Performance of next-generation molecular methods in the diagnosis of pleural space infections and their aetiology

Pleural space infections (PSIs) are common and associated with substantial healthcare cost, morbidity, and mortality. Accurate PSI diagnosis remains challenging due to low culture positivity rates, frequent polymicrobial involvement, and non-specific diagnostic biomarkers. To better understand the comparative diagnostic yield of culture-independent technologies for PSI diagnosis, we prospectively characterised 26 clinically suspected PSIs and 10 control patients with non-infective pleural effusions using shotgun metagenomics, bacterial metataxonomics, quantitative PCR, and conventional culture. We demonstrate that culture-independent molecular techniques have superior sensitivity and negative predictive values for PSI diagnosis compared with conventional culture. Bacterial metataxonomics and metagenomics were both sensitive to the detection of polymicrobial infections, and unveiled microbial heterogeneity. Metagenomics also detected fungi and DNA viruses within pleural fluid. Dominant pathogens in the PSI cohort according to the highest resolution method, metagenomics, included streptococci ( S. intermedius , S. pyogenes , S. mitis ), Prevotella spp. ( P. oris , P. pleuritidis ), staphylococci ( S. aureus , S. saprophyticus ), and Klebsiella pneumoniae. Taken together, these results demonstrate the potential utility of molecular methods in the accurate diagnosis of PSI, which represents an important step towards improving personalised treatment decision-making and antimicrobial stewardship in suspected PSI. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by Wishlist Sunshine Coast Hospital Foundation (award 2021-04-CRG to PTB, TB, OO, JG, DSS, and EPP) and Advance Queensland (awards AQIRF0362018 to EPP and AQIRF095-2020-CV to OSO). ### 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: The study was approved by The Prince Charles Hospital (TPCH) Human Research Ethics Committee (HREC) HREC/2022/QPCH/81858 and the Sunshine Coast Hospital and Health Service Research Governance Office. All participants provided written informed consent. 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 All sequence data generated in this study are available via NCBI BioProject PRJNA972883; genome assemblies are available via NCBI BioProject PRJNA970939.