Digital PCR linkage analysis resolves Streptococcus pneumoniae signature from commensal interference in saliva...

Serotyping of Streptococcus pneumoniae (pneumococcus) is essential for evaluating the effects of conjugate polysaccharide vaccines on asymptomatic colonization (carriage) of vaccine-targeted capsular variants (serotypes). However, culture-independent application of molecular methods is hampered by genetic exchange between pneumococcus and oral streptococci co-residing within the airways. Unlike quantitative PCR (qPCR), digital PCR (dPCR) enables linkage analysis that allows for the identification of gene co-occurrence within individual bacterial cells. To minimize false-positive classification with molecular serotyping of highly polymicrobial saliva samples, a duplexed dPCR protocol for linkage analysis was developed. The performance of the protocol was evaluated by determining linkage between piaB and lytA, as well as between piaB and genes coding for serogroups 6, 9, and serotype 4 capsular polysaccharides in 141 saliva samples from children and adults from children and adults residing in the Netherlands, to identify pneumococcal strains present during natural carriage. Identification of linkage between distant genes required intact pneumococcal cells. The 95% limit of detection for linkage analysis between piaB and lytA was determined to be 9.2 CFU/reaction. Co-occurrence of piaB and lytA within single bacterial cells was consistently observed in saliva samples. Similarly, linkage was identified between serogroup 6 and piaB in the majority of saliva samples from serogroup 6 carriers. Application of dPCR distinguished pneumococcus from oral Streptococcus species in saliva tested for serogroup 9 and serotype 4. Duplex dPCR linkage analysis allowed for differentiation of pneumococcus from oral streptococci with homologous capsular loci. By improving the diagnostic accuracy of molecular surveillance, linkage analysis is a promising technique for culture-independent epidemiologic surveillance of pneumococcal serotype carriage.IMPORTANCEAccurate monitoring of pneumococcal conjugate vaccine effects requires reliable detection of Streptococcus pneumoniae serotype carriage in community-dwelling populations. However, sequences shared between pneumococcal strains and related commensal oral streptococci complicate this procedure. Here, we describe a method that takes advantage of the unique capacity of digital PCR (dPCR) to identify non-random associations among molecular targets in a sample, indicative of a single cell origin. Proof of principle was demonstrated using mock bacterial communities, followed by application to 141 saliva specimens from community-dwelling individuals. This methodological advance provides a reliable means of determining serotype-specific S. pneumoniae carriage using oral fluids, an approach particularly valuable for vaccine impact studies in adult populations. Given the growing public health interest in adult pneumococcal vaccination programs and the lack of validated methods for oral mucosa, we believe our protocol represents a timely and important contribution to the field. In addition, our findings help resolve longstanding debates on the utility of oral fluids for pneumococcal surveillance.