Within-host SARS-CoV-2 diversity in immunocompromised patients during acute infection

ABSTRACT Within-host SARS-CoV-2 diversity during acute infection is largely characterized by low-frequency intra-host single-nucleotide variants (iSNVs), but whether early iSNV patterns differ by immune status remains unclear. We analyzed biobanked diagnostic specimens from adults with RT-qPCR-confirmed SARS-CoV-2 infection collected in Cali, Colombia, between 2020 and 2022. Immunocompromised cases sampled during the acute phase were classified into four clinical categories, and immunocompetent cases from the same catchment area were used for comparison. Whole-genome sequencing (Illumina) yielded 102 high-quality genomes (≥85% coverage; immunocompetent, n = 60; immunocompromised, n = 42) from 114 samples. iSNVs were identified using iVar with stringent thresholds (depth ≥400× , Phred ≥20, allele frequency 3%–97%). Primary outcomes included iSNV counts, allele-frequency distributions, within-host complexity (Shannon entropy), normalized polymorphic sites, and Hamming distance. Analyses were conducted across all circulating lineages and within the Mµ (B.1.621) variant. Across all lineages, normalized polymorphic sites differed by immune status, with higher values in immunocompetent individuals ( P = 0.0064), whereas iSNV counts, allele-frequency distributions, and Shannon entropy were comparable between groups. When restricted to Mµ, immunocompetent individuals exhibited higher iSNV allele frequencies in the Spike region ( P = 0.0016). Within Mµ, mutational profiles differed by immune status: S-region iSNVs were predominantly synonymous in immunocompromised individuals and non-synonymous in immunocompetent individuals. Recurrent iSNVs revealed a shared core mutational signature across immune groups, with no Mµ-specific recurrent iSNVs exclusive to immunocompromised patients. Overall, intra-host diversity during acute infection was broadly similar by immune status, while lineage- and region-specific signals in the S region highlight nuanced early sub-consensus dynamics warranting functional investigation in immunophenotype-informed studies. IMPORTANCE Understanding how immune status shapes within-host SARS-CoV-2 diversity is key for interpreting transmission risk and evolutionary potential. Immunocompromised patients are often presumed to harbor more diverse viral populations, yet evidence early in infection remains limited. Analyzing samples, we compared minor variant (intra-host single-nucleotide variant [iSNV]) patterns between immunocompromised and immunocompetent individuals and evaluated lineage-specific effects. We found comparable within-host diversity, but normalized polymorphic sites were higher in immunocompetent than in immunocompromised individuals when considering all lineages. We observed an Mµ-restricted S-region signal: immunocompetent hosts showed higher S-region iSNV allele frequencies, whereas immunocompromised hosts exhibited a shift toward synonymous S-region iSNVs. These results refine assumptions about early infection in immunocompromised hosts, emphasize the value of reporting synonymous and non-synonymous changes, and highlight that conclusions can depend on viral lineage and the diversity metric considered. Our study underscores the need for immunophenotype-informed studies to test whether synonymous iSNVs modulate viral expression, replication, and transmission.