Intra-Host Evolution of SARS-CoV-2 During Persistent Infection of Pediatric COVID-19 Patients

Abstract

Children typically experience milder SARS-CoV-2 infection than adults, yet a subset—especially those with immune compromise—develop persistent infection. This study examined clinical characteristics and intra-host viral evolution in pediatric patients with prolonged COVID-19, comparing immunocompetent and immunocompromised hosts. Residual nasopharyngeal specimens from patients <20 years old with ≥3 samples ≤90 days apart underwent qRT-PCR confirmation and whole-genome sequencing using tiled amplicon Illumina methods. Demographic, clinical, and immune-status data were extracted through chart review. Viral load trajectories and longitudinal genomic changes were analyzed. Sixteen patients met inclusion criteria: 5 immunocompetent and 11 immunocompromised children. Groups showed similar demographic distributions (mean ages 6 vs. 9.2 years; 60% vs. 64% male; 60% vs. 45% Hispanic). Vaccination was rare, with all immunocompetent and 82% of immunocompromised patients unvaccinated. COVID-19 treatment differed markedly: 1 immunocompetent (20%) versus 8 immunocompromised patients (73%) received therapies including monoclonal antibodies or remdesivir. Clinical severity varied, with immunocompetent children largely asymptomatic or managed as outpatients, whereas immunocompromised children more frequently required hospitalization (28%), ICU care (9%), or died (18%). Despite this, WHO severity scores were similar (2.4 vs. 2.2). Sequencing yielded 15 isolates from immunocompetent and 35 isolates from immunocompromised patients. Viral loads generally declined over time, though several immunocompromised children demonstrated prolonged high viral loads. Longitudinal sequencing identified intra-host viral diversification in both groups, including nonsynonymous Spike mutations and minority variants associated with immune escape. Some immunocompromised patients accumulated mutations at positions linked to therapeutic resistance. Pediatric persistent SARS-CoV-2 infection is associated with measurable intra-host viral evolution. Immunocompromised children demonstrated longer infection duration, more frequent treatment exposure, and extended viral shedding, creating conditions that may favor viral diversification. These findings highlight the need for continued genomic surveillance in vulnerable pediatric populations.