An abundance of aliC and aliD genes were identified in saliva using a novel multiplex qPCR to characterize group II non-encapsulated pneumococci with improved specificity

Pneumococcal surveillance studies are reporting increasing prevalence of non-encapsulated pneumococci (NESp). NESp are an important reservoir for genetic exchange among streptococci, including for antimicrobial resistance, and are increasingly implicated in disease. Disease-associated NESp commonly carries the virulence genes <i>pspK</i>, or <i>aliC</i> and <i>aliD</i> in their <i>cps</i> locus instead of capsule genes. While molecular methods targeting the cps region are widely used for serotyping encapsulated strains, there are few assays available for the classification of NESp, meaning it is not widely undertaken. Therefore, we exploited these genes as targets for a novel qPCR assay for detecting and classifying NESp strains with improved efficiency and specificity. We conducted bioinformatic analysis on sequences from 402 NESp and 45 other mitis-group streptococci and developed a multiplex-qPCR, targeting <i>pspK</i>, <i>aliD</i> and two regions of <i>aliC</i>. The assay was validated using 16 previously identified NESp isolates. We then applied the assay to DNA extracted from culture-enriched saliva and isolated and characterized suspected NESp colonies, with confirmation by whole genome sequencing. Bioinformatic analyses demonstrated that previously published primers for <i>aliC</i> and <i>aliD</i> had low pneumococcal specificity but indicated that targeting two regions of <i>aliC</i> would improve species specificity, without compromising sensitivity. Our novel multiplex assay accurately typed all isolates. When screening saliva, we found a high prevalence of <i>aliC</i> and <i>aliD</i>, even in samples negative for pneumococcal genes <i>lytA</i> and <i>piaB</i>. Isolated colonies which were <i>aliC</i> and <i>aliD</i> positive could be differentiated as non-pneumococcal streptococci using our assay. Our multiplex-qPCR assay can be used to efficiently screen even highly polymicrobial samples, such as saliva, for NESp genes, to detect and differentiate potentially pathogenic NESp clades from closely related mitis-group streptococci. This will allow for a better understanding of the true prevalence of NESp and its impact on pneumococcal carriage and disease.