There is little data published on pneumococcal carriage in Poland. Over the past two decades reports of only two Polish studies investigating the carriage of S. pneumoniae have been published. The first study was conducted between November 2000 and May 2001 in the capital city of Warsaw [14, 15]. The second study took place between November 2002 and June 2003 in Lublin [16, 17]. Both these studies have been conducted before the first of the PCVs, the heptavalent vaccine, became available commercially in the country. In light of obligatory pneumococcal vaccination for children born after 31st of December 2016, data on pneumococcal carriage is needed. Here, we investigated S. pneumoniae carriage in unvaccinated toddlers in the country with PCV10 and PCV13 commercially available for over five years and at the time of PCV10 implementation into infants’ immunization program. Our first goal was to establish a baseline for future studies investigating an impact of PCV10 introduction into NIP on S. pneumoniae carriage. The second goal was to assess suitability of testing oral fluids for pneumococcal carriage detection by comparing saliva testing with the gold standard method of conventional diagnostic culture of NPS.
Conventional diagnostic culture was the only method used to detect S. pneumoniae in previous studies conducted in Poland between 2000 and 2003 [14–17]. The carriage rate of 25.4% we detected with that method in our cohort was lower compared with 45.7–54.6% reported by Sulikowska et al. [14, 15] and 33.1–44.4% by Korona-Glowniak et al. [16, 17]. However, the differences in cohorts’ demographic composition, methods of sampling, and seasons of samples collection, make interpretation of differences in S. pneumoniae carriage difficult.
It is documented that DCC attendance increases pneumococcal transmission [29–31]. Also in all three Polish studies, our study included, the risk of pneumococcal carriage was significantly higher in children attending DCCs compared to staying home. Carriage rate of 31.9% detected by us with conventional culture of NPS in children attending DCCs was significantly different from 54.2–56.5% by Sulikowska et al. [14, 15] (Chi-square, p < 0.005). Hence, the higher proportion of children staying home in our study (47.5%) compared with cohort investigated by Sulikowska et al. [14, 15] (25.7%) and by Korona-Glowniak et al. [16, 17] (22.3%), per se contributed to lower rate of S. pneumoniae carriage. Interestingly, there was no significant difference between rate of 15.0% we detected in home-setting children compared with 19.3–25.9% reported by Sulikowska et al. (Chi-square, p > 0.1). Also, children have been enrolled into our study all year long whereas in the previous two exclusively during autumn and winter, the seasons when carriage rates are higher [31, this study]. It seems very likely that the continuous enrolment contributed further to lower carriage rates in our study. Finally, higher S. pneumoniae carriage reported by Korona-Glowniak et al. [16, 17] can be attributed to oropharyngeal samples (OPS) being cultured on the top of NPS in their study. When excluding results for OPS, there was no difference in point-prevalence of 25.4% detected with NPS culture by us and 24.4% detected by Korona-Glowniak et al. [16, 17] (n = 94 of 394 versus n = 228 of 933, Chi-square, p = 0.88). In conclusion, there is no evidence for any substantial indirect effects of commercial use of PCVs on rates of overall pneumococcal carriage in unvaccinated children (detected by culture).
There was, however, a reduction in proportions of PCV10-VTs (23.2% versus 73.4%) and PCV13-VTs (26.3% versus 80.4%) among the strains cultured in our study, when compared to the 2002–2003 study conducted by Korona-Glowniak et al. [16, 17] prior to PCVs entering the Polish market. This reduction in VTs carriage in unvaccinated children represents a heard effect of commercial vaccination with PCVs prior to and independent from PCV10 implementation into the NIP. Also, instead of a decline of PCV10-VTs carriage over the study period there was, albeit not statistically significant, an increase in a fraction of VTs among all strains cultured. It also points at commercial vaccination prior to PCV implementation into NIP as the dominant force behind the herd effect. It also suggests to us that study results can be considered to represent the baseline for future surveillance of impact of PCVs introduction into NIP on carriage.
One noticeable result was low prevalence of serotype 19A in carriage. Serotype 19A has been reported to emerge at various sites in replacement after PCV7 implementation [7, 32] and also to persist in carriage in populations with infants vaccinated with PCV10 [33, 34]. Here, carriage of 19A was detected in only two children and exclusively with the molecular method. With estimated 25–30% of infants being vaccinated in Poland with PCV13 outside NIP, low presence of this PCV13-VT could reflect indirect PCV13 effects. It also indicates a high IPD cases to carriers rate (CCR), thus high invasiveness of serotype 19A strains in Poland, because between 2016 and 2020 (time of present study) serotype 19A was, after serotype 14, the second most common in IPD in Polish children aged 12–59 months, whereas in our study serotype 19A ranked 19th in frequency in carriage [6].
Importantly, low numbers of VTs cultured from unvaccinated children make future assessment of direct effects of PCV implementation into NIP on VTs carriage difficult. The solution could be increasing the power of the next study by sampling higher numbers of subjects and/or detecting carriage of serotypes with a substantially more sensitive diagnostic approach. The latter can be addressed by testing multiple samples per child [16, 17] and/or employing molecular detection methods.
In our study, application of molecular diagnostics and testing saliva on the top of NPS increased the number of carriers detected by a factor of 1.7, from ninety-four identified by gold standard of conventional culture of NPS to an overall number of 158. In both materials, NPS and saliva, the application of molecular methods significantly increased the sensitivity of S. pneumoniae detection. This is in line with results reported by Wyllie et al. in studies applying a similar protocol to test NPS from children and to test NPS and saliva samples from adults, conducted between 2014 and 2016 in the Netherlands [20, 33].
NPS is the specimen recommended by WHO in pneumococcal carriage detection in children [19] and it has been reported that NPS is a more valuable material than OPS [35]. In our study, the culture of NPS was far superior to culture of saliva and also, 39.6% of carriers (61 of 154) identified by qPCR were detected by NPS only. However, with 21.5% (34 of 158) of carriers detected exclusively in saliva, testing oral fluids substantially increased the number of carriers detected. In line with this finding, Korona-Glowniak et al. [16, 17] also reported that testing OPS along NPS significantly increased the number of carriers detected, and that there was no difference between the number of carriers detected by culturing NPS compared with OPS [16, 17]. Therefore, the best carriage detection might be achieved by testing from each individual multiple specimens, e.g. NPS, OPS and saliva, or a combination of any two of these.
Molecular methods appeared to be superior to conventional culture in detecting co-carriage of multiple serotypes in this study (2.1% in culture versus 26.6% in qPCR). Wyllie et al. [33] and Kandasamy et al. [36] obtained similar levels of multiple serotype carriage using molecular methods. The higher sensitivity of any minority serotype detection in multi-serotype carriage allowed for a more detailed analysis of the occurrence of serotypes. Since available qPCR assays did not cover all serotypes, and not always distinguished serotypes within a serogroup, the number of multi-serotype carriers still might be understated.
Among our study limitations was a lack of molecular assays that would detect carriage of every circulating serotype. For example, strains of serotypes 24F, 28F, 35A, 35F, and 38 have been cultured from children, yet none of these serotypes were targeted with qPCR. Another limitation was low resolution of certain qPCRs not discriminating between serotypes within a serogroup, with 10 out of 27 qPCR assays targeting more than one serotype. A limitation was also the low sensitivity of conventional diagnostic culture. It concerns both sample types, but due to very rich bacterial growth, including many non-pneumococcal α-hemolytic colonies, it was particularly difficult to isolate live S. pneumoniae from saliva. With large numbers of serotype-carriage events detected exclusively with qPCR, and in the light of reports on non-pneumococcal streptococci expressing the pneumococcal capsular polysaccharides [28, 37], we paid particular attention to the specificity of assays. We addressed it by testing for serotype samples negative for S. pneumoniae and excluding the results of assays that generated a positive result (serotype 4 and serotype 5 specific qPCRs). Nevertheless, we can’t exclude that some of the results represent carriage of confounded non-pneumococcal bacteria detected with qPCRs. For example, when applied to oropharyngeal and saliva samples from adults, a diminished specificity of serogroup 9-specific assay has been reported [26, 28] and serogroup 9 was the clear outlier when culture data was compared with qPCR results in our study (Table 2 and Fig. 2a and 2c). However, since we did not observe positivity in this assay among samples negative for S. pneumoniae, nor was there a difference between the number of NPS and saliva samples positive for this serogroup by qPCR, and we are not aware of any reports on the assay’s poor specificity in NPS from children, we consider results for serogroup 9 as reliable.
In summary, pneumococcal carriage rate detected with WHO’s recommended method in Polish children was lower compared with studies conducted prior to the introduction of commercial PCVs in the country, yet we attribute it to differences in set-ups of studies rather than the effect of PCVs. On the other hand, the decline in prevalence of PCV10-VTs and PCV13-VTs carriage compared with the pre-PCV period suggests strong herd effects of commercial vaccination independent of NIP in Poland. According to the results obtained in our study, NPS was a more valuable material in carriage detection in children and qPCR was the more sensitive method in pneumococcal and pneumococcal serotype carriage detection. Also, information about carriage rate and serotype distribution among unvaccinated Polish children gained during this study can be used as a baseline in future carriage projects. The knowledge concerning the methods used in pneumococcal carriage detection gained during our study can be used for further research.