ABSTRACTWhile 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 usedlytAqPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (piaB). 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 (n= 34) and >64 (n= 69) years (n= 344 total samples). Following culture enrichment, extracted DNA was tested using qPCR forpiaBandlytA. By sequencing the variable region ofrpsB(S2 typing), we identified the species of bacteria isolated from samples testinglytA-positive only. While 30 of 344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for bothpiaBandlytA, 52 (15.1%) samples testedlytA-positive only. No samples testedpiaB-positive only. Through extensive reculture attempts of thelytA-positive samples collected in 2018/2019, we isolated 23 strains (in 8 samples from 5 individuals) that were also qPCR-positive for onlylytA. Sequencing determined thatStreptococcus mitisandStreptococcus infantiswere predominantly responsible for thislytA-positive qPCR signal. We identified a comparatively large proportion of samples generating positive signals with the widely usedlytAqPCR and identified nonpneumococcalStreptococcusspecies 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.IMPORTANCETesting saliva samples with quantitative PCR (qPCR) improves the sensitivity of detection of pneumococcal carriage. The qPCR assay targetinglytA, 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 nonpneumococcalStreptococcusspp. that generate positive signals in this widely used assay. By testing also forpiaB(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 onlylytAmay lead to an overestimation of true carriage rates.