Streptococcus pneumoniae carriage studies in adults: Importance, challenges, and key issues to consider when using quantitative PCR-based approaches

Abstract

Streptococcus pneumoniae causes significant morbidity and mortality among older adults. Detection of pneumococcal carriage is an accepted endpoint in pneumococcal conjugate vaccine studies. However, low sensitivity of culture-based approaches and nasopharyngeal samples have hampered adult S. pneumoniae carriage studies in the past. In contrast, detection of adult S. pneumoniae carriers with qPCR-based approaches can achieve high sensitivity and specificity and qPCR-based testing of oral samples improves accuracy of adult carriage detection. In this Viewpoint we outline a strategy for accurate qPCR-based testing. We recommend a dual-target approach for S. pneumoniae qPCR detection as no genetic target is universally present among or solely unique to it. Furthermore, we advise the evaluation of concordance among quantified qPCR targets to improve the accuracy of S. pneumoniae testing and qPCR-based serotyping. We do not recommend omission of qPCR-based oral sample testing as it will likely result in an underestimation of true adult carrier rates.

A spitting image: molecular diagnostics applied to saliva enhance detection of Streptococcus pneumoniae and pneumococcal serotype carriage

“…When qPCR detection was applied to culture-enriched instead of MP saliva samples the number of child and adult carriers identified increased by 41 and 111%, respectively. It underlines the importance of testing oral samples for sensitive surveillance of pneumococcal carriage among adults ( Miellet et al, 2023 ).…”

“…When compared with the gold standard method, qPCR applied to culture-enriched saliva samples displayed significantly enhanced sensitivity of detection for both children and adults. Application of molecular methods to saliva samples allows us to capture an image of the S. pneumoniae carriage that mirrors the accuracy of nasopharyngeal sample testing in children and is superior in adults ( Miellet et al, 2023 ). However, 19 and 37% of pneumococci detected by qPCR were unique to nasopharyngeal cultures and culture-enriched saliva samples, respectively.…”

“…It implies that a negative nasopharyngeal sample does not necessarily preclude a positive saliva sample, and vice versa. As such, careful interpretation of carriage detection results is important ( Miellet et al, 2023 ).…”

“…Detection of S. pneumoniae and pneumococcal serotypes in nasopharyngeal and oropharyngeal samples, including qPCRguided culturing, has been detailed previously (Miellet et al, 2022(Miellet et al, , 2023. Saliva samples stored frozen with 10% glycerol were thawed and diluted in an equal volume of PBS.…”

“…Importantly, we ( Wyllie et al, 2017 ) and others ( Carvalho et al, 2012 , 2013 ; Boelsen et al, 2020 ) have cautioned that assays used for molecular detection and serotyping of pneumococcus may exhibit reduced specificity in oral samples due to the presence of pneumococcal genes among commensal oral streptococci. Consequently, a robust experimental strategy is needed to maintain high specificity of pneumococcal detection in polymicrobial samples ( Miellet et al, 2023 ). In a recent study we have described quantifying and comparing in an agreement analysis ( Ranganathan et al, 2017 ) pneumococcal genes, piaB and lytA , and serotype/serogroup-specific genes in nasopharyngeal samples from children, and nasopharyngeal and oropharyngeal samples from adults ( Miellet et al, 2022 ).…”