Molecular surveillance of pneumococcal carriage in all ages

The human upper respiratory tract is the main niche of common commensal, Streptococcus pneumoniae. However, acquisition of S. pneumoniae may also progress to pneumococcal disease, which despite being vaccine-preventable, remains a leading killer of infants and elderly. Up to 80% of children are colonised with one or more pneumococcal strains in their first years of life. They are therefore regarded the main reservoir for pneumococci in the population and the primary demographic group targeted for pneumococcal vaccination. Carriage rates rapidly decrease with increasing host age, with carriage in adults and elderly now rarely detected, despite historical records reporting carriage in approximately 50% of all adults when oral samples were tested in sensitive animal inoculation assays. Since the early 1900s, sampling techniques have changed. The current gold standard method for pneumococcal detection is the culture of a nasopharyngeal swab, with nasopharyngeal carriage of S. pneumoniae now an accepted endpoint in studies on the effects of pneumococcal vaccines (PCVs). Sensitive detection of pneumococcal carriage and serotypes circulating across the whole population is of great importance for understanding the full effects of PCV-introduction and also when new vaccination strategies are being considered, especially since vaccination of at-risk adults, elderly in particular, is advocated. Extensive surveillance on both pneumococcal disease and carriage in the Dutch population has seen the Netherlands become a major player in pneumococcal research, particularly following the introduction of PCVs. Since the reliance on culture-based detection methods presents a major limitation in accurate reporting, these surveillance studies provided us with a unique opportunity to directly compare the currently recommended gold standard culture-based method to recently developed molecular methods for pneumococcal carriage detection. Moreover, we investigated alternative samples from the upper respiratory tract for enhanced pneumococcal detection in ages spanning from infants, through adulthood to elderly. As compared to conventional culture, application of molecular methods significantly increase the detection of pneumococcal carriage detection overall, as well as the frequency of individual serotypes circulating, in all age groups. However, no single upper respiratory tract sample is optimal across all ages. In adults, testing nasopharyngeal samples alone grossly underestimates carriage rates whereas testing saliva is superior to any other sample. Our findings from Dutch surveillances have implications for future carriage evaluation studies. While preference remains with nasopharyngeal sampling in infants, for all older ages we advocate saliva as the preferred sample in carriage surveillances. In addition, with little to no discomfort when sampling saliva, this encourages greater adherence to repeated sampling routines. Regardless of sample type collected, culture-enrichment is required for sensitive pneumococcal detection, particularly for pneumococci present at a lower relative abundance, or for secondary (or lesser) serotypes when serotype surveillance is being considered. Since we proved that culture-based detection of pneumococci in oropharyngeal and saliva samples is near impossible, culture-enriched samples should be tested with validated molecular methods targeting two pneumococcal-specific gene sequences; we recommend lytA and piaB. We highlight important considerations which must be undertaken for sensitive and specific detection of S. pneumoniae and pneumococcal serotypes