High levels of detection of non-pneumococcal species ofStreptococcusin saliva from adults in the USA

ABSTRACT Background While the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with qPCR, concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely-used lytA qPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay ( piaB) . Methods During the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple timepoints from 103 healthy adults aged 21-40 (n=34) and ≥64 (n=69) years. Following culture-enrichment, extracted DNA was tested using qPCR for piaB and lytA . By sequencing the variable region of rpsB (S2-typing), we identified the species of bacteria isolated from samples testing lytA -positive only. Results While 30/344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for both piaB and lytA , 52 (15.1%) samples tested lytA -positive only. No samples tested piaB -positive only. Through extensive re-culture of the 32 lytA -positive samples collected in 2018/2019, we isolated 23 strains (from 8 samples, from 5 individuals) that were also qPCR-positive for only lytA . Sequencing determined that Streptococcus mitis and Streptococcus infantis were predominantly responsible for this lytA -positive qPCR signal. Conclusions We identified a comparatively large proportion of samples generating positive signals with the widely used lytA -qPCR and identified non-pneumococcal streptococcal species responsible for this signal. This highlights the importance of testing for the presence of multiple gene targets in tandem for reliable and specific detection of pneumococcus in respiratory tract samples.