key: cord-0850612-1xbg59ob authors: Pisano, M. B.; Sicilia, P.; Zeballos, M.; Lucca, A.; Fernandez, F.; Castro, G.; Goya, S.; Viegas, M.; Lopez, L.; Barbas, G.; Re, V. title: SARS-CoV-2 genomic surveillance enables the identification of Delta/Omicron co-infections in Argentina. date: 2022-03-10 journal: nan DOI: 10.1101/2022.03.08.22270920 sha: ea677189c356c692e99893cf23abdb1e8916ba68 doc_id: 850612 cord_uid: 1xbg59ob Molecular surveillance of SARS-CoV-2 is crucial to early detect new variants and lineages. In addition, detection of coinfections with more than one SARS-CoV-2 lineage have been sporadically reported. In this work, surveillance of SARS-CoV-2 variants was performed on 2067 RNA samples (Ct>30) obtained during December 2021 and January 2022 from Cordoba province, Argentina, by real time RT-PCR specific for VOC/VOI relevant mutations (TaqMan SARS-CoV-2 Mutation Panel, Applied Biosystems). The following distribution of variants was obtained: Omicron (54.9%), Delta (44.2%) and Lambda (0.8%). Three samples (0.1%), obtained the last week of December, presented a profile compatible with a Delta/Omicron co-infection. One of them was sequenced by NGS-Illumina, obtaining reads for both VOCs. One of the studied patients presented severe symptoms, although he was not vaccinated and presented risk factors (older than 60 years, arterial hypertension). We describe for the first time in Argentina, the identification of cases of co-infection with two SARS-CoV-2 lineages, VOCs Delta and Omicron, during the third COVID-19 wave in the country (a high viral circulation period), when Delta and Omicron co-circulated. Our findings highlight the importance of continuing with molecular surveillance and co-detection studies of VOC/VOIs, in order to elucidate possible recombination events and the emergence of new variants. During the two years of the COVID-19 pandemic, the original SARS-CoV-2 that was identified at the end of 2019 has evolved into various lineages (He et al. 2021) , presenting characteristic mutations. Among them, variants that posed an increased risk to global . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 10, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 public health have been identified as variants of interest (VOI) and variants of concern (VOC), which present a defined pattern of mutations (WHO 2022) . Five VOCs -Alpha (lineage B.1.1.7), Beta (lineage B.1.351), Gamma (lineage P.1), Delta (lineage B.1.617.2) and Omicron (lineage B.1.1.529)-, and two VOIs -Lambda (C.37) and Mu (B.1.621)-have been reported up to date (WHO 2022) . Whole genome sequencing (WGS) has been widely used since the beginning of the pandemic to monitor virus variants, to get a better understanding of the virus biology and epidemiology (Hosch et al. 2022) . However, it is a time-consuming and expensive technique, that requires trained staff and specific equipment, restricting its access in resource-limited settings (Blairon et al. 2021) . As an alternative, reverse transcription real time polymerase chain reactions (RT real time PCR) assays for detection of relevant mutations associated with SARS-CoV-2 variants have been developed, to typify circulating variants, being a more accessible tool for the monitoring of VOCs (Ong et al. 2021 , Blairon et al. 2021 . Molecular SARS-CoV-2 surveillance has allowed to identify the simultaneous infection (coinfection) of a single individual by two distinct SARS-CoV-2 lineages, an event that has been sporadically reported (Dezordi et al. 2021 , Zhou et al. 2021 , Hosch et al. 2022 ). These cases constitute an opportunity for viral genetic recombination and the emergence of new lineages with differential phenotype (Dezordi et al. 2021) , which may cause more severe clinic symptoms (Zhou et al. 2021) . The frequency of co-infected patients and its role to promote recombination-driven SARS-CoV-2 evolution is still unknown and poorly understood (Dezordi et al. 2021) . In Argentina, the profile of circulating lineages and variants has been changing throughout the pandemic, as has happened in the rest of the world (Outbreak info 2022). Molecular surveillance in the country started with WGS carried out by the Ministries of Science and Technology, and Health, at the national level (PAIS 2022, Ministerio de Salud de la Nacion 2022). But then, given the increase in the number of COVID-19 cases, the appearance of VOC/VOIs and the need for rapid results that enable the public health decision-making, some provinces implemented different strategies based on real-time RT-PCRs for . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In this report, we describe for the first time in Argentina, the identification of cases of coinfection with two SARS-CoV-2 lineages, particularly with VOCs Delta and Omicron, occurred in December 2021, detected within the molecular surveillance carried out during the third COVID-19 wave in the country. A strategy of SARS-CoV-2 VOC/VOI typing was implemented in the province of Cordoba, Argentina, during 2021, which varied throughout the year according to the the local profile of circulating variants (Castro et al. 2021) . By the beginning of December 2021, with a major presence of Delta circulating in the community, the strategy included a first . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22270920 doi: medRxiv preprint screening of L452R and P681R mutations, followed by detections of P681H, K417N, L452Q (the second round of mutation detection was carried out depending on the results of the first screening) (Castro et al. 2021, Gobierno de la provincia de Cordoba 2022). TaqMan™ SARS-CoV-2 Mutation Panel (Applied Biosystems) was used for detection of the relevant mutations/deletions. Each reaction of real time RT-PCR is performed as multiplex, including probes detecting the wildtype (wt) as well as probes detecting the mutant nucleotide sequences, used to enable detection of wildtype and mutated sequence simultaneously. Briefly, 7µL of RNA were added to 8 µL of a mixture containing TaqPath TM 1-Step RT-qPCR Master Mix, CG (4X), TaqMan TM SARS-CoV-2 Mutation Panel Assay (40X) and nuclease-free water. Samples that resulted compatible with a co-infection profile in the real-time RT-PCRs for VOC/VOI screening, were subjected to WGS by Illumina platform, using the Illumina COVIDSeq RUO kit, version COVIDSeq Test Kit. Manual inspection of variant-specific mutation sites were accessed using the program Tablet (Milne et al. 2013) . Sequenced sample was submitted to GISAID database under the accession number EPI_ISL_8938300. From the 2067 samples analyzed using the mutation-specific real-time PCR strategy for detection of VOCs/VOIs, 913 (44.2%) belonged to VOC Delta, 1135 (54.9%) to VOC Omicron, 16 (0.8%) to VOI Lambda and 3 (0.1%) presented profiles compatible with coinfections. The distribution of variants varied throughout the 2 months studied, with an abrupt change in viral circulation, being Omicron all detections performed at the end of January 2022 (Figure 1 ). Main features of the 3 co-infected patients are shown in Table 1 . Figure 2A . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22270920 doi: medRxiv preprint Amplification of P681R resulted positive for the mutation, but negative for the wt sequence ( Figure 2A, Supplementary Table 1 ), which could indicate the presence of another mutation in that position. Mutation of P681H resulted positive for the mutation and negative for wt (Figure 2A, Supplementary Table 1) . Finally, the mutation K417N was detected, together with the wt sequence, which was weakly amplified in the three cases ( Figure 2A, Supplementary Table 1 ). To rule out possible cross-contaminations, the 3 samples were repeated from the original samples (nucleic acid extraction and specific RT-PCRs for VOC/VOI detection was performed), arriving at the same result. Sample N°1 presented sufficient amount of RNA and high viral load, so the whole genome sequence could be generated for further investigation. The sequence obtained contained 29867 nucleotides, from which 0.87% were Ns and 0.34% were mutations (compared to the reference sequence WIV04). The average percentage of reads matching VOC Omicron was higher than reads matching VOC Delta ( Figure 2B ). Positions in which Delta was majority presented low coverage. Pangolin COVID-19 Lineage Assigner (Pangolin v3.1.19) could not assign a lineage to this sequence. Coinfection with distinct SARS-CoV-2 lineages is considered a rare phenomenon. However, it is likely and is thought to be underestimated (Dezordi et al. 2021) . In this work we report the coinfection with Delta and Omicron VOCs. This is the first description of co-infected individuals carrying two distinct lineages of SARS-CoV-2 in Argentina. VOC Delta was first described in our province on July 2021, when it was detected in a traveler and his close contacts. Due to the efforts carried out by the health authorities of the province, which included tracking and isolating Delta positive cases and its close contacts, the spread of this VOC was delayed, so its increase was gradual, until reaching its highest proportion of circulation (85%) in November 2021 (Castro et al. 2021), but without a substantial increase in the number of cases (Gobierno de la provincia de Córdoba 2022). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22270920 doi: medRxiv preprint VOC Omicron was detected in Argentina -and particularly in Córdoba province-the first days of December 2021, in a traveler from Dubai, and it quickly spread throughout the province (Gobierno de la provincia de Córdoba 2022). The sharp increase in Omicron frequency was accompanied by an increase in the number of cases, giving rise to the third wave of COVID-19 in the province and the entire country (Gobierno de la provincia de Córdoba 2022, Ministerio de Salud de la Nacion 2022). In this context of co-circulation of variants, 3 samples with Delta/Omicron SARS-CoV-2 coinfection were identified, all of them detected the last 2 weeks of December, when cocirculation of Delta and Omicron was registered (Gobierno de la provincia de Córdoba reported, also in a very low proportion of the tested samples (Dezordi et al. 2021 , Hosch et al. 2022 , Zhou et al. 2021 . Although these events are rare, they are believed to be CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22270920 doi: medRxiv preprint the prevalence and circulation of SARS-CoV-2 will minimize the chance of forming recombinant lineages with genetic combinations that could potentially increase virus fitness (Jackson et al. 2021) . Until now, no major clinical implications have been described in patients with co-infection with more than one SARS-CoV-2 lineage (Hosch et al. 2022) . In this study, only one of the patients presented severe symptoms (pneumonia, dyspnea), although they were probably due to the fact that he was not vaccinated and to the presence of risk factors (over 60 years of age, arterial hypertension) rather than the co-infection. However, more clinical research is needed and should be carried out on these patients. In conclusion, we found, for the first time in Argentina, co-infections by two SARS-CoV-2 lineages (Delta/Omicron) during the third wave of COVID-19, the largest in our country (Ministerio de Salud de la Nacion 2022). This highlights the importance of continuing molecular surveillance, especially in moments of high viral circulation, to detect both coinfections as well as recombinations. It is also important to continue studying clinical cases related to coinfections, in order to elucidate possible severe cases. All authors have seen and approved the manuscript. The Government of the Province of Córdoba through the Ministry of Health determined that the ethical review, approval and written informed consent are not required for the study of oropharyngeal swab samples obtained from human participants in the study "SARS-CoV-2 genomic surveillance enables the identification of Delta/Omicron coinfections in Argentina" in accordance with local legislation and institutional requirements. Furthermore, the data of this study were openly available to the public before the initiation of the study. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 10, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 of interlineage recombinants in the SARS-CoV-2 pandemic. Cell. 2021. 184(20) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 10, 2022. ; https://doi.org/10.1101/2022.03.08.22270920 doi: medRxiv preprint Yes . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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