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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 lytA qPCR 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 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. While 30 of 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 reculture attempts of the lytA -positive samples collected in 2018/2019, we isolated 23 strains (in 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. We identified a comparatively large proportion of samples generating positive signals with the widely used lytA 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. IMPORTANCE Testing saliva samples with quantitative PCR (qPCR) improves the sensitivity of detection of pneumococcal carriage. The qPCR assay targeting lytA , 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 piaB (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 lytA may lead to an overestimation of true carriage rates., Competing Interests: The authors declare a conflict of interest. A.L.W has received consulting and/or advisory board fees from Pfizer, Diasorin, PPS Health, Co-Diagnostics, and Global Diagnostic Systems for work unrelated to this project, and is Principal Investigator on research grants from Pfizer, Merck, and Flambeau Diagnostics to Yale University. D.M.W has received consulting fees from Pfizer, Merck, GSK, Affinivax, and Matrivax for work unrelated to this project and is Principal Investigator on research grants and contracts with Pfizer and Merck to Yale University. All other co-authors declare no potential conflict of interest.

Recent discoveries demonstrate a critical role for circadian rhythms and sleep in immune system homeostasis. Both innate and adaptive immune responses - ranging from leukocyte mobilization, trafficking, and chemotaxis to cytokine release and T cell differentiation -are mediated in a time of day-dependent manner. The National Institutes of Health (NIH) recently sponsored an interdisciplinary workshop, "Sleep Insufficiency, Circadian Misalignment, and the Immune Response," to highlight new research linking sleep and circadian biology to immune function and to identify areas of high translational potential. This Review summarizes topics discussed and highlights immediate opportunities for delineating clinically relevant connections among biological rhythms, sleep, and immune regulation.

In Italy, Toscana virus is the most common cause of meningitis from May to October. Though only a few cases have been reported in U.S. travelers returning from Europe, most cases are likely unrecognized due to lack of familiarity with the disease. Here, we describe the case of an 82-year-old man presenting with fever, profound weakness, and hearing loss after returning to the United States following a 2-week summertime vacation in southern Italy who was ultimately diagnosed with Toscana virus encephalitis. This case should alert clinicians to the possibility of Toscana virus infection in returning travelers and provides information on how to obtain testing if Toscana virus is suspected., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)