http://orcid.org/0000-0002-1619-1919
Figures
Abstract
Streptococcus pneumoniae is a common cause of infectious diseases such as pneumonia and sepsis. Its colonization is thought to be the first step in the development of invasive pneumococcal diseases. This study aimed to investigate pneumococcal colonization patterns in early childhood. A longitudinal birth cohort study was conducted for investigating nasopharyngeal colonized pneumococci at 1, 6, 12, 18, 24, and 36 months of age, particularly focusing on the serotype distribution and antimicrobial susceptibilities. Pneumococcal conjugate vaccine (PCV) effect on nasopharyngeal colonization was also assessed. During 2013–2017, 855 infants were enrolled and a total of 107 isolates were recovered from 95 infants during the first three years of life. In this period, the prevalence of pneumococcal colonization increased, with values ranging from 0.2% (2/834) at 1 month of age to 5.9% (19/323) at 36 months of age. The investigation of serotype revealed that 81.1% (73/90) belonged to the non-PCV13 serotypes–23A, 15A, 15C, and 15B. Moreover, PCV13 serotypes significantly decreased during 2014–2015, when routine PCV13 vaccination was initiated in Taiwan. PCV13 introduction may lead to the reduction in the rates of pneumococcal isolates resistant (R) to penicillin. Under conditional PCV13 vaccination, pneumococcal isolates primarily belonged to non-PCV13 serotypes. This non-PCV13 serotype replacement exhibited lower rates of penicillin R isolates, suggesting that PCV13 administration may reduce the antibiotic-nonsusceptible pneumococcal disease burden and antibiotic use.
Citation: Tsai M-H, Liao S-L, Chiu C-Y, Shih H-J, Hua M-C, Yao T-C, et al. (2020) Longitudinal investigation of nasopharyngeal pneumococcal carriage in early childhood: The PATCH birth cohort study. PLoS ONE 15(8): e0237871. https://doi.org/10.1371/journal.pone.0237871
Editor: Herman Tse, Hong Kong Children's Hospital, HONG KONG
Received: April 7, 2020; Accepted: August 4, 2020; Published: August 20, 2020
Copyright: © 2020 Tsai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors do not have permission to share the data. The institutional review boards of Keelung Chang Gung Memorial Hospital imposed sharing restriction on the minimal data set, the grounds for restriction, and the nature of restriction. Data are available from the Department of Medical Research of Keelung Chang Gung Memorial Hospital Institutional Data Access (contact via Ms. Kao +886-2-24313131 ext.3395; kaoting@cgmh.org.tw) for researchers who meet the criteria for access to confidential data.
Funding: The study was supported by grants from Keelung Chang Gung Memorial Hospital, Taiwan (CMRPG2E0121, CMRPG2E0122, CMRPG2E0123, CMRPG2E0124, CMRPG2E0125). The funder, Keelung Chang Gung Memorial Hospital, had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: PCV, pneumococcal conjugate vaccine; MIC, minimum inhibitory concentration; I, intermediate; R, resistant; PCR, polymerase chain reaction; MLST, multi-locus sequence typing; ST, sequence type
Introduction
Streptococcus pneumoniae is one of the main causes of infectious diseases, including pneumonia, meningitis, and sepsis, which are associated with high morbidity and mortality [1,2]. It is estimated that S. pneumoniae is responsible for 1.6 million deaths annually, particularly affecting young children and elderly persons [3]. S. pneumoniae is commonly found in the nasopharyngeal flora of healthy persons; pneumococcal colonization is thought to be the first step in the development of invasive diseases [4,5]. In addition, the widespread use of antimicrobial agents has led to the high prevalence of antimicrobial-resistant pneumococci, which has made treatment of pneumococcal infections challenging [2]. Therefore, vaccines have been developed to prevent pneumococcal infections.
In 2000, pneumococcal conjugate vaccine (PCV) was globally introduced to combat the invasive pneumococcal disease in young children [6]. The first PCV was the 7-valent PCV, which contained serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Subsequently, the vaccine was expanded to cover ten (PCV7 and serotypes 1, 5, and 7F) and thirteen (PCV10 and serotypes 3, 6A, and 19A) serotypes [7]. In Taiwan, PCV7 was introduced for private use in 2005 [8,9]. Nonetheless, an increasing number of invasive infections occurred due to the non-vaccine serotype 19A [10]. Therefore, PCV10 was introduced in Taiwan in 2010, with minimal or no notable improvement, possibly because these three serotypes (serotypes 1, 5, and 7F) exhibited a low occurrence in Taiwan [11]. In 2011, PCV13 was introduced for private use, as an initial step. A national catch-up PCV13 program was launched in March 2013 for children aged 2–5 years and was then expanded, from January 2014 to December 2014, to also cover children aged 1–5 years [12]. By the end of 2014, the age-appropriate immunization rate reached 80%, according to the report from the Center for Disease Control in Taiwan [2]. PCV13 was not introduced as a routine immunization for all infants until January 2015 [12].
A longitudinal birth-cohort study was conducted to improve the understanding of pneumococcal colonization in children in the context of high antibiotic selection pressure and conditional use of pneumococcal vaccination in Taiwan. Infants were prospectively examined for nasopharyngeal colonized bacteria at specified time points. Both the distribution of serotypes and the evolution of pneumococcal antibiotic susceptibility were assessed.
Methods
Study population
The Prediction of Allergen in Taiwanese Children (PATCH) Study, an ongoing prospective study launched in 2013 following a birth cohort of infants, was designed to investigate the bacterial colonization and the factors related to the development of asthma and other allergic diseases. Women in the third trimester of pregnancy, admitted at the Obstetrics Clinic of Keelung Chang Gung Memorial Hospital, were invited to participate in the study. Infants exhibiting severe congenital abnormalities, low birth weight (<2000 g), or requiring mechanical ventilation at any point since birth were excluded. The institutional review boards of Keelung Chang Gung Memorial Hospital approved the study project and a written informed consent was obtained from the mother of each participant.
Sample and clinical parameter collection
Mothers were requested to bring the enrolled infants (1, 6, 12, 18, 24 and 36 months of age) to the Pediatric Clinic of Keelung Chang Gung Memorial Hospital for S. pneumoniae detection from nasopharyngeal swabs. One swab per infant per visit study was taken. Questionnaire surveys were conducted at each visit to obtain information regarding demographic data, housing and living conditions, socioeconomic status, risk factors for colonization, history of respiratory tract infection and pneumococcal vaccination, and other clinical parameters.
Nasopharyngeal swabs were collected with separate cotton-tipped swabs (Copan Swab Applicator, Copan Diagnostics Inc., Brescia, Italy), through the nose, into the nasopharyngeal spaces at the scheduled visits. After placed into a transported medium, swabs were transported to the microbiology laboratory within 2 h after collection and cultured for bacteria by using standard methods for identification [13]. All swabs were plated within 6 hours of sampling on blood agar plates with 5% sheep’s blood to isolate S. pneumoniae.
Antimicrobial susceptibility testing
Pneumococcal isolates were examined for antimicrobial susceptibility to penicillin, levofloxacin, ceftriaxone, and trimethoprim-sulfamethoxazole using the E-test method (bioMerieux, Durham, USA). Susceptibility and resistance were defined in accordance with the criteria suggested by the Clinical and Laboratory Standards Institute (CLSI) [14]. The minimum inhibitory concentrations (MICs) used were based on the criteria recommended by the CLSI for meningeal and non-meningeal infections. For consistency of data interpretation in the study, isolates were categorized as susceptible (S; penicillin, ≤ 0.06 mg/L; ceftriaxone, ≤ 0.5 mg/L), intermediate (I; penicillin, 0.12–1 mg/L; ceftriaxone, 1 mg/L), and resistant (R; penicillin, > 2 mg/L; ceftriaxone, > 1 mg/L). According to the CLSI criteria, although susceptibility in the I category indicated that β-lactam remained effective for non-meningeal infections, the result should be interpreted as resistant for meningeal infections.
Serotyping of pneumococci and multi-locus sequence typing
Serotypes of pneumococcal isolates were determined using commercialized antisera (Statens Serum Institut, Copenhagen, Denmark) and polymerase chain reaction (PCR) methods [15,16]. Multi-locus sequence typing (MLST) was performed by PCR-sequencing of a set of pneumococcal housekeeping genes, including ardE, gdh, gki, recP, spi, xpt, and ddl [17]. The sequence data were compared with the MLST database (http://pubmlst.org/spneumoniae/). New alleles and allelic profiles were submitted to the database curator for assignment of the sequence type (ST) numbers. eBURST analysis was utilized to group STs into clonal complexes (STs shared six of the seven MLST loci).
Statistical analysis
Data were analyzed using SPSS, version 12.0 (SPSS inc., Chicago, IL). Student’s t-test was used to analyze numerical data. If the data were not normally distributed, the Mann-Whitney U-test was used for analysis. Chi-square test or Fisher’s exact test, when appropriate, was used to analyze categorical data. In addition, the linear trend of the proportion of isolates with PCV13 coverage and rates of penicillin R isolates across the study period were tested using the Cochran–Armitage chi-square test. To investigate factors associated with pneumococcal carriage by univariate analysis, each colonized infant was frequency-matched with the non-colonized infants on variables of year of study, season, age and gender. Variables for which P<0.1 were chosen for model selection of multivariate analysis. P<0.05 was considered statistically significant.
Results
Demographics and factors associated with pneumococcal colonization among the study population
From March 2013 to December 2017, 900 prenatal infants were recruited and 45 postnatal were excluded: 40, low birth weight (<2000 g); four, ventilator had been used after birth; and one, cerebral palsy. Hence, 855 infants were enrolled, with the numbers of collected samples in different age groups being 834 (1 month), 749 (6 months), 677 (12 months), 504 (18 months), 449 (24 months), and 323 (36 months). Nasopharyngeal culture was used for pathogen detection of all collected samples, and 107 pneumococcal isolates were recovered from 95 cases during the study period. Of the pneumococcal colonization time points during the first three years of infant life, pneumococcal colonization was found in 0.2% (2/834) at 1 month, followed by 2.5% (19/749) at 6 months, 3.1% (21/677) at 12 months, 3.0% (15/504) at 18 months, 3.1% (14/449) at 24 months, and 5.9% (19/323) at 36 months. Fig 1 shows the trend of S. pneumoniae carriage at the scheduled sampling time points during the first 36 months of infant life.
Of the 95 cases with pneumococcal colonization, 82 (86.3%) had received PCV13 and 8 (9.8%, 8/82), 31 (37.8%), 20 (24.4%), 23 (28.0%) of them had one, two, three and four doses of PCV13, respectively. Then we investigated factors associated with the occurrence of pneumococcal colonization and the result is shown in Table 1. For the selection of non-pneumococcal carriers as the control group, each pneumococcal carrier was frequency-matched with the non-carrier on variables of year of study, season, age and gender. Finally, 92 carriers and 172 non-carriers were chosen for the investigation. Three factors including demographic characters, environmental features and health conditions, were compared between pneumococcal carriers and non-pneumococcal carriers. Except day-care center attendance (11.6% versus 3.8%, p = 0.04), no other variable was found to be significantly associated with the occurrence of pneumococcal carriers. Furthermore, multiple logistic regression analysis also revealed some evidence that day-care center attendance was associated with pneumococcal carriage (OR 3.16; 95% CI 0.99–10.11; p = 0.05).
Serotype distribution among the pneumococcal isolates during the study period
The distribution of serotypes of S. pneumoniae isolates is shown in Table 2. Of the total 107 isolates, 90 belonged to 13 different serotypes but the other 17 were non-typeable. In the 90 isolates with different serotypes, 81.1% (73/90) were non-PCV13 serotypes, with the most common serotype being 23A (33.3%, 30/90), followed by 15A (22.2%, 20/90), 15C (8.9%, 8/90), 15B (7.8%, 7/90), and 19A (7.8%, 7/90). We further analyzed the proportion of isolates with PCV13 coverage during 2013–2017 and showed that PCV13 serotype isolates were 33.3% (3/9) in 2013 and 85.7% (6/7) in 2014, respectively. However, a decrease occurred from 2014 to 2017: from 85.7% to 3.8% (1/26) and the decreasing trend during this period was statistically significant (p = 0.001) (Fig 2).
Antimicrobial susceptibility of pneumococcal isolates during the study period
Of the 90 pneumococcal isolates with identified serotypes, eight were unrecoverable and 82 were collected for antimicrobial susceptibility examination. Except 10 cases with antimicrobial agent prescription due to respiratory tract infection, most of the cases (87.8%, 72/82) did not receive antibiotic therapy within 2 weeks before sampling. For penicillin, the rates of I and R isolates were 76.8% (63/82) and 23.2% (19/82), respectively, and no S isolate was found. For ceftriaxone, the rates of S, I, and R isolates were 64.6% (53/82), 31.7% (26/82), and 3.7% (3/82), respectively. Table 3 shows the results for the penicillin susceptibility analysis of pneumococcal isolates during the study period.
We further found that rates of penicillin R isolates decreased from 85.7% (6/7), 25.0% (6/24), and 17.6% (3/17) to 0% during 2014–2017. Furthermore, the rates of I isolates increased from 14.3% (1/7), 75.0% (18/24), and 82.4% (14/17) to 100% during the same period. The decrease of penicillin R isolate rates during this period was statistically significant (p<0.001). Of the penicillin R isolates during 2014–2017, 60% (9/15) belonged to PCV13 serotypes (19A in 6 isolates and 19F in 3 isolates). However, most (91.4%, 53/58) of the penicillin I isolates were non-PCV13; 23A (45.3%, 24/53) was the most common serotype, followed by 15A (24.5%, 13/53), 15C (11.3%, 6/53), 15B (7.5%, 4/53), and 34 (7.5%, 4/53), respectively.
Distribution of sequence types and serotypes during the study period
Eighteen different STs were identified during the study period, with 3 major STs being found in 63.3% (57/90) of the isolates, including ST 63 (30.0%, 27/90), ST 338 (21.1%, 19/90) and ST 166 (12.2%, 11/90). The largest clone, ST 63, included the majority of isolates from serotype 15A (74.1%, 20/27). All isolates from the second (ST 338) and third (ST 166) clones belonged to serotype 23A. Both serotype 15A and serotype 23A were non-PCV13 serotypes. Table 4 shows the distribution of sequence types and their association with serotypes during the study period.
Discussion
Our study showed that the prevalence of pneumococcal colonization gradually increased during the first three years of life, which is consistent with other studies [18,19]. Moreover, in our previous report, the prevalence of Staphylococcus aureus decreased in the same period, and an inverse relationship between the prevalence of S. pneumoniae and S. aureus was also reported by Labout, et al. [19,20]. This phenomenon might be due to pneumococcal competition, which is mediated via the production of hydrogen peroxide by S. pneumoniae or interference by S. aureus present in the nasopharyngeal spaces [21,22].
We also found that the overall prevalence of S. pneumoniae colonization was lower in our cohort than that has been reported in other studies [11,18]. One cause of this discrepancy may be due to different age groups between our study and others. Kuo, et al. reported that 14.1% of children had pneumococcal colonization and 57.6% were aged 2 to 5 years, the highest colonization age group of children less than 5 years [18]. However, in our series, most of them (65.3%, 62/95) were aged less than 2 years. Another cause may be because various PCV immunization programs had been implemented in Taiwan prior to our study. Of the statistical data reported by Wei, et al., a total of 128,680 and 114,140 doses of PCV10 were imported in 2010 and 2011, which would provide complete immunization for 20.5% and 15.2% of infants born in the corresponding years with the 3+1 schedule, respectively [23]. Wei, et al. also reported that 2,027,160 doses of PCV13 were imported during 2011–2013, which would provide complete immunization for 85.9% of infants born during the respective period using the 3+1 schedule [24]. We thought that these implementations prior to 2013 might have an impact on the pneumococcal colonization rate during the study period from 2013 to 2017. Besides, in our study, day-care center attendance was shown as a factor associated with the occurrence of pneumococcal carriage, which increased the risk of pneumococcal carriage by around threefold. It suggested that horizontal transmission among the pneumococcal colonized infants through the surrounding carriers, especially day-care center members, may be important for the acquisition of pneumococci in early childhood.
Serotype epidemiology of invasive pneumococcal disease (IPD) in Taiwan prior to 2013 revealed that unlike the decreasing trend of IPD caused by PCV7 serotypes, IPD caused by PCV13 serotypes decreased in the first years after PCV7 introduction in late 2005 and restored in 2008–2013 [24]. The discrepancy of trends of IPD caused by PCV7 and PCV13 serotypes was related to the emergence of IPD caused by the replacement serotypes not in PCV7 but was covered by PCV13, especially serotype 19A. Besides 19A (38.2%), PCV7 serotypes including 14 (11.6%), 23F (10.7%), 19F (10.1%) and 6B (8.3%), were the most common ones prior to 2013 [23]. Of our study, with respect to the serotype distribution during 2013–2017, the decrease of PCV13 serotypes occurred from 2014 to 2015, when the universal PCV13 vaccination program was initiated in Taiwan. In addition, 81.1% were non-PCV13 serotypes, with the most common ones being 23A, 15A, 15C, and 15B. We also found that the clonality of the leading non-PCV13 serotypes to be ST33823A and ST6315A, and this result is consistent with the report by Su, et al. [2]. In the United States, ST 33823A was observed in the pre-PCV7 era through capsular switch or structural change from the pre-existing ST33823F strain [25]. In Taiwan, serotype 23F was the most common serotype before PCV13 introduction, despite limited information on the ST distribution [11]. Limited amount of ST33823A distribution was documented during the same period [8,9]. All these findings suggested that similar to the findings in the US, the capsular switch from ST33823F to ST33823A may also have occurred in Taiwan, with increasing use of PCV13 resulting in an increase in ST33823A.
Regarding the association of PCV13 with antimicrobial susceptibility, we found that PCV13 administration might have reduced the rates of penicillin R isolates among pneumococcal isolates. Moreover, most of the penicillin R isolates belonged to PCV13 serotypes. Notably, Degan, et al. found that PCV7/PCV13 serotypes 6B, 9V, 14, 19F, and 23F and PCV13-specific serotypes 6A and 19A were the most common observed antibiotic-resistant serotypes [26]. Their study also showed that the immune responses against 6A, 19A, and 19F were significantly higher after PCV13 administration than those after PCV7 administration [27]. Thus, PCV13 had an added benefit over PCV7 in reducing the carriage of antibiotic-nonsusceptible S. pneumoniae.
There were some limitations in our study. First, it is an ongoing prospective study but the research period was limited from 2013 to 2017; thus, the initially enrolled cases could not all complete the first 36-month study. Second, the number of isolates was relatively small in our series. Based on these reasons, the selection bias might occur and thus the current result might be interpreted with caution. However, our study, a prospective cohort-designed study, may reduce the possibility of selection bias for the comparison because in a cohort study, the outcome is not known at case enrollment when exposure status is established. Nevertheless, selection bias can occur in retrospective studies since the outcomes have already occurred at the time of selection.
Conclusions
Our study showed that under conditional PCV13 vaccination, the replacement of serotypes from PCV13 to non-PCV13 serotypes might be found along with the evolution of pneumococcal antibiotic susceptibility. The replacement non-PCV13 serotypes yielded lower rates of penicillin R isolates, suggesting that PCV13 may reduce the carriage of antibiotic-nonsusceptible S. pneumoniae, the burden of antibiotic-nonsusceptible pneumococcal diseases, and broad-spectrum antibiotic use.
Acknowledgments
We thank all members of the Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan and Department of Medical Laboratory, Chang Gung Memorial Hospital, Keelung, Taiwan for their suggestions and assistance in this study. We also thank Ms Cristina (the Editage company, Taipei, Taiwan) for her professional English language editing.
References
- 1. Simell B, Auranen K, Kayhty H, Goldblatt D, Dagan R, O’Brien KL. Pneumococcal Carriage Group. The fundamental link between pneumococcal carriage and disease. Expert Rev Vaccines 2012; 11:841–855. pmid:22913260
- 2. Su LH, Kuo AJ, Chia JH, Li HC, Wu TL, Feng Y, et al. Evolving pneumococcal serotypes and sequence types in relation to high antibiotic stress and conditional pneumococcal immunization. Sci Rep 2015; 5:15843. pmid:26522920
- 3. Black RE, Cousens S, Johnson HL, Lawn JE, Rudan I, Bassani DG, et al. Global, regional, and national causes of child mortality in 2008: a systematic analysis. Lancet 2010; 375:1969–1987. pmid:20466419
- 4. Syrjanen RK, Kilpi TM, Kaijalainen TH, Herva EE, Takala AK. Nasopharyngeal carriage of Streptococcus pneumoniae in Finnish children younger than 2 years old. J Infect Dis 2001; 184:451–459. pmid:11471103
- 5. Kwambana BA, Barer MR, Bottomley C, Adegbola RA, Antonio M. Early acquisition and high nasopharyngeal co-colonisation by Streptococcus pneumoniae and three respiratory pathogens amongst Gambian new-borns and infants. BMC Infect Dis 2011; 11:175. pmid:21689403
- 6. Hausdorff WP, Bryant J, Paradiso PR, Siber GR. Which pneumococcal serotypes cause the most invasive disease implications for conjugate vaccine formulation and use, part I. Clin Infect Dis 2000; 30: 100–121. pmid:10619740
- 7. Wyllie AL, Wijmenga-Monsuur AJ, van Houten MA, Bosch AATM, Groot JA, van Engelsdorp Gastelaars J, et al. Molecular surveillance of nasopharyngeal carriage of Streptococcus pneumoniae in children vaccinated with conjugated polysaccharide pneumococcal vaccines. Sci Rep 2016; 6:23809. pmid:27046258
- 8. Chen CJ, Lin CL, Chen YC, Wang CW, Chiu CH, Lin TY, et al. Host and microbiologic factors associated with mortality in Taiwanese children with invasive pneumococcal diseases, 2001 to 2006. Diagn Microbiol Infect Dis 2009; 63: 194–200. pmid:19150710
- 9. Hsieh YC, Huang YC, Lin HC, Ho YH, Chang KY, Huang LM, et al. Characterization of invasive isolates of Streptococcus pneumoniae among Taiwanese children. Clin Microbiol Infect 2009; 15: 991–996. pmid:19392891
- 10. Kyaw MH, Lynfield R, Schaffner W, Craig AS, Hadler J, Reingold A, et al. Effect of introduction of the pneumococcal conjugate vaccine on drug-resistant Streptococcus pneumoniae. N Eng J Med 2006; 354: 455–463. pmid:16598044
- 11. Hsieh YC, Lin PY, Chiu CH, Huang YC, Chang KY, Liao CH, et al. National survey of invasive pneumococcal diseases in Taiwan under partial PCV7 vaccination in 2007: emergence of serotype 19A with high invasive potential. Vaccine 2009; 27: 5513–5518. pmid:19615960
- 12. Su WJ, Lo HY, Chang CH, Chang LY, Chiu CH, Lee PI, et al. Effectiveness of pneumococcal conjugate vaccines of different valences against invasive pneumococcal disease among children in Taiwan: a nationwide study. Pediatr Infect Dis J 2016; 35: e124–33. pmid:26974752
- 13.
Murray PR, Bron EJO, Pfaller MA. Manual of Clinical Microbiology, 6th ed; American Society of Microbiology, Washington DC, US, 1980.
- 14.
Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; 23rd informational supplement. Document M100-S18. Wayne, PA, USA: CLSI, 2013.
- 15. Pai R, Gertz RE, Beall B. Sequential multiplex PCR approach for determining capsular serotypes of Streptococcus pneumoniae isolates. J Clin Microbiol 2006; 44: 124–131. pmid:16390959
- 16. Yun KW, Cho EY, Hong KB, Choi EH, Lee HJ. Streptococcus pneumoniae type determination by multiplex polymerase chain reaction. J Korean Med Sci 2011; 26: 971–978. pmid:21860544
- 17. Enright MC, Spratt BG. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 1998; 144: 3049–3060. pmid:9846740
- 18. Kuo CY, Hwang KP, Hsieh YC, Cheng CH, Huang FL, Shen YH, et al. Nasipharyngeal carriage of Streptococcus pneumoniae in Taiwan before and after the introduction of a conjugate vaccine. Vaccine 2011; 29: 5171–5177. pmid:21621578
- 19. Labout JA, Duijts L, Arends LR, Jaddoe VW, Hofman A, de Groot R, et al. Factors associated with pneumococcal carriage in healthy Dutch infants: the Generation R study. J Pediatr 2008; 153: 771–776. pmid:18621390
- 20. Tsai MH, Chiu CY, Shih HJ, Liao SL, Hua MC, Huang SH, et al. Longitudinal investigation of nasopharyngeal methicillin-resistant Staphylococcus aureus colonization in early infancy: the PATCH birth cohort study. Clin Microbiol Infect 2017; 23: 121.e1–7. pmid:27793735
- 21. Pettigrew MM, Gent JF, Revai K, Patel JA, Chonmaitree T. Microbial interactions during upper respiratory tract infections. Emerg Infect Dis 2008; 14: 1584–1591. pmid:18826823
- 22. Xu Q, Almudervar A, Casey JR, Pichichero ME. Nasopharyngeal bacterial interactions in children. Emerg Infect Dis 2012; 18: 1738–1745. pmid:23092680
- 23. Wei SH, Chiang CS, Chen CL, Chiu CH. Pneumococcal disease and use of pneumococcal vaccines in Taiwan. Clin Exp Vaccine Res 2015; 4: 121–129. pmid:26273570
- 24. Wei SH, Chiang CS, Chiu CH, Chou P, Lin TY. Pediatric invasive pneumococcal disease in Taiwan following a national catch-up program with the 13-valent pneumococcal conjugate vaccine. Pediatr Infect Dis J 2015; 34: e71–e77. pmid:25247584
- 25. Pai R, Gertz RE, Whitney CG, Beall B. Clonal association between Streptococcus pneumoniae serotype 23A, circulating within the United States, and an internationally dispersed clone of serotype 23F. J Clin Microbiol 2005; 43: 5440–5444. pmid:16272467
- 26. Dagan R, Juergens C, Trammel J, Patterson S, Greenberg D, Givon-Lavi N, et al. Efficacy of 13-valent pneumococcal conjugate vaccine (PCV13) versus that of 7-valent PCV (PCV7) against nasopharyngeal colonization of antibiotic-nonsusceptible Streptococcus pneumoniae. J Infect Dis 2015; 211: 1144–1153. pmid:25355940
- 27. Dagan R, Patterson S, Juergens C, Greenberg D, Givon-Lavi N, Porat N, et al. Comparative immunogenicity and efficacy of 13-valent and 7-valent pneumococcal conjugate vaccines in reducing nasopharyngeal colonization: a randomized double-blind trial. Clin Infect Dis 2013; 57: 952–962. pmid:23804191