Diagnostic Utility of Saliva in Molecular Carriage Surveillance of Vaccine-Targeted Bacterial Pathogens: A Spitting Image

The primary objective of this dissertation was to assess the diagnostic utility of various upper airway specimens, especially saliva, combined with molecular methods for the surveillance of asymptomatic colonization (carriage) by vaccine-targeted bacterial pathogens in both paediatric and adult populations. A central aim was the creation of an accurate and robust carriage surveillance protocol capable of (i) identifying community reservoirs of Streptococcus pneumoniae and Neisseria meningitidis and (ii) evaluating the impact of conjugate vaccine programs on vaccine-type carriage, a widely used endpoint for vaccine impact studies. In addition, the dissertation sought to (iii) identify risk factors for carriage and (iv) investigate viral-bacterial interactions among community-dwelling individuals.

Comparative analyses of paired nasopharyngeal and saliva samples from children together with nasopharyngeal, oropharyngeal and saliva samples from adults, demonstrated that the application of molecular methods to culture-enriched saliva revealed a markedly higher prevalence of pneumococcal carriage (~29%) in adults compared with diagnostic culture alone, and detected of up to five serotypes per carrier among toddlers. Molecular testing of oral samples proved particularly important in adults, in whom colonization was largely confined to the oral cavity and oropharynx. Without saliva testing, more than 80% of adult carriers would have been missed. While molecular testing significantly improved diagnostic accuracy, and specificity could be routinely monitored though a dual-target approach with agreement analysis, several serotype-specific assays displayed suboptimal specificity, underscoring the need for further method refinement.


Application of the proposed protocol enabled evaluation of pneumococcal conjugate vaccine (PCV) introduction in England and the Netherlands, as well as meningococcal MenACWY vaccine implementation in the Netherlands. The findings demonstrated diminished vaccine-type-meningococci carriage following MenACWY introduction and a variation in non-vaccine-type pneumococcal circulation between countries with different PCVs. As differences in pneumococcal serotype composition across sample types were limited by richness, and vaccine-type carriage remained universally low, no evidence was found for masked circulation of vaccine-targeted serotypes.

The protocol also facilitated examination of viral-bacterial associations across populations, including older adults. In this age group, frequent exposure to young children and rhinovirus infection were identified as risk factors for colonization, while influenza-like illness was associated with prolonged increase in pneumococcal abundance within the upper airways of previously non-colonized individuals.

In conclusion, the findings of this dissertation highlight the oral mucosa as an underappreciated reservoir of bacterial pathogens. This work provides strong evidence that saliva-based molecular surveillance represents a robust and scalable approach to studying pneumococcal and meningococcal carriage. With the introduction of higher-valency PCVs into adult immunization programs, the surveillance protocol developed here provides a much-needed tool for vaccine impact studies in non-paediatric populations. It also holds significant promise for informing vaccine policy in low- and middle-income countries, where disease-based surveillance is often infeasible. As vaccination strategies evolve, such approaches will be pivotal for guiding evidence-based policy, ensuring vaccine effectiveness, and strengthening global preparedness against respiratory bacterial pathogens.