Association between disease severity and co-detection of respiratory pathogens in infants with RSV infection

BACKGROUND Respiratory syncytial virus (RSV) is the leading cause of hospitalisation associated with acute respiratory infection in infants and young children, with substantial disease burden globally. The impact of additional respiratory pathogens on RSV disease severity is not completely understood. OBJECTIVES The objective of this study was to explore the associations between RSV disease severity and the presence of other respiratory pathogens. METHODS Nasopharyngeal swabs were prospectively collected from two infant cohorts: a prospective longitudinal birth cohort study and an infant cross-sectional study recruiting infants <1 year of age with RSV infection in Spain, the UK, and the Netherlands during 2017–20 [part of the REspiratory Syncytial virus Consortium in EUrope (RESCEU) project]. The samples were sequenced using targeted metagenomic sequencing with a probe set optimised for high-resolution capture of sequences of over 100 pathogens, including all common respiratory viruses and bacteria. Viral genomes and bacterial genetic sequences were reconstructed. Associations between clinical severity and presence of other pathogens were evaluated after adjusting for potential confounders, including age, gestational age, RSV viral load, and presence of comorbidities. RESULTS RSV was detected in 433 infants. Nearly one in four of the infants (24%) harboured at least one additional non-RSV respiratory virus, with human rhinovirus being the most frequently detected (15% of the infants), followed by seasonal coronaviruses (4%). In this cohort, RSV-infected infants harbouring any other virus tended to be older (median age: 4.3 vs. 3.7 months) and were more likely to require intensive care and mechanical ventilation than those who did not. Moraxella, Streptococcus , and Haemophilus species were the most frequently identified target bacteria, together found in 392 (91%) of the 433 infants ( S. pneumoniae in 51% of the infants and H. influenzae in 38%). The strongest contributors to severity of presentation were younger age and the co-detection of Haemophilus species alongside RSV. Across all age groups in both cohorts, detection of Haemophilus species was associated with higher overall severity, as captured by ReSVinet scores, and specifically with increased rates of hospitalisation and respiratory distress. In contrast, presence of Moraxella species was associated with lower ReSVinet scores and reduced need for intensive care and mechanical ventilation. Infants with and without Streptococcus species (or S. pneumoniae in particular) had similar clinical outcomes. No specific RSV strain was associated with co-detection of other pathogens. CONCLUSION Our findings provide strong evidence for associations between RSV disease severity and the presence of additional respiratory viruses and bacteria. The associations, while not indicating causation, are of potential clinical relevance. Awareness of coexisting microorganisms could inform therapeutic and preventive measures to improve the management and outcome of RSV-infected infants. ### Competing Interest Statement G.-L. L. is currently an employee of Mundipharma Research Ltd., a position he commenced subsequent to the completion of this study. S. B. D. has been an investigator for clinical trials of vaccines and antimicrobials for pharmaceutical companies, including AstraZeneca, Merck, Pfizer, Valneva, Iliad, Sanofi, and Janssen, and previously sat on RSV advisory boards for Sanofi and Merck. M. D. S. has been an investigator on behalf of the University of Oxford for studies funded or supported by vaccine manufacturers, including Pfizer, GSK, Novavax, MCM vaccines and Janssen. M. D. S. is currently an employee of Moderna Biotech UK, a position he commenced subsequent to the completion of this study. M. A. A. is supported by a Sir Henry Dale Fellowship, jointly funded by the Royal Society and Wellcome Trust (220171/Z/20/Z). J. E. B. and K. A. J. are funded on a Wellcome Trust Biomedical Resources Grant (218205/Z/19/Z, PubMLST: Disseminating and exploiting bacterial diversity data for public health benefit). J. A. is an employee of Janssen Pharmaceutica NV. P. J. M. O. has received honoraria from GSK, Pfizer Inc, Sanofi Pasteur, Seqirus, and Janssen for taking part in advisory boards and expert meetings and for acting as a speaker in congresses outside the scope of the submitted work. P. J. M. O. is also a National Institute for Health and Care Research (NIHR) Senior Investigator. F. M.-T. has received honoraria from GSK, Pfizer Inc, Sanofi Pasteur, MSD, Seqirus, and Janssen for taking part in advisory boards and expert meetings and for acting as a speaker in congresses outside the scope of the submitted work. F. M.-T. has also acted as principal investigator in randomised controlled trials of the above-mentioned companies as well as Ablynx, Regeneron, Roche, Abbott, Novavax, and MedImmune, with honoraria paid to his institution. F. M.-T. receives support for his research activities from the Instituto de Salud Carlos III (Proyecto de Investigación en Salud, Acción Estratégica en Salud): Fondo de Investigación Sanitaria (FIS;PI1601569/PI1901090/PI22/00406) del plan nacional de I+D+I and 'fondos FEDER'. H. N. received grants from Innovative Medicine Initiative, NIHR, Bill and Melinda Gates Foundation, WHO, and Pfizer. H. N. also received consultancy or honoraria and speaker fees from Sanofi, Merck, Novavax, ReViral and GSK (all paid to institution). A. J. P. is chair of the UK Department of Health and Social Care's Joint Committee on Vaccination and Immunisation. A. J. P. has also provided advice to Shionogi on COVID-19 vaccines and his institution receives research funding from NIHR, Bill & Melinda Gates Foundation, Wellcome Trust, Medical Research Council and AstraZeneca for vaccine research. All other authors report no potential conflicts. The views expressed in this article are those of the authors and may not be understood or quoted as being made on behalf of or reflecting the position of the organizations with which the authors are employed/affiliated. ### Funding Statement This work was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, the NIHR Thames Valley and South Midlands Clinical Research Network, the British Research Council, and the Respiratory Syncytial Virus Consortium in Europe (RESCEU) project. RESCEU has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (grant number 116019). This Joint Undertaking receives support from the European Union Horizon 2020 Research and Innovation Program and European Federation of Pharmaceutical Industries and Associations. ### Author Declarations 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: Comité de ética de la Investigación de Santiago-Lugo of Hospital Clínico Universitario de Santiago de Compostela, Spain, gave ethical approval for this work (no. 2017/395). Ethics committee of the University of Oxford, UK, gave ethical approval for this work. Health Research Authority, UK, gave ethical approval for this work (no. 231136). NHS National Research Ethics Service Oxfordshire Research Ethics Committee A, UK, gave ethical approval for this work (no. 15/SC/0335). NHS National Research Ethics Service South Central - Hampshire A, UK, gave ethical approval for this work (no. 17/SC/0522). Medical Ethical Committee of the University Medical Center Utrecht, the Netherlands, gave ethical approval for this work (no. 17/563). 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 and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All raw sequencing reads of the samples included in this study are accessible at the European Nucleotide Archive under the study accession PRJEB34042 (). The RSV consensus sequences included in the phylogenetic analyses have been deposited in GenBank. The accession numbers can be found in Table S14.