No 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 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.

Carriage of Streptococcus pneumoniae (pneumococcus) in the upper respiratory tract is considered a prerequisite to invasive pneumococcal disease. During the first year of the COVID-19 pandemic, markedly lower rates of invasive pneumococcal disease were reported worldwide.

Background. Reported rates of invasive pneumococcal disease were markedly lower than normal during the 2020/2021 winter in the Northern Hemisphere, the first year after the start of the COVID-19 pandemic. However, little is known about rates of carriage of pneumococcus among adults during this period. Methods. Between October 2020-August 2021, couples living in the Greater New Haven Area were enrolled if both individuals were aged 60 years and above and did not have any individuals under the age of 60 years living in the household. Saliva samples and questionnaires regarding social activities and contacts and medical history were obtained every 2 weeks for a period of 10 weeks. Following culture-enrichment, extracted DNA was tested using qPCR for pneumococcus-specific sequences piaB and lytA. Individuals were considered positive for pneumococcal carriage when Ct-values for piaB were less than 40. Results. We collected 567 saliva samples from 95 individuals aged 60 years and above (47 household pairs and one singleton). Of those, 7.1% of samples tested positive for pneumococcus by either piaB only (n=6) or both piaB and lytA (n=34), representing 22/95 (23.2%) individuals and 16/48 (33.3%) households over the course of the 10-week study period. Study participants attended few social events during this period. However, many participants continued to have regular contact with children. Individuals who had regular contact with preschool and school aged children (i.e., 2-9 year olds) had a higher prevalence of carriage (15.9% vs 5.4%). Conclusions. Despite COVID-19-related disruptions, a large proportion of older adults carried pneumococcus at least once during the 10-week study period. Prevalence was particularly high among those who had contact with school-aged children, but carriage was not limited to this group.

There is often a need to separate a specific serotype of pneumococcus from a complex mixture of other pneumococcal strains. For instance, there could be a minority strain present at a low frequency in the nasopharynx that is difficult to identify and isolate by traditional culture. Or there might be a need to detect a minority serotype that has switched capsules in clinical samples or in laboratory experiments. We therefore developed a novel assay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. We show using qPCR and colony counting methods, that six serotypes (12F, 23F, 3, 14, 19A and 15A) present as ~0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. This method is an important development for laboratory techniques which select for certain target serotype such as transformation and co-colonization experiments. With further development, this method has potential for clinical applications such as isolating pneumococcal strains from clinical samples.