Rapid FTIR-based serotyping of Streptococcus pneumoniae using the IR Biotyper: a comparative study against WGS and PCR-hybridization for clinical algorithm integration

ABSTRACT Serotyping of Streptococcus pneumoniae is essential for epidemiological surveillance and vaccine formulation. While the Quellung reaction remains the gold standard, it is limited by its technical demands and cost. Genomic approaches such as whole-genome sequencing (WGS) offer high accuracy, but are resource-intensive and complex to implement routinely. The IR Biotyper, based on Fourier transform infrared spectroscopy (FTIR), provides a rapid, simple, and cost-effective alternative. A total of 324 invasive S. pneumoniae isolates collected between January and December 2024 were included in this study. Each strain was serotyped using FTIR, WGS, and PCR with reverse hybridization (PCR/HI). We assessed agreement at both the serogroup and serotype levels using Cohen’s Kappa index and evaluated FTIR’s ability to detect serotypes included in current pneumococcal vaccines. FTIR showed good agreement with WGS (Kappa = 0.782; P < 0.001) and with PCR/HI (Kappa = 0.777; P < 0.001). It accurately identified prevalent serotypes, such as 3, 8, 33F, and 9N, with strong performance for vaccine-included serotypes. The main limitation was intra-serogroup discrimination, particularly in structurally similar serotypes. FTIR is a reliable and reproducible method for preliminary serotyping of S. pneumoniae , suitable for integration into tiered diagnostic algorithms. Its rapid turnaround and alignment with molecular/genomic methods support its use as a screening tool in clinical microbiology laboratories, particularly in high-demand settings. IMPORTANCE Serotyping of Streptococcus pneumoniae is crucial for monitoring vaccine coverage and guiding public health interventions; yet, current gold-standard methods are costly, time-consuming, or technically demanding. This study demonstrates that Fourier transform infrared spectroscopy (FTIR)-based serotyping using the IR Biotyper system is a reliable, rapid, and cost-effective alternative for routine use in clinical laboratories. By validating FTIR against both whole-genome sequencing (WGS) and PCR-hybridization across a large cohort of invasive isolates, we provide compelling evidence for its inclusion in diagnostic workflows. The results support a practical, tiered algorithm where FTIR serves as an efficient first screening method, reserving molecular or genomic techniques for complex or ambiguous cases. This approach balances diagnostic performance with clinical feasibility and has direct implications for improving real-time pneumococcal surveillance and optimizing vaccine strategies.