ARTIC-based amplicon sequencing assay evaluated for respiratory syncytial virus detection accuracy and lineage identification.

This study evaluated the performance of an ARTIC-based amplicon sequencing assay for detecting respiratory syncytial virus (RSV). The analysis utilized 214 deidentified remnant clinical specimens obtained from the Georgia Public Health Laboratory. These specimens served as the population for assessing assay characteristics, with custom primer sets serving as the comparator for specificity testing.

The primary outcomes measured included assay accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), sequencing depth, and genomic coverage. The assay demonstrated an overall accuracy of 92.8%, with a sensitivity of 96.2% and a specificity of 87.2%. The positive predictive value was 92.6% and the negative predictive value was 93.2%. Intra- and inter-run precision yielded nearly 100% consensus genome identity, with 0 to 5 nucleotide differences observed across 16 and 53-57 genomes assessed.

Sequencing depth was slightly higher for RSV-A (median 53,433x; mean 51,076x) compared to RSV-B (median 49,699x; mean 46,945x). Conversely, genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Specificity testing involving 31 non-RSV specimens revealed no false-positive detections. Safety data, including adverse events and tolerability, were not reported.

Key limitations include the reliance on remnant specimens rather than a prospective cohort, which may limit generalizability to current clinical workflows. The slight variations in sequencing depth and coverage between viral lineages warrant attention when interpreting genomic data. Given the observational nature of the specimen evaluation, these findings support the assay's utility but require cautious application regarding lineage identification without further validation in active patient populations.