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The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely genomic sequencing of SARS-CoV-2 viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. Additionally, the enormous demand of genomic sequencing on supply chains and the resulting shortages of laboratory supplies necessitate the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method where the same protocol can be used in a STAT scenario, from sample to sequencer in as little as eight hours, and a high-throughput scenario, where one technologist can perform 576 library preparations over the course of one 8-hour shift. This new method uses Freed et al.s 1200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and truncated Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original, application of this new method in hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results suggest that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 whole genome sequencing protocol.
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BackgroundThe Canadian COVID-19 immunization strategy deferred second doses and allowed mixed schedules. We compared two-dose vaccine effectiveness (VE) by vaccine type (mRNA and/or ChAdOx1), interval between doses, and time since second dose in two of Canadas larger provinces. MethodsTwo-dose VE against infections and hospitalizations due to SARS-CoV-2, including variants of concern, was assessed between May 30 and October 2, 2021 using test-negative designs separately conducted among community-dwelling adults [≥]18-years-old in British Columbia (BC) and Quebec, Canada. FindingsIn both provinces, two doses of homologous or heterologous SARS-CoV-2 vaccines were associated with [~]95% reduction in the risk of hospitalization. VE exceeded 90% against SARS-CoV-2 infection when at least one dose was an mRNA vaccine, but was lower at [~]70% when both doses were ChAdOx1. Estimates were similar by age group (including adults [≥]70-years-old) and for Delta-variant outcomes. VE was significantly higher against both infection and hospitalization with longer 7-8-week vs. manufacturer-specified 3-4-week interval between doses. Two-dose mRNA VE was maintained against hospitalization for the 5-7-month monitoring period and while showing some decline against infection, remained [≥]80%. InterpretationTwo doses of mRNA and/or ChAdOx1 vaccines gave excellent protection against hospitalization, with no sign of decline by 5-7 months post-vaccination. A 7-8-week interval between doses improved VE and may be optimal in most circumstances. Findings indicate prolonged two-dose protection and support the use of mixed schedules and longer intervals between doses, with global health, equity and access implications in the context of recent third-dose proposals.
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IntroductionIn randomized controlled trials, single-dose efficacy against SARS-CoV-2 illness exceeded 90% for mRNA vaccines (BNT162b2 and mRNA-1273), and 75% for ChAdOx1. In British Columbia (BC), Canada second doses were deferred up to 16 weeks and ChAdOx1 was only initially recommended for adults 55 years of age and older. We compared single-dose vaccine effectiveness (VE) during the spring 2021 wave in BC when Alpha and Gamma variants of concern (VOC) predominated. MethodsVE was estimated against infection and hospitalization by test-negative design: cases were RT-PCR test-positive for SARS-CoV-2 and controls were test-negative. Adults 50-69 years old with specimen collection between April 4 and May 22 (weeks 14-20) were included. Variant-specific VE was estimated between weeks 17-20 when genetic characterization of all case viruses was performed, primarily through whole genome sequencing. ResultsVE analyses included 7,116 (10%) cases and 60,958 controls. Three-quarters of vaccinated participants received mRNA vaccine (60% BNT162b2, 15% mRNA-1273) and 25% received ChAdOx1. Half of genetically characterized viruses were Alpha, with 38% Gamma, 4% Delta and 8% non-VOCs. Single-dose VE against any infection was 75% (95%CI: 72-78) for BNT162b2, 82% (95%CI: 76-87) for mRNA-1273 and 61% (95%CI: 54-66) for ChAdOx1. VE against hospitalization was 83% (95%CI: 76-89), 85% (95%CI: 63-94) and 96% (95%CI: 86-99), respectively. VE against Alpha vs. Gamma infections did not differ among mRNA (78%;95%CI: 73-82 and 80%;95%CI: 74-85) or ChAdOx1 (66%;95%CI: 57-74 and 60%;95%CI: 48-69) recipients. ConclusionsA single dose of mRNA vaccine reduced the SARS-CoV-2 infection risk by at least 75%, including infections due to early VOC. Although effectiveness of a single dose of ChAdOx1 was lower at 60% against infection, just one dose of any vaccine reduced the hospitalization risk by more than 80%. In the context of constrained vaccine supplies, these findings have implications for global vaccine deployment to reduce the overall burden of infections and hospitalizations due to SARS-CoV-2.
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IntroductionRandomized-controlled trials of mRNA vaccine protection against SARS-CoV-2 included relatively few elderly participants. We assess singe-dose mRNA vaccine effectiveness (VE) in adults [≥]70-years-old in British Columbia (BC), Canada where the second dose was deferred by up to 16 weeks and where a spring 2021 wave uniquely included co-dominant circulation of B.1.1.7 and P.1 variants of concern (VOC). MethodsAnalyses included community-dwelling adults [≥]70-years-old with specimen collection between April 4 (epidemiological week 14) and May 1 (week 17). Adjusted VE was estimated by test-negative design through provincial laboratory and immunization data linkage. Cases were RT-PCR test-positive for SARS-CoV-2 and controls were test-negative. Vaccine status was defined by receipt of a single-dose [≥]21 days before specimen collection, but a range of intervals was assessed. In variant-specific analyses, test-positive cases were restricted to those genetically-characterized as B.1.1.7, P.1 or non-VOC. ResultsVE analyses included 16,993 specimens: 1,226 (7.2%) test-positive cases and 15,767 test-negative controls. Of 1,131 (92%) viruses genetically categorized, 509 (45%), 314 (28%) and 276 (24%) were B.1.1.7, P.1 and non-VOC lineages, respectively. VE was negligible at 14% (95% CI 0-26) during the period 0-13 days post-vaccination but increased from 43% (95% CI 30-53) at 14-20 days to 75% (95% CI 63-83) at 35-41 days post-vaccination. VE at [≥]21 days was 65% (95% CI 58-71) overall: 72% (95% CI 58-81), 67% (95% CI 57-75) and 61% (95% CI 45-72) for non-VOC, B.1.1.7 and P.1, respectively. ConclusionsA single dose of mRNA vaccine reduced the risk of SARS-CoV-2 in adults [≥]70-years-old by about two-thirds, with protection only minimally reduced against B.1.1.7 and P.1 variants. Substantial single-dose protection in older adults reinforces the option to defer the second dose when vaccine supply is scarce and broader first-dose coverage is needed.
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Mutations in emerging SARS-CoV-2 lineages can interfere with the laboratory methods used to generate high-quality genome sequences for COVID-19 surveillance. Here, we identify 46 mutations in current variant of concern lineages affecting the widely used laboratory protocols for SARS-CoV-2 genomic sequencing by Freed et al. and the ARTIC network. We provide laboratory data showing how three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada, and we also demonstrate how we modified the Freed et al. protocol to restore performance.
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Wastewater-based genomic surveillance of the SARS-CoV-2 virus shows promise to complement genomic epidemiology efforts. Multiplex tiled PCR is a desirable approach for targeted genome sequencing of SARS-CoV-2 in wastewater due to its low cost and rapid turnaround time. However, it is not clear how different multiplex tiled PCR primer schemes or wastewater sample matrices impact the resulting SARS-CoV-2 genome coverage. The objective of this work was to assess the performance of three different multiplex primer schemes, consisting of 150bp, 400bp, and 1200bp amplicons, as well as two wastewater sample matrices, influent wastewater and primary sludge, for targeted genome sequencing of SARS-CoV-2. Wastewater samples were collected weekly from five municipal wastewater treatment plants (WWTPs) in the Metro Vancouver region of British Columbia, Canada during a period of increased COVID-19 case counts from February to April, 2021. RNA extracted from clarified influent wastewater provided significantly higher genome coverage (breadth and median depth) than primary sludge samples across all primer schemes. Shorter amplicons appeared more resilient to sample RNA degradation, but were hindered by greater primer pool complexity in the 150bp scheme. The identified optimal primer scheme (400bp) and sample matrix (influent) was capable of detecting the emergence of mutations associated with genomic variants of concern, of which the daily wastewater load significantly correlated with clinical case counts. Taken together, these results provide guidance on best practices for implementing wastewater-based genomic surveillance, and demonstrate its ability to inform epidemiology efforts by detecting genomic variants of concern circulating within a geographic region. ImportanceMonitoring the genomic characteristics of the SARS-CoV-2 virus circulating in a population can shed important insights into epidemiological aspects of the COVID-19 outbreak. Sequencing every clinical patient sample in a highly populous area is a difficult feat, and thus sequencing SARS-CoV-2 RNA in municipal wastewater offers great promise to augment genomic surveillance by characterizing a pooled population sample matrix, particularly during an escalating outbreak. Here, we assess different approaches and sample matrices for rapid targeted genome sequencing of SARS-CoV-2 in municipal wastewater. We demonstrate that the optimal approach is capable of detecting the emergence of SARS-CoV-2 genomic variants of concern, with strong correlations to clinical case data in the province of British Columbia. These results provide guidance on best practices on, as well as further support for, the application of wastewater genomic surveillance as a tool to augment current genomic epidemiology efforts.
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Comprehensive and timely testing is required for SARS-CoV-2 variant of concern (VoC) screening. Whole genome sequencing (WGS) provides the broadest means to detect circulating VoCs, but requires longer turnaround time than targeted molecular testing by quantitative polymerase chain reaction (qPCR). We demonstrated the feasibility of a combined testing approach for VoC prevalence assessment in British Columbia, and showed high concordance between qPCR testing and WGS. This directly informed wider VoC screening strategy implementation, and public health efforts.
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Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to {pound}10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.
