From Quellung to Genomics: Comparative Evaluation of Six WGS-Based Serotyping Tools for Streptococcus pneumoniae Surveillance in High-Burden Settings

Introduction: Streptococcus pneumoniae remains a major cause of severe, life-threatening disease across all age groups. Effective surveillance of serotype distribution is crucial for informing nationwide vaccine policy, particularly in high-burden countries such as India. While the traditional Quellung reaction is the gold standard for pneumococcal serotyping, its implementation is limited by high costs, labor intensity, and restricted accessibility. Whole genome sequencing (WGS)-based approaches have emerged as scalable alternatives, promising greater efficiency.   Methods: Six leading WGS-based pneumococcal serotyping tools - SeroBA, Pneumo-Typer, PneumoKITy, PfaSTer, SeroCall, and PneumoCaT - were evaluated using a large collection of Streptococcus pneumoniae isolates obtained from publicly available datasets (supplementary table S1). Serotype level and serogroup level concordance with reference phenotypic serotyping methods was calculated as proportions with 95% confidence intervals (CIs) using the Wilson score method. Results: A total of 1,629 Streptococcus pneumoniae genomes were analyzed, of which 1,595 met assembly-level quality control (QC) criteria. The remaining 34 QC-failed genomes were retained for downstream analysis, as WGS-based serotyping tools continued to generate consistent and reliable serogroup-level assignments despite reduced assembly quality. Overall, Pneumo-Typer (93.86%) and SeroBA (91.16%) achieved the highest concordance with reference phenotypic serotyping methods identifying major serotypes. However, despite its strong concordance rate, Pneumo-Typer exhibited reduced performance in species-level discrimination.   Conclusions: WGS-based serotyping represents a robust, scalable alternative to antisera-based classification for pneumococcal surveillance, with particular relevance for high-burden settings where conventional typing methods remain inaccessible. SeroBA provided the overall balance between serotype concordance, serogroup concordance, species-level discrimination, and computational performance. Integrating WGS-driven surveillance with global genomic datasets will strengthen epidemiological understanding of serotype dynamics, track vaccine-driven serotype replacement, and underpin evidence-based pneumococcal vaccine policy for high-burden countries.