Enhanced LAMP-based assay for rapid detection of non-vaccine Streptococcus pneumoniae capsular types in meningitis

ABSTRACT Streptococcus pneumoniae remains a major pathogen responsible for invasive pneumococcal diseases (IPDs), such as meningitis and bacteremia, particularly affecting children under five and older adults. The introduction of pneumococcal conjugate vaccines (PCVs) has led to significant reductions in IPD incidence, yet the challenge of capsular-type replacement persists. Conventional serological typing methods, such as the Quellung reaction, are limited by their requirement for technical expertise and specialized resources, especially in low- and middle-income countries. To address these challenges, we previously developed loop-mediated isothermal amplification (LAMP)-based capsular typing assays for PCV13 and PPSV23. This study aimed to develop improved LAMP assays targeting fifteen non-vaccine capsular types (6C, 7C, 13, 15A, 16F, 21, 23A, 23B, 24F, 31, 34, 35B, 35F, 38, 39), and to assess their suitability for detecting these capsular types in cerebrospinal fluid (CSF) and blood. Each assay was evaluated to determine analytical reactivity, analytical specificity, and analytical sensitivity, and its performance was compared with that of CDC conventional multiplex PCR. To assess their applicability in patients with suspected meningitis, analytical detection limits were determined using bacterial genomic DNA-spiked CSF and blood specimens. The analytical specificity of the LAMP assays was confirmed using 50 capsular types of pneumococcal strains. The assays detected as few as 100 copies of purified DNA as well as DNA spiked into CSF and blood, demonstrating greater analytical sensitivity than conventional PCR. This highly specific, sensitive, and robust LAMP assay provides substantial advantages for large-scale screening, particularly in resource-limited settings. IMPORTANCE This is the first report of LAMP assays for these 15 capsular types of S. pneumoniae . LAMP assays demonstrated 10–1,000-fold improved analytical sensitivity over conventional PCR for several capsular types. Established LAMP assays targeting non-vaccine pneumococcal capsular types offer substantial value for public-health surveillance of IPDs. Monitoring these capsular types is particularly important for assessing the impact of newly introduced vaccines, such as PCV20 and PCV21. This approach enables efficient IPD surveillance even in resource-limited settings, supports timely optimization of vaccination strategies, and contributes to reducing the global IPD burden. This method may serve as a pivotal tool for tracking vaccine impacts in regions where non-vaccine-targeted capsular types represent an emerging threat.