Following the introduction of heptavalent 7-valent pneumococcal conjugate vaccine (PCV7) in 2000, the rates of invasive pneumococcal disease (IPD) in US children have decreased substantially.1–5 Rates of other childhood pneumococcal illnesses such as otitis media and noninvasive pneumonia have also decreased.6–8 The adoption of PCV7 was also followed by reduced IPD rates in older children, adults, and immunocompromised hosts through herd immunity.3,5,9,10
Nine years after introduction of PCV7, IPD rates in children remain lower than prevaccine levels,11 but reductions in PCV7-type IPD have been partially offset by increases in serotypes not targeted by the vaccine.3,11–22 Nonvaccine serotypes, notably 19A, have rapidly filled the ecologic niche previously occupied by PCV7 serotypes, primarily by clonal expansion and, to a lesser extent, capsular switching; they now account for a substantial fraction of pneumococcal colonization and IPD in children and adults.3,12–19,21,23–25 Furthermore, antibiotic resistance has increasingly emerged among nonvaccine serotypes through both clonal expansion of previously resistant serotypes and acquisition of resistance by serotypes that were mainly susceptible in the past.21,23,26
Nonvaccine serotype replacement and the continued burden of pneumococcal disease have stimulated the development of a vaccine that targets a broader range of serotypes. In February 2010, the Food and Drug Administration–approved licensure of the 13-valent PCV (PCV13),27 which targets the same 7 serotypes as PCV7 and 6 additional serotypes (1, 3, 5, 6A, 7F, and 19A), accounting for an estimated 63% of IPD in US children aged <5 years in 2007.11 Ongoing evaluation of the pneumococcal serotypes carried in a defined geographic area allows us to assess whether S. pneumoniae carriage serotypes have stabilized several years after PCV7 introduction and to anticipate the impact widespread use of PCV13 could have on carriage and disease.
Our prior work in Massachusetts communities in 2001,28 2003–2004,13 and 2006–200721 showed that replacement of vaccine serotypes by nonvaccine serotypes in the nasopharynx was rapid and virtually complete by 2007.21 Certain clones previously associated with vaccine serotypes persisted in the postvaccine era by acquiring a nonvaccine serotype, including the multiply resistant ST 320 clone, which newly expressed a 19A capsule.29–31 Certain nonvaccine serotype clones associated with lowered susceptibility to penicillin (ST 199, ST 558) rapidly increased in carriage prevalence.29 We now report the results of ongoing surveillance of S. pneumoniae carriage in the same Massachusetts communities. We evaluate ongoing changes in serotype distribution and antibiotic resistance in the context of universal use of PCV7 before the introduction of PCV13.
MATERIALS AND METHODS
Study Design and Population
Between October 2008 and March 2009, children aged 3 months to <7 years seen in pediatric practices in 8 Massachusetts communities were enrolled during the “cold and flu” season. Children in these 8 communities were similarly recruited for pneumococcal colonization assessments in young healthy children during comparable periods in 2001, 2003–2004, and 2006–2007.13,21,28 Children from an additional 8 Massachusetts communities were also included in 2001 and 2003–2004.
Parental consent was obtained by study staff members, and nasopharyngeal specimens were obtained by trained nurses during well-child or sick visits. Parents of participating children completed a brief questionnaire regarding exposure to some potential predictors of pneumococcal carriage, including group child care, young siblings, recent antibiotic use, and concurrent illness.28 Participants' medical records were also reviewed for information on vaccines received, recent antibiotic use, and symptoms and diagnoses at the time of the visit. All study procedures were approved by the Harvard Pilgrim Health Care Institutional Review Board.
Identification of Serotypes and Antimicrobial Resistance Profiles
Nasopharyngeal swabs were plated within 24 hours for identification of S. pneumoniae. Serotype was primarily determined using the Quellung reaction with commercially available antisera (Serum Statens Institute, Copenhagen, Denmark), with confirmation by multilocus sequence typing. All putative 6A specimens were then tested using monoclonal antibodies to identify which were actually the closely related 6C serotype.32 Antibiotic susceptibility testing was performed using E-tests. Clinical and Laboratory Standards Institute susceptibility breakpoints were used to classify organisms as susceptible, intermediate, or resistant based on 2008 criteria to the following antibiotics: penicillin, amoxicillin, ceftriaxone, erythromycin, clindamycin, trimethoprim-sulfamethoxazole, levofloxacin, and vancomycin.33,34 Clinical and Laboratory Standards Institute susceptibility breakpoints for penicillin were the following: susceptible (minimal inhibitory concentration [MIC ] of ≤2 mg/L), intermediate (4 mg/L), and resistant (≥8.0 mg/L).32 For comparison purposes across sampling periods, we also used pre-2008 penicillin susceptibility breakpoint criteria: susceptible (MIC of ≤0.06 mg/L), intermediate (>0.06–1.0 mg/L), and resistant (≥2.0 mg/L).33 Penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) was defined as any intermediate or resistant isolate when using either PNSP>0.06 or PNSP>2.0 breakpoints
The proportions of children colonized with (a) any S. pneumoniae, (b) PCV7 serotypes (4, 6B, 9V, 14, 18C, 19F, 23F), (c) additional PCV13 serotypes (1, 3, 5, 6A, 7F, 19A), and (d) non-PCV13 serotypes were calculated for the 2008–2009 season and compared with data from 2001, 2003–2004, and 2006–2007. As described previously, data from all available communities in each sampling period (16 communities in 2001 and 2003–2004; subset of 8 communities in 2006–2007 and 2008–2009) were used to evaluate trends over time.21 Because serotype information was missing for 35 S. pneumoniae isolates (which could not be regrown) from 2001 and 7 isolates from 2003–2004, the proportion of carriage among isolates with known serotypes are reported over time. Trends in antibiotic susceptibility were also described for S. pneumoniae across the 4 sampling periods; statistical tests for differences and for linear trends over time were performed using generalized linear mixed models controlling for community.
Potential predictors of overall pneumococcal carriage and PNSP carriage were gathered from questionnaires and chart review data. These included age-group (3–<6 months, 6–<24 months, 24–<36 months, 36–<84 months), gender, group child care attendance, presence of young siblings <6 years, recent antibiotic use in last 2 months and acute respiratory tract infection (RTI) at time of sampling.21,28 Acute RTI was defined by reported symptoms on questionnaires or a diagnosis in the medical record of any upper or lower RTI, including otitis media, sinusitis, bronchitis, pharyngitis, cough illness, or pneumonia. Because carriage patterns were similar in 2006–2007 and 2008–2009 in the context of widespread PCV7 use, predictors of carriage were evaluated for both seasons combined. Bivariate and multivariate logistic regression analyses used generalized linear mixed models to account for clustering by community (SAS 9.2, SAS Institute, Cary, NC).
Nasopharyngeal specimens were collected from 1011 children and compared with prior sampling periods. Characteristics of the communities across the 4 sampling periods are described in Table, Supplemental Digital Content 1, https://links.lww.com/INF/B19, along with the proportion of study participants who carried S. pneumoniae. Study participants were similar in age distribution, gender, group child care attendance, and number of young siblings across sampling periods (Table, Supplemental Digital Content 2, https://links.lww.com/INF/B20). The proportion of children with acute RTI (overall F statistic, P < 0.001) and recent antibiotic use (overall F statistic, P < 0.001) differed across the 4 sampling periods. PCV7 vaccine coverage (receipt of ≥1 dose) among participants increased from 50% in 2001 to 82% in 2003–2004, before becoming nearly universal by 2006–2007 (98%) and 2008–2009 (99%).
Nasopharyngeal Carriage of S. pneumoniae
S. pneumoniae carriage in Massachusetts children (Table, Supplemental Digital Content 1, https://links.lww.com/INF/B19) varied significantly over time (P = 0.006), decreasing in 2003–2004 before returning to baseline values and remaining stable between 2006–2007 and 2008–2009. The trend in carriage across sampling periods was similar for children 3 to <24 months (2001: 26%; 2003–2004: 25%; 2006–2007: 32%; 2008–2009: 31%) and children ≥24 months (2001: 28%; 2003–2004: 22%; 2006–2007: 28%; 2008–2009: 27%).
Figure 1 shows the proportion of pneumococcal carriage (among isolates with known serotypes) attributable to vaccine and nonvaccine serotypes. The proportion of PCV7 serotypes decreased significantly over the first 3 sampling periods (P < 0.001) and remained extremely low in 2008–2009. Serotype replacement with strains not included in the PCV7 vaccine was virtually complete by 2006–2007. Additional PCV13 serotypes (1, 3, 5, 6A, 7F, 19A) remained a stable proportion of all pneumococcal isolates across sampling periods, ranging from 20% to 23%. Carriage with non-PCV13 serotypes increased significantly from 2001 to 2006–2007 (P < 0.001) and remained stable between 2006–2007 and 2008–2009 (P = 0.32).
Figure 2 shows trends in the proportions of the most commonly identified serotypes in 2008–2009. Altogether, these 16 serotypes accounted for 89% of isolates during the 2008–2009 sampling period. Serotype 19A (14%) was the most frequently identified pneumococcal type in 2008–2009, followed by 6C (11%), 15B/C (11%), 23A (7%), 23B (7%), and 35B (7%). Of these, only 19A is directly targeted by PCV13. Significant increases in prevalence over time were noted for serotypes 19A (P = 0.005), 7F (P = 0.009), 6C (P < 0.001), 23A (P = 0.02), and 23B (P = 0.002). In contrast, 6A (P < 0.001) and 19F (P < 0.001) declined across sampling periods.
PCV7 serotypes continued to circulate in 2006–2007, including 19F (2.4%), 23F (0.3%), 6B (0.7%), and 9V (0.3%), all of which were in children who were up-to-date on their PCV7 immunization series for their age. In 2008–2009, the following PCV7 isolates were identified: 19F (1.4%), 6B (0.3%), and 14 (0.3%). All 4 children colonized with 19F in 2008–2009 were up-to-date on PCV7 for their age, whereas the 2 children colonized with 14 and 6B were not.
The overall fraction of PNSP did not vary significantly over time using either PNSP>0.06 (33% in 2001, 38% in 2003–2004, 37% in 2006–2007, and 34% in 2008–2009, P = 0.73) or PNSP>2.0 breakpoints (11% in 2001, 3% in 2003–2004, 4% in 2006–2007, and 6% in 2008–2009, P = 0.56). By 2006–2007 or 2008–2009; however, PCV13 serotypes were significantly less likely to be susceptible to penicillin (PNSP>0.06), erythromycin, clindamycin, trimethoprim-sulfamethoxazole, and ceftriaxone when compared with non-PCV13 serotypes (Table, Supplemental Digital Content 3, https://links.lww.com/INF/B21). Although there was no discrimination in penicillin nonsusceptibility using current PNSP>2.0 breakpoints, evidence of incremental rises in MIC were evident using PNSP>0.06 breakpoints. Using PNSP>0.06 breakpoints, increases in penicillin nonsusceptibility were noted for 6C (P = 0.09) and 15 B/C (P < 0.001) across sampling periods (Table 1). However, the majority of penicillin nonsusceptibility was due to increased prevalence of 19A and 35B. Among 19A isolates, nonsusceptibility significantly increased over time for the following antibiotics: PNSP>2.00 (11%, 4%, 17%, 38%; P = 0.01), erythromycin (0%, 36%, 30%, 69%; P = 0.001), clindamycin (0%, 4%, 15%, 38%; P < 0.001), and ceftriaxone (13%, 8%, 20%, 52%; P = 0.003).
Predictors of Carriage
Potential risk factors associated with S. pneumoniae are described in Table 2. Younger age-group, group child care attendance, young siblings, and RTI were associated with pneumococcal carriage in multivariate models. Antibiotic exposure in the past 2 months was associated with decreased rates of pneumococcal carriage. We also evaluated predictors of carriage with penicillin-nonsusceptible pneumococci, using PNSP>2.0 breakpoints, among children swabbed in 2006–2007 and 2008–2009 (Table 2). Younger age and child care attendance were significantly associated with PNSP>2.0 carriage in multivariate models.
In serially sampling Massachusetts communities during the past decade, we have noted a steady decline and near elimination of carriage of PCV7 serotypes in healthy children. However, after an initial decline in overall carriage due to loss of PCV7 serotypes, carriage returned to pre-PCV7 levels (30%) as a result of replacement by nonvaccine serotypes. Serotype 19A carriage steadily increased to become a common colonizing pneumococcal serotype and the dominant source for IPD.21 As PCV7 vaccination rates reached a plateau, the pneumococcal population structure in Massachusetts reached a new equilibrium composed of non-PCV7 strains. Similar replacement with 19A has been reported after vaccination with PCV7 in the Netherlands, although it has not been uniform, and the factors that determine the precise serotypes involved in replacement in different populations remain poorly understood.35,36
In early 2010, PCV13 was approved for use in the United States to expand vaccine coverage to serotypes 1, 3, 5, 6A, 7F, and 19A. This new selective pressure will again alter the current equilibrium among pneumococcal carriage isolates. As PCV13 use increases during the next several years, we anticipate that overall rates of colonization may transiently drop, but eventual non-PCV13 serotype replacement may occur. The greatest impact of PCV13 in the United States is expected to be reductions in carriage and disease attributable to 19A since other PCV13 serotypes are infrequent sources of carriage and disease.11,24,25,37
The common replacement serotypes found here (19A, 6C, 15B/C, 23A, 23B, 35B) have arisen by expansion of existing clones, emergence of new clones, and serotype switching, whereby the capsular genes that determine serotype are replaced by those of another serotype.28 As serotype 6A declined significantly, 6C became increasingly prevalent and is now the second most common serotype in our sample. The decline in 6A has been attributed to cross-protection from PCV7, which includes 6B as a target.32,38 In similar manner, PCV13 may provide cross-protection against 6C due to the inclusion of the closely related serotype 6A.38 If this occurs, additional reductions in pneumococcal colonization and IPD may occur. Nonetheless, we anticipate continued replacement of nasopharyngeal carriage by non-PCV13 serotypes as PCV13 use expands. Increases in IPD may or may not occur as a consequence, and will depend in part on the relative invasiveness of the most prevalent replacement serotypes.39–41
Widespread use of PCV7 led to an initial decline in antibiotic resistance, as PCV7 serotypes previously constituted the majority of antibiotic resistant strains.5,42,43 However, the emergence of non-PCV7 antibiotic resistant strains, particularly serotypes19A, 35B, and 15B/C, have mitigated the reduction of antibiotic resistance by PCV7. Serotype 19A now represents the most commonly carried nonsusceptible serotype for penicillin, erythromycin, and ceftriaxone resistance. Because 19A is now responsible for the largest fraction of PNSP>0.06 and PNSP>2.0 carriage, we anticipate that PCV13 use may result in a decline in overall antibiotic resistance rates. However, the long-term impact of PCV13 use on antibiotic resistance remains uncertain.23,44
In the post-PCV7 era and before PCV13 use, the major predictors of pneumococcal carriage have remained similar, including young age, group child care attendance, having young siblings, concomitant RTI, and lack of recent antibiotic exposure.21 It appears from this and prior sampling periods that vaccination has not altered common risk factors for pneumococcal carriage.13,21
Almost all carriage strains in our sample are classified as penicillin susceptible by the revised 2008 breakpoints, which require a MIC of at least 2.0 μg/mL for nonsusceptibility. However, young age and group child care were significantly associated with carriage of strains with higher penicillin MICs within the revised susceptible range. Under antibiotic pressure, such strains may increase following PCV13, similar to what occurred following PCV7.21,22,44
The results of this study should be interpreted in the context of several limitations. First, we did not examine the possibility that children carried multiple strains simultaneously, which might have altered the reported distribution of serotype and antibiotic resistance. Second, as we sampled during “cold and flu” seasons, we do not know whether S. pneumoniae carriage patterns are similar throughout the year, or whether we adjusted completely for differences in respiratory illness rates between sampling periods. Third, we assume that carriage among children seeking care in physician offices reflects carriage in their community, and our method of sampling may bias our data toward serotypes that are cultured more easily during respiratory illnesses. Nevertheless, the frequency of participants having well-child visits and our adjustment for the presence of respiratory illness should have mitigated these effects. Furthermore, our ability to serially sample the same Massachusetts communities allows us to provide unique information regarding the pneumococcal population structure in colonized children.
In conclusion, PCV7 has resulted in near complete replacement with nonvaccine serotypes since 2007. Serotype 19A remains the predominant serotype since 2007 and is responsible for the majority of antibiotic resistance in our isolates, although other common replacement serotypes, 6C, 15B/C, and 35B, have begun to show evidence of increasing MICs to penicillin. We anticipate PCV13 use will reduce carriage with 19A and may decrease the prevalence of antibiotic-resistant pneumococci in the community, at least for some time. Serotype replacement and other adaptive changes will need to be monitored in the presence of this newest selective pressure on S. pneumoniae.
The authors thank Katie Haffenreffer, Kristine Robin, and Verna Moran, RN, for their dedication to this project, and extend our appreciation to the following participating practices: Alan Bulotsky MD & Associates (Brockton, MA), Berkshire Pediatric Associates (Pittsfield, MA), Cape Ann Pediatricians (Gloucester, MA), Children's Health Care (Newburyport, MA), Harvard Vanguard Medical Associates (Chelmsford, MA), Medical Associates Pediatrics (Leominster, MA), Middleboro Pediatrics (Lakeville, MA), and Needham Pediatrics (Needham, MA). They also thank the many parents and children who made this study possible.
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