Duplex ddPCR detection of lytA and piaB in Streptococcus pneumoniae from nasopharyngeal swabs

Detecting Streptococcus pneumoniae traditionally relies on complex and time-consuming culture methods, limiting sensitivity, especially in samples with low bacterial loads. Molecular diagnostics, particularly real-time PCR (qPCR), have significantly improved detection thresholds compared to culture. This pilot study aimed to assess the sensitivity and specificity of a duplex droplet digital PCR (ddPCR) assay targeting pneumococcal-specific genes lytA and piaB in nasopharyngeal swabs from asymptomatic children. A total of 165 nasopharyngeal swab samples were collected, of which ten samples were excluded during quality control, leaving 155 for analysis. These included 73 culture-positive (47%) and 82 culture-negative (53%) samples. The ddPCR analysis yielded 105 samples (68%) positive for both lytA and piaB, representing a statistically significant increase in detection compared to culture (McNemar's test, p < 1 × 10⁻⁶). An additional 12 samples exhibited single-gene positivity (lytA only: 7, piaB only: 5) but were not classified as definitive positives due to specificity concerns. Duplex ddPCR demonstrated enhanced sensitivity, particularly in samples with low pneumococcal density, and the requirement of dual-target positivity ensured high specificity. These results underline duplex ddPCR as a promising and robust diagnostic tool for pneumococcal carriage detection, with potential implications for epidemiological surveillance and clinical diagnostics. Future research directions include assay optimization, quantitative bacterial load analysis, and evaluation of the method's applicability for rapid pneumococcal detection in invasive disease contexts.