Systematic genomic analysis of SARS-CoV-2 co-infections throughout the pandemic and segregation of the strains involved
Authors: Peñas-Utrilla Daniel , Pérez-Lago Laura , Molero-Salinas Andrea , Estévez Agustín , Sanz Amadeo , Herranz Marta , Martínez-Laperche Carolina , Andrés-Zayas Cristina , Veintimilla Cristina , Catalán Pilar , Alonso Roberto , Muñoz Patricia , García de Viedma Darío ,   on behalf of the Gregorio Marañón Microbiology-ID COVID 19 Study Group, Alcalá Luis , Aldámiz Teresa , Álvarez-Uría Ana , Bermúdez Elena , Bouza Emilio , Buenestado-Serrano Sergio , Burillo Almudena , Carrillo Raquel , Cercenado Emilia , Cobos Alejandro , Díez Cristina , Escribano Pilar , Fanciulli Chiara , Galar Alicia , García Mª Dolores , Gijón Paloma , Guillén Helmuth , Guinea Jesús , Irigoyen Álvaro , Kestler Martha , López Juan Carlos , Machado Marina , Marín Mercedes , Martín-Rabadán Pablo , Montilla Pedro , Padilla Belén , Palomino-Cabrera Rosalía , Palomo María , Pérez-Granda María Jesús , Pérez Leire , Reigadas Elena , Rincón Cristina , Rodríguez Belén , Rodríguez Sara , Rodríguez-Grande Cristina , Rojas Adriana , Ruiz-Serrano María Jesús , Sánchez Carlos , Sánchez Mar , Serrano Julia , Tejerina Francisco , Valerio Maricela , Vesperinas Lara , Vicente Teresa , de la Villa Sofía 
Published: July 2023

Abstract
Background
SARS-CoV-2 recombinants involving the divergent Delta and Omicron lineages have been described, and one of them, “Kraken” (XBB.1.5), has recently been a matter of concern. Recombination requires the coexistence of two SARS-CoV-2 strains in the same individual. Only a limited number of studies have focused on the identification of co-infections and are restricted to co-infections involving the Delta/Omicron lineages.

Methods
We performed a systematic identification of SARS-CoV-2 co-infections throughout the pandemic (7609 different patients sequenced), not biassed towards the involvement of highly divergent lineages. Through a comprehensive set of validations based on the distribution of allelic frequencies, phylogenetic consistency, re-sequencing, host genetic analysis and contextual epidemiological analysis, these co-infections were robustly assigned.

Results
Fourteen (0.18%) co-infections with ≥ 8 heterozygous calls (8–85 HZs) were identified. Co-infections were identified throughout the pandemic and involved an equal proportion of strains from different lineages/sublineages (including pre-Alpha variants, Delta and Omicron) or strains from the same lineage. Co-infected cases were mainly unvaccinated, with mild or asymptomatic clinical presentation, and most were at risk of overexposure associated with the healthcare environment. Strain segregation enabled integration of sequences to clarify nosocomial outbreaks where analysis had been impaired due to co-infection.

Conclusions
Co-infection cases were identified throughout the pandemic, not just in the time periods when highly divergent lineages were co-circulating. Co-infections involving different lineages or strains from the same lineage were occurring in the same proportion. Most cases were mild, did not require medical assistance and were not vaccinated, and a large proportion were associated with the hospital environment.