RESUMO
To control the SARS-CoV-2 pandemic, healthcare systems have focused on ramping up their capacity for epidemiological surveillance through viral whole genome sequencing. In this paper, we tested the performance of two protocols of SARS-CoV-2 nucleic acid enrichment, an amplicon enrichment using different versions of the ARTIC primer panel and a hybrid-capture method using KAPA RNA Hypercap. We focused on the challenge of the Omicron variant sequencing, the advantages of automated library preparation and the influence of the bioinformatic analysis in the final consensus sequence. All 94 samples were sequenced using Illumina iSeq 100 and analysed with two bioinformatic pipelines: a custom-made pipeline and an Illumina-owned pipeline. We were unsuccessful in sequencing six samples using the capture enrichment due to low reads. On the other hand, amplicon dropout and mispriming caused the loss of mutation G21987A and the erroneous addition of mutation T15521A respectively using amplicon enrichment. Overall, we found high sequence agreement regardless of method of enrichment, bioinformatic pipeline or the use of automation for library preparation in eight different SARS-CoV-2 variants. Automation and the use of a simple app for bioinformatic analysis can simplify the genotyping process, making it available for more diagnostic facilities and increasing global vigilance.
Assuntos
COVID-19 , Sequenciamento de Nucleotídeos em Larga Escala , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Humanos , COVID-19/epidemiologia , COVID-19/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Genoma Viral , RNA Viral/genética , Mutação , Monitoramento Epidemiológico , Biologia Computacional/métodos , Sequenciamento Completo do Genoma/métodosRESUMO
BACKGROUND: To infer a reliable SARS-CoV-2 antibody protection level from a serological test, an appropriate quantitative threshold and solid equivalence across serological tests are needed. Additionally, tests should show a solid correlation with neutralising assays and with the protection observed in large population cohorts even against emerging variants. OBJECTIVES: We studied convalescent and vaccinated populations using 11 commercial antibody assays. Results were compared to evaluate discrepancies across tests. Neutralisation capacity was measured in a subset of the samples with a lentiviral-based assay. METHODS: Serum from convalescent (n = 121) and vaccinated individuals (n = 471, 260 with Comirnaty, 110 with Spikevax, and 96 with Vaxzevria) was assessed using 11 different assays, including two from Abbott, Euroimmun, Liaison, Roche, and Vircell, and one from Siemens. A spike protein-lentiviral vector with a fluorescent reporter was used for neutralisation assay of serum from convalescent (n = 26) and vaccinated (n = 39) individuals. RESULTS: Positivity ranged between 81.3 and 94.3% after infection and 99.4 and 99.7% after vaccination, depending on the assay. Both cohorts showed a high level of qualitative agreement across tests (Fleiss' kappa = 0.598 and 0.719 for convalescent and vaccinated respectively). Spikevax vaccine recipients showed the highest level of antibodies in all tests. Effectiveness of each test predicting SARS-CoV-2 neutralising capacity depended on assay type and target, with CLIA and anti-S being more effective than ELISA and anti-N assays, respectively. CONCLUSIONS: High-throughput immunoassays are good predictors of neutralising capacity. Updated targets and better standardisation would be required to find an effective correlate of protection, especially to account for antibodies against new variants.