Pneumococcal carriage and serotype distribution in Portuguese children six months after the lifting of COVID-19 restrictions: rise in serotype 3 amid stable non-vaccine serotypes

Streptococcus pneumoniae remains a major cause of infectious disease globally, with young children serving as key reservoirs for transmission. This study evaluated pneumococcal carriage and serotype distribution among young children in Portugal, six months after the lifting of COVID-19 public health measures and seven years after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the National Immunization Plan. A cross-sectional study was conducted in late 2022 among children aged 18 months to 6 years attending day-care centers in an urban region. Saliva samples, vaccination records, and demographic/clinical data were collected. Pneumococcal carriage was determined by qPCR targeting lytA and piaB genes. Molecular serotyping of 65 serotypes/serogroups was performed. New primers/probes were designed and validated for serotypes/serogroups 5, 7A/F, 9A/V, 9N/L, 17F and 33A/F/37, expanding the range of serotypes that could be reliably identified. Among 584 children, 34.9% were pneumococcal carriers. Carriage of PCV13 serotypes was low, except for serotypes 3 (7.8%) and 19F (5.4%). Among non-PCV13 serotypes, 23A (10.8%), 15B/C (10.3%), 23B (9.8%), and 11A/D (9.3%) were the most frequent. Theoretical coverage of PCV13, PCV15, and PCV20 was 17.6%, 23.0%, and 46.1%, respectively. Children 4-6 years were nearly 15 times more likely to carry PCV13 serotypes than those aged 18-24 months (p = 0.012). Six months post-COVID-19 restrictions lifting, serotype distribution largely resembled the pre-pandemic period. Although saliva sampling had lower sensitivity than nasopharyngeal swabbing for estimating overall pneumococcal carriage prevalence, it was particularly useful to assess serotype distribution. Importantly, in a context where nasopharyngeal swabbing was met with reluctance, saliva sampling emerged as a practical, effective, and non-invasive alternative for pneumococcal serotype carriage surveillance.