In this study of pneumococcal carriage in children attending DCCs in Norway, a high overall carriage rate of 78.4% was found, with a peak prevalence of 88.7% in children 2 to 3 years old. These frequencies are higher than previously described in Norway, as well as in similar studies performed in other Scandinavian countries or elsewhere in Western Europe (5
). This is probably due to the highly sensitive sampling method used in this study, i.e., giving the bacteria optimal conditions in an enrichment broth directly after sampling, allowing a short interval before plating, and performing serotyping directly from the incubated enrichment broth to identify multiple serotypes. The use of serum broth as a sensitive method for recovery of pneumococci has been described by Kaltoft (26
). Detection of capsular antigen after enrichment has been demonstrated as a sensitive way to identify pneumococci in carriage studies, yielding more than culture on agar plates alone and with no further gain from adding gentamicin in the enrichment broth (27
). In the present study, the presence of pneumococci was indicated by agglutination in 33 samples that were initially culture negative on blood agar. In 30 of these samples, growth was obtained after replating of the enrichment broth, increasing the yield by 7.2%, from 419 to 449 positive samples. The lack of recovery of strains from three antigen-positive samples might be due to a small amount of bacteria in the sample, autolysis of the pneumococci or overgrowth by other species. Hence, transport in an enrichment broth, followed by incubation and serotyping directly from the broth, is a sensitive method for isolation of pneumococci. However, the method is suitable only when plating can be performed within a short time interval after sample collection.
Longitudinal studies have demonstrated that children are successively colonized with multiple strains of pneumococci (18
). However, identification of simultaneous carriage of multiple serotypes is laborious, and the yield varies according to the method used (24
). Serotyping of multiple colonies is time-consuming, and if the second serotype constitutes 4 to 27% of the population, 11 to 59 randomly picked CFU would have to be subcultured and serotyped for identification of the two serotypes (24
). An immunoblot method designed to detect multiple serotypes in carriage studies was used in a study in Navajo and White Mountain Apache reservations. Multiple serotypes were detected in 8.1% of positive samples (6
). In a study of pneumococcal carriage in the Gambia, serotyping was performed by latex agglutination, and multiple serotypes were identified in 11.5% of positive samples (30
). We performed a serotype screening by latex agglutination on incubated enrichment broths, followed by subcultivation of up to 16 colonies from blood agar to isolate multiple strains. By this method, more than one serotype was recovered in 57 (12.7%) of 449 positive specimens. Additional serotypes were indicated in 19 positive specimens by broth agglutination, but these organisms were not identified on agar plates, possibly due to small fractions of the pneumococcal populations being constituted by these strains. It is possible that additional pneumococcal strains in a sample might be lost as a result of negative agglutination. In fact, two isolates were identified as additional strains as a result of differing inhibition zones on susceptibility testing. No agglutination-negative samples showed growth on blood agar, indicating that agglutination is highly sensitive. Direct agglutination from an enrichment broth thus has the advantage of being an easy and inexpensive method for identification of more than one pneumococcal strain per sample. By this method, random colony selection can be limited to samples where carriage of multiple serotypes is indicated by agglutination.
Crowding of children in DCCs gives ample opportunities for transmission of bacteria, and DCC attendance is considered a strong risk factor for carriage of pneumococci (37
). This high-risk setting might in part explain the high prevalence of carriage found in this study. Nasopharyngeal sampling was performed in the autumn, shortly after the summer holidays and at the beginning of a new season in the DCCs with new children attending. This might be a vulnerable time point for the acquisition of pneumococci because of a disturbance of the DCC epidemiological unit and good opportunities for exchange of bacteria between the children not yet immunologically adapted to this new environment. The prevalence of carriage was significantly associated with low age, as was colonization with PCV-7 serotypes, an association that has been documented by others (22
). Although RTIs and young siblings have been identified as risk factors and use of antibiotics and breast feeding have been negatively associated with carriage (14
), no statistically significant associations with these risk factors were found in this study. This is not surprising, as the population studied is believed to be very homogenous and considering the high prevalence of carriage, making the identification of potential risk factors for carriage problematic.
The distribution of serotypes is consistent with results of other studies of nasopharyngeal carriage, with serotypes 6B, 6A, 19F, and 23F being the most prevalent (4
). The proportion of PCV-7 serotypes among isolated pneumococci was 45%. This proportion is consistent with results from The Netherlands (42%) and from a previous study from Norway (42%), although a higher PCV-7 coverage (64%) has been reported in the United Kingdom (4
). The potential coverage of an 11-valent vaccine (including serotypes 1, 3, 5, and 7F) would be only slightly higher (47.3%).
In our study, 8 isolates (1.6%) belonged to serotype 1. This serotype is associated with epidemic outbreaks of pneumococcal disease, though it is rarely recovered from asymptomatic carriers (8
). The prevalence of serotype 1 pneumococci increased in Scandinavian countries during the 1990s and accounted for 11.1% of cases of systemic pneumococcal disease in children aged 0 to 5 years in Norway in 1995 to 2001, making it the third most prevalent serotype causing invasive disease in children (36
). This increase was primarily due to the emergence of one antibiotic-susceptible clone, ST306 (21
), a clone that seems to be genetically stable and that was found to be the most prevalent serotype 1 clone in Europe in the past decade (8
). All serotype 1 pneumococci isolated in the present study belonged to this ST. Serotype 1 pneumococci are rare in the United States, and this serotype is not included in PCV-7. Thus, the selective pressure caused by mass vaccination in Norway might have implications for the future occurrence of this serotype.
To our knowledge, this is the largest study of carriage in which MLST has been performed on all isolates. The genotyping of 509 isolates showed that the pneumococcal population was highly heterogeneous; 102 STs were identified, and the most frequent contributed to fewer than 8% of the isolates. Of the 15 clones most frequently recovered in this study, all have previously been described in other countries, including 2 of the 26 worldwide-spread resistant pneumococcal clones currently accepted by PMEN, England14
-9 (ST9) and Portugal19F
-21 (ST177). Nearly one-third of the STs were novel, the majority of which were recovered from only one child. The proportion of novel STs, and the variation of STs, was highest among the most frequently recovered serotypes, i.e., 6A, 6B, 19F, and 23F. These serotypes are carried for longer time periods than other serotypes (18
), and they might have a greater opportunity for horizontal transfer of genetic material, giving rise to new STs. However, this could also be influenced by a sampling bias, with the highest clonal diversity being found among the most frequently recovered serotypes. The novel STs might represent a Norwegian pneumococcal population. However, as the majority of these novel clones were recovered from only a few children each, they might be clones with a limited success in transmission.
The antimicrobial susceptibility among pneumococci recovered in Norway is favorable; the isolates are generally sensitive to the drugs tested. Penicillin nonsusceptibility was found in a small fraction of isolates, i.e., nine isolates (1.8%) belonging to eight clones, of which four displayed PCV-7 serotypes. The isolates that were nonsusceptible to macrolides and tetracycline were more homogenous and were assigned to a few CGs displaying PCV-7 serotypes. Thus, a reduction, or limited spread, of these clones is expected after the introduction of the conjugate vaccine in the Norwegian vaccination program.
The rapid increase in macrolide resistance observed in Norway among IPD isolates from 2001 and onwards was attributable mainly to the England14
-9 clone, displaying low-level resistance to erythromycin (44
). However, from 2004, the fraction of pneumococci with coresistance to erythromycin and clindamycin, indicating a macrolide-lincosamine-streptogramin B-type resistance, increased, and in 2006 this phenotype constituted 15% of erythromycin-nonsusceptible invasive isolates in Norway (33
). Among the pneumococci recovered in this study, 10 (33%) of the erythromycin-nonsusceptible isolates were coresistant to clindamycin. Of these, eight isolates (80%) belonged to the Portugal19F
-21 CG. In addition, this phenotype was found in one of the eight serotype 6A, ST490 isolates recovered in this study; a clone unrelated to the PMEN clones and not previously associated with this resistance pattern. The macrolide-lincosamine-streptogramin B phenotype was also found in a novel ST (ST2624), represented by the most extensively nonsusceptible isolate in this material, which was intermediately susceptible to penicillin and resistant to erythromycin, clindamycin, tetracycline, and trimethoprim-sulfamethoxazole. CG177, consisting of Portugal19F
-21 and its SLVs, constituted 19 (18.1%) of the nonsusceptible isolates in this study. These isolates display serotype 19F, a serotype included in PCV-7. However, this serotype has been associated with vaccine failures (2
), and in a community-randomized trial, carriage of this serotype has actually been found to be higher among children who had received four doses of PCV-7 than in controls (34
). The effects of a three-dose vaccination regimen on the carriage and spread of these clones need to be followed closely.
The clone most frequently isolated from the children was ST199, displaying serotypes 15B, 15C, and 19A. Increasing prevalences of IPD caused by the nonvaccine serotypes 19A, 15B, and 15C in the United States have been described in the years following licensure of PCV-7 (17
). This replacement of PCV-7 serotypes in IPD has occurred 3 to 4 years after implementation of widespread vaccination and has been found to be mainly due to an expansion of CG199, i.e., ST199 and newly described SLVs (35
). Although the invasiveness of this clone is believed to be moderate (7
), its high prevalence among asymptomatic carriers in Norway, already before the introduction of PCV-7, raises concern that a similar replacement in IPD might occur.
Multiple pneumococcal strains simultaneously carried by the individual children were genetically unrelated to each other, as shown by MLST. However, serotype 3 pneumococci were isolated at a significantly higher frequency from carriers of multiple serotypes than from the total population, with ST180 being the most frequently identified ST from multiple carriage. Serotype 3 has been found to be positively associated with acute otitis media and acute conjuncitivitis (40
), but with a low potential for causing invasive disease (7
). It is possible that the capsule of serotype 3 is favorable for colonization of mucosal surfaces, but the reason for its overrepresentation in cocolonization with other pneumococci is unclear. The mucoid appearance of serotype 3 colonies might make them more discernible in a mixed population and might bias this observation. However, it is possible that this particular serotype has a strong genetic integrity and ability to survive in the competition with other pneumococci for colonization of the nasopharyngeal niche.
Pneumococci recovered within a single DCC were genotypically less diverse than the total pneumococcal population, and one or two dominating clones were identified in most DCCs. The composition of pneumococcal populations within each DCC consisted of the dominating clones, either predominantly found in that DCC or recovered from several DCCs, and of clones, often novel STs, unique for the DCC. In this way the clonal distribution of pneumococci was unique for each DCC, a pattern described as an autonomous epidemiological unit by Sa-Leao et al. (38
). The observed distribution of clones within DCCs, as contrasted to the total population, is consistent with the suggested model of the emergence of a neutral bacterial population structure from overlapping microepidemics within clustered host populations (15
). However, longitudinal studies of DCC populations are needed to evaluate the stability of the DCC epidemiological unit, as a cross-sectional study will catch only a glimpse of a probably highly dynamic interplay between children and the pneumococci.
The high prevalence of pneumococcal carriage among children in DCCs described in this study underlines the importance of this population as a reservoir for spread of the bacterium to the whole community. In addition, the high level of DCC attendance among Norwegian children might augment the importance of these epidemiological settings in the spread of pneumococci. Consequently, the reduced colonization, and the resulting herd immunity, is an advantage of conjugate vaccination. To evaluate the effect of a three-dose PCV-7 immunization regimen on carriage and spread of susceptible and nonsusceptible clones of S. pneumoniae, a follow-up study is planned for 2008.