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. 2021 Jan 26;16(1):e0245789. doi: 10.1371/journal.pone.0245789

Pneumococcal nasopharyngeal carriage in Indonesia infants and toddlers post-PCV13 vaccination in a 2+1 schedule: A prospective cohort study

Ari Prayitno 1,*, Bambang Supriyatno 1, Zakiudin Munasir 1, Anis Karuniawati 2, Sri Rezeki S Hadinegoro 1, Joedo Prihartono 3, Dodi Safari 4, Julitasari Sundoro 5, Miftahuddin Majid Khoeri 4
Editor: Ray Borrow6
PMCID: PMC7837470  PMID: 33497405

Abstract

Background

The PCV13 immunization demonstration program began in October 2017 in Indonesia. The aim of this study is to assess the dynamic changes of pneumococcal serotype before and after PCV13 administration, with two primary and one booster doses.

Methods

The prospective cohort study was conducted as a follow up study measuring the impact of PCV13 demonstration program by the Indonesian Ministry of Health in Lombok Island, West Nusa Tenggara, Indonesia, from March 2018 to June 2019. The subjects were two-month-old healthy infants who were brought to the primary care facility for routine vaccination and followed until 18 months of age. We use convenience sampling method. There were 115 infants in the control group and 118 infants in the vaccine group, and the PCV immunization was given on a 2+1 schedule. Nasopharyngeal (NP) swabs were collected four times during the vaccination periods by trained medical staff. Specimens were analyzed by culture methods to detect S. pneumonia colonization and multiplex polymerase chain reaction (mPCR) to determine serotype. The most frequently detected serotypes will be named as dominant serotypes. Descriptive analysis of demographic characteristics, the prevalence of overall and serotype colonization, and the distribution of serotypes were performed. The prevalence of both cohort groups were compared using chi-square test. Statistical significance was set at p < 0.05.

Results

Two hundred and thirty three infants age two months old were recruited, with 48.9% of the subjects were male and 51.1% of the subjects were female. Sociodemographic data in both cohort groups were relatively equal. Nasopharyngeal pneumococcal colonization before PCV13 administration occurred in 19.1% of the control and 22.9% of the vaccine group. The prevalence increased with increasing age in both groups. The prevalence of VT serotypes in control groups aged 2 months, 4 months, 12 months, and 18 months was 40.9%, 44.2%, 53.8%, and 54.3%, respectively, and in the vaccine group, 25.9%, 40.4%, 38.0%, and 22.6%, respectively. The most common VT serotypes in both groups were 6A/6B, 19F, 23F, and 14. The prevalence of VT serotypes decreased significantly compared to non-vaccine type serotypes after three doses of the PCV13 vaccine (p < 0.001). Another notable change was the decline in prevalence of serotype 6A/6B after PCV13 administration using the 2+1 schedule.

Conclusions

This study shows lower prevalence of VT and 6A/6B serotypes in the nasopharynx among children who were PCV13 vaccinated compared with those who were unvaccinated. The result from this study will be the beginning of future vaccine evaluation in larger population and longer period of study.

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

Study design and participants

A prospective cohort study was conducted as a follow up study measuring the impact of PCV13 demonstration program in Lombok Island, West Nusa Tenggara, Indonesia, from March 2018 until June 2019. In 2017, total population of under five children in Lombok Island was 290,866 with estimated 18,557 children with pneumonia (6.38%). The administration of PCV13 on a 2+1 schedule was carried out by the Indonesian Ministry of Health as demonstration program in Lombok (HK.01.07 / MENKES / 199 / 2017). The Ministry of Health administered PCV13 when subjects 2, 3, and 12 months old, whereas this study obtained nasopharyngeal swab before the first dose of PCV13, at the age of 4 months old, before booster dose administration at 12 months old, and 6 months after the last dose. The demonstration program started in 2017 from 2 districts in West Nusa Tenggara Province (West Lombok and East Lombok as the vaccine group) to 251 infants, where the most cases of pneumonia were found, and expanded in 2018 to the entire Lombok and Bangka district.

The study has been reviewed and approved by ethical committee of Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia (1053/UN2.F1/ETIK/2017). Written informed consent was obtained from parents/guardians of the infants before the study. In addition, parents/guardians were also asked for the full information of address and phone number of parents or guardian as follow-up methods to encourage them to come when the infants were 4, 12, and 18 months old.

The eligibility criteria for subjects of this study were two-month-old healthy infants who were brought to the primary care facility for routine vaccination in Lombok Island. Medical history, demographics data, living conditions, vital signs assessment, and anthropometric measurement were recorded on a case report form before the vaccination given. We use convenience sampling method with at least 90 subjects per group based on sample size (n) formula for cohort studies n=(Zα(1+1/m)p(1-p)+Zβp0(1-p0)/m+p1(1-p1))2(p0-p1)2, with p=p1+mp0m+1. Here, Zα is standard normal variate for level of significance (α = 5%; Zα = 1.96), m = number of control subject per vaccinated subject (m = 1), Zβ is standard normal variate for power or type 2 error (β = 80%; Zβ = 0.84), p is the average proportion exposed, p0 is the probability of events in control group based on previous research prevalence (26%) [9], and p1 is the probability of events in vaccinated group based on previous research (10%) [12, 13]. Ten percent of drop out estimation (f) was calculated using formula n'=n(1-f), then the minimum subjects for each group are 110 subjects. Subjects lived in Central Lombok have not immunized for PCV at the mean time and were enrolled to control group (n = 115), whereas subjects lived in West or East Lombok received PCV13 immunization (as the Indonesian Ministry of Health program) and were enrolled to vaccine groups (n = 118).

The exclusion criteria were infants with acute infection (such as fever, acute otitis media, respiratory tract illness) within 7 days of recruitment, chronic illness, immunocompromise, or received antibiotic treatment during the 3 days before recruitment. Control and vaccine groups were age and location matched. If a recruited child became unwell over the period they aged from 2 months to 18 months, the nasopharyngeal swab would be performed after the catch-up immunization.

Sample collection

Nasopharyngeal (NP) swab specimens were collected from children by trained medical staff using a flexible nasopharyngeal flocked nylon swab (Copan, Italy; #503SC01), as recommended by WHO [14]. We collected four swabs during the vaccination periods: before vaccination (two months of age; the first dose), one month after the second dose (four months of age), before the third dose (12 months of age), and six months after the booster (18 months of age). The NP swabs were placed in cryotubes containing 1 mL skimmed milk, tryptone, glucose, and glycerol (STGG) as a transport medium and placed in a cooler with icepacks. Within 4 hours of collection, the inoculated STGG samples were vortexed for 10–20 sec prior to storage in a −80°C freezer at the Biomedical Laboratory General Hospital of West Nusa Tenggara Province [15, 16]. All NP-STGG specimens were transported in dry ice to the Eijkman Institute for Molecular Biology, Jakarta, Indonesia, for further analysis.

Laboratory procedures

Isolation and identification of S. pneumoniae were performed using methods previously described [17]. Briefly, 100 μL aliquots of each NP-STGG sample were added to 5 mL Todd-Hewitt broth supplemented with 0.5% yeast extract and 1 mL of rabbit serum, and the mixture was incubated for 5 hours at 37°C in a CO2 incubator. We cultured 10 μL of enriched NP swab on a sheep blood agar plate (BAP), which was incubated at 37°C with 5% CO2 for 18–20 hours. The colonies with pneumococcal characteristics (watery, alpha-hemolytic, flat-depressed center) were sub-cultured on BAP. An optochin disk (30 μg) was then placed on the surface of the inoculated blood agar plate for optochin susceptibility testing. The colonies susceptible to optochin (diameter > 14 mm) were defined as S. pneumoniae [15]. We performed bile solubility testing to confirm colonies with pneumococcal characteristics but with optochin resistance. Pneumococcal isolates were serotyped by conventional multiplex PCR. The microbiologists undertaking laboratory procedures were blinded to participant cohort and demographics [18].

Data analysis

Data from case report forms and laboratory was entered to Microsoft Excel 365. Datasets were imported and statistical analyzed using Graphpad Prism 8. Descriptive analysis of demographic characteristics, the prevalence of overall and serotype colonization, and the distribution of serotypes was performed in the beginning of the study to determine if control and vaccine groups were different at baseline. The differentiation at baseline may leads to different outcomes. Categorical variables were summarized by counts and percent. Inferential analyses using the chi-square test and determining the odds ratio were performed to compare data between control and vaccine group. Statistical significance was assigned at p < 0.05. However, this study will not be assessing vaccine effectiveness due to small sample size and limited time.

PCV13 serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F are considered vaccine-type (VT) serotypes, while others are non-vaccine type (NVT) serotype. If an isolate was detected, but typing was not possible due to biological reasons or technical issues, isolate will be assigned as untypeable serotype which included in NVT serotype. If multiple pneumococcal serotypes are identified, the infant will be recorded and observed based on the number of serotypes. Therefore, there will be higher number of serotypes than the number of infants. However, the data of each infants on carrying how many serotypes and the changes will not be reported in this study due to some considerations. The most frequently detected serotypes will be named as dominant serotypes. The following variables were assessed: gender, nutritional status, primary health care area, number in household, and cigarette smoke exposure as confounding variables. Nutritional status was assessed according to WHO guidelines (weight-for-age Z-score).

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.

Fig 1

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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
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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
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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.

Fig 2

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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.

Fig 3

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The distribution of S. pneumoniae VT and NVT serotype

The most common S. pneumoniae VT serotypes for both groups were 6A/6B, 19F, 23F, and 14 in all age groups. In the control group at two months of age, the most common serotype was 14 (4/9) followed by serotype 3 (2/9), whereas in the vaccine group, it was serotype 19F (3/7) followed by 6A/6B (3/7). The prevalence changed over time. Serotypes 19A, 1, and 5 were not found in the control group at two months of age. Serotype 19A appeared later at four months of age, while serotype 1 and 5 were present at 18 months of age. In the vaccine group, serotypes 3, 19A, and 5 were not found at two and four months of age. Serotypes 3 and 19A were present at 12 months of age, while serotype 5 appeared at 18 months of age in a small number of subjects. The most common NVTs in the control group were serotypes 15B/15C, 16F, 34, and 13, whereas, in the vaccine group, the most common were 15B/15C followed by 6C/6D, 23A, and 13.

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.

Supporting information

S1 Table. Vaccine type serotype distribution by age.

(DOCX)

Click here for additional data file. (19.5KB, docx)
S2 Table. Non-vaccine type serotype distribution by age.

(DOCX)

Click here for additional data file. (25.2KB, docx)

Acknowledgments

We would like to express our gratitude to all staff from the Ministry of Health in West Nusa Tenggara, Biomedical Laboratory General Hospital of West Nusa Tenggara Province, Eijkman Institute for Molecular Biology for the collection and processing of study data, and all research assistants who have supported throughout the study processes.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Ray Borrow

27 Aug 2020

PONE-D-20-14898

Dynamic changes in nasopharyngeal pneumococcal serotype before and after pneumococcal comjugate vaccine administered on a 2+1 schedule

PLOS ONE

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Reviewer #1: This paper aims to assess changes of pneumococcal carriage pre and post PCV13 administration, as a 2+1 schedule, in Indonesia. The key findings were that PCV13 as a 2+1 schedule reduced VT serotype carriage and reduced carriage of 6A/B as dominant serotypes. Overall, the scientific background and rationale for the investigation being reported could be strengthened and given further specificity. Strength of rationale would benefit is lacking. Overall, there is insufficient information regarding methods (e.g., sample size calculation, inclusion / exclusion criteria). It would be helpful to include further details in the results, such as a flow chart of participants, and a tabular comparison of participant characteristics stratified by control and vaccine cohorts. Largely the strengths and limitations of the paper and unaddressed, and should be reflected upon. In addition to the strong suggestion of using the STROBE checklist for observational studies, specific comments are below:

The study design is a prospective cohort – it would be of benefit to include this in the Title.

1. Title: Page 1: This title is descriptive and would draw the attention of those working in infectious diseases epidemiology, vaccinology, public health, and microbiology. Those working in the pneumococcal field would be drawn in particular. I would recommend specifying which PCV is under investigation. Further, it would be helpful to include the study design in the title. For example, something like “Pneumococcal nasopharyngeal carriage in Indonesian infants and toddlers post-PCV13 vaccination in a 2+1 schedule: a prospective cohort study.”

2. Abstract Page 2 Methods: Would suggest inclusion of a few more details in the Methods section, including how participants were recruited; how pneumococci were detected and isolated; how was dominance of serotypes determined; and what statistical methods were used for inferential analyses?

3. Abstract Page 2 Methods: Here and throughout the paper, would recommend using the term prevalence if carriage is reported as % in this paper, rather than proportion. A proportion can only have values from zero to one.

4. Abstract Page 2 Methods: Would recommend including the age eligibility of participants and any other broad inclusion criteria – e.g., this study recruited infants 2 months of age, followed until 18 months of age.

5. Here and throughout the paper, would recommend using the term prevalence if carriage is reported as % in this paper, rather than proportion. A proportion can only have values from zero to one.

6. Abstract Page 2 Results: Would suggest including a summary key demographics (e.g., x% of the participants were female), and any differences in participant characteristics by control and vaccine groups.

7. Abstract Page 2 Results: Would suggest including units and % after each reported age and prevalence, i.e., “the [prevalence] of VT serotypes in control groups aged 2 months, 4, months, 12 months, and 18 months was 40.9%, 44.2%, 53.8%, and 54.3%, respectively.

8. Abstract Page 2 Results: How was the p value determined, with regard to the reported significant decrease in VT serotypes compared with NVT serotypes? Further, would recommend reporting the p value itself, rather than p<0.05. In addition, it would be expected that the decline in VT serotypes would be greater than any decline in NVT serotypes, given the introduction of PCV13.

9. Abstract Page 3 Conclusion: I am unsure that the stated conclusion can be drawn from what appear to be crude analyses.

10. Introduction Page 3: Here and throughout, references are not cited where required – e.g., a reference for the statement that pneumonia is the leading cause of death in children under five years old worldwide – could recommend McAllister et al The Lancet, 2018 available here: https://www.thelancet.com/journals/langlo/article/PIIS2214-109X(18)30408-X/fulltext.

11. Introduction Page 3: Would recommend editing “The most common cause of pneumonia is Streptococcus pneumoniae” to “A leading cause of pneumonia is Streptococcus pneumoniae”.

12. Introduction Page 3: Would suggest rewording “PCV administration can change …may be relative unchanged”. 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, serotype replacement may occur.

13. Introduction Page 3 and 4: Would suggest editing “In Bandung City, Indonesia, the pneumococcal carriage prevalence was 22.0% at the beginning of the study and increased to 68.4% after 10 months” to reflect that reported prevalence includes data from children recruited from both Bandung city and from a semi-rural area in Padalarang district ~25km from Bandung city – this will help provide some further context for the reader.

14. Introduction Page 4: In the paragraph describing the recommendations of the WHO with regard to schedules, it would be useful to include the information provided on page 13 as to where and why the WHO recommends different schedules. This will help provide background and information as to the current study setting.

15. Introduction Page 4: To provide some further specificity, would recommend indicating that “the goal of this study is to assess the effectiveness of PCV13” rather than just PCV; and including the age of healthy children; similarly, would recommend indicating the age of children, e.g., “… in healthy children aged 2 – 18 months in Indonesia.”

16. Introduction: were there any prespecified hypotheses?

17. Introduction Page 4: To provide some further specificity, would recommend indicating that “the goal of this study is to assess the effectiveness of PCV13” rather than just PCV; and including the age of healthy children; similarly, would recommend indicating the age of children, e.g., “… in healthy children aged 2 – 18 months in Indonesia.”

18. Methods Page 4, Study Design and Participants: The description of the study design, indication of location, and time frame of study were very clear. Further, the overt statement of ethical approval for the study is important, and described clearly.

19. Methods Page 4/5, Study Design and Participants: Some further details describing the setting and location would be welcome – for example, the population size, whether access to healthcare is universal, and rates of pneumonia among children aged under 5

20. Methods Page 4/5, Study Design and Participants: The description of the study design, indication of setting, and time frame of study were very clear. Further, the overt statement of ethical approval for the study is important, and described clearly.

21. Methods, Study Design and Participants Page 5: Some key information regarding participants would be of value to include. For example, the eligibility criteria, sources and methods of participant recruitment, and methods of follow-up. Were control and vaccine groups matched? It appears that they may be age matched – where there any other matching criteria if relevant?

22. Methods, Study Design and Participants Page 5: The paper states that a consecutive sampling method was used, i.e., one in which every subject meeting inclusion criteria is selected until the required sample size is achieved. However, no sample size justification / calculation is provided, and neither are inclusion or exclusion criteria. Therefore it is unknown if the sample size provides sufficient power for this study. Further, if “healthy” children were recruited, what were the exclusion criteria – i.e., how was “healthy” determined? Were temperatures assessed and any child with a fever excluded? Were children with comorbidities, AOM, respiratory tract infections, taking antibiotics excluded? If a recruited child became unwell over the period they aged from 2 months to 18 months were they excluded?

23. Methods – would recommend a section that describes and defines all variables. That is, define clearly, all outcomes, exposures, predictors, potential confounders, and effect modifiers as relevant. For example, “VT carriage was defined as carriage of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F, 18C, and 23F”.

24. Methods, Sample collection, page 5: would recommend specifying the swab material – presumably flocked nylon, as opposed to just flocked.

25. Methods, Sample collection, page 5: would recommend reviewing and referencing Satzke et al, Vaccine 2013 available here: https://www.clinicalkey.com.au/#!/content/playContent/1-s2.0-S0264410X13011742?returnurl=https:%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0264410X13011742%3Fshowall%3Dtrue&referrer=https:%2F%2Fpubmed.ncbi.nlm.nih.gov%2F24331112%2F with regard to microbiological methods. It is important to cite this reference regarding the methods of NP sampling being in accordance with WHO guidelines.

26. Methods, Laboratory procedures, page 6: How was the dominant serotype determined?

27. Methods, Laboratory procedures, page 6: What kind of blood was used in the blood agar plates?

28. Methods, Data analysis page 6: Was it possible to type all isolates? Were there any instances where an isolate was detected, but typing was not possible due to biological reasons or technical issues? If so, how was this missing data handled?

29. Methods, Data analysis page 6: It is unclear why participants with multiple carriage were assigned either VT or NVT only. Where participants carry both VT and NVT, it should be possible to record an observation of carriage for both VT and NVT serotypes.

30. Methods, Data analysis page 6: It is unclear how the statistical methods employed address the stated goal of assessing vaccine effectiveness of PCV13 as a 2+1 schedule in health Indonesian children. It seems crude analysis only has been undertaken. Is this because the sample size was insufficient to permit more robust analysis, such as the standard 1 – odds ratio multiplied by 100, to determine VE? Further, would recommend that the analysis takes into account the significant differences in terms of participant characteristics by vaccine and control group, to determine the odds ratio for such analysis.

31. Methods, Data analysis page 6: It is important to describe any efforts made to reduce any potential sources of bias – these may arise through selection processes, measurement processes, and differences in groups. Please could it be stated whether the microbiologists undertaking laboratory procedures were blinded to participant cohort and demographics?

32. Methods, Data analysis page 6: Following the previous point, it is strongly recommended, that the two groups, i.e., vaccine and control, have participant demographics summarized via standard methods (i.e., categorical variables by counts and percentages, continuous variables by mean and standard deviation or median and interquartile range, as appropriate to the distribution) and then compared using appropriate methods. This is because it is important to determine if control and vaccine groups were different at baseline – differences in outcome may be due to differences at baseline, rather than the exposure of interest, and this should be considered.

33. Methods: It is important to describe any efforts made to reduce any potential sources of bias – these may arise through selection processes, measurement processes, and differences in groups.

34. Methods: Which software was used for databases, and for descriptive and inferential analyses?

35. Results: demographic characteristics, page 7: Please include a flow chart of participants

36. Results: demographic characteristics, page 7: A summary of the participant characteristics by control and vaccine groups is essential. Would strongly recommend including this in tabular form, along with relevant comparisons. It would be of benefit to report characteristics such as sex distribution across groups, and any differences in characteristics by group, along with a statistical comparison.

Results: demographic characteristics, page 7: The counts and percentages reported with regard to nutritional status and exposure to cigarette smoke by vaccine and control group appear to be quite different. Both of these factors have been shown in previous studies to be associated with pneumococcal carriage. For this reason, it is important to compare participant demographics by cohort group, and then adjust as necessary in inferential analyses.

37. Results: demographic characteristics, page 7: Recommend replacing table 1 with a comparison of characteristics by cohort group, then undertaking formal analyses of association of factors associated with carriage, and using the results to determine effectives of PCV13.

38. Results: The proportion of S. pneumoniae, page 8: Here, and throughout, as in the Abstract, suggest replacing “proportion” with prevalence, and including a 95% confidence interval.

39. Results: The pattern of S. pneumoniae serotype colonization, page 9: Suggest moving “Multiple colonization is colonization by more than one ….during the entire study.” to Methods, in a section on Variables.

40. Results: The pattern of S. pneumoniae serotype colonization, page 10: This paper states that “After vaccination with PCV13, we expected that VT serotypes would be replaced by NVT serotypes or become negative, and did not expect that NVT serotypes would be replaced by VT or there would be no colonization by VT. “ Any such statements could usefully be reworded as pre-specified, hypotheses and included in the Introduction. However, the duration of this study is very short (16 months) which is may not be sufficient to for serotype replacement in a vaccine naïve population to emerge.

41. Results: The pattern of S. pneumoniae serotype colonization, page 10: This paper refers to “subjects with negative colonization chang[ing] to VT’ etc. It would be of value to describe carriage as observations. For example, “We observed participants in the control group who were non-pneumococcal carriers at age 2 months to 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.”

42. Discussion, page 10: Recommend beginning the discussion with a summary of the key results along with reference to the study objectives. For example, “this study aimed to assess the effectiveness off of PCV13 …. We found ….”

43. Discussion, page 11: This paper states that “This means that PCV13 administration on a 2 + 1 schedule did not prevent an increase in pneumococcal colonization.” It is not clear whether this study has a sufficient sample size, or statistical methods that were undertaken, that support this statement.

44. Discussion, page 11: This paper states that “These findings prove that there are other factors beyond PCV vaccination which affect pneumococcal colonization in the nasopharynx of infants and children…” This study does not examine factors associated with pneumococcal carriage. As such, it does not provide evidence to support this statement. However, many previous studies have investigated factors associated with pneumococcal carriage, including Fadlyana et al Pneumonia, 2018 (available here https://link.springer.com/article/10.1186/s41479-018-0058-1) which describes factors associated with pneumococcal nasopharyngeal carriage in young children living in Indonesia. Further, given it has been recognised that factors other than vaccination affect pneumococcal carriage, it would be worthwhile conducting analyse that a) determine factors relevant to carriage in the current study sample and b) take such factors into account in analyses.

45. Discussion, page 12: The paper reports that “PCV13 is expected the result in no colonization of VT S.pneumoniae serotypes or replacement by NVT serotype. Although a microsimulation model from Finland predicted elimination of VT carriage among vaccinated individuals within 5 – 10 years of PCV introduction (under the assumption of a 90% vaccine coverage with 50% vaccine efficacy against acquisition) (see Nurhonen et al PLOS ONE 2013 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056079), other evidence suggests that vaccine impact from transmission models based on data from low carriage prevalence settings, may translate to high carriage prevalence settings. Further, any elimination of VT carriage is likely to occur in settings with a mature PCV program, rather than 16 months of PCV, as supported by the paper’s inclusion of reference to the PRIME finding that there is persistent carriage of VT even after more than three years of post-PCV13 administration. As such, it is inconsistent to expect “PCV13 vaccination …to result in no colonization of VT S.pneumoniae serotypes…”

46. Discussion, page 13: I am unconvinced that this study provides evidence to support the statement that “two primary doses are not enough to reduce VT serotypes in nasopharyngeal colonization.”

47. Discussion, page 13: The paper states that a “booster dose will only be effective if it is after two primary doses.” It should be noted that this is likely context dependent. For example, in a setting with a mature PCV program, it may be possible to move to a 1+ 1 schedule, as the UK has done. See Flasche et al PLOS Mid, 2015 (https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1001839) and Choi et al PLOSMed, 2019 (https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1002845).

48. Study limitations, page 14: I agree that a limitation of this study is the difficulty in determining duration of serotype acquisition – more frequent swabbing would be necessary to achieve this. Other limitations should also be acknowledged and reflected upon, including: the lack of generalizability beyond other healthy children; the differences between control and vaccine groups; reflections upon the sampling method; if the sample size is of sufficient size to provide power.

49. Conclusions, page 14: The effectiveness of 2+1 studies is known. This study has not provided evidence for this, and does not assesses vaccines.

50. It may be of use to read and reflect upon the following articles:

a. Wahl et al, Lancet Glob Health 2018 https://www.thelancet.com/journals/langlo/article/PIIS2214-109X(18)30247-X/fulltext

b. Fadlyana et al Pneumonia, 2018 https://link.springer.com/article/10.1186/s41479-018-0058-1

c. Satzke et al, PLOS Med, 2015 https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1001903

d. Greenberg et al, Clin Infect Dis 2016 https://academic.oup.com/cid/article/42/7/897/322584

e. Farida et al, PLOS ONE 2014 https://journals.plos.org/plosone/article?id=10.1371/jousrnal.pone.0087431

Reviewer #2: Important note: This review pertains only to ‘statistical aspects’ of the study and so ‘clinical aspects’ [like medical importance, relevance of the study, ‘clinical significance and implication(s)’ of the whole study, etc.] are to be evaluated [should be assessed] separately/independently. Further please note that any ‘statistical review’ is generally done under the assumption that (such) study specific methodological [as well as execution] issues are perfectly taken care of by the investigator(s). This review is not an exception to that and so does not cover clinical aspects {however, seldom comments are made only if those issues are intimately / scientifically related & intermingle with ‘statistical aspects’ of the study}. Agreed that ‘statistical methods’ are used as just tools here, however, they are vital part of methodology [and so should be given due importance].

COMMENTS: Study design is fairly simple; however, I have certain questions/doubts: In ‘Methods’ section, ‘Study design and participants’ subsection you said “Control group members were not immunized for PCV and lived in Central Lombok, while vaccine group members lived in West or East Lombok and received PCV13 immunization” which is O.K., but later you say “We use consecutive sampling method”, how is that done? What exactly you want to convey?

There are no comments on ‘Sample collection’ and ‘Laboratory procedures’ because they are beyond my area of knowledge. ‘Data analysis’ again is very simple [like simple study design]. You say ‘the Kolmogorov-Smirnov test was performed when some assumptions of chi-square were not satisfied’. The question is ‘which assumptions (of chi-square) were not satisfied?’ [note that Kolmogorov-Smirnov test is only a goodness-of-fit test; whereas chi-squared is a goodness-of-fit test as well as a test of association]

If only ‘0’ frequency in two cells [table-1] is the issue, I recommend to use RIDIT analysis [Bross IDJ. ‘How to use RIDIT analysis’. Biometrics, 1958; 14:18-38. There are many recent references, but they are on application results. This is (an original, old classic and) gives details on ‘how to use’ the technique].

Since the ‘Article Type’ is ‘Clinical Trial’ it is expected to give details of required sample size estimation. Even ‘allocation’ procedure is desirable to be describe adequately. As said in ‘Methods’ section of abstract it is actually the ‘prospective cohort study’, but it is still desirable to discuss about ‘required sample size estimation’. I am not sure regarding important ‘clinical significance’ and ‘implication(s)’ of this study and so in my opinion it is vital to assess the study in this context by subject expert. ‘What the study contributes?’ is always the important question.

‘Study limitations’ mentioned are not very clear. As said earlier ‘Study design’ as well as ‘Data analyses’ are simple and therefore, not much to comment.

Reviewer #3: The authors present the results of a study investigating nasopharyngeal colonization rates with vaccine and non-vaccine type pneumococci following vaccination of infants with PCV in Indonesia, with the finding of lower VT colonization rates in the vaccinated group at 18 months. As mentioned by the authors, pneumonia and pneumococcal infections are a major cause of global mortality among infants and young children, and such studies are important to understand pneumococcal epidemiology and inform immunization programs in different regions.

Major comments:

1. Revise/reorganize abstract and body of manuscript to clarify the relationship between the present study (which is referred to as a prospective cohort) and the clinical trial with vaccine and control groups. Was NP colonization a prespecified endpoint in a clinical trial, or was this a separate/ancillary cohort study of clinical trial participants?

2. A table or figure depicting pneumococcal serotype distribution would be helpful, either in the main manuscript or as supplemental material

3. Limitations also appear to include relatively small sample size and single geographic area with distinct pneumococcal epidemiology, which may limit application to broader areas/populations

Minor comments:

1. Title-spelling of pneumococcal conjugate vaccine

2. Introduction-clarify if pneumococcus is the most common cause of bacterial pneumonia (viruses generally more common overall)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachment

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Click here for additional data file. (14.6KB, docx)

Decision Letter 1

Ray Borrow

4 Nov 2020

PONE-D-20-14898R1

Pneumococcal nasopharyngeal carriage in Indonesia infants and toddlers post-PCV13 vaccination in a 2+1 schedule: a prospective cohort study

PLOS ONE

Dear Dr. Prayitno,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all of the points raised below during the review process.

Please submit your revised manuscript by Dec 19 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Ray Borrow, Ph.D., FRCPath

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: (No Response)

Reviewer #3: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: (No Response)

Reviewer #3: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: (No Response)

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper aims to assess changes of pneumococcal carriage pre and post PCV13 administration, as a 2+1 schedule, in Indonesia. The key findings were that PCV13 as a 2+1 schedule reduced VT serotype carriage and reduced carriage of 6A/B as dominant serotypes. The scientific background and rationale for the investigation being reported has been strengthened. The flow chart of participants is very useful, as is the description of the setting. The tabular comparison of participant characteristics stratified by control and vaccine cohorts is a welcome inclusion. Further specificity regarding methods would be very helpful – for example statistical methods and sample size calculation. Of concern is the appropriateness of the statistical methods to address the stated aim of the study. Some specific comments and concerns are outlined below.

1. Abstract Page 3 Conclusion: I remain unsure that the stated conclusion can be drawn from these analyses. It may be more appropriate to edit to something akin to “This study describes lower prevalence of VT and 6A/6B serotypes among children who were PCV13 vaccinated compared with those who were unvaccinated”

2. Introduction: With regard to the statement “The transmission of various pneumococcal infections can be prevented by the administration of the pneumococcal conjugate vaccine in infants under one year of age, I would recommend editing in line with the WHO definition of PCV vaccination, i.e., Two doses of PCV10 given before the age of 12 months, or one or more doses of PCV10 given at or after 12 months of age (see World Health Organization. Pneumococcal vaccines WHO position paper - 2012 - recommendations. Vaccine. 2012;30(32):4717-8. Epub 2012/05/25. doi: 10.1016/j.vaccine.2012.04.093. PubMed PMID: 22621828.]

3. Introduction: Could the authors please amend the section regarding previous research on NP carriage in children in Indonesia, to introduce the Bandung city study. This will help aid flow for the reader.

1. Introduction: The inclusion of pre-specified hypotheses in the introduction are very useful. I was unclear what the author’s meant by NVT serotypes would become “negative”. Please could this be revised?

2. Methods, Study Design and Participants Page 5: The authors have included some further details regarding sampling method, however no sample size justification / calculation has been provided. Please could the authors address this?

3. Methods, Study Design and Participants Please could the authors provide some further clarity regarding eligibility, inclusion, and exclusion criteria?

4. Methods, Data analysis page 6: Could the authors please clarify how participants with multiple carriage were recorded? For example, did the authors record observations of positive to VT and NVT serotypes if participants carried both? Further, did the authors record observations of positive to serotype specific carriage where multiple serotypes were carried by the same participant?

5. Methods, Data analysis page 6: It is unclear how the statistical methods employed address the stated goal of assessing vaccine effectiveness of PCV13 as a 2+1 schedule in health Indonesian children. It seems crude analysis only has been undertaken. Is this because the sample size was insufficient to permit more robust analysis, such as the standard 1 – odds ratio multiplied by 100, to determine VE? Further, would recommend that the analysis takes into account the significant differences in terms of participant characteristics by vaccine and control group, to determine the odds ratio for such analysis. It is not necessary to measure the antibody level or decreasing of disease burden to conduct such analysis. Some further clarity regarding how demographic characteristics were summarized would be of value. For example, “categorical variables were summarized by counts and percent, and continuous variables were summarized by median and (IQR).”

6. Methods. The authors describe a prospective cohort study. Factors affecting recruitment and enrolment of participants into a prospective cohort study are unlikely to introduce selection bias. This is because in order for selection bias to occur, the selection has to be related to both exposure and outcomes. But in a prospective cohort study, participants are enrolled before the outcome of interest has occurred (in this case, carriage). Although enrolment may be related to exposure status, the prospective nature of the study makes it difficult to consider the outcome (carriage) as influencing enrolment.

7. Results: Useful to see a comparison of characteristics by cohort group. It would be useful to include analysis of association of factors associated with carriage, and using the results to determine effectives of PCV13 – this could be achieved by building logistic regression models to investigate the association of PCV13 vaccination with the outcome of interest (carriage) adjusting for potential confounders, and then estimating vaccine effectiveness using 1 – odds ratio x 100

8. Discussion; This paper states that “This means that PCV13 administration on a 2 + 1 schedule did not prevent an increase in pneumococcal colonization.” It is not clear whether this study has a sufficient sample size, or statistical methods that were undertaken, that support this statement.

9. Discussion: I remain unconvinced that this study provides evidence to support the statement that “two primary doses are not enough to reduce VT serotypes in nasopharyngeal colonization”.

10. Discussion, page 13: The paper states that a “the booster dose will only be effective if it is after two primary doses and will not be effective after three primary doses.” The authors indicate that this statement is not from their current study, but from the PRIME study. In the paper cited (Whitney et al Pediatr Infect Dis J 2014), the authors state “overall booster doses are clearly beneficial for programs that use only 2 primary doses, but the clinical benefit of a booster dose remains uncertain for programs that achieve high coverage with 3 primary doses”. This is different from suggesting that a booster dose will only be effective after two primary doses and will not be effective after three primary doses. Effectiveness of booster doses and primary doses are context dependent. In a setting with a mature PCV program, it may be possible to move to a 1+ 1 schedule, as the UK has done. See Flasche et al PLOS Mid, 2015 (https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1001839) and Choi et al PLOSMed, 2019 (https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1002845). I would recommend some amendment to reflect the context specific nature of the effectiveness of the number of primary and booster doses.

Reviewer #2: COMMENTS: Since most of the comments (atleast major ones) made on earlier draft by me (and hopefully by other respected reviewers also as there were many) are attended positively/adequately, and, in my opinion, the manuscript is improved a lot. I feel that now the manuscript has achieved acceptable level of our journal.

Reviewer #3: With some additional clarification of the study design and characteristics of the study cohorts, several additional questions arise that should be addressed:

1. The authors now refer to the sampling method as a “convenience sample” and provide some information regarding the initial clinical trial. However key details regarding the selection methods are still lacking. Specifically:

a. How many participants were included in the original clinical trial? Recommend adding the numbers enrolled in the initial vaccine trial to the top of the flow diagram shown in Figure 1.

b. Based on the first version of the manuscript and subsequent revision, it can be inferred that consecutive participants in the clinical trial were enrolled into the two cohorts based on geographic location/vaccine group assigment, however this is still not explicitly stated. Is this accurate? Please make selection methods clear and specific in the manuscript.

c. Please include more details regarding the power analysis used to determine the size of the cohorts in this study. What endpoint(s) was the study powered for? This seems important in determining whether the study was sufficiently powered to support the authors' conclusions re: overall carriage rates.

2. While additional demographic information is now included, the analysis does not take potential confounders into account, and is, as mentioned by others, a crude analysis. For example, the characteristics of the cohorts indicate a difference in rates of malnutrition between groups-while the authors state that baseline differences could affect outcome measures (and that there is a previously reported association between pneumococcal carriage and nutritional status), the statistical models do not account for such variables as covariates. Have the authors attempted to perform a multivariate analysis to investigate this?

Minor comments:

1. Page 4: Where mentioned, provide a brief definition of serotype replacement and the general timeframe in which it typically occurs, for readers who may not be familiar with this phenomenon.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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PLoS One. 2021 Jan 26;16(1):e0245789. doi: 10.1371/journal.pone.0245789.r004

Author response to Decision Letter 1

18 Dec 2020

Dear Dr./Mr./Ms. Reviewers

Thank you for giving us the opportunity to submit a revised draft of our manuscript titled Pneumococcal nasopharyngeal carriage in Indonesia infants and toddlers post-PCV13 vaccination in a 2+1 schedule: a prospective cohort study to PLOS ONE. We appreciate the time and effort that you and the reviewers have dedicated to provide us with insightful comments on our paper. We have edited the manuscript to address the suggestions provided by the reviewers. You can read our responses to all comments and questions from reviewers in the "Responses to Reviwers" file.

Attachment

Submitted filename: Response to Reviewers [2] Ver 2.docx

Click here for additional data file. (23.8KB, docx)

Decision Letter 2

Ray Borrow

8 Jan 2021

Pneumococcal nasopharyngeal carriage in Indonesia infants and toddlers post-PCV13 vaccination in a 2+1 schedule: a prospective cohort study

PONE-D-20-14898R2

Dear Dr. Prayitno,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ray Borrow, Ph.D., FRCPath

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: As said earlier, all the comments were already attended positively/adequately, now the manuscript is improved a lot. No major issue left, in my opinion.

Reviewer #3: The authors have addressed my previous comments and the manuscript is overall much improved from the original submission.

A minor item that could be clarified is the statement that multivariate analysis produced "a very big number." I'm not sure what number this refers to, for clarity it is likely sufficient to simply state as a limitation that the analysis as described did not account for potential confounders.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Dr. Sanjeev Sarmukaddam

Reviewer #3: No

Acceptance letter

Ray Borrow

15 Jan 2021

PONE-D-20-14898R2

Pneumococcal nasopharyngeal carriage in Indonesia infants and toddlers post-PCV13 vaccination in a 2+1 schedule: a prospective cohort study

Dear Dr. Prayitno:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Kind regards,

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on behalf of

Prof. Ray Borrow

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Vaccine type serotype distribution by age.

    (DOCX)

    Click here for additional data file. (19.5KB, docx)
    S2 Table. Non-vaccine type serotype distribution by age.

    (DOCX)

    Click here for additional data file. (25.2KB, docx)
    Attachment

    Submitted filename: renamed_bed1d.docx

    Click here for additional data file. (14.6KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (30.1KB, docx)
    Attachment

    Submitted filename: Response to Reviewers [2] Ver 2.docx

    Click here for additional data file. (23.8KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting information files.

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