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1.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.10.09.21264695

ABSTRACT

The SARS-CoV-2 ARTIC amplicon protocol is the most widely used genome sequencing method for SARS-CoV-2, accounting for over 43% of publicly-available genome sequences. The protocol utilises 98 primers to amplify ~400bp fragments of the SARS-CoV-2 genome covering all 30,000 bases. Understanding the analytical performance metrics of this protocol will improve how the data is used and interpreted. Different concentrations of SARS-CoV-2 control material were used to establish the limit of detection (LoD) of the ARTIC protocol. Results demonstrated the LoD was a minimum of 25-50 virus particles per mL. The sensitivity of ARTIC was comparable to the published sensitivities of commercial diagnostics assays and could therefore be used to confirm diagnostic testing results. A set of over 3,600 clinical samples from three UK regions were then evaluated to compare the protocols performance to clinical diagnostic assays (Roche Lightcycler 480 II, AusDiagnostics, Roche Cobas, Hologic Panther, Corman RdRp, Roche Flow, ABI QuantStudio 5, Seegene Nimbus, Qiagen Rotorgene, Abbott M2000, Thermo TaqPath, Xpert). We developed a Python tool, RonaLDO, to perform this validation (available under the GNU GPL3 open-source licence from https://github.com/quadram-institute-bioscience/ronaldo). Positives detected by diagnostic platforms were generally supported by sequencing data; platforms that used RT-qPCR were the best predictors of whether the sample would subsequently sequence successfully. To maximise success of sample sequencing for phylogenetic analysis, samples with Ct <31 should be chosen. For diagnostic tests that do not provide a quantifiable Ct value, adding a quantification step is recommended. The ARTIC SARS-CoV-2 sequencing protocol is highly sensitive, capable of detecting SARS-CoV-2 in samples with Cts in the high 30s. However, to routinely obtain whole genome coverage, samples with Ct <31 are recommended. Comparing different virus detection methods close to their LoD was challenging and significant discordance was observed.

2.
ssrn; 2021.
Preprint in English | PREPRINT-SSRN | ID: ppzbmed-10.2139.ssrn.3864067

ABSTRACT

Background: Controlling the spread of the COVID-19 pandemic relies heavily on worldwide immunization programs. Most of the current approved vaccines utilise a two-dose strategy to prime-boost the immune system. To enable larger groups of patients to receive the first dose, the UK government increased the gap between the two doses from three to 12 weeks. Here we report on the immunogenicity of the first dose.Methods: Anti SARS-CoV-2 IgG production (quantitative Abbott immunoassays) and vitamin D (25-hydroxyvitamin D (25(OH)D; LCMS) and 1-α,25-dihydroxyvitamin D (1,25(OH) 2 D, LCMS)) were measured in a group of 105 HCW, post immunization with the Pfizer/BioNTech vaccine.Findings: The anti-Spike IgG Ab response showed minimal change within the first week, rapidly peaked 3·2 weeks post immunization, subsequently decreasing to the nadir at 8 weeks prior to the scheduled second dose being administered. All subjects who responded with positive anti-Spike IgG Abs, retained significant levels at 8 weeks. Response to immunization was significantly modified by age and replete vitamin D status (25(OH)D>50 nmol/L).Interpretation: The results indicate that in this cohort 1) Ab levels are greatest 3-4 weeks post the first dose of the Pfizer/BioNTech vaccine 2) Abs are still circulating eight weeks post first dose 3) age significantly modifies the initial Ab response, the youngest decile having a greater peak and faster decline, resulting in 4) no significant difference in Ab concentration with age at W8 and 5) baseline vitamin D affected the peak of response, with vitamin D replete individuals showing the strongest Ab response.Funding Information: Abbott Diagnostics Ltd supplied the kits used to quantify the anti-SARS -CoV-2 Spike IgG and technical support as well as provided financial support for sample collections.Declaration of Interests: Abbott Diagnostics Ltd provided the necessary immunoassay for analysis of the samples for SARS-CoV-2 IgG and financial support for collection of samples. SR and MB work at Abbott Diagnostics Ltd. All other authors have no conflict of interest. Ethics Approval Statement: All participants provided written informed consent. The study was approved by the Health Research Authority Health and Care Research Wales ethical committee (IRAS#292799).


Subject(s)
COVID-19
3.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.09.28.20201475

ABSTRACT

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3,200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organisations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1,565 positive samples (172 per 100,000 population) from 1,376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6% of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. 1,035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically-distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a sublineage associated with 6 care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients indicating infection control measures were effective; found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.


Subject(s)
COVID-19
4.
biorxiv; 2020.
Preprint in English | bioRxiv | ID: ppzbmed-10.1101.2020.06.24.162156

ABSTRACT

The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 24 samples at a time. There is a need for a higher throughput method to reduce cost per genome. Here we present CoronaHiT, a method capable of multiplexing up to 95 small genomes on a single Nanopore flowcell, which uses transposase mediated addition of adapters and PCR based addition of symmetric barcodes. We demonstrate the method using 48 and 94 SARS-CoV-2 genomes per flowcell, amplified using the ARTIC protocol, and compare performance with Illumina and ARTIC ONT sequencing. Results demonstrate that all sequencing methods produce inaccurate genomes when the RNA extract from SARS-CoV-2 positive clinical sample has a cycle threshold (Ct) >= 32. Results from set same set of 23 samples with a broad range of Cts show that the consensus genomes have >90% coverage (GISAID criteria) for 78.2% of samples for CoronaHiT-48, 73.9% for CoronaHiT-94, 78.2% for Illumina and 73.9% for ARTIC ONT, and all have the same clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 94 SARS-CoV-2 genomes per nanopore flowcell without compromising the quality of the resulting genomes while reducing library preparation complexity and significantly reducing cost. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.


Subject(s)
COVID-19
5.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.06.11.20128306

ABSTRACT

We evaluated the FDA approved SARS-CoV-2 immunoassay (developed at Mount Sinai, by Krammer and colleagues) for the identification of COVID-19 seroconversion and potential cross-reactivity of the assay in a United Kingdom (UK) National Health Service (NHS) hospital setting. In our "set up" cohort we found that the SARS-CoV-2 IgG was detectable in 100% of patients tested 14 days post positive COVID-19 nucleic acid test. Serum samples taken from pregnant women in 2018 were used as a negative control group with zero false positives. We also analysed samples from patients with non-COVID-19 viral infections, paraproteinaemia or autoantibodies and found false positive results in 6/179. Modification of the sensitivity threshold to five standard deviations from the mean of the control group eliminated all false positive result in the set up cohort. We confirmed the validity of the test with a revised threshold on an independent prospective "validation cohort" of patient samples. Taking data from both cohorts we report a sensitivity of the Mount Sinai assay of 96.6% (28/29) and specificity of 100% (299/299) using a revised threshold cut-off, at a time point at least 14 days since the diagnostic antigen test. Finally, we conducted a health economic probabilistic sensitivity analysis (PSA) on the costs of producing the tests, and the mean cost we estimate to be 13.63 pounds sterling (95%CI 9.63 - 18.40), allowing its cost effectiveness to be tested against other antibody tests. In summary, we report that the Mount Sinai IgG ELISA assay is highly sensitive test for SARS-Cov-2 infection, however modification of thresholding was required to minimise false positive results.


Subject(s)
COVID-19 , Virus Diseases
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