Nasopharyngeal colonization by Streptococcus pneumoniae in children and adults before the introduction of the 10-valent conjugate vaccine, Paraguay

Gustavo Chamorro; Aníbal Kawabata; María da Gloria Carvalho; Fabiana C Pimenta; Fernanda C Lessa; Carlos Torres; María José Lerea; María Eugenia León

doi:10.1371/journal.pone.0280722

. 2023 Feb 16;18(2):e0280722. doi: 10.1371/journal.pone.0280722

Nasopharyngeal colonization by Streptococcus pneumoniae in children and adults before the introduction of the 10-valent conjugate vaccine, Paraguay

Gustavo Chamorro ¹, Aníbal Kawabata ¹, María da Gloria Carvalho ², Fabiana C Pimenta ², Fernanda C Lessa ², Carlos Torres ³, María José Lerea ³, María Eugenia León ^1,^*

Editor: Arghya Das⁴

PMCID: PMC9934358 PMID: 36795644

Abstract

Streptococcus pneumoniae is a cause of invasive diseases such as pneumonia, meningitis, and other serious infections among children and adults in Paraguay. This study was conducted to establish S. pneumoniae baseline prevalence, serotype distribution, and antibiotic resistance patterns in healthy children aged 2 to 59 months and adults ≥60 years of age prior to the introduction of PCV10 in the national childhood immunization program in Paraguay. Between April and July 2012, a total of 1444 nasopharyngeal swabs were collected, 718 from children aged 2 to 59 months and 726 from adults ≥60 years of age. The pneumococcal isolation, serotyping, and antibiotic susceptibility testing were performed using standard tests. Pneumococcal colonization prevalence was 34.1% (245/718) in children and 3.3% (24/726) in adults. The most frequent pneumococcal vaccine-types (VT) detected in the children were 6B (42/245), 19F (32/245), 14 (17/245), and 23F (20/245). Carriage prevalence with PCV10 serotypes was 50.6% (124/245) and PCV13 was 59.5% (146/245). Among colonized adults, prevalence of PCV10 and PCV13 serotypes were 29.1% (7/24) and 41.6% (10/24), respectively. Colonized children were more likely to share a bedroom, have a history of respiratory infection or pneumococcal infection compared to non-colonized children. no associations were found in adults. However, no significant associations were found in children and neither in adults. Vaccine-type pneumococcal colonization was highly prevalent in children and rare in adults in Paraguay prior to vaccine introduction, supporting the introduction of PCV10 in the country in 2012. These data will be useful to evaluate the impact of PCV introduction in the country.

Introduction

Streptococcus pneumoniae remains one of the most important causes of morbidity and mortality in children and adults around the world [1]. According to the World Health Organization (WHO), of the estimated 5.83 million deaths among children <5 years of age globally in 2015, 294,000 (uncertainty range [UR], 192 000–366 000) were caused by pneumococcal infections [2]. The burden of pneumococcal disease is high in children, older adults, and those who are immunosuppressed, and most of the deaths occur in developing countries [3].

S. pneumoniae normally colonizes in the human upper respiratory tract. The frequency of nasopharyngeal colonization by S. pneumoniae varies depending on age, health, and socioeconomic status of the study population [4], but it is estimated to be between 5.0% to 75.0% [5] and the distribution of the serotypes in nasopharyngeal carriage or invasive disease varies according to geographic location [6]. Children are more commonly colonized with S. pneumoniae (20–50%) than adults (5–20%), and the highest colonization rates are found among children less than five years of age, which corresponds precisely with the highest incidence of pneumococcal disease [7]. The prevalence of pneumococcal colonization increases in the first years of life, reaching a peak of approximately 50.0% to 80.0% in children aged 2 to 3 years and decreasing thereafter until stabilizing at 5.0% to 10.0% in children >10 years of age [8]. Children have been suggested to be the source of transmission in the household and also be involved more in community transmission than adults [9].

Unlike the amount of information available on colonization in children, less is known about pneumococcal colonization in older adults, especially in low- and middle-income countries even though studies have reported that it is lower [7] than the rate found in children [10]. Colonization is a precondition for pneumococcal disease [11].

Paraguay introduced the 10-valent pneumococcal conjugate vaccine (PCV10) in 2012. The introduction of PCV10 reduces vaccine-type pneumococcal colonization in vaccinated individuals, which leads to a decrease in transmission and indirect protection of unvaccinated individuals (herd immunity). However, it can lead to a gradual increase in non-vaccine serotypes [12–14].

We conducted a cross-sectional pneumococcal colonization survey among children and adults to better understand pneumococcal colonization prevalence, serotype distribution, and antibiotic resistance patterns to establish baseline pneumococcal carrier status.

Materials and methods

Setting, design and study period

As part of the national surveillance program for meningitis and pneumonia, a cross-sectional study was carried out, whose study period was from 2012 to 2020.

Study population and samples

We enrolled all children aged 2 to 59 months and adults ≥60 years of age who met the inclusion criteria, from April through July 2012 across 10 hospitals in urban area, from outpatient corresponding to the following departments: Central (Regional Hospital of Luque in Luque City, Acosta Ñú General Pediatric Hospital in San Lorenzo City, Distrital Hospital Ñemby in Ñemby City); Amambay (Regional Hospital Pedro Juan Caballero in Pedro Juan Caballero City); Itapúa (Regional Hospital Encarnación in Encarnación City); Caaguazú (Regional Hospital Coronel Oviedo in Coronel Oviedo City); Alto Paraná (Regional Hospital Ciudad del Este in Ciudad del Este City); and the capital Asunción City (Barrio Obrero Hospital, San Pablo Hospital). A nasopharyngeal (NP) swab was collected for each participant after written consent from parent (children) or the adult. In December 2012, the Ministry of Health and Social Welfare of Paraguay introduced PCV10 to the regular vaccination schedule of the Expanded Program on Immunizations, using the three-dose schedule (Scheme 2 + 1): two primary doses in children <1 year (2 to 11 months) and a booster at one year of age (12 months or 6 months after the second dose).

Inclusion criteria

Individuals presented to outpatient clinics for conditions other than an acute illness and who did not have fever or immune disorders.

Exclusion criteria

Documentation of any dose of pneumococcal vaccine or incomplete information to accurately determine immunization status (PCV7 was already licensed in Paraguay and available in the private sector).

Isolation, identification, and serotyping

For each participant, a trained staff collected specimen from the nasopharynx using calcium alginate swabs (Fisher Brand). The swab was placed in a cryotube containing STGG (skim milk, tryptone, glucose, glycerin) media, vortexed, and stored at -80°C [15]. A total of 10% of the NP swabs (random sample) were cultured upon arrival at the Central Laboratory of Public Health for initial quality control using the methodology described below.

All the NP swabs were stored at -80°C and shipped in October 2014 to the Streptococcus Laboratory at the U.S. Centers for Disease Control and Prevention (CDC) in dry ice for processing. Pneumococci were isolated from STGG-NP by inoculating 200 μL into 5 mL Todd-Hewitt broth with 0.5% yeast extract plus 1 mL of rabbit serum (enrichment) followed by colony isolation on 5% blood agar plates [16]. Suspect alpha-hemolytic colonies were confirmed by standard microbiological tests, including colony morphology, optochin susceptibility, and bile solubility tests [17].

The serotypes of the pneumococcal isolates were identified by Quellung reaction and multiplex PCR [18]. The serotypes were classified as vaccine type (VT): PCV10 serotypes (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F), PCV13 serotypes (PCV10 serotypes plus serotypes 3, 6A, and 19A), PCV15 serotypes (PCV13 plus 22F and 33F), PCV20 serotypes (PCV15 plus 8, 10A, 11A, 12F, and 15B), or non-vaccine type (NVT, all other serotypes). When more than one potential pneumococcal colony type was identified, each colony morphology was tested, and all serotypes identified from the unique specimen were included in the analysis.

Antibiotic susceptibility

Pneumococcal isolates were tested for susceptibility to commonly used antibiotics [amoxicillin/clavulanic acid, azithromycin, cefepime, cefotaxime, ceftriaxone, cefuroxime, chloramphenicol, clindamycin, daptomycin, ertapenem, erythromycin, levofloxacin, linezolid, penicillin, meropenem, moxifloxacin, tetracycline, trimethoprim/sulfamethoxazole, vancomycin] using broth microdilution method (Trek Diagnostics, Cleveland OH) at the Streptococcus Laboratory at CDC Atlanta in December 2020. Isolates were classified as susceptible, intermediate, or resistant based on the non-parenteral breakpoints of the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines [19].

Risk factors

The following risk factors were considered in healthy children aged 2 to 59 months of age and adults ≥ 60 years: sharing a room, attending daycare, infectious processes of the upper respiratory tract in the last month, congenital disease, history of a previous episode of pneumonia, and if the person enrolled (≥60 years old) smokes or if there are smokers in the house. These data were collected in a standardized form.

Statistical analysis

The data obtained in the investigation were entered into a database in Excel format. Statistical analyzes were performed using STATA software [20], version 11.0 (Stata Corp, College Station, TX). Initially, a general analysis of the age of the population included in the sample was made, using the measures of central tendency and dispersion.

Demographic, clinical, and socio-economic characteristics were compared using chi-square for categorical variables and t-test for continuous variable. Two-sided P value of 0.05 was considered statistically significant. Multivariable analysis, was performed to evaluate independent risk factors associated with pneumococcal colonization. Separate multivariable models were constructed for each outcome using automated stepwise logistic regression (congenital disease, pneumonia and presence of smokers in the household). Variables with P values< 0.30 in univariable analysis were included as candidate variables for the model. Multivariable analyses was not conducted if univariable analyses showed <2 variables with P<0.30 for the outcome of interest.

Ethical aspects

This study was approved by the research ethics committee of the Central Laboratory of Public Health (Cod.: 014/28112011). CDC provided technical assistance and tested coded specimens, and therefore was considered not engaged in research. A non-disclosure agreement was signed between CDC and the Paraguay MHWS Central Public Health Laboratory. Informed consent was obtained from children’s guardians and from adults. The NP swabs and survey questionnaires were numerically coded and processed anonymously. The data were stored in a database and use restricted for this analysis.

Results

Demographic data

A total of 1444 participants who attended the 10 health services were included in this study, and 97 individuals were excluded due to sample contamination or missing demographic data. Half of the participants were children [718 (49,72%)], of those, 350 (48.74%) were male. Most of the children were from Central (n = 362), Asunción (n = 135), and Caaguazú (n = 93). Among the 726 adults, 472 (65.0%) were female, 362 (49.8%) were 60–69 years old, 283 (39.0%) were 70–79 year old, and 81 (11.2%) were ≥80 years of age. The majority of the adults were from Central (n = 299), Asunción (n = 188), Alto Parana (n = 59). The overall prevalence of pneumococcal colonization in children was 34.1% (95% CI 31.69–36.65) and in adults was 3.3% (95% CI 2.46–4.38). The prevalence of pneumococcal colonization was highest among children aged 2–11 months and lowest among those 36–59 months old (Table 1).

Table 1. Prevalence of pneumococcal colonization of the study population by age group (n = 1444).

Variable	Category	2–59 months				Category	≥ 60 years
Variable	Category	Total tested	Pneumococcal carriers	%	IC95%	Category	Total tested	Pneumococcal carriers	%	IC95%
Age group	2–11 months	88	44	50.0	47.39–52.61	60–69 years	362	13	4.0	2.70–4.69
	12–23 months	196	70	35.7	33.26–38.27	70–79 years	283	9	3.2	2.34–4.23
	24–35 months	147	51	34.7	32.23–37.21	≥ 80 years	81	2	2.5	1.75–3.43
	36–59 months	287	80	27.9	25.54–30.22	≥ 80 years	81	2	2.5	1.75–3.43
Gender	Male	350	123	32.6	31.60–38.71	Male	274	9	3.3	2.13–4.88
Gender	Female	368	122	31.1	29.71–36.72	Female	452	15	3,3	2.13–4.88
Department	Asunción (Capital)	135	49	36.3	32.82–39.98	Asunción (Capital)	188	8	4,3	2.92–6.01
	Central	362	111	30.7	27.28–34.16	Central	299	12	4,0	2.69–5.68
	Alto Paraná	55	15	27.3	24.07–30.71	Alto Paraná	59	0	0
	Itapúa	22	7	31.8	28.36–35.30	Itapúa	20	1	5	3.49–6.79
	Amambay	36	19	52.7	49.06–56.49	Amambay	35	1	2.9	1.79–4.38
	Caaguazú	93	39	41.9	38.28–45.63	Caaguazú	120	2	1.7	0.85–2.87
	Others¹	15	5	33.3	29.84–36.87	Others¹	5	0	0

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Others¹ San Pedro (n = 3), Cordillera (n = 11), Misiones (n = 2), Paraguarí (n = 1), Presidente Hayes (n = 2), Boquerón (n = 1).

Pneumococcal colonization prevalence was similar between gender in both children and adults. Children from the Department of Amambay presented the highest pneumococcal carriage rate with 52.7% (19/36), followed by the children from the Department of Caaguazú 41.9% (39/93) and the Central Department with 30.7% (111/362).

Among the 245 children colonized with pneumococci, 38 serotypes were identified. Among the 24 adults colonized with pneumococci, 15 serotypes were identified (Fig 1). The VT pneumococcal serotypes in children accounted for 49.4% (PCV10), 57.9% (PCV13), 60.0% (PCV15), and 70.2% (PCV20). The most frequent serotypes were 6B, 19F, 23F, and 14. The most frequent non-PCV13 serotypes in children in this study were 15B, 11A, 16F, 23B, and 13.

Fig 1 — ¹PCV10: 10 valent Pneumococcal conjugate vaccine. ²PCV13: 13 valent Pneumococcal conjugate vaccine. ³NPCV: Serotypes not included in PCV10 and PCV13. NT: Non typeable. n: Isolates numbers.

Children who shared a bedroom had 2.3 times increased odds of being a pneumococcal carrier compared to those who did not share a bedroom (35% vs.18%, p = 0.03) (Table 2). However, this association was no longer significant after adjusting for share room (0.072), smokers present in the household (p = 0.194), congenital condition (p = 0.569), and pneumonia (p = 0.118). In adults, no statistically significant associations with pneumococcal carriage were observed (Table 3).

Table 2. Association between risk factors and nasopharyngeal colonization with S. pneumoniae, according to age group.

Risk factor	Total	OR	95% CI	p-value
2–59 months (n = 245)
Share room	718	2.38	1.03–5.49	0.036
Smokers present	715	1.31	0.93–1.84	0.124
Attends daycare	547	1.31	0.93–1.84	0.928
Congenital condition	718	1.54	0.69–3.45	0.289
Respiratory infection	718	1.30	0.95–1.77	0.095
Illness/Pneumonia	713	2.18	0.95–5.02	0.060
≥ 60 years (n = 24)
Share room	726	0.89	0.39–2.08	0.804
Smokers in the house	726	1.47	0.76–2.69	0.184
You smoke	709	1.33	0.30–5.86	0.704
Congenital condition	726	.	.	0.501
Respiratory infection	726	1.62	0.66–3.99	0.286
Illness/Pneumonia	660	0.71	0.09–5.45	0.743

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Table 3. Multivariable analysis for pneumococcal colonization risk factors among children (n = 245).

Risk factor	OR (95% CI)	P-adjusted
Share room	2.38 (1.03–5.49)	0.072
Smokers present	1.31 (0.93–1.84)	0.194
Congenital condition	1.54 (0.69–3.45)	0.569
Illness/Pneumonia	2.18 (0.95–5.02)	0.118

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All S. pneumoniae isolates were susceptible to levofloxacin, ertapenem, moxifloxacin, linezolid, and vancomycin. Out of 269 pneumococcal isolates, 64 (23.9%) were resistant to trimethoprim/sulfamethoxazole, 50 (18.7%) to tetracycline, 49 (18.3%) to azithromycin and erythromycin, 32 (11.9%) to cefuroxime, 26 (9.7%) to clindamycin, and 10 (3.7%) to chloramphenicol. Some pneumococcal isolates presented intermediate susceptibility to cefepime 17 (6.3%), penicillin 10 (3.7%), and ceftriaxone 14 (5.2%). In children, of the 245 pneumococcal isolates, 19 (7.7%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline, 18 (7.3%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline-cefuroxime, 18 (7.3%) were resistant to erythromycin-clindamycin-trimethoprim/sulfamethoxazole-tetracycline, and 6 (2.4%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline-chloranphenicol. In adults, of the 24 pneumococcal isolates, 3 (12.5%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline, 2 (8.3%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline-cefuroxime, and 2 (8.3%) were resistant to erythromycin-clindamycin-trimethoprim/sulfamethoxazole-tetracycline. (Table 4).

Table 4. Distribution of antimicrobial resistance by serotypes.

2–59 months
Antibiotics	PCV10										PCV13			p
Antibiotics	4	6B	9V	14	18C	19F	23F	1	5	7F	3	6A	19A	p
Penicillin G	0	0	0	0	0	0	0	0	0	0	0	0	0	0.923
Ceftriaxone	0	0	0	0	0	0	0	0	0	0	0	0	0	0.504
Azithromycin	0	7(16.67)	0	7(41.18)	0	18(56.25)	4(20.00)	0	0	0	0	7(16.67)	0	0.002
Tetracycline	0	6(14.29)	0	7(41.18)	1(25.00)	16(50.00)	5(25.00)	0	0	0	1(50.00)	0	0	<0.001*
Erythromycin	0	7(16.67)	0	7(41.18)	0	18(56.25)	4(20.00)	0	0	0	0	7(4375)	0	0.002
TMP–SMX	0	8(19.05)	3(75.00)	11(64.71)	0	18(56.25)	7(35.00)	0	0	0	0	1(6.25)	0	0.001
Clindamycin	0	6(14.29)	1(25.00)	1(5.88)	1(25.00)	16(50.00)	0	0	0	0	0	0	0	0.221
Chloramphenicol	0	2(4.76)	0	0	0	1(3.13)	2(10.00)	0	0	0	0	0	0	0.030
AMC	0	0	0	0	0	1(3.13)	0	0	0	0	0	0	0	1.000
Cefuroxime	0	2(4.76)	0	4(23.53)	0	16(50.00)	3(15.00)	0	0	0	0	0	0	0.523
≥ 60 years
Penicillin G	0	0	0	0	0	0	0	0	0	0	0	0	0	-
Ceftriaxone	0	0	0	0	0	0	0	0	0	0	0	0	0	-
Azithromycin	0	0	0	0	0	2(100)	0	0	0	0	0	2(66.67)	0	0.144
Tetracycline	0	0	0	1(100)	0	2(100)	0	0	0	0	0	1(33.33)	0	0.107
Erythromycin	0	0	0	0	0	2(100)	0	0	0	0	0	2(66.67)	0	0.144
TMP–SMX	0	0	0	0	0	2(100)	0	0	0	0	0	1(33.33)	0	0.648
Clindamycin	0	0	0	0	0	0	0	0	0	0	0	1(100)	0	0.823
Chloramphenicol	0	0	0	0	0	0	0	0	0	0	0	0	0	0.904
AMC	0	0	0	0	0	0	0	0	0	0	0	0	0	-
Cefuroxime	0	2(66.67)	0	0	0	2(100)	0	0	0	0	0	0	0	0.144

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TMP–SMX: Trimethoprim-sulfamethoxazole.

AMC: Amoxicillin/Clavulanic Acid.

Discussion

Before the introduction of PCV10 in Paraguay, 31.86% of children aged ≤59 months were colonized with pneumococci, and to the best of our knowledge this is the first study providing baseline data prior to PCV introduction and that might allow vaccine impact evaluation.

In this study, the VT pneumococcal serotypes were the most frequent. The prevalence of VT is similar to other pre-vaccine introduction reports from other Latin American countries. In Cuba, pneumococcal colonization was found in 21.60% of children aged >18 months and the most frequently found serotypes were 6A, 23F, 6B, 19F, and 14 [21]. The colonization rate in children from Niterói, Brazil, was 49.2% and the most prevalent serotypes were 6B, 19F, 6A, 14, 15C, and 23F [22]. Fernandez et al described a colonization of 49.7% in children <2 years of age in Paysandú, Uruguay, with predominance of serotypes 6A, 6B, 14, and 19F [23].

A study conducted in children <5 years of age from southeast Brazil before the introduction of PCV10 also detected serotypes 11A, 23B, and 16F [24].

In this study, colonization in children was 10 times higher than in adults. There are few previously published studies of colonization in adults and the rates reported were 2 to 6 times higher in children than in adults [25]. A study in Kenya found among subjects ages 0–4, 5–9, and 10–85 years, that the prevalence of pneumococcal colonization was 57.0%, 41.0%, and 6.4%, respectively [26].

Currently, the available conjugate vaccines reduce the colonization of vaccine serotypes, as well as the burden of pneumococcal disease [27].

In children, sharing a room was the only variable associated with colonization of S. pneumoniae in this study. A study in five cities in Argentina found that children who shared a room with 3 or more people had an increased risk of colonization [28]. Unlike, another study from South Africa found a high prevalence of S. pneumoniae in patients with respiratory conditions [29]. Several studies have reported the intrafamilial spread of S. pneumoniae [30,31].

In this study, no association was found between attending daycare centers and colonization. However, the risk of nasopharyngeal colonization by potential pathogens, such as S. pneumoniae, in healthy children is higher in children who regularly attend daycare centers than in those who are cared for at home as has previously been documented [32]. This higher incidence could be explained by the characteristics and environmental conditions that exist in these institutions for children, as well as by the age of the population group. The prevalence of carriage is independent of geographical region but strongly associated with accumulated risk factors, such as young age, high-density living conditions, and poor health conditions [33,34].

In adults, no significant differences were observed between colonized and non-colonized subjects by age, gender, presence of comorbidities, vaccination status, previous respiratory infection, previous episode of pneumonia, and smokers. In Alaska overcrowding in households was associated with increased colonization at all ages, including adults. Also, the prevalence of colonization increased with the increasing number of occupants of the home and decreased with the increasing number of rooms in a home [35]. The presence of children in a home was associated with a higher prevalence of colonization for all ages, reflecting the importance of young children as potential transmitters of pneumococcus [36,37].

Our data indicated that the rates of trimethoprim-sulfamethoxazole, tetracycline, erythromycin, azithromycin, cefuroxime, and chloramphenicol resistance among isolates were found more frequently in the serotypes included in PCV10than in non-vaccine serotypes. In addition, no resistance to penicillin or third-generation cephalosporin was found. Unlike another similar study, no penicillin-resistant isolates were found [4]. High antibiotic resistance rates in S. pneumoniae may facilitate transmission of this pathogen among young children [38].

Pneumococci resistant to the different antibiotics were detected mainly in vaccine serotypes. The reduced susceptibility may be especially related to the circulation of vaccine serotypes typically associated with antimicrobial resistance, such as 14, 19F, and 23F [39]. PCVs can reduce antibiotic-resistant infections through direct reductions in the presence of vaccine serotypes and thus decrease the use of antibiotics [40].

Our study has some limitations. First, the data evaluated on the behaviors were based on self-reported data and could not be verified, but precautions were taken to minimize bias by cross questions. Second, although the results of this study show the period prior to vaccination in Paraguay, the long storage time of the samples prior to processing and then the analysis time, could not provide pre-vaccine information for decision-making. This study could be a baseline for future cross-sectional studies aimed at monitoring changes in the prevalence of serotype in the same population.

Conclusion

Serotypes found among pneumococci in the nasopharynx include the most common serotypes that caused invasive disease in Paraguay in the years prior to vaccine introduction (2010–2012) [41]. In addition, in this study, strains with resistance to antimicrobials were found more frequently in serotypes included in PCV10.The data on isolates from nasopharyngeal colonization can provide relevant information on the potential burden of pneumococcal disease and are important for understanding circulation before vaccination programs are implemented as well as for evaluating the impact after vaccine introduction in the country.

Supporting information

S1 Table. Pneumococcal carriage dataset Paraguay.

(XLSX)

Click here for additional data file.^{(93.1KB, xlsx)}

Data Availability

The minimal dataset underlying the results described in the paper can be found at supporting Information files and at 10.6084/m9.figshare.21089815.

Funding Statement

This work was supported by grants from the Mercosur Structural Convergence Fund (FOCEM) -Mercosur, FOCEM agreement N° 03/11 Project "Research, Education and Biotechnologies Applied to Health (COF 03/11). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was largely supported with own funds from the Ministry of Public Health and Social Welfare through the PNEI / PAI and the LCSP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0280722.r001

Decision Letter 0

Arghya Das

28 Aug 2022

PONE-D-22-20913Nasopharyngeal colonization by Streptococcus pneumoniae in children and adults before the introduction of the 10-valent conjugate vaccine, ParaguayPLOS ONE

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Additional Editor Comments:

The study period is not clear from the text as the authors have mentioned it to be between June and December 2012 in abstract while in materials and methods it has been mentioned to be from April through December 2012.

The authors need to clarify why the exclusion criteria includes any documented dose of pneumococcal vaccine given the fact that PCV-10 was introduced in December 2012 and the study period is before the introduction of PCV-10.

The authors need to clarify the use of CLSI 2020 guidelines for the study done in the year 2012.

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors of the present manuscript intends to find S. pneumoniae baseline prevalence, serotype distribution, and antibiotic resistance patterns in healthy children aged 2 to 59 months and adults ≥60 years of age prior to the introduction of PCV10 in the national childhood immunization program in Paraguay. Although the study seems interesting at the first glance, there some key issues related to the selection of subjects and statistical analyses which need to be addressed by the authors. Moreover, the study was conducted almost a decade ago. It is not clear whether the study is part of an ongoing surveillance program or a standalone project. Without the follow up data after introduction of the PCV-10, the findings of the study may not be impactful.

Abstract

Lines 17-19: Colonized children were more likely to share a bedroom (P=0.03) and have a history of respiratory infection (P=0.095) or pneumococcal infection (P=0.06) compared to non-colonized children,….

Comment: The above statistics are not worth mentioning because for variables like history of respiratory infection and pneumococcal infection, the p values are higher that is assumed significant for the present study (i.e. <0.05). Moreover, sharing of bedroom has not been found significant (as mentioned under Results) after adjusting for congenital disease, pneumonia, and smokers present in the household.

Introduction

Lines 30-32: According to the World Health Organization (WHO), of the estimated 5.83 million deaths among children <5 years of age globally in 2015, 294,000 were caused by pneumococcal infections.

Comment: Please add the uncertainty interval within parenthesis after the estimated figure of 294,000.

Lines 32-34: The burden of pneumococcal disease is high in children, older adults, and those who are immunosuppressed, and it occurs in high-, low-, and middle-income countries.

Comment: The authors may consider slightly modifying the later part of the above sentence.

‘…..and most of the deaths occur in developing countries’ instead of ‘….and it occurs in high-, low-, and middle-income countries’.

Reference- https://www.cdc.gov/pneumococcal/global.html

Lines 39-42: Children are more commonly colonized with S. pneumoniae than adults, and the highest colonization rates are found among children less than five years of age, which corresponds precisely with the highest incidence of pneumococcal disease.

Comment: The statistical estimate for carriage related prevalence among both children and adults from reference no. 07 by Brooks et al. could be incorporated for the comparative depiction between the mentioned groups.

Lines 45-46: Children have been suggested to be the source of transmission in the household.

Comment: Although true, the above statement seems incomplete without mentioning the other findings from the cited literature. Like, children are also found to be involved more in community transmission than adults.

Materials and methods

Lines 63 to 76: We enrolled all children aged 2 to 59 months and adults ≥60 years of age………………………… through the Ministry of Health and Social Welfare in the health units and immunization centers in Paraguay.

Comment: The study design is poorly understood. The authors should make a flow diagram for allowing better understanding of the selection of subjects.

There are several queries which need to be clarified.

As per the title and aim of the present manuscript the authors intend to demonstrate the pneumococcal colonization prevalence, serotype, distribution, etc. before the introduction of PCV-10 vaccine. The study subjects were included from April to December 2012. As per the authors, the PCV 10 was introduced in December 2012.

However, as per a previously published study, PCV10 has been introduced much earlier than the study period.

Kieninger MP, Caballero EG, Sosa AA, Amarilla CT, Jáuregui B, Janusz CB, Clark AD, Castellanos RM. Cost-effectiveness analysis of pneumococcal conjugate vaccine introduction in Paraguay. Vaccine. 2015 May 7;33 Suppl 1:A143-53.

The relevance of the following sentence is not at all understood. “PCV10 was introduced for all eligible children (two primary doses in children <1 year and one booster at one year of age, in December 2012”

The PCV-10 vaccination schedule followed in Paraguay can be incorporated as supplementary file. If the vaccine has been introduced in December 2012 for the first time, how can the booster be considered for few children at the same time.

Lines 81-82: Documentation of any dose of pneumococcal vaccine or incomplete information to accurately determine immunization status.

Comment: Some of the important exclusion criteria have not been considered. For example, recent administration of antibiotics which might influence the S. pneumoniae colonizations. Please justify.

Line 97: Pneumococcal isolates were serotyped by Quellung reaction and multiplex PCR.

Comment: Modify the above sentence like “The serotypes of the pneumococcal isolates were identified by Quellung reaction and multiplex PCR”. The methodology of multiplex PCR should be mentioned or referenced.

Lines 106-114: Pneumococcal isolates were tested for susceptibility…… breakpoints of the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines.

Comment: The reviewer feels curious to know as when the susceptibility testing was actually performed. Because the samples were collected in 2012 and the interpretation of the test was done as per the CLSI guidelines published in 2020. Moreover, was susceptibility testing individually for all morphotypes of the colonies were performed if more than one colony type was isolated (as it is done for serotyping)?

Lines 170-173: Children who shared a bedroom had 2.3 times increased odds of being a pneumococcal carrier compared to those who did not share a bedroom (35% vs.18%, p=0.03). However, this association was no longer significant after adjusting for congenital disease, pneumonia, and smokers present in the household.

Comment: The adjusted p values could not be found in the Table 02.

How was the adjustment carried out? Was a multivariate analysis performed? More details on the statistical analyses are absent under Materials and Methods.

Lines 187-195: In children, multidrug resistance (MDR) was found in 19/245 (7.7%) of……….. and 2/24 (8.3%) of erythromycin-clindamycin-trimethoprim/sulfamethoxazole-tetracycline.

Comment: Please consider rephrasing of the two sentences mentioned in the above paragraph. It will be better if the authors present the susceptibility profile in form of graphs (showing susceptibility/resistance proportions) separately in children and adults. The above description of MDR (presumably as at least one member of ≥ 3 classes of antibiotics) seems to be complex. The authors should find easier way of depiction of the above statistics either in tables or figures.

Lines 196-198: The serotypes found most frequently with resistance to trimethoprim/sulfamethoxazole, tetracycline, erythromycin, cefuroxime, and clindamycin were 19F, 14, and 6B.

Comment: It is advisable to provide detailed statistical information before mentioning the above statement.

General comments:

The manuscript should be checked and edited by a native English speaker.

The collected data is almost a decade old. How will the authors justify the importance of the findings in recent time (given the colonization pattern have been likely changed over the past decade)? The reviewer is of the opinion that the authors may conduct a follow up surveillance to depict the changes in the colonization after 10 years of PCV-10 introduction in Paraguay.

Reviewer #2: The manuscript is written in crisp language and focused to answer the research question. But this study should be followed by another follow up study to assess the response of nationwide PCV 10 vaccination on nasopharyngeal colonization of streptococcus pneumoniae.

**********

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Reviewer #1: Yes: Anwita Mishra, MD

Reviewer #2: Yes: Saikat Mondal

**********

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PLoS One. 2023 Feb 16;18(2):e0280722. doi: 10.1371/journal.pone.0280722.r002

Author response to Decision Letter 0

3 Nov 2022

Letter to Academic Editor and Revieweres

Respectfully, all the changes made to the manuscript and the pertinent clarifications are detailed below.

Journal Requirements:

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https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Reponse:

We have reviewed the PLOS ONE style requirements.

Could you therefore please include the title page into the beginning of your manuscript file itself, listing all authors and affiliations.

Reponse:

We have included the title page at the beginning of the manuscript file, listing all authors and affiliations.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Reponse:

This research has not received any specific scholarship from public sector agencies of the Ministry of Public Health and Social Welfare, PNEI-PAI or the LCSP. Therefore they do not have a grant number and we have decided to withdraw this information.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Reponse:

We have uploaded the minimum set of data underlying your study as supporting data files.

Also, the minimal dataset underlying the results described in the manuscript can be found at: 10.6084/m9.figshare.21089815 (The DOI becomes active when the item is published).

Additional Editor Comments:

Reponse: The samples were collected from april to july 2012.

Reponse: The pneumococcal vaccine data was collected because the PCV7 was probably already licensed in Paraguay and available in the private sector.

The authors need to clarify the use of CLSI 2020 guidelines for the study done in the year 2012.

Reponse: The pneumococcal isolation was done and the isolates stored. However, the MIC testing was performed only in 2020 at CDC.

Responses to Reviewer #1

1. The authors of the present manuscript intends to find S. pneumoniae baseline prevalence, serotype distribution, and antibiotic resistance patterns in healthy children aged 2 to 59 months and adults ≥60 years of age prior to the introduction of PCV10 in the national childhood immunization program in Paraguay. Although the study seems interesting at the first glance, there some key issues related to the selection of subjects and statistical analyses which need to be addressed by the authors. Moreover, the study was conducted almost a decade ago. It is not clear whether the study is part of an ongoing surveillance program or a standalone project. Without the follow up data after introduction of the PCV-10, the findings of the study may not be impactful.

Reponse:

Although it was planned to carry out the post-vaccination study, unfortunately, for financial reasons we have not been able to carry it out. In the same way, we believed it convenient to present the results of the pre-vaccination study, since this is the only study that has been carried out in healthy carriers in Paraguay.

Reponse: The study is part of an ongoing surveillance program. Although it was planned to carry out the post-vaccination study, unfortunately, for financial reasons we have not been able to carry it out. In the same way, we believed it convenient to present the results of the pre-vaccination study, since this is the only study that has been carried out in healthy carriers in Paraguay.

2. Abstract

Reponse:

We have eliminated the statistics being as follows:

Colonized children were more likely to share a bedroom, have a history of respiratory infection or pneumococcal infection compared to non-colonized children. No associations were found in adults. However, no significant associations were found in children and neither in adults.

3. Introduction

Lines 30-32: According to the World Health Organization (WHO), of the estimated 5.83 million deaths among children <5 years of age globally in 2015, 294,000 were caused by pneumococcal infections.

Comment: Please add the uncertainty interval within parenthesis after the estimated figure of 294,000.

Reponse:

We have added the range of uncertainty: (uncertainty range [UR], 192 000–366 000)

4. Lines 32-34: The burden of pneumococcal disease is high in children, older adults, and those who are immunosuppressed, and it occurs in high-, low-, and middle-income countries.

Comment: The authors may consider slightly modifying the later part of the above sentence.

…..and most of the deaths occur in developing countries’ instead of ‘….and it occurs in high-, low-, and middle-income countries’. Reference- https://www.cdc.gov/pneumococcal/global.html

Reponse: we have modified according to suggestion “and most of the deaths occur in developing countries”

5. Lines 39-42: Children are more commonly colonized with S. pneumoniae than adults, and the highest colonization rates are found among children less than five years of age, which corresponds precisely with the highest incidence of pneumococcal disease.

Reponse:

We have added the requested prevalence, in children 20-50% and adults 5-20%.

6. Lines 45-46: Children have been suggested to be the source of transmission in the household.

Reponse:

We have added according to the mentioned literatura and suggested “also be involved more in community transmission than adults”.

7. Materials and methods

Comment: The study design is poorly understood. The authors should make a flow diagram for allowing better understanding of the selection of subjects.

There are several queries which need to be clarified.

a) As per the title and aim of the present manuscript the authors intend to demonstrate the pneumococcal colonization prevalence, serotype, distribution, etc. before the introduction of PCV-10 vaccine. The study subjects were included from April to December 2012. As per the authors, the PCV 10 was introduced in December 2012.

However, as per a previously published study, PCV10 has been introduced much earlier than the study period.

Reponse:

According to Castellanos RM. Cost-effectiveness analysis of pneumococcal conjugate vaccine introduction in Paraguay. Vaccine. 2015 May 7;33 Suppl 1:A143-53.

TRIVAC model version 2.0 is a tool that evaluates vaccine impact and incremental costeffectiveness ratios (ICER), providing a coherent and transparent framework for each vaccine, with comparable and standardized outcomes. Utilizing parameters, such as demography, disease burden, vaccine costs, vaccine coverage, vaccine efficacy, vaccine serotype coverage, health service utilization and costs.

In order to estimate coverage per dose in the year of introduction, EPI information on Pentavalent and MMR1 dose coverage over the last 5 years was used.

Therefore, for this mentioned study, the real application of any PCV has not been implemented.

b)The relevance of the following sentence is not at all understood. “PCV10 was introduced for all eligible children (two primary doses in children <1 year and one booster at one year of age, in December 2012”

Reponse:

We have modified according to suggestion:

“In December 2012, the Ministry of Health and Social Welfare of Paraguay introduced PCV10 to the regular vaccination schedule of the Expanded Program on Immunizations, using the three-dose schedule (Scheme 2 + 1): two primary doses in children <1 year ( 2 to 11 months) and a booster at one year of age (12 months or 6 months after the second dose)”.

8. Lines 81-82: Documentation of any dose of pneumococcal vaccine or incomplete information to accurately determine immunization status.

Comment: Some of the important exclusion criteria have not been considered. For example, recent administration of antibiotics which might influence the S. pneumoniae colonizations. Please justify.

Reponse:

We agree with the reviewer that recent exposure to antibiotics may affect the colonization results either leading to lower detection of colonization or detection of resistance pneumococci. Many colonization studies do not exclude patients on antibiotics since you can still detect pneumococci among patients with recent antibiotic exposure.

9. Line 97: Pneumococcal isolates were serotyped by Quellung reaction and multiplex PCR.

Reponse:

We have modified according to suggestion: The serotypes of the pneumococcal isolates were identified by Quellung reaction and multiplex PCR.

And we added the referencePai R, Gertz RE, Beall B. Sequential multiplex PCR approach for determining capsular serotypes of Streptococcus pneumoniae isolates. J Clin Microbiol. 2006; 44(1):124-31. doi: 10.1128/JCM.44.1.124-131.2006.

10. Lines 106-114: Pneumococcal isolates were tested for susceptibility…… breakpoints of the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines.

Reponse:

The pneumococcal isolation was done and the isolates stored. However, the MIC testing was performed only in 2020 at CDC.

Yes, all morphotypes of colonies with diferente serotypes were tested for antimicrobial susceptibility test by broth microdilution.

11. Lines 170-173: Children who shared a bedroom had 2.3 times increased odds of being a pneumococcal carrier compared to those who did not share a bedroom (35% vs.18%, p=0.03). However, this association was no longer significant after adjusting for congenital disease, pneumonia, and smokers present in the household.

Comment: The adjusted p values could not be found in the Table 02.

How was the adjustment carried out? Was a multivariate analysis performed? More details on the statistical analyses are absent under Materials and Methods.

Reponse:

We have added to methods: “Multivariable analysis, where appropriate, was performed to evaluate independent risk factors associated with pneumococcal colonization. Variables with P values< 0.30 in univariable analysis were included as candidate variables for the model. Multivariable analyses was not conducted if univariable analyses showed <2 variables with P<0.30 for the outcome of interest”.

We have modified the results:

However, this association was no longer significant after adjusting for congenital disease (p=0.569), pneumonia (p=0.118), and smokers present in the household (p=0.194). In adults, no statistically significant associations with pneumococcal carriage were observed.

Table 3. Multivariable analysis for pneumococcal colonization risk factors among children (n=245).

Risk factor OR (95% CI) P-adjusted

Share room 2.38 (1.03-5.49) 0.072

Smokers present 1.31 (0.93-1.84) 0.194

Congenital condition 1.54 (0.69-3.45) 0.569

Illness/Pneumonia 2.18 (0.95-5.02) 0.118

12. Lines 187-195: In children, multidrug resistance (MDR) was found in 19/245 (7.7%) of……….. and 2/24 (8.3%) of erythromycin-clindamycin-trimethoprim/sulfamethoxazole-tetracycline.

Reponse: We have considered reformulating the two mentioned sentences, remaining as follows:

In children, of the 245 pneumococcal isolates, 19 (7.7%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline, 18 (7.3%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline-cefuroxime, 18 (7.3%) were resistant to erythromycin-clindamycin-trimethoprim/sulfamethoxazole-tetracycline, and 6 (2.4%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline-chloranphenicol. In adults, of the 24 pneumococcal isolates, 3 (12.5%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline, 2 (8.3%) were resistant to erythromycin-trimethoprim/sulfamethoxazole-tetracycline-cefuroxime, and 2 (8.3%) were resistant to erythromycin-clindamycin-trimethoprim/sulfamethoxazole-tetracycline.

Table 4. Distribution of antimicrobial resistance by serotypes.

2 - 59 months

Antibiotics PCV10 PCV13 p

4 6B 9V 14 18C 19F 23F 1 5 7F 3 6A 19A

Penicillin G 0 0 0 0 0 0 0 0 0 0 0 0 0 0.923

Ceftriaxone 0 0 0 0 0 0 0 0 0 0 0 0 0 0.504

Azithromycin 0 7(16.67) 0 7(41.18) 0 18(56.25) 4(20.00) 0 0 0 0 7(16.67) 0 0.002

Tetracycline 0 6(14.29) 0 7(41.18) 1(25.00) 16(50.00) 5(25.00) 0 0 0 1(50.00) 0 0 <0.001*

Erythromycin 0 7(16.67) 0 7(41.18) 0 18(56.25) 4(20.00) 0 0 0 0 7(4375) 0 0.002

TMP–SMX 0 8(19.05) 3(75.00) 11(64.71) 0 18(56.25) 7(35.00) 0 0 0 0 1(6.25) 0 0.001

Clindamycin 0 6(14.29) 1(25.00) 1(5.88) 1(25.00) 16(50.00) 0 0 0 0 0 0 0 0.221

Chloramphenicol 0 2(4.76) 0 0 0 1(3.13) 2(10.00) 0 0 0 0 0 0 0.030

AMC 0 0 0 0 0 1(3.13) 0 0 0 0 0 0 0 1.000

Cefuroxime 0 2(4.76) 0 4(23.53) 0 16(50.00) 3(15.00) 0 0 0 0 0 0 0.523

≥ 60 years

Penicillin G 0 0 0 0 0 0 0 0 0 0 0 0 0 -

Ceftriaxone 0 0 0 0 0 0 0 0 0 0 0 0 0 -

Azithromycin 0 0 0 0 0 2(100) 0 0 0 0 0 2(66.67) 0 0.144

Tetracycline 0 0 0 1(100) 0 2(100) 0 0 0 0 0 1(33.33) 0 0.107

Erythromycin 0 0 0 0 0 2(100) 0 0 0 0 0 2(66.67) 0 0.144

TMP–SMX 0 0 0 0 0 2(100) 0 0 0 0 0 1(33.33) 0 0.648

Clindamycin 0 0 0 0 0 0 0 0 0 0 0 1(100) 0 0.823

Chloramphenicol 0 0 0 0 0 0 0 0 0 0 0 0 0 0.904

AMC 0 0 0 0 0 0 0 0 0 0 0 0 0 -

Cefuroxime 0 2(66.67) 0 0 0 2(100) 0 0 0 0 0 0 0 0.144

TMP–SMX: Trimethoprim-sulfamethoxazole

AMC: Amoxicillin/Clavulanic Acid

13. The manuscript should be checked and edited by a native English speaker.

Reponse:

The authors agree on the importance of conducting follow-up surveillance to represent changes in colonization 10 years after the introduction of PCV-10 in Paraguay.

General comments:

The manuscript should be checked and edited by a native English speaker.

Reponse:

We agree with the reviewer. Although it was planned to carry out the post-vaccination study, unfortunately for economic reasons we have not been able to carry it out. Similarly, we thought it appropriate to present the results of the pre-vaccination study, since this is the only study that has been carried out in healthy carriers in Paraguay. We will do our best to carry out a postvaccinal colonization study.

Responses to Reviewer #2

Reponse:

King regards

María Eugenia León

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(32.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0280722.r003

Decision Letter 1

Arghya Das

15 Dec 2022

PONE-D-22-20913R1Nasopharyngeal colonization by Streptococcus pneumoniae in children and adults before the introduction of the 10-valent conjugate vaccine, ParaguayPLOS ONE

Dear Dr. León Ayala,

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 the points raised during the review process.Please submit your revised manuscript by Jan 29 2023 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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Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Multivariable analysis, where appropriate, was performed to evaluate independent risk factors associated with pneumococcal colonization.

Comment: Please specify the method that was adopted for multivariable analysis.

The serotypes found most frequently with resistance to trimethoprim/sulfamethoxazole, tetracycline, erythromycin, cefuroxime, and clindamycin were 19F, 14, and 6B (Table 4).

Comment: The inference drawn in the sentence just before Table 4 does not completely match with the statistics mentioned in the table. Hence, the above sentence should be deleted.

[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: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have responded to the queries or comments satisfactorily. However the following needs to be incorporated in the manuscript: under material and methods the study period has still not been mentioned clearly. Kindly give specific study period details.

Reviewer #3: The authors conducted the study to establish S. pneumoniae baseline prevalence, serotype distribution, and antibiotic resistance patterns in healthy children and adults prior to the introduction of PCV10 in the national childhood

immunization program in Paraguay. And concluded with the observation of Vaccine-type pneumococcal colonization were prevalent in children and rare in adults in Paraguay prior to vaccine introduction, supporting the introduction of PCV10 in the country in 2012.

The manuscript describe a technically sound piece of scientific research with data that supports the conclusions.

**********

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Reviewer #1: Yes: Anwita Mishra, MD

Reviewer #3: Yes: Rahul Garg

**********

PLoS One. 2023 Feb 16;18(2):e0280722. doi: 10.1371/journal.pone.0280722.r004

Author response to Decision Letter 1

6 Jan 2023

Reponse to reviewers:

1. Journal Requirements:

Reponse: We have reviewed the reference list and it is complete. No new articles have been cited.

2. Multivariable analysis, where appropriate, was performed to evaluate independent risk factors associated with pneumococcal colonization.

Comment: Please specify the method that was adopted for multivariable analysis.

Reponse: The method adopted for the multivariate analysis is specified as follows:

“Multivariable analysis, was performed to evaluate independent risk factors associated with pneumococcal colonization. Separate multivariable models were constructed for each outcome using automated stepwise logistic regression (congenital disease, pneumonia and presence of smokers in the household)”.

3. The serotypes found most frequently with resistance to trimethoprim/sulfamethoxazole, tetracycline, erythromycin, cefuroxime, and clindamycin were 19F, 14, and 6B (Table 4).

Comment: The inference drawn in the sentence just before Table 4 does not completely match with the statistics mentioned in the table. Hence, the above sentence should be deleted.

Reponse: Because the inference drawn in the sentence just before Table 4 does not fully match the statistics mentioned in the table.

Deleted sentence: The serotypes found most frequently with resistance to trimethoprim/sulfamethoxazole, tetracycline, erythromycin, cefuroxime, and clindamycin were 19F, 14, and 6B.

4. Reviewer #1: The authors have responded to the queries or comments satisfactorily. However the following needs to be incorporated in the manuscript: under material and methods the study period has still not been mentioned clearly. Kindly give specific study period details.

Reponse: In order to provide specific details of the study period, the following is added in material and methods:

Setting, design and study period

As part of the national surveillance program for meningitis and pneumonia, a cross-sectional study was carried out, whose study period was from 2012 to 2020.

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PLoS One. doi: 10.1371/journal.pone.0280722.r005

Decision Letter 2

Arghya Das

8 Jan 2023

Nasopharyngeal colonization by Streptococcus pneumoniae in children and adults before the introduction of the 10-valent conjugate vaccine, Paraguay

PONE-D-22-20913R2

Dear Dr. León Ayala,

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: I Don't Know

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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Reviewer #1: (No Response)

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Reviewer #1: Yes: ANWITA MISHRA MD

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PLoS One. doi: 10.1371/journal.pone.0280722.r006

Acceptance letter

Arghya Das

7 Feb 2023

PONE-D-22-20913R2

Nasopharyngeal colonization by Streptococcus pneumoniae in children and adults before the introduction of the 10-valent conjugate vaccine, Paraguay

Dear Dr. León:

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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on behalf of

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Associated Data

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

Supplementary Materials

S1 Table. Pneumococcal carriage dataset Paraguay.

(XLSX)

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Data Availability Statement

The minimal dataset underlying the results described in the paper can be found at supporting Information files and at 10.6084/m9.figshare.21089815.

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PERMALINK

Nasopharyngeal colonization by Streptococcus pneumoniae in children and adults before the introduction of the 10-valent conjugate vaccine, Paraguay

Gustavo Chamorro

Aníbal Kawabata

María da Gloria Carvalho

Fabiana C Pimenta

Fernanda C Lessa

Carlos Torres

María José Lerea

María Eugenia León

Roles

Abstract

Introduction

Materials and methods

Setting, design and study period

Study population and samples

Inclusion criteria

Exclusion criteria

Isolation, identification, and serotyping

Antibiotic susceptibility

Risk factors

Statistical analysis

Ethical aspects

Results

Demographic data

Table 1. Prevalence of pneumococcal colonization of the study population by age group (n = 1444).

Fig 1. Distribution of pneumococcal serotypes by age group in Paraguay (n = 269).

Table 2. Association between risk factors and nasopharyngeal colonization with S. pneumoniae, according to age group.

Table 3. Multivariable analysis for pneumococcal colonization risk factors among children (n = 245).

Table 4. Distribution of antimicrobial resistance by serotypes.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Arghya Das

Roles

Author response to Decision Letter 0

Decision Letter 1

Arghya Das

Roles

Author response to Decision Letter 1

Decision Letter 2

Arghya Das

Roles

Acceptance letter

Arghya Das

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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