 prepandemic negative specimens collected in 2014, and (2) a set of 122 specimens from 87 hospitalized COVID-19 patients in the US and UK that were confirmed with a positive SARS-CoV-2 RNA test result. RESULTS: The ArchitectTM test had a specificity of 100 % and sensitivity of 99.1 % and 93.9 % when excluding or including immunocompromised patients, respectively for specimens collected >14 days post symptom onset or >5 days post-RNA testing. The PanbioTM test had 99.3 % agreement to ArchitectTM. Notably, N = 6 immune-compromised individuals were identified that did not develop detectable antibodies by day 30. CONCLUSION: There is good concordance between the ArchitectTM SARS CoV-2 IgG Assay and PanbioTM COVID-19 IgG/IgM Rapid Test Device for the detection of SARS CoV-2 IgG.

Real-world evaluation of a novel technology for quantitative simultaneous antibody detection against multiple SARS-CoV-2 antigens in a cohort of patients presenting with COVID-19 syndrome.

An evaluation of a rapid portable gold-nanotechnology measuring SARS-CoV-2 IgM, IgA and IgG antibody concentrations against spike 1 (S1), spike 2 (S) and nucleocapsid (N) was conducted using serum samples from 74 patients tested for SARS-CoV-2 RNA on admission to hospital, and 47 historical control patients from March 2019. 59 patients were RNA(+) and 15 were RNA(-). A serum (±) classification was derived for all three antigens and a quantitative serological profile was obtained. Serum(+) was identified in 30% (95% CI 11-48) of initially RNA(-) patients, in 36% (95% CI 17-54) of RNA(+) patients before 10 days, 77% (95% CI 67-87) between 10 and 20 days and 95% (95% CI 86-100) after 21 days. The patient-level diagnostic accuracy relative to RNA(±) after 10 days displayed 88% sensitivity (95% CI 75-95) and 75% specificity (95% CI 22-99), although specificity compared with historical controls was 100% (95%CI 91-100). This study provides robust support for further evaluation and validation of this novel technology in a clinical setting and highlights challenges inherent in assessment of serological tests for an emerging disease such as COVID-19.