Introduction

Pneumonia is the leading cause of death in children under five years old worldwide [1]. More than two million children die from pneumonia every year, which exceeds the total number of deaths caused by AIDS, malaria, and measles combined. Worldwide, one out of five deaths of children under five years old is caused by pneumonia. The Indonesian Health Profile reported that 922,000 Indonesian children under five years of age died from pneumonia in 2015 and West Nusa Tenggara is in first place with total cases in children under five was 33,291 cases [2, 3].

The most common cause of bacterial pneumonia is Streptococcus pneumoniae, which lives in colonies in the human nasopharynx. The transmission of various pneumococcal infections can be prevented by the administration of the pneumococcal conjugate vaccine in infants under one year of age. PCV10 dan PCV13 are licensed to reduce invasive pneumococcal disease (IPD). Two doses of PCV10 or PCV13 given before the age of 12 months at an interval of 4–8 weeks or more and one booster doses given between 9–15 months of age [4]. The effectiveness of PCV is important because it correlates with a reduced disease burden of IPD causes by vaccine-type (VT) serotype. Harboe et al. [5] showed that IPD incidence was reduced 21% in the total population and 71% in children under five years old after PCV13 administration in the Netherlands. PCV reduces carriage, and thereby transmission of VT pneumococci, providing both direct and indirect protection from common disease-causing serotypes. As a PCV program matures, VT serotype may reduce and replacement may occur by non-vaccine type serotype (NVT) [6, 7].

In Indonesia, research on the nasopharyngeal colonization in children was limited. The earliest pneumococcal study conducted on Lombok Island in 1997 and followed up in 2012 with PCV13 administration. The results of both studies preceding PCV and after use of PCV13 showed no difference in the prevalence of nasopharyngeal colonization of S. pneumoniae (48% in 1997 and 46% in 2012) [8, 9]. Other study conducted in Bandung City and semi-rural area in Padalarang district (~25km from Bandung City), regarding pneumococcal carriage prevalence, mentioned the prevalence was 22.0% at the beginning of the study and increased to 68.4% after 10 months from study [10].

In 2012, the World Health Organization Strategy Advisory Group of Experts (WHO SAGE) recommended the administration of PCV for national immunization programs worldwide. Specifically, the WHO recommended three doses of PCV, either three primary doses (3+0) or two primary doses plus booster (2+1). There are multiple factors to consider in implementing a national immunization program, such as the interval of primary doses, the use of booster, and cost-effectiveness. There is no single schedule that is optimal for every setting. According to WHO, the 3+0 vaccination schedule of PCV may be preferred in countries in which disease rates peak well before the end of the first year of life or when the immunization coverage is low if given after the first year, whereas the 2+1 schedule is given when the duration of protection becomes a concern or for specific protection against serotypes 1, 3, or 5. Under the observation of an invasive pneumococcal disease, the 2+1, 3+0, and 3+1 schedules are equally effective. In addition, PCV immunization will help to increase immunization coverage if the schedule coincides with the established national immunization schedule, as it will be more convenient both for the parents and health workers [11].

Indonesia requires an optimal schedule to gain maximum protection for vulnerable age groups. Since 2017, the national PCV immunization program conducted in Indonesia has been via the demonstration program, in which PCV immunization begins in one small area then gradually expands to the entire country. The immunization schedule used is 2+1, adjusted to the national immunization schedule. The first dose of PCV13 is administered to two-month-old children simultaneously with DPT/HepB/Hib1 and OPV2, and the second dose of PCV13 is administered to children three months old along with DPT/HepB/Hib1 and OPV3. The third PCV13 injection (booster) is given to children 12 months old. Evaluation of the program effectiveness is necessary as the basis for continuing the PCV immunization program to other provinces. Pneumococcal vaccine effectiveness can be assessed by measuring changes in S. pneumoniae serotype colonization in the nasopharynx, which compares the prevalence of VT serotypes to NVT serotypes.

The goal of this research is to assess the dynamic changes of S. pneumoniae colonization before and after PCV13 administration using two primary doses (in a one-month interval) and one booster dose in healthy infants and toddlers aged 2–18 months in Indonesia. After vaccination with PCV13, we hypothesized that VT serotypes would be replaced by NVT serotypes or become no colonization.

Methods

Results

Demographic characteristic

The first nasopharyngeal swab was collected from 233 two-month-old infants (115 subjects in control groups and 118 subjects in vaccine groups). The second and third nasopharyngeal swabs were obtained from 230 four-month-old infants and 223 12-month-old infants, respectively. The last nasopharyngeal swab was collected from 233 18-month-old children. Ten infants dropped out of the study, six infants moved, three infants refused to continue the study, and one died of febrile convulsions (Fig 1). All infants were included in the analysis as originally allocated to control and vaccine groups.

Fig 1

Flow chart of participants in the study.

Gender prevalence in both groups were relatively equal: in the control group, 46.1% were male, and 53.9% were female, and in the vaccine group, 51.7% were male, and 48.3% were female. Most commonly, subjects lived in a household of 4–6 people (63.5% in the control group and 68.6% in the vaccine group). Most 2-month-old infant subjects were exposed to cigarette smoke (99 of 115 [86.1%] in the control group and 92 of 118 [78%] in the vaccine group). However, there was a significant difference of nutritional status between cohort groups (p < 0.001). Most subjects in the control group (106 of 115 subjects, 92.2%) and the vaccine group (86 of 122 subjects, 72.9%) had good nutritional status (Table 1).

Table 1

Demographic characteristics by cohort groups.

Characteristics	Control			Vaccine			Overall p-value
	n (%)	Odds Ratio (95% CI)	p-value	n (%)	Odds Ratio (95% CI)	p-value
Gender							0.392
Male	53 (46.1)	2.42 (0.93–6.34)	0.066	61 (51.7)	2.77 (1.10–6.98)	0.027
Female	62 (53.9)	Reference		57 (48.3)	Reference
Nutritional Status							< 0.001
Overweight + Obese	9 (7.8)	N/A	0.129	21 (17.8)	N/A	0.003
Normal weight	106 (92.2)	Reference		86 (72.9)	Reference
Underweight	0 (0)	N/A	N/A	11 (9.3)	N/A	0.029
Number in household							0.123
1–3	27 (23.5)	0.34 (0.07–1.81)	0.195	16 (13.6)	1.07 (0.24–4.84)	0.933
4–6	73 (63.5)	0.71 (0.19–2.55)	0.600	81 (68.6)	0.91 (0.29–2.84)	0.880
≥ 7	15 (13)	Reference		21 (17.8)	Reference
Cigarette smoke exposure							0.107
Yes	99 (86.1)	4.08 (0.50–32.35)	0.158	92 (78.0)	0.46 (0.18–1.19)	0.107
No	16 (13.9)	Reference		26 (22.0)	Reference

Table 2 shows the demographic characteristics in S. pneumoniae positive and negative groups. There were significant differences in gender between the positive and no colonization groups, yet other demographic characteristics were not significantly different.

Table 2

Demographic characteristics in S. pneumoniae positive and negative groups.

Characteristics	S. pneumoniae		Odds Ratio (95% CI)	p-valuea
	Positive (n = 49)	No Colonization (n = 184)
Gender				0.004
Male	33 (28.9%)	81 (71.1%)	2.62 (1.35–5.09)
Female	16 (13.4%)	103 (86.6%)	Reference
Nutritional Status				0.255
Overweight+Obese	0	30 (100%)	N/A
Normal weight	49 (25.5%)	143 (74.5%)	N/A
Underweight	0	11 (100%)	N/A
Primary Health Care Area				0.261
Praya	22 (19.1%)	93 (80.9%)	0.99 (0.36–2.69)
Gerung	10 (18.2%)	45 (81.8%)	0.93 (0.30–2.85)
Lenek	11 (34.4%)	21 (65.6%)	2.18 (0.69–6.90)
Selong	6 (19.4%)	25 (80.6%)	Reference
Number in household				0.625
1–3	7 (16.3%)	36 (83.7%)	0.58 (0.19–1.76)
4–6	33 (21.4%)	121 (78.6%)	0.82 (0.35–1.91)
≥ 7	9 (25%)	27 (75%)	Reference
Cigarette smoke exposure				0.625
Yes	39 (20.4%)	152 (79.6%)	0.82 (0.37–1.81)
No	10 (23.8%)	32 (76.2%)	Reference

^aChi-Square test.

The prevalence of S. pneumoniae

Streptococcus pneumoniae colonization was found in 49 of 233 (21%) two-month-old infants. The youngest subject who carried S. pneumoniae in the nasopharynx was 46 days old and in the control group. This subject carried serotype 3 in the first nasopharyngeal swab, then 6A/6B in the second nasopharyngeal swab, 6B in the third nasopharyngeal swab, and was negative at 18 months of age.

The prevalence of S. pneumoniae carriage increased with age (Fig 2), with no significant difference between the control group and vaccine group (p > 0.05). The prevalence in 12-month-old subjects positive for S. pneumoniae was significantly higher than in two- month-olds in the control and vaccine groups (p < 0.01).

Fig 2

The prevalence of S. pneumoniae-positive subjects by age in control and vaccine groups.

VT and NVT serotypes prevalence by age

Fig 3 shows the prevalence of VT and NVT serotypes in both control and vaccine groups at 2, 4,12, and 18 months old. There was no significant difference between both groups at 2, 4, and 12 months old (p > 0.05). At 18 months old, the prevalence of VT serotypes in the vaccine group was significantly lower than the control group (p < 0.01). Furthermore, we observed participants in the control group who were non-pneumococcal carriers at age 2 months have a higher prevalence of VT carriage at age 12 months, compared with participants in the vaccine group who were non-pneumococcal carriers at age 2 months.

Fig 3

VT and NVT serotype prevalence by age in control and vaccine groups.

Discussion

The study aimed to assess the effectiveness of PCV13 administration in altering nasopharyngeal S. pneumoniae colonization using two primary doses (in a one-month interval) and one booster dose in healthy infants and toddlers aged 2–18 months in Indonesia. We found that the sociodemographic data in both groups were relatively equal as baseline, except in nutritional status. The vaccine groups were significantly more likely than control groups to be malnourished (underweight or overweight/obese). PCVs have been found to have lower effectiveness among malnourished children. The explanation for this effect remains unknown [19, 20].

We found that the rates of S. pneumoniae colonization in this study was slightly higher than in other studies conducted in Indonesia. A study from Bandung in 2005 showed that the prevalence of pneumococci at six weeks was 12% and 13.9% at eight weeks old [21]. Murad et al. [10], in the same city, found that the prevalence increased to 15% within nine years, but was still lower than in this study. This difference may be influenced by people’s behavior patterns, geographical factors, exposure to cigarette smoke, and other factors. In the Murad study, 63% of subjects were exposed to cigarette smoke, whereas in this study, 78–86.1% had been exposed. The prevalence of subjects who experienced pneumococcal colonization in this study was also higher than those found in studies in Finland (9%) and Turkey (9.7%) [22, 23]. However, the prevalence was still lower than in subjects six weeks of age in Ethiopia in 2013 (32.7%) [24].

The prevalence of S. pneumoniae increased with age in the control and vaccine groups, and there was no significant difference in either group in changes of S. pneumoniae colonization by age. Although before PCV13, the vaccine group had a higher prevalence of pneumococcal colonization than the control group, after nine months (from the second dose), it was slightly lower than in the control group and persisted up to six months after the booster administration, although without a statistically significant difference. Furthermore, there are other factors beyond PCV vaccination which affect pneumococcal colonization in the nasopharynx of infants and children, such as immune system maturity, physical activity, number of household members, frequency of respiratory tract infections, exposure to cigarette smoke, and others [25].

These findings are supported by other published studies. Heinsbroek et al. [26] stated that three years after the introduction of PCV13 in 2011 in Malawi, pneumococcal prevalence rates remained high at 68%, whereas initially at six weeks of age, the prevalence was 43.8%. Meanwhile, an Ethiopian study by Sime et al. [24] demonstrated the prevalence of pneumococcal colonization remained high (49.1%) at two years of age after the administration of PCV10 using a 3+0 schedule when the initial pneumococcal prevalence rate at six weeks of age was 32.7%.

The persistence of nasopharyngeal pneumococcal rates a few years after the introduction of the PCV vaccine did not follow the expected trend of a higher prevalence of VT than NVT serotypes. Pneumococcal colonization was dominated by NVT serotypes with a decreasing prevalence of VT serotypes after the completion of the PCV immunization. In Ethiopia, Sime et al. [24] found that the rate of VT was 18.4% at six weeks of age and decreased to 7.1% at two years of age after PCV10 immunization. Likewise, Heinsbroek et al. [26] reported three years after the introduction of PCV13 in 2011 in Malawi, the rate of VT colonization decreased from 13% to 9.1% at 18 weeks, slightly increased to 16.5% at 1–4 years of age and stably decreased (7.9% at five years of age) after PCV13 immunization. A microsimulation model from Finland predicted elimination of VT carriage among vaccinated individuals within 5–10 years of PCV introduction [27].

We found four VT serotypes that always colonized the nasopharynx since before PCV13 immunization until after completion of the PCV13 schedule: 6A/6B, 19F, 23F, and 14. Serotypes 6A/6B, 23F, and 19F were found predominantly at the same location in Central Lombok in the previous study by Hadinegoro et al. [9] in 2011. This means that after seven years and even after PCV13 immunization on a 2+1 schedule, the dominance of these three serotypes in Lombok did not change. The Pneumococcal Conjugate Vaccine Review of Impact Evidence (PRIME) [28] stated there is a persistent carriage of VT even after more than three years of post-PCV13 administration.

Serotype 6A/6B was consistently dominant in the control and vaccine groups, both before and after completion of the PCV13 injection. It appears that PCV13 has begun to play a role in suppressing serotype 6A/6B colonization after the second dose, while for those who were unimmunized with PCV13, the prevalence of serotypes 6A/6B will continue to increase by age. The consistent presence of serotypes 6A/6B in the nasopharynx is understandable because both serotypes 6A and 6B are 2 of the 3 serotypes with the longest acquisition duration among all pneumococcal serotypes. Serotype 6A has a colonization duration of 237 days and 6B, 184 days [29].

The persistence of serotypes 6A/6B, 23F, 19F, and 14 six months after the third dose of the PCV13 administration (at 18 months old) is supported by the results of Sime et al. [24] Song et al. [30] reported that serotype 14 is highly invasive, whereas 6A, 6B, and 23F were generally less invasive, as stated in most studies. There are different clinical impacts of each serotype, such as serotype 19F, which is associated with higher mortality. Serotypes 6A/6B became the most common etiology of pneumococcal meningitis (40%) in Ugandan children under five years of age, according to their surveillance study, followed by serotypes 22A, 23F, 14, and 19A [31]. A meta-analysis study showed that serotype 14 was the most prevalent etiology of pneumococcal community-acquired pneumonia [32].

This study did not show a reduction in VT serotypes either one month or eight months after the administration of the second primary dose. The decrease in VT serotypes occurred after booster administration. A short period of the PRIME observation [28] showed that 2+1 provided a higher antibody response for most serotypes compared to the 3+0 schedule, but with no statistically significant differences, except for serotype 6B. The administration of the booster dose provides a higher antibody concentration compared to only primary doses. The PRIME [28] also showed that the 2+1 schedule provides a greater reduction of VT serotypes compared to 3+0, although this reduction was not statistically significant. However, Whitney et al. [11] mentioned that the effectiveness between schedules only relevant in the early PCV program and context dependent. Furthermore, Flasche et al. [33] and Choi et al. [34] stated that it may even be sufficient to move to a 1+1 schedule in mature PCV program.

Goldblatt et al. [35] stated that the two-month interval provided a better immunological response than the one-month interval if immunization was started at two months of age. Whitney et al. [11] showed in a review that the IgG geometric mean concentrations of two primary doses using the two-month interval was higher than with the one-month interval, but the difference was not statistically significant. The PRIME [28] could not assess the impact of the interval schedule on reduced nasopharyngeal carriage due to a lack of data. Our study showed that two primary doses administered with a one-month interval could reduce the VT serotype compared to the control group in nasopharyngeal carriage.

The Indonesian Ministry of Health decided to implement the 2+1 schedule for PCV immunization, given simultaneously with the pentavalent vaccine (DTP/hepatitis-B-Hib & OPV) in the National Immunization Program, according to Indonesia Technical Advisory Group on Immunization recommendation. This schedule will increase the immunization coverage and compliance of the parent due to a reduction in visits to the health facility.

Study limitations

The limitation of this study is the difficulty in determining the duration of serotype acquisition of nasopharyngeal S. pneumoniae due to a wide range and inconsistent interval of nasopharyngeal swab collection. In addition, this study includes relatively small sample size and only take place in single geographic area with distinct pneumococcal epidemiology based on national demonstration program, which may limit application to broader areas/populations in Indonesia but will help to evaluate the program. The logistic regression models were also tried to be performed to investigate the potential confounders, but the analysis as described did not account for potential confounders.

Conclusions

This study shows lower prevalence of VT and higher prevalence of NVT serotypes among children who were PCV13 2+1 vaccinated compared with those who were unvaccinated. Another notable finding is that serotypes 6A / 6B in the nasopharynx, as the most dominant serotypes found in infants, were lower among children who were PCV13 vaccinated. The result from this study will be the beginning of future vaccine evaluation. Further studies are needed in larger population and longer period of study.