High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States.

While the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with quantitative PCR (qPCR), concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely used qPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (). During the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple time points from 103 healthy adults aged 21 to 39 ( = 34) and >64 ( = 69) years ( = 344 total samples). Following culture enrichment, extracted DNA was tested using qPCR for and . By sequencing the variable region of (S2 typing), we identified the species of bacteria isolated from samples testing -positive only. While 30 of 344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for both and , 52 (15.1%) samples tested -positive only. No samples tested -positive only. Through extensive reculture attempts of the -positive samples collected in 2018/2019, we isolated 23 strains (in 8 samples from 5 individuals) that were also qPCR-positive for only . Sequencing determined that Streptococcus mitis and Streptococcus infantis were predominantly responsible for this -positive qPCR signal. We identified a comparatively large proportion of samples generating positive signals with the widely used qPCR and identified nonpneumococcal Streptococcus 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 polymicrobial respiratory tract samples. Testing saliva samples with quantitative PCR (qPCR) improves the sensitivity of detection of pneumococcal carriage. The qPCR assay targeting , the gene encoding the major pneumococcal autolysin, has become widely accepted for the identification of pneumococcus and is even considered the "gold standard" by many. However, when applying this approach to investigate the prevalence of pneumococcal carriage in adults in New Haven, CT, USA, we identified nonpneumococcal Streptococcus spp. that generate positive signals in this widely used assay. By testing also for (encoding the iron acquisition ABC transporter lipoprotein, PiaB), our findings demonstrate the importance of testing for the presence of multiple gene targets in tandem for reliable molecular detection of pneumococcus in respiratory tract samples; targeting only may lead to an overestimation of true carriage rates.