Rapid screening of SARS-CoV-2 variants, an important tool for pandemic surveillance

setting and design

This observational study was conducted at the 12 October University Hospital (Madrid, Spain), a tertiary reference hospital for SARS-CoV-2 variant analysis. This study was conducted in accordance with the 2013 revised Declaration of Helsinki. Regarding the protection and assurance of digital data, no research ethics committee approval was required, as stated in the Organic Law 3/2018 enacted on 5 December. right. This law allows health authorities and public bodies with public health oversight powers to conduct scientific research without the consent of the data subject in situations of exceptional relevance and seriousness to public health.

In 2021, prospective SARS-CoV-2 variant analysis will be performed on most of the initial positive samples, depending on the number of positive samples obtained during the peak wave and the availability of reagents. I was. Mutant screening was performed over a total of 22 weeks (1–11 weeks, 28 ~37 weeks, and 50–52 weeks). ). In the remaining 30 weeks (weeks 12-27 and 38-49), all positive initial samples were subjected to variant screening..

Research procedure

Analysis of SARS-CoV-2 variants by RT-PCR was performed using different diagnostic tests based on their availability and utility in detecting emerging variants. A summary of the reagents used throughout the study is presented in the table. 1.

Results were interpreted as: alpha mutants (S gene deletion or N501Y and del H69/V70 positive). Beta/gamma (positive for N501Y and E484K). delta (plus on L452R). Omicron (positive for N501Y, del H69/V70, and K417N).

Whole-genome sequencing (WGS) is considered the gold standard technique and was performed on samples previously analyzed for variants using RT–PCR and had Ct values ​​<25. The decision to perform WGS also depended on the number of positive samples obtained. Wave peak times and availability of reagents. As a result, WGS was performed on 10.8% of samples that underwent SARS-CoV-2 variant analysis by RT-PCR. Furthermore, when it was not possible to perform WGS on all samples previously analyzed by RT-PCR (during the peak wave), the selection of samples for WGS was based on random criteria. These selection processes ensured representation from a diverse set of samples. WGS was performed from original samples using the Ion Torrent (Thermo Fisher Scientific, Waltham, MA, USA) or ABL SA Group (Luxembourg/Illumina, San Diego, CA, USA) platform, depending on reagent availability. was done (table 1).Both methods followed his ARTIC nCoV-2019 sequencing protocol⁷. Specifically, the Ion Torrent platform performed base calling, trimming, and quality control using the Ion Reporter™ software’s built-in pipeline. The reads were then mapped against the reference genome Wuhan-Hu-1 (GenBank accession number: MN908947.3) using the IRMA assembler.⁸ Plugin for Ion Reporter™ software (ThermoFisher Scientic, Carlsbad, CA, USA). Variant calling was performed in parallel with his Variant Caller plugin for Ion Reporter™ software and Snippy v.4.6.0.⁹, with default settings (minimum coverage 10-fold, minimum allele frequency 90%). Variant annotation was based on the reference genome. The Variant Caller plugin was also used to search for minor variants. To filter these, Geneious Prime software (version 2020.0.4, Biomatters Ltd., New Zealand) was used to map reads to reference strains and manually check for variants on the alignment. All variants with low coverage, present in less than 15% of reads, located near the ends of reads (amplicons), or with poor read quality were discarded. A variant identified as problematic by De Maio et al.^Ten was also discarded. Various stages of the pipeline were run on the ABL platform using MicrobioChek software (ABL SA Group, Luxembourg).¹¹. Strains were assigned according to the PANGO scheme¹² Powered by Pangolin 2.0 web app¹³the NextStrain era scheme on the NextClade web page¹⁴.All SARS-CoV-2 sequences were deposited in the GISAID database¹⁵ (Code: h-CoV-19/Spain/MD-H12O).

statistical analysis

Qualitative variables are displayed as numbers and percentages. The figure was created using GraphPad Prism version 6.