Culture-independent identification and serotyping of Streptococcus pneumoniae by targeted metagenomics in pleural fluid samples

2.Streptococcus pneumoniae is the leading cause of empyema and pneumonia in children, and monitoring of effectiveness of polyvalent pneumococcal vaccines has been essential for controlling invasive pneumococcal disease (IPD) in children and elderly adults. Conventional serotyping of pneumococci has relied on Quellung reaction following laboratory culture, however more recently whole genome sequencing (WGS) has been implemented in many reference laboratories to enhance traditional typing. Pleural fluid samples from cases with empyema are often culture negative, limiting the utility of WGS and requiring polymerase chain reaction (PCR) or 16S rRNA sequencing to detect S. pneumoniae. These molecular methods have limited sensitivity and capacity to characterise pneumococcus in clinical samples, especially in specimens with a low pathogen abundance. This study applied capture-based enrichment (tNGS) to identify and characterise S. pneumoniae directly from pleural fluid samples. A total of 51 pleural fluid samples were subjected to tNGS with a custom probe panel, for 39 known positive fluids collected from IPD cases between 2018-2025 in New South Wales, Australia. tNGS results were benchmarked against molecular-based serotyping. Our tNGS achieved 100% sensitivity and specificity in detecting S. pneumoniae. Serotyping results were concordant with PCR and 95% (37/39) of S. pneumoniae PCR positive pleural fluid cases could be serotyped using tNGS. Standard molecular methods however could only determine serotype in 56% (22/39) of samples. This tNGS enabled 39% improvement in ability to directly identify and serotype IPD-associated serotypes of S. pneumoniae in difficult-to-culture pleural fluids can significantly enhance laboratory surveillance of IPD as well as our understanding of vaccine effectiveness. 3. Impact statementThere is currently a gap in understanding the pneumococcus serotype diversity causing infection within the pleural fluid space. The gold-standard Quellung method to determine serotype relies on culturing the pneumococci first. However, pleural fluids often remain culture-negative, and cases of pneumococcal empyema have been a historical blind spot in pneumococcal surveillance. This study offers a new methodology to close this gap and allow serotyping of previously untypable cases. The study demonstrated a targeted next generation sequencing (tNGS) approach to determine serotype without the need to first culture the bacteria. This novel use of tNGS targets part of the cps gene cluster, which determines serotype. To the best of our knowledge this is the first panel to do so. We have successfully serotyped 95% of pleural fluid S.pneumoniae PCR positive samples, where previously only 56% could be determined using conventional PCR typing methods. This demonstrates for the first time a novel tNGS method capable of determining the full serotype landscape causing pleural fluid infection. This development will enhance the understanding of vaccine effectiveness and contribute to the prevention of invasive pneumococcal disease. 4. Data summarySupplementary data containing reference cpsB genomes are available within this article. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. 1.5 RepositoriesENA project accession number PRJEB111154. All supporting data has been provided within the article or in supplementary data files. One supplementary data file is available with the online version of this article.