Molecular surveillance of pneumococcal carriage following completion of immunization with the 13-valent pneumococcal conjugate vaccine administered in a 3 + 1 schedule

Abstract

In a cross-sectional study, with the use of molecular methods, we aimed to gain insight into oropharyngeal pneumococcal colonization over time in 1212 Greek children recruited in general pediatric settings throughout the country; they were fully vaccinated with PCV13 (3 + 1 schedule). A single sample was obtained from each child at a time interval of 26 days to 70 months after administration of the 4th (booster) PCV13 dose; sampling time was divided into six time intervals. Carriage of Streptococcus pneumoniae was detected by real-time PCR targeting the lytA gene and isolates were serotyped by singleplex real-time PCR assays. Multiple control procedures to avoid false-positive results were applied. We showed an overall S. pneumoniae carriage rate of 48.6%. Serotyping identified typeable isolates in 82% of the total lytA-positive samples. Non-PCV13 serotypes represented 83.8% of total isolates when excluding serogroups with mixed PCV13 and non-PCV13 serotypes. In multivariate analysis daycare/school attendance emerged as the main contributing factor. Notably, serotypes 19A and 3 were the only two PCV13 serotypes the colonization rate of which increased over time (χ² for trend P < 0.001 and P = 0.012, respectively). The application of the SP2020 gene on lytA-positive serotyped samples showed pneumococcal colonization in 97% of cases, and the overall colonization profile over time closely resembled that of the lytA gene. With the provisions of the methodological approach and age group of our study, the use of the oropharynx emerges as a reliable alternative to the nasopharynx in estimating pneumococcal carriage in epidemiological studies.

Figure 1

Colonization rate of children with typeable isolates according to increasing interval since the last dose of PCV13 (total, healthy and children with respiratory tract infection).

Figure 2

Study serotypes/serogroups revealed according to their inclusion in the PCV13.

Figure 3

Percentage of colonized children with non-PCV13 serotypes, PCV13 serotypes, and serogroups 6A/B/C/D and 9A/L/N/V according to time elapsed since the 4th (booster) dose. There is significant increase of colonization with non-PCV13 and PCV13 serotypes with increasing time interval.

Figure 4

Percentage of colonized children with serotypes 19A, 3 and 19F according to time elapsed since the 4th (booster) dose. There is a significant increase of colonization of serotypes 19A and 3 with increasing time interval.

Figure 5

Samples tested positive for both genes: typeable (n = 424) (a) and samples not assigned to a specific serotype (n = 84) (b). Each blue circle indicates the CT values of samples tested positive for both lytA and SP2020 genes. The size of the blue circles (‘bubbles’) is proportionate to the number of samples which presented with the specific CT combination.

Figure 6

lytA and SP2020 gene positivity status of samples, according to time elapsed since the 4th (booster) dose (six interval durations spanning 26 days to 70 months).