Background Nirsevimab is a long-acting monoclonal antibody used to prevent respiratory syncytial virus (RSV) infection in infants and high-risk children. During the 2024 RSV season in Western Australia, 23,525 doses were administered to infants entering their first season, and 1,233 doses to at-risk children. In this context, the selection and spread of escape variants is a potential concern. This study aimed to investigate nirsevimab-binding site mutations using both clinical and wastewater data.

Methods We performed whole-genome sequencing on 382 clinical RSV samples and 12 wastewater samples collected between September 2023 and October 2024. RSV subtypes, genetic diversity, and mutations within the nirsevimab-binding region of the F protein were analysed. Phylogenetic analysis was conducted to assess lineage dynamics and the potential emergence of escape variants.

Results RSV-A was the dominant subtype (61.8%), with RSV-B accounting for 38.2% of cases. No lineage shifts were observed following nirsevimab introduction and none of the known mutations associated with high-level nirsevimab resistance were detected in either clinical or wastewater samples. The prevalent RSV-B mutation combination (F:I206M:Q209R:S211N) was observed consistently but is not associated with reduced nirsevimab efficacy. Wastewater sampling, covering approximately 2 million people from Perth metropolitan region, confirmed findings from clinical sequences, reinforcing the absence of resistance mutations.

Conclusion No evidence of nirsevimab escape mutations was found in either clinical or wastewater samples during the 2024 RSV season. Continued genomic surveillance, including wastewater monitoring, is essential to detect emerging resistance and ensure the long-term efficacy of prophylactic interventions.

The authors have declared no competing interest.

CCB is supported by an NHMRC Investigator Grant (APP1173163) and HCM is supported by a Stan Perron Foundation. The REVIVE study is supported by the Perth Children Hospital Foundation and the STAMP RSV program is supported by the Stan Perron Charitable Foundation.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study was carried out under REVIVE (REspiratory syncytial Virus Immunisation program - eValuating Effectiveness and impact) protocol which was approved by Child and Adolescent Health Service Human Research Ethics Committee (Research Governance Service 6789) and Ramsay Health Care WA/SA Human Research Ethics Committee (2024/ETH/0027).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

The RSV sequences used in this study will be made publicy available with publication.