key: cord-0937505-uj9pig9l authors: Wawina-Bokalanga, Tony; Logist, Anne-Sophie; Sinnesael, Robbe; Van Holm, Bram; Delforge, Marie-Luce; Struyven, Pierre; Cuypers, Lize; André, Emmanuel; Baele, Guy; Maes, Piet title: Genomic evidence of co-identification with Omicron and Delta SARS-CoV-2 variants: A report of two cases date: 2022-05-20 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2022.05.043 sha: fcdf974384f4e361fdb73e4a0e114a24b815e8b1 doc_id: 937505 cord_uid: uj9pig9l On November 24th, 2021, a new SARS-CoV-2 variant assigned to the lineage B.1.1.529 (Omicron) was first reported to WHO from South Africa. Despite the co-circulation of several SARS-CoV-2 variants, co-infection by different variants is not commonly identified. Here, we report two cases of SARS-CoV-2 co-identifications with the Omicron and Delta variants. SARS-CoV-2 variants, co-infection by different variants is not commonly identified. Here, we report two cases of SARS-CoV-2 co-identifications with the Omicron and Delta variants. SARS-CoV-2 co-infection, Omicron, Delta, COVID-19, Whole-genome sequencing A new SARS-CoV-2 variant of concern (VOC) assigned to the lineage B.1.1.529, designated as Omicron, has been reported to the World Health Organization (WHO) by South Africa scientists on November 24 th , 2021 (World Health Organization 2021). The first cases of SARS-CoV-2 infection caused by the Omicron variant were reported to originate from Botswana and South Africa (World Health Organization 2021). In Belgium, the first case of SARS-CoV-2 infection caused by the Omicron variant was identified in late November 2021 (Vanmechelen et al. 2022 ). Since mid-January 2022, the Omicron variant has become the dominant lineage in most countries worldwide with a growing tendency to displace lineage B.1.617.2, designated as the Delta variant (Nextstrain SARS-CoV-2 resources 2022). Despite the increasing number of SARS-CoV-2 infections and the continuous emergence of new VOCs, there are only few studies on the occurrence of SARS-CoV-2 co-infection caused by different variants (Francisco et al. 2021; Lythgoe et al. 2021) . Here, we report the co-identification of SARS-CoV-2 variants B.1.1.529 (Omicron) and B.1.617.2 (Delta) in two geographically unrelated cases, detected simultaneously using the Oxford Nanopore Technologies (ONT) GridION. On December 23 rd , 2021, a 38-year-old male living in the municipality of Anderlecht, in the Brussels Capital Region (Belgium), presented himself to a general practitioner (GP) with a congested nose, sore throat and fatigue. He had no history of clinically significant underlying medical conditions and reported no recent travel history. He had previously been tested positive for SARS-CoV-2 on March 11 th , 2021 and was fully vaccinated with the Pfizer-BioNTech BNT162b2 mRNA Covid-19 vaccine (first and second dose received on the 5 th and 26 th of June 2021, respectively). As suspected of being infected by SARS-CoV-2, a nasopharyngeal swab was collected on December 23 rd , 2021, which resulted in a positive test using TaqPath Covid-19 RT-PCR kit (ThermoFisher Scientific). He was not hospitalized and his symptoms resolved after one week. The sample was selected for whole-genome sequencing (WGS) as it entailed a breakthrough SARS-CoV-2 infection with a high viral load (Cycle threshold (Ct) value of 8.1) in a vaccinated individual. On January 5 th , 2022, a 34-year-old female living in the municipality of Ganshoren, in the Brussels Capital Region (Belgium), presented herself at the emergency unit of the Centre Hospitalier de Wallonie Picarde (CHwapi) with fever for two days, muscle aches and shortness of breath. She had a medical history of multiple sclerosis and was treated with Dimethyl fumarate (Tecfidera 240 mg/day). She had no recent travel history and was vaccinated with Johnson & Johnson's COVID-19 vaccine on November 9 th , 2021. The patient brought with her a positive SARS-CoV-2 antigenic test performed the same day in the morning. She reported that her partner had tested positive on January 1 st , 2022. On her physical examination she was not hypoxaemic with oxygen saturation of 100%. The infectiologist re-assessed and allowed the patient to go back home the same day after administration of a single-dose intravenous infusion of Sotrovimab (GlaxoSmithKline), a SARS-CoV-2 monoclonal antibody. A nasopharyngeal swab was collected on January 5 th , 2022, and tested SARS-CoV-2 positive (Ct value of 18.7) with GeneXpert using the Xpress SARS-CoV-2/Flu/RSV plus assay (Cepheid). The two samples were collected as part of active surveillance of COVID-19 diagnostics and selected for WGS. Viral RNAs were extracted using the automated nucleic acid extraction KingFisher TM Flex system (ThermoFisher). We performed WGS using the GridION sequencing platform and obtained a total of 1,994,707 and 4,876,752 filtered reads from case 1 and 2, respectively. The resulting full genome sequences have approximatively 29,900 nucleotides with an average coverage depth of 1700x and 3500x, using the rapid and ligation barcoding kit respectively. Although we have been able to obtain the full genome sequences, it was not possible to assign the two sequences to a specific SARS-CoV-2 variant or lineage using either the Nextclade tool or the Pangolin lineage classification software (Aksamentov 2021; O'Toole et al. 2021) due to the high number of sites where different nucleotides were called at one strictly map reads to an Omicron reference sequence since it was the most predominant circulating strain at the time. A total of 1,776,231 (89,1%) and 4,168,164 (85,5%) reads from case 1 and 2 respectively, strictly mapped to Omicron genome reference. Subsequently, sequencing reads that did not match (10,9% from case 1 and 14,5% from case 2) with Omicron were mapped to a Delta reference sequence. Interestingly, they all In this study, we simultaneously detected SARS-CoV-2 co-infection with 21K Omicron and 21J Delta variants in two geographically unrelated patients using ONT GridION sequencing. Although a limited number of studies have previously reported the co-infection events between SARS-CoV-2 Alpha and Delta variants (Hosch et al. 2021; Zhou HY. 2021 Importantly, SARS-CoV-2 co-infection with different variants may lead to the emergence of novel SARS-CoV-2 recombinant variants, because of its high recombination rates, which might influence the viral transmission, disease severity and vaccine efficacy (Rehman et al. 2020 ). Indeed, due to the small number of infected patients in this study, it was not possible to draw a reliable conclusion about the impact of Omicron and Delta co-infection on disease severity and vaccine efficacy. Furthermore, there was not sufficient detailed information available concerning contact tracing for understanding how the two patients acquired the SARS-CoV-2 co-infection. Moreover, we did not rule out contamination issues because by the time we sequenced and identified the two variants of SARS-CoV-2, more than 30 days had already passed since the onset of symptoms, and one of the two patients had been treated with Sotrovimab (GlaxoSmithKline). In addition, two main arguments support the co-infection events: (i) the total number of reads supporting each variant was high (around 1,78 million reads mapping to Omicron vs 218 thousand reads mapping to Delta for case 1; and 4,17 million reads to Omicron vs 708 thousand reads mapping to Delta for case 2) and (ii) sampling and PCR diagnostic tests of samples were performed at different moments and in different laboratories. However, despite the co-circulation of several SARS-CoV-2 variants in Belgium, Omicron was the most dominant variant and represented over 96% of total samples that where sequenced at the time. In conclusion, our findings highlight the importance of genomic surveillance to diagnose SARS-CoV-2 co-infection with different variants and emphasize that more needs to be learned about these co-infection events, and their influence on COVID-19 outcome and vaccine efficacy. TWB and PM conceived the study. TWB, ASL, RS, BS and PM performed the experiments. MLD and PS performed the RT-PCR diagnostic test and sent the samples for WGS. TWB prepared the first draft of the manuscript. LC, EA, GB and PM read and critically revised the manuscript for publication. All authors have read and approved the final manuscript. The authors declare that they have no conflict of interest. This work and the sequencing capacity were supported in part by a COVID-19 research grant of 'Fonds Wetenschappelijk Onderzoek'/Research Foundation Flanders (grant G0H4420N) and by an 'Internal Funds KU Leuven' awarded to PM (grant 3M170314). Nextclade: clade assignment, mutation calling and quality control for viral genomes Evidence of co-infection during Delta and Omicron variants of concern co-circulation Pervasive transmission of E484K and emergence of VUI-NP13L with evidence of SARS-CoV-2 co-infection events by two different lineages in Rio Grande do Sul, Brazil Genomic Surveillance Enables the Identification of Co-infections With Multiple SARS-CoV-2 Lineages in Equatorial Guinea Rambaut, A. nCoV-2019 novel coronavirus bioinformatics protocol SARS-CoV-2 within-host diversity and transmission Nextstrain SARS-CoV-2 resources Assignment of epidemiological lineages in an emerging pandemic using the pangolin tool Evolutionary Trajectory for the Emergence of Novel Coronavirus SARS-CoV-2 Identification of the First SARS-CoV-2 Lineage B.1.1.529 Virus Detected in Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant of Concern Genomic evidence for divergent co-infections of SARS-CoV-2 lineages. bioRxiv GB acknowledges support from the Internal Funds KU Leuven (grant C14/18/094) and the Research Foundation -Flanders ("Fonds voor Wetenschappelijk Onderzoek -Vlaanderen," G0E1420N, G098321N). UZ Leuven, as national reference center for respiratory pathogens, is supported by Sciensano, which is gratefully acknowledged. The study was approved by the KU/UZ Leuven Clinical Trial and Ethical review board (approval number S66037).