Efforts to reduce the burden of pneumococcal infection have intensified over the past decade with the development of conjugate vaccines and attempts to reduce antimicrobial resistance by promoting prudent antibiotic use. To assess the impact of the 7-valent (PCV7, covering serotypes 4, 6B, 9V, 14, 18C, 19F, 23F) and 13-valent (PCV13, additionally covering serotypes 1, 3, 5, 6A, 7F, 19A) pneumococcal conjugate vaccines (PCVs) included in the French National vaccination program in 2003 and 2010, respectively, on nasopharyngeal pneumococcal carriage, serotype distribution and antimicrobial susceptibility among children attending daycare centers in Southeastern France were studied. Surveillance has been ongoing since 1999, through repeated, identical cross-sectional surveys.1 We report the results of our latest survey, in 2012, which was compared with our previous findings.
A cross-sectional survey was conducted on a random sample of children 3–40 months of age attending daycare centers (DCCs) in the Alpes Maritimes district from January to April 2012, according to the same methodology and seasonality as previously reported.1
Nasopharyngeal samples were analyzed at Nice University Hospital (CHUN) microbiology laboratory and pneumococcal (SP) isolates identified as previously described. Isolates were considered penicillin nonsusceptible (PDSP) for minimum inhibitory concentrations > 0.06 mg/L, and penicillin-resistant for minimum inhibitory concentrations > 2 mg/L. Results were compared with identical surveys conducted in 1999, 2002, 2004, 2006 and 2008.
The study protocol was approved by the Nîmes University Hospital Sud Mediterranée III Ethics committee, according to French regulations.
Until 2008, nonvaccine-type isolates were determined using pooled latex agglutination kits. In 2012, specific serotypes were identified at the French reference centre for pneumococci and nontypeable (NT) isolates were confirmed by the Statens Serum Institute and analyzed by multilocus sequence typing (MLST) in the CHUN laboratory. MLST types were determined as described (http://spneumoniae.mlst.net/misc/info.asp). Allele and sequence type (ST) assignments were determined using the MLST database (http://spneumoniae.mlst.net), and clonal complexes using eBURST (www.eburst.mlst.net).2
To estimate the proportion of SP carriers within 5% accuracy and with 95% confidence, with a hypothesis of 18% carriers of PDSP isolates, that is, an unchanged rate since the previous 2008 survey, a sample size of 375 was calculated.
Data were analyzed using EpiInfo software. The χ2 test was used in univariate analysis to identify potential differences between the pre-PCV13 and post-PCV13 era and the χ2 test for trend to assess trends over time since the initial 1999 survey, with a 5% level of significance.
Between January and April 1999, 2002, 2004, 2006, 2008 and 2012, nasopharyngeal aspirates were obtained from children 3–40 months of age attending 25 randomly selected DCCs in the Alpes Maritimes area in Southeastern France. Population characteristics, carriage and pneumococcal immunization rates, antibiotic susceptibility and serotype distribution are shown in Table 1. The proportion of SP carriers fell significantly, from 54.0% in 1999 to 46.0% in 2012 (χ2 for trend: P < 10–3). The proportion of PDSP carriers dropped from 33.9% in 1999 to 15.6% in 2012 (χ2 for trend: P < 10–3), but the lowest carrier rate was observed in 2006 (17%) and did not decrease significantly further following the implementation of PCV13 in 2010 (18.6% in 2008 and 15.6% in 2012; P = 0.28).
PCV Immunization Status
In 2012, PCV coverage was documented in 347 children (95.1%). Of these, 287(82.7%) had received ≥1 dose of PCV13 and 60 (17.3%) PCV7 only. Carriage rate was comparable between children immunized with ≥1 dose of PCV13 (137/287: 47.7%) and those exclusively immunized with PCV7 (24/60: 40.0%) (P = 0.27). Likewise, carriage of PSDP was similar in both groups: 9/60 (15.0%) for those who received only PCV7 and 47/287 (16.4%) of those who received PCV13 (P = 0.76).
No PCV-7 strain was isolated in 2012, and only 11 (6.5%) children harbored PCV13 serotypes, all 19A; among these, 1 child had undocumented vaccination status, 1 had not been immunized, 1 had exclusively received PCV7 and 4, 2 and 2 children had received 1, 2 and 3 doses of PCV13, respectively (Table 1). Seventeen different known serotypes were identified in 2012, among which 15B/C, 23A/B, 35B, 15A, 11A, 17F, 6C and NT accounted for 82% of isolates. Two children attending the same DCC each harbored 2 different strains (serotypes 15A and NT). Serotype 6C carriers had all received at least 1 dose of PCV13.
MULTILOCUS SEQUENCE TYPING
MLST of the 16 NT isolates from 2012 identified 4 STs: 6 ST4149 strains, all recovered in a single DCC, 5 ST344 in 2 DCCS and 4 ST3097 in 3 DCCs. ST4149 and ST3097 differ from ST344 by a single locus. All these STs are grouped within the CC344 clonal complex. In the MLST database, ST4149 and ST3097 have only been found in NT isolates, whereas ST344 has also been described among serotype 6A, 19F and 35B isolates. ST63 was recovered in a single child and is related to several serotypes, that is, 14, 15, 15A, 19A, 19F, 23A, 23F.
Antimicrobial susceptibility improved between 1999 and 2012, although rates achieved in 2006 for penicillin and erythromycin stabilized thereafter. Proportions of PDSP among pneumococcal isolates were comparable in 2008 and 2012: 42.6% versus 35.3%, respectively; P = 0.21. In 2012, all serotype 19A (11/11) and serotype 29 (2/2) isolates were intermediately susceptible to penicillin, as were 93% of NT (15/16), 81% of 15A (13/16), 78% of 35B (14/18) and 55% of 6C (5/9) isolates. No isolate was penicillin-resistant. Eight isolates (4.7%) were intermediately susceptible to amoxicillin: serotypes 15A (n = 2), 19A (n = 3) and 35B (n = 3); 5(2.9%) were intermediately susceptible to ceftriaxone: serotypes 19A (n = 4) and 15A (n = 1). These proportions were significantly lower than in 2008: 27/159 (17.0%; P < 10–3) and 33/159 (20.7%; P < 10–3), respectively.
MDR isolates in 2012 (n = 61; 35.8%), included serotypes 19A (11/11), NT (15/16), 15A (14/16), 6C (6/9), 23A (6/15), 11A (2/12), 35B (5/18), 17F (1/11) and 29 (1/2).
In 2012, 146 children (40.0%; 95% CI: 34.2–46.2) received ≥1 course of antibiotic treatment, and 185 courses were prescribed altogether: amoxicillin+/−clavulanate (56.7% of prescriptions, up from 39.0% in 2008; P = 0.001), and third generation cephalosporins (34.0%, down from 47.8% in 2008; P = 0.006).
The 6 surveys conducted among children attending DCCs since 1999 revealed major changes in serotype distribution of pneumococcal carriage strains following the introduction of PCV7 and PCV13. Of PCV13-types, only serotype 19A remained in 2012 among our study population, while overall pneumococcal carriage was comparable to 2008, as replacement serotypes appeared. The proportion of penicillin-susceptible strains increased significantly up to 2006, but remained subsequently stable, certain replacement serotypes displaying reduced susceptibility to several antibiotics.
Only pooled sero-group determination was available for several nonvaccine serotypes before 2012, and in 2012 MLST was performed on NT strains only, while this procedure might have yielded relevant information concerning 19A STs.
Lee et al3 report stable colonization rates among children who were 6–23 months of age in Massachusetts between 2007 and 2011, with significant increases in non-PCV13 serotypes, following PCV13 availability in 2010, 15B/C being the most common, but PCV13-type 19A remaining second. Conversely, Ricketson et al4 found a 33% decrease in overall colonization among children 1–4.5 years attending community health centers in Calgary in 2012 following the introduction of PCV13, nonvaccine serotypes accounting for 94% of isolates. The dominant serotypes observed in these surveys, namely 6C, 11A, 15A/B/C, 23A/B, 35B/F, are consistent with our findings and have also been reported in Italy by Zuccotti et al5 in 2011. In a randomized control trial, Dagan et al6 showed a protective effect of PCV13 on colonization with serotype 6C. However, all carriers of serotype 6C in our study had received at least 1 dose of PCV13.
Cohen et al7 also report a decrease in carriage and penicillin nonsusceptibility among children with acute otitis media following PCV13 immunization, dominant replacement serotypes partly reflecting our findings, that is, 15A/B/C, 35B and 11A.
Higher colonization rates are expected in day-care settings due to horizontal spread. Day-care attendance is described as a risk factor for carriage of serotype 19A, known to cause invasive disease.8 In our survey, only serotype 19A isolates remained among PCV13 serotypes, and were non-susceptible to several antibiotics, including penicillin. According to a recent report from the United States, serotype 19A accounted for 34.1% of all isolates from children younger than 5 in 2010–2011 and was the most frequent serotype among both noninvasive (32.8%) and invasive (43.9%) strains, over 50% of isolates displaying penicillin non-susceptibility.9 Serotype 35B ranked second both in frequency and penicillin nonsusceptibility.
Unlike other reports, a significant proportion of NT strains were isolated, mostly nonpenicillin-susceptible. The majority shared 6/7 investigated sequences and were therefore related. All identified STs were already recorded in the MLST database.
Antimicrobial prescriptions did not decline further beyond 2006, albeit with fewer third generation cephalosporins, known to promote microbial resistance to a higher extent than amoxicillin.10
Our findings reveal sustained colonization and antibiotic nonsusceptibility rates from 2008, when >90% of children had received PCV7, to 2012, when 82% had received PCV13, and confirm major serotype replacement. The combined effects of vaccine and antibiotic-driven selective pressure warrant continued assessment of pneumococcal evolution.
These surveys were made possible thanks to grants provided by Produits Roche (1999), Wyeth Pharmaceuticals (2002, 2004 and 2006), and the French national hospital program for clinical research: Programme hospitalier de recherche clinique (2008 and 2012). We wish to thank all those who contributed to this survey: the daycare staff, parents and children; Microbiological investigations: Anne Leydier, Sandrine Courtial and Sylvie Lemée; Serotype determination: Dr. Emmanuelle Varon, Centre National de Référence des pneumocoques, Hôpital G Pompidou, Paris, France; Data management: Christine Boulle and Sabine Anthony; Sample and data collection: Marie-Fleur Alquier, Stéphanie Baudisson, Salim Fakir; Administrative and financial issues: Vanina Oliveri and Vanessa Espin and Local public health authorities for maternal and child protection and health care (Conseil general des Alpes Maritimes): Mai-Ly Durant, Marie-Agnès Grinneiser, Françoise Bevançon.
1. Dunais B, Bruno-Bazureault P, Carsenti-Dellamonica H, et al. A decade-long surveillance of nasopharyngeal colonisation with Streptococcus pneumoniae
among children attending day-care centres in south-eastern France: 1999-2008. Eur J Clin Microbiol Infect Dis. 2011;30:837–843
2. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol. 2004;186:1518–1530
3. Lee GM, Kleinman K, Pelton SI, et al. Impact of 13-valent pneumococcal conjugate vaccination on Streptococcus pneumoniae
carriage in young children in Massachusetts. J Pediatric Infect Dis Soc. 2014;3:23–32
4. Ricketson LJ, Wood ML, Vanderkooi OG, et al.for the Calgary Streptococcus pneumoniae
Epidemiology Research (CASPER) investigators. Trends in asymptomatic nasopharyngeal colonization with Streptococcus pneumoniae
after introduction of the 13-valent pneumococcal conjugate vaccine in Calgary, Canada. Pediatr Infect Dis J. 2014;33:724–730
5. Zuccotti G, Mameli C, Daprai L, et al.PneuMi Study Group (PMSG). Serotype distribution and antimicrobial susceptibilities of nasopharyngeal isolates of Streptococcus pneumoniae
from healthy children in the 13-valent pneumococcal conjugate vaccine era. Vaccine. 2014;32:527–534
6. Dagan R, Patterson S, Juergens C, 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
7. Cohen R, Levy C, Bingen E, Koskas M, Nave I, Varon E. Impact of 13-valent pneumococcal conjugate vaccine on pneumococcal nasopharyngeal carriage in children with acute otitis media. Pediatr Infect Dis J. 2012;31:297–301
8. Kaplan SL, Barson WJ, Lin PL, et al. Serotype 19A Is the most common serotype causing invasive pneumococcal infections in children. Pediatrics. 2010;125:429–436
9. Richter SS, Heilmann KP, Dohrn CL, et al. Pneumococcal serotypes before and after introduction of conjugate vaccines, United States, 1999–2011(1.). Emerg Infect Dis. 2013;19:1074–1083
10. Dagan R, Barkai G, Leibovitz E, et al. Will reduction of antibiotic use reduce antibiotic resistance? The pneumococcus paradigm. Pediatr Infect Dis J. 2006;25:981–986