The main reservoir of Streptococcus pneumoniae is the nasopharynx of young children.1,2 When the balance between host and pathogen is disturbed, the pneumococcus (Pn) can spread to adjacent mucosal tissues to cause infections such as acute otitis media (AOM) and pneumonia or enter the bloodstream causing invasive infections such as sepsis and meningitis.3 Young children attending day-care centers (DCCs) commonly carry pneumococci, especially drug-resistant strains that can easily be transmitted by person-to-person contacts in the environment of DCCs where antibiotic use is frequent and leads to selection for resistant strains.4–6
β-Lactam antibiotics have been the most commonly used antimicrobials for treatment of pneumococcal disease. Since the mid-1960s, the prevalence of penicillin-resistant strains increased worldwide complicating disease management7 and providing an incentive to address prevention of pneumococcal disease through immunization.
Pneumococci comprise more than 90 serotypes defined by the capsular polysaccharide.8 The pneumococcal conjugate vaccine, Prevenar, is a 7-valent formulation that includes polysaccharides of types 4, 6B, 9V, 14, 18C, 19F and 23F linked to the nontoxic diphtheria variant protein carrier CRM197 (7-valent pneumococcal conjugate vaccine). The vaccine was introduced in the United States in 20009 and became available in Portugal in July 2001. This conjugated protein carrier, CRM197, elicits a T cell-dependent response, making it immunogenic and efficacious in children younger than 2 years.10,11
In clinical trials, the conjugate vaccine induced high concentrations of serum anticapsular antibodies12 and reduced nasopharyngeal carriage of serotypes included in the vaccine [vaccine types (VT)].13–18
Conjugate vaccines are efficacious against invasive disease and modestly beneficial against acute otitis media.10,11,19 Antibacterial-resistant strains of pneumococci most often are serotypes included in 7-valent pneumococcal conjugate vaccine.20,21 Vaccination has a direct effect on reducing carriage of drug-resistant pneumococci (DRPn) and an indirect effect on preventing dissemination of resistant strains in the community.22–24 Children protected through vaccination against VT strains are more likely to carry nonvaccine type (NVT) strains.25 The capacity of these NVT pneumococci to acquire antimicrobial resistance traits and cause disease is not known.
The purposes of our study conducted between 2001 and 2003 were to evaluate the impact of the 7-valent conjugate vaccine on the nasopharyngeal carriage of drug-susceptible and drug-resistant pneumococci and to evaluate the serotypes, drug resistance pattern and genetic backgrounds of drug-resistant pneumococci colonizing healthy children attending DCCs in the Lisbon area of Portugal.
MATERIALS AND METHODS
The target population consisted of 695 healthy children 6 months–6 years old attending 8 DCCs in the Lisbon area of Portugal. The DCCs were selected to reflect the demography of the community. DCCs were divided into 2 groups: an intervention group (vaccinees) with 238 children in 5 DCCs; and a control group with 457 children in 3 DCCs. Written and signed informed consent for vaccination and sampling procedures was obtained from parents or guardians of all participating children. The absolute number of samples in the intervention group from the first to the sixth sampling period (2003) decreased to about one-half, because every year older children who had been vaccinated in the first period (2001) left the DCC and consequently could not be followed.
This prospective study was conducted between 2001 and 2003 (3-year surveillance period). The vaccination was performed in the intervention group (vaccinees) in May, June and November 2001. The nasopharyngeal (NP) flora of the vaccinees was analyzed during 6 sampling periods: in May, June and November 2001, immediately before the vaccine dose administration; and afterward in February and November 2002 and February 2003. Children of the control group were sampled for pneumococcal carriage during the same sampling periods except in the second period (June 2001) when they were not sampled.
Antibiotic consumption and numbers of episodes of respiratory tract infections (RTI) were evaluated through questionnaires filled out by parents or guardians before each sampling period.
All Pn isolates were tested for antimicrobial resistance and all further characterizations were restricted to drug-resistant S. pneumoniae (DRPn) strains. All data including antimicrobial consumption, frequency of RTI as well as serotype and genetic data of S. pneumoniae isolates were introduced into a database of special design26 for further analysis.
The 7-valent pneumococcal conjugate vaccine, Prevenar, was provided by Wyeth Lederle Portugal (Farma), Lda. According to the specifications of the manufacturer, the vaccine contains 2 μg of each of the polysaccharides of serotypes 4, 9V, 14, 18C, 19F and 23F and 4 μg of 6B coupled to the nontoxic diphtheria-toxin analogue CRM197 and absorbed on aluminum phosphate. Infants 6–11 months of age received 3 vaccine doses, children 12–24 months of age received 2 doses and children older than 2 years received a single dose as recommended by the vaccine manufacturer. The 238 children enrolled in the intervention group received a total number of 305 doses of vaccine during the 3 vaccination periods. Intramuscular injection of 0.5 mL of vaccine was performed by a pediatric nurse in the deltoid muscle of the upper arm of each child.
Specimen Collection and Identification.
Sampling was performed by trained pediatric nurses. A single nasopharyngeal flexible swab (BBL Culture Swab; Becton-Dickinson, Sparks, MD) was inserted through each child's nostril until it touched for a few seconds the posterior wall of the nasopharynx. The swab was removed, then introduced into the transport medium (Stuart medium) and transported at ambient temperature to the Laboratory of Molecular Genetics at Instituto de Tecnologia Química e Biológica. Bacterial samples were processed within 4 hours after arrival at the laboratory as described previously.27 Single colonies streaked on blood agar plates and identified as S. pneumoniae were grown in tryptic soy agar (Difco, Detroit, MI), and isolates were frozen and stored at −70°C.27
Antimicrobial Susceptibility Testing.
All Pn strains were tested by the Kirby-Bauer technique, according to National Committee for Clinical Laboratory Standards recommendations,28 against oxacillin, chloramphenicol, erythromycin, clindamycin, tetracycline, trimethoprim-sulfamethoxazole and levofloxacin (Becton-Dickinson). Minimum inhibitory concentrations (MICs) of penicillin and ceftriaxone were determined by E-test (AB Biodisk, Solna, Sweden) according to the manufacturer's recommendations.
Pneumococcal strains resistant to at least one of the antimicrobial agents tested were designated as DRPn. DRPn (except strains resistant only to oxacillin and/or trimethoprim-sulfamethoxazole) were further characterized for serotype, and genetic background [pulsed field gel electrophoresis (PFGE) pattern].
DRPn were serotyped by the quellung reaction with the use of commercially available antisera (Statens Seruminstitut, Copenhagen, Denmark).29
Strains that gave negative or positive reaction for agglutination with all the pooled sera were named nontypable (NT). Pn strains with serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, included in the vaccine, were defined as VT strains. Strains with other serotypes, were defined as nonvaccine type (NVT) strains.
PFGE was performed after digestion of genomic DNA with SmaI using Pn strain R6 and PFGE λ marker (New England Biolabs) as molecular weight standards according to an earlier protocol.30
Classification of PFGE patterns was performed by visual comparison according to accepted criteria.31 We compared our patterns with the ones identified in studies from our group conducted between 1996 and 1999 (S. Nunes et al, unpublished data)30,32 and also with the patterns of 26 international clones included in the Pneumococcal Molecular Epidemiology Network (PMEN).33
The effect of vaccination was evaluated on vaccinated and nonvaccinated cohorts. Contingency table analysis was conducted with the χ2 test, both for individual sampling periods and for the pairwise comparison of all periods. The χ2 test was performed for association between vaccination and carriage of Pn or occurrence of afflictions episodes. A maximum error type I of 0.05 was considered for recognition of a significant vaccine effect.
Demographic and Clinical Data.
We enrolled 695 children between May 2001 and February 2003. There were no significant differences concerning demographic and clinical characteristics between the intervention group and control group.
The intervention group (n = 238) included 118 male and 120 female subjects. The control group (n = 457) included 237 boys and 220 girls.
Antibiotic consumption at sampling or 1 month before sampling was high in both groups during all sampling periods, as reported by the parents through the questionnaires. Twenty-eight children (4%) in the intervention group and 32 children (5%) in the control group have taken antibiotics at the time of sampling. One hundred nine children (17%) in the intervention group and 74 children (11%) in the control group took antibiotics 1 month before sampling.
Otitis media, throat infection and low respiratory infections were indicated as the afflictions that most frequently caused antimicrobial consumption in both groups. Forty-seven children (34%) in the intervention group and 40 children (29%) in the control group took antibiotics because of otitis media. Forty-seven children (34%) in the intervention group and 58 children (42%) in the control group took antibiotics because of throat infection. Concerning lower respiratory infection, only 9% (13 children) and 6% (9 children) of children included, respectively, in the intervention and control groups took antibiotic because of this affliction.
Carriage of S. pneumoniae Strains.
Of the 1950 NP samples obtained, 1014 were from children in the intervention group (vaccinees) and 936 from the control group. Total carriage rate of Pn strains was similar in intervention and control groups during all sampling periods. As many as 68% of the NP samples carried S. pneumoniae in both the control (633 Pn strains isolated from 936 NP samples) and the intervention (692 Pn strains isolated from 1,014 NP samples) groups. The greatest difference in Pn carriage between the 2 groups was 9% (Table 1). The carriage rate of Pn fluctuated in a very similar manner among controls and vaccinees during the sampling periods.
The carriage rate of DRPn was similar in intervention and control groups throughout the sampling periods. Among the total of 692 Pn strains collected from the intervention group, 37% were DRPn; among the 633 Pn strains recovered from the control group, 39% were DRPn (Table 1).
Carriage rates of penicillin-nonsusceptible strains were also similar and constant during the sampling periods and no significant differences were found between intervention and control groups. Of 692 Pn strains isolated in the intervention group, 24% were nonsusceptible to penicillin; of 633 Pn strains isolated in the control group, 23% were penicillin-nonsusceptible (Table 1). Within the intervention group, a decline was observed in penicillin nonsusceptibility from >24% of isolates in 2001 to 17–19% in 2002–2003, which was not observed in the control group, although that difference was not statistically significant (P > 0.05). All pneumococcal strains were susceptible to levofloxacin and the overall prevalence of strains resistant to the other antimicrobials tested was similar between control and intervention samples. The differences in susceptibility to each one of the antimicrobial tested between intervention and control were not statistically significant (P > 0.05) except for chloramphenicol (Table 2).
Serotypes of DRPn.
Although the total number of DRPn strains was similar between the control and intervention samples, differences became apparent once the strains were separated into bacteria expressing VT and NVT capsular polysaccharides (Fig. 1). In the intervention group (vaccinees), the frequency of VT strains decreased significantly, from 81% to 5%, throughout the 6 sampling periods (P < 0.05). At the same time, the frequency of NVT strains increased significantly from 19% to 95% (P < 0.05). In the control group, the carriage rate of VT strains remained high (between ∼59 and 75%) and the carriage rate of NVT strains remained between 25 and 46% and showed no trends of consistent increase or decline during the surveillance period (Table 3 and Fig. 1).
Before vaccine administration (first sampling period), the carriage rate of VT DRPn strains was 81% in the intervention group and 59% in the control group. During the remaining sampling periods, carriage rates of DRPn with vaccine serotypes continued to decrease in the intervention group reaching 5%; whereas the frequency of these strains remained above 53% in the controls. The prevalence of NVT strains in the first sampling period was 19% in the intervention group and 41% in the control group. During the remaining sampling periods, carriage rates of NVT strains increased in the intervention group, reaching 95% in the sixth sample, whereas the frequency of these serotypes remained on average ∼33% of the DRPn strains in the control samples (Table 3). Of all the DRPn strains with VT serotypes that were present in the first sample from the intervention group, a single serotype 9V remained by the final sampling period. DRPn strains expressed a wider range of NVT serotypes that included serotypes 6A, 10A, 15A, 15C, 19A, 23A and 33F. Serotype 15A was recovered in all sampling periods (Fig. 1). In the control samples, the NVT serotypes were 6A, 9A, 15A, 17, 18, 19A and 33F with the most frequent one being 19A (Fig. 1). NT strains were detectable in the intervention (25 strains) and control (28 strains) groups.
Penicillin-Nonsusceptible S. pneumoniae Strains Expressing Vaccine and Nonvaccine Serotypes.
Among DRPn strains, penicillin-nonsusceptible strains with VTs decreased from 59% to 5% and NVT strains increased from 15% to 67% in the intervention group. In the control group, penicillin-nonsusceptible strains most frequently expressed VT serotypes (Fig. 2). High level resistance to penicillin (MIC ≥ 1.5 μg/ml) was only observed in few VT isolates. Penicillin MIC50 was 0.094 μg/ml in VT isolates, either in the intervention or control, and among the NVT isolates it was 0.125 μg/ml in the intervention and 0.032 μg/ml in the control. Penicillin MIC90 was 1 μg/ml in VT isolates of the intervention and 0.75 μg/ml in the control; the MIC90 was 0.5 μg/ml among the NVT isolates of intervention and control groups.
PFGE Patterns of DRPn Strains.
Most of the vaccine-type DRPn, either in the intervention (vaccinees) or control groups, belonged to 9 internationally spread epidemic clones identified and deposited in the collection of the PMEN (Figs. 3 and 4). These clonal types were as follows: Spain23F-1, Spain6B-2, Spain9V-3, England14-9, Poland6B-20, Portugal19F-21, Greece6B-22, Sweden15A-25 and Colombia23F-26.
In the intervention group, during the first sampling period, 81% of the 79 DRPn isolates expressed VT capsules. The majority of vaccine type strains belonged to one or the other of 9 international epidemic clones (PMEN clones), and only 3 VT strains showed PFGE types different from the PMEN clones. The rest of the 15 DRPn from this sampling period expressed NVT capsules: 5 strains belonged to the serotype 15A clone of Sweden15A-25; and 6 of the other 10 strains showed 4 non-PMEN clonal types.
In this period, epidemic PMEN clonal types were dominant over the non-PMEN clonal types. Of 79 DRPn strains isolated, 89% (70 strains) belonged to 8 epidemic PMEN clonal types and 11% only (9 strains) belonged to 7 non-PMEN clonal types. By the last sampling period, the number of VT strains in the intervention group was reduced to a single serotype 9V strain with the PFGE pattern of the international clone Spain9V-3. All the rest of the 20 DRPn expressed NVT capsules and were represented by 7 PFGE patterns of which only 1 is a PMEN clone. In this last period, the non-PMEN clones became dominant over the epidemic PMEN clones. Of 21 DRPn strains isolated, 2 strains (10%) belonged to PMEN clonal types (2 clones) and 19 strains (90%) belonged to 7 non-PMEN clonal types.
In the control group during the first sampling period, when the majority (41 of 70) of DRPn strains expressed VT capsules, all of these belonged to 7 internationally spread clones identified in the PMEN. The representation of these internationally spread epidemic clones remained high among the DRPn strains with VT capsular types collected throughout the sampling periods.
The epidemic PMEN clonal types were dominant over the non-PMEN clonal types in the first period. Of 70 DRPn strains isolated, 84% (59 strains) belonged to 8 epidemic PMEN clonal types and 16% (11 strains) belonged to 8 non-PMEN clonal types. By the last sampling period, PMEN clonal types and non-PMEN clones were detected in similar ratios, concerning both the number of clonal types and the number of strains. Of 45 DRPn strains isolated, 49% (22 strains) belonged to PMEN clonal types (8 clones) and 51% (23 strains) belonged to 12 non-PMEN clonal types.
In summary, of 231 DRPn strains from the intervention group, 71% (165 strains) had PFGE patterns identical with those of 9 international clones included in the PMEN.33 The remaining 66 strains (29%) presented 19 different PFGE patterns (non-PMEN clones). In the control group, 143 of 222 DRPn strains (64%) had PFGE patterns identical with those of 8 international clones included in the PMEN.33 The remaining 79 strains (36%) presented 29 different PFGE patterns (non-PMEN clones).
Serotyping and molecular typing by PFGE identified a few cases that might represent capsular transformation. Among the isolates recovered from the control group at the first sampling period, 6 strains showed the PFGE pattern of the international clone Spain23F-1 that typically expresses serotype 23F capsule. These particular 6 strains expressed serotype 19A. Another group of 10 strains showed the PFGE pattern of the international clone Poland6B-20 which typically expresses serotype 6B. The 10 particular strains expressed serotype 6A. Additional examples for putative capsular switch were also detected among some strains with familiar PFGE type but expressing no identifiable capsular types (nontypable strains). There was no evidence for an increased frequency of strains with such suspected capsular changes among the DRPn isolated from the nasopharyngeal samples of vaccinees. Therefore capsular switch did not appear to be a major mechanism contributing to the change from the vaccine to the nonvaccine serotype of the DRPn isolates.
We evaluated the impact of the 7-valent pneumococcal vaccine on the carriage of drug-resistant S. pneumoniae strains in the nasopharynx of children attending DCCs in Portugal. Because most drug-resistant pneumococci express a limited number of serotypes that are present in the 7-valent vaccine, the carriage rates of drug-resistant pneumococci was expected to decrease in the vaccinees as part of the reduction in all pneumococcal VTs that colonized the nasopharynx of the children.
In agreement with several studies on the impact of pneumococcal conjugate vaccines, our results also showed that vaccination with 7-valent pneumococcal vaccine did not affect the global carriage of Pn strains (susceptible and resistant). Also in agreement with previous studies,13,14,16–18 the carriage of vaccine type DRPn strains decreased markedly from 81% to 5% in the vaccinated children. In contrast to findings reported in some earlier studies,13,14,16–18,34,35 the total carriage rate of DRPn was not reduced. In parallel with the decline in the frequency of DRPn expressing the vaccine serotypes, there was an increase in DRPn with nonvaccine serotypes. As a result, the carriage rate of DRPn strains has remained virtually unchanged during the 3 years of follow-up sampling of the NP flora of vaccinees. Furthermore the DRPn with the nonvaccine serotypes were represented in almost all cases by bacteria exhibiting genetic backgrounds different from the PMEN clones, as identified by their unusual PFGE patterns.
We explain the replacement of vaccine type DRPn by drug-resistant pneumococci with different genetic backgrounds and expressing NVT serotypes as the combined effect of vaccine pressure and undiminished use of antibacterial agents. Vaccination against the VT serotypes must exert selective pressure on the colonizing flora of pneumococci preventing the cross-infection and multiplication of the vaccine type strains, which provides a competitive advantage to pneumococci expressing other capsular types not present in the vaccine. The emergence of drug-resistant strains among these NVT type bacteria could be because of the acquisition of drug-resistant determinants from the DRPn strains with the VT capsules that are receding under the vaccine pressure. Alternatively drug-resistant strains with the NVT capsules may preexist as minor components of the nasopharyngeal flora because of multiple carriage of pneumococci.36 This latter alternative appears likely given that DRPn with serotypes 6A, 15A, 15C and 33F were identified in the NP flora at the onset of the study.
Vaccine efficacy is closely linked to a decrease in vaccine type strains that normally implies a decrease in resistance and virulence.20,21 Drug resistance, especially to penicillin, is found almost exclusively among isolates of the vaccine serotypes.21,34 In our study, we found that penicillin-nonsusceptible strains with nonvaccine serotypes became frequent in vaccinees with time (Fig. 2). Our findings are consistent with the predictions of mathematical modeling studies suggesting that the probability for the occurrence of long term replacement of DRPn would increase after vaccination.37
The pathogenic potential of nonvaccine serotypes is not known, although the capacity of these strains for effective colonization might imply pathogenic potential.38 This has been shown by the Finnish Otitis Media Study Group,11 who found a 33% increase in AOM episodes caused by NVT in a group of vaccinated children. In our study, nonvaccine serotypes isolated from vaccinees became prominent in the nasopharynx (95%) with some serotypes (eg, 15A, 33F) repeatedly recovered from children along the sampling periods (Fig. 1). High frequency and prolonged duration of carriage has been associated with frequent pneumococcal disease.39 The serogroups and serotypes identified in our study, such as 6A, 10A, 15A/C, 19A, 23A and 33F, have been associated with AOM episodes in Finland, the United States and Israel11,40,41 and with invasive disease episodes in France (6A, 10A, 15C, 19A),42 United Kingdom (6A, 15C, 19A, 33F)43 and Portugal (6A, 10A, 19A, 23A, 33F).44
The nasopharyngeal flora of DRPn of vaccinated children in our setting appeared to undergo a progressive compositional change over time because of replacement of the initially dominant epidemic DRPn clones by other clonal types of pneumococci expressing new serotypes that are not present in the vaccine.
The origin and nature of resistance determinants and pathogenic potential of the DRPn clones replacing the vaccine type pneumococci in the nasopharyngeal flora are unknown. However, the results of our study seem to imply that reduction of the carriage of drug-resistant strains of S. pneumoniae requires a combination of the conjugate vaccine and reduction in the use of antimicrobial agents.
We thank the directors and staff of the participating day-care centers as well as all the parents and children who collaborated in this study. We also thank the 2 experienced state registered pediatric nurses, Anabela Gonçalves and Paula Gonzaga, for their excellent vaccination and sampling procedures and also all the members of the Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa who participated in this work. We thank Sara Silva, Rodrigo Oliveira and António Maretzek for building the database and the web interface; Francisco Pinto and Susana Vinga for discussion on statistical analysis; and Alexander Tomasz for helping with the study design, interpretation of the results and extensive revision of the manuscript.
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