Integration of bioinformatic tools for the detection of SARS-CoV-2 co-infection cases

Co-infection with multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, though rare, may have clinical and public health implications, including facilitating variant recombination. Early detection of co-infections is, therefore, crucial. In this study, we report two probable cases of co-infection identified during routine genomic surveillance. Initially suspected as cross-contamination due to the presence of private mutations and nucleotide mixtures flagged by Nextclade and bammix, the samples were re-extracted and re-sequenced after workspace decontamination, yet the anomalies persisted. To investigate further, we developed a bioinformatics pipeline (Katmon) incorporating various tools such as Freyja, with lineage abundance results that illustrated the presence of multiple variants, and VirStrain, which confirmed inconsistent lineage assignments. We also visualized the alternative allele fractions for each lineage-defining mutation and amplicon, showing evidence of two variants, Delta and Omicron, co-existing within a single amplicon. Amplicon sorting effectively separated reads corresponding to the two variants, and the resulting consensus sequences aligned with their respective lineage assignments. These findings suggest that the first sample, PH-RITM-1395, involved a Delta-Omicron co-infection, while the second sample, PH-RITM-4146, probably contains both a co-infection and a recombinant variant. To further support the second sample's recombinant nature, we employed sc2rf, which identified Delta-Omicron breakpoints. Retrospective analysis of 1,078 samples from July 2021 to July 2022, encompassing the period of co-circulation of different variants in the Philippines, flagged four additional co-infection cases, including Delta-Omicron and Beta-Omicron, suggesting a lower bound co-infection prevalence of 0.27% and 0.19%, respectively. Furthermore, the pipeline was used to test previously identified co-infections of different variants from different countries. Our findings underscore the critical importance of real-time genomic surveillance and advanced bioinformatics pipelines in detecting SARS-CoV-2 co-infections and variant recombination.