Evidence of co-infections during Delta and Omicron SARS-CoV-2 variants co-circulation through prospective screening and sequencing

Abstract

Objectives: To describe Delta/Omicron SARS-CoV-2 variants co-infection detection and confirmation during the fifth wave of COVID-19 pandemics in France in 7 immunocompetent and epidemiologically unrelated patients.

Methods: Since December 2021, the surveillance of Delta/Omicron SARS-CoV-2 variants of concern (VOC) circulation was performed through prospective screening of positive-samples using single nucleotide polymorphism (SNP) PCR assays targeting SARS-CoV-2 S-gene mutations K417N (Omicron specific) and L452R (Delta specific). Samples showing unexpected mutational profiles were further submitted to whole genome sequencing (WGS) using three different primer sets.

Results: Between weeks 49-2021 and 02-2022, SARS-CoV-2 genome was detected in 3831 respiratory samples, of which 3237 (84.5%) were screened for VOC specific SNPs. Unexpected mutation profiles suggesting a dual Delta/Omicron population were observed in 7 nasopharyngeal samples (0.2%). These co-infections were confirmed by WGS. For 2 patients, the sequence analyses of longitudinal samples collected 7 to 11 days apart showed that Delta or Omicron can outcompete the other variant during dual infection. Additionally, for one of these samples, a recombination event between Delta and Omicron was detected.

Conclusions: This work demonstrates that SARS-CoV-2 Delta/Omicron co-infections are not rare in high virus co-circulation periods. Moreover, co-infections can further lead to genetic recombination which may generate new chimeric variants with unpredictable epidemic or pathogenic properties that could represent a serious health threat.

Keywords: Delta and Omicron VOC; Recombination; SARS-CoV-2 co-infection; Single nucleotide polymorphism screening; Whole genome sequencing.

Screening was performed with the TaqMan™ SARS-CoV-2 mutation panel molecular assay (ThermoFisher Scientific, Massachusetts, USA). For each mutation of interest, probes labelled with VIC or FAM detect the wild type (WT) and the mutated sequence, respectively. WT samples are represented as red dots along the x-axis and mutated samples as blue dots along the y-axis. A dual population is suspected by intermediate mutation profile with detection of WT and mutated sequences in the same sample and is represented by green dots. The figure at the bottom represents the variant allele frequency clade-defining mutations for 21J Delta and 21K Omicron along the SARS-CoV-2 genome for one patient (P6). Mutations are represented with the variant allele frequency on the first Y axis and the depth of each genomic position on the secondary Y axis. Recombination points are indicated by blue arrows and were confirmed by Single Molecule Real Time sequencing.Abbreviations: SNP, Single Nucleotide Polymorphism

Fig. 1

Screening profiles for VOC-specific amino-acid substitution S:L452R and S:K417N suggestive of a dual population Screening was performed with the TaqMan™ SARS-CoV-2 mutation panel molecular assay (ThermoFisher Scientific, Massachusetts, USA). For each mutation of interest, probes labelled with VIC or FAM detect the wild type (WT) and the mutated sequence, respectively. WT samples are represented as red dots along the x-axis and mutated samples as blue dots along the y-axis. A dual population is suspected by intermediate mutation profile with detection of WT and mutated sequences in the same sample and is represented by green dots. Abbreviations: SNP, Single Nucleotide Polymorphismcreening profiles for VOC-specific amino-acid substitution S:L452R and S:K417N suggestive of a dual population. Screening was performed with the TaqManÔ SARS-CoV-2 mutation panel molecular assay (ThermoFisher Scientific, Massachusetts, USA). For each mutation of interest, probes labelled with VIC or FAM detect the wild type (WT) and the mutated sequence, respectively. WT samples are represented as red dots along the x-axis and mutated samples as blue dots along the y-axis. A dual population is suspected by intermediate mutation profile with detection of WT and mutated sequences in the same sample and is represented by green dots. Abbreviations: SNP, Single Nucleotide Polymorphism

Fig. 2

Representation of variant allele frequency clade-defining mutations for 21J Delta and 21K Omicron along the SARS-CoV-2 genome. All 21J and 21K clade defining mutations along the genome are presented for P1-D1 (A) and P1-D7 (B), P5-D1 (C) and P5-D7 (D) and P6-D1 (E). Mutations are represented with the variant allele frequency on the first Y axis and the depth of each genomic position on the secondary Y axis. If the mutation was not present in the vcf file, the value was set at zero. The black arrows indicate clade defining mutations or deletions excluded for the proportion’s estimation of co-infection (no more than 13 per sample for the artic v4). P6-D1 recombination points were confirmed by Single Molecule Real Time sequencing.