Identification of pneumococcal serotypes with individual recognition of vaccine types by a highly multiplexed real-time PCR-based MeltArray approach

Pneumococcus serotyping is important for monitoring serotype epidemiology, vaccine-induced serotypes replacement and emerging pathogenic serotypes. However, the lack of high-resolution serotyping tools has hindered its widespread implementation. We devised a single-step, multiplex real-time polymerase chain reaction (PCR)-based MeltArray approach termed PneumoSero that can identify 92 serotypes with individual recognition of 54 serotypes, including all 24 currently available vaccine types. The limit of detection (LOD) and the ability to coexisting serotypes were studied, followed by analytical evaluation using 92 reference pneumococcal strains and 125 non-pneumococcal strains, and clinical evaluation using 471 pneumococcus isolates and 46 pneumococcus-positive clinical samples. The LODs varied with serotypes from 50 to 100 copies per reaction and 10% of the minor serotypes were detectable in samples containing two mixed serotypes. Analytical evaluation presented 100% accuracy in both 92 reference pneumococcal strains and 125 non-pneumococcal strains. Clinical evaluation of 471 pneumococcus isolates displayed full concordance with Sanger sequencing results. The 46 clinical specimens yielded 45 typeable results and one untypeable result. Of the 45 typeable samples, 41 were of a single serotype and four were of mixed serotypes, all of which were confirmed by Sanger sequencing or separate PCR assays. We conclude that the PneumoSero assay can be implemented as a routine tool for pneumococcal serotyping in standard microbiology laboratories and even in clinical settings.