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1 Department of Pediatrics, Erasmus MC-Sophia, Room Ee 1500, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands
2 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, The Netherlands
3 Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK
4 Scottish Meningococcus and Pneumococcus Reference Laboratory, Stobhill Hospital, Glasgow, UK
5 Department of Pediatrics, Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands
Correspondence
D. Bogaert
d.bogaert{at}erasmusmc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In addition, the increasing (multi)drug resistance among pneumococcal isolates hampers adequate treatment (Centers for Disease Control and Prevention, 1997; Crook & Spratt, 1998; Klugman, 1996; Tomasz, 1997).
Nasopharyngeal colonization with pneumococci is common; in general, humans are colonized at least once early in life. Although colonization with pneumococci is mostly asymptomatic, it can progress to respiratory or even systemic disease as a result of a (temporary) defect in the mucosal barrier function, e.g. as a result of a viral infection. Importantly, pneumococcal disease has to be preceded by nasopharyngeal colonization with the homologous strain (Faden et al., 1990; Gray et al., 1980). Moreover, pneumococcal colonization causes horizontal spread of this pathogen within the community; for example, crowding, which occurs in hospitals, day-care centres and jails, enhances horizontal spread of pneumococcal strains (Hoge et al., 1994; Kristinsson, 1995; Mandigers et al., 1994; Muñoz et al., 1991; Principi et al., 1999). Because the highest incidence of pneumococcal colonization, and the highest crowding index, are found in young children, this risk group is considered to be the most important vector for horizontal dissemination of pneumococcal strains within the community (Leiberman et al., 1999). New pneumococcal conjugate vaccines are highly effective against invasive disease in young children (Black et al., 2002). Furthermore, a protective effect against mucosal infections, such as (recurrent) otitis media, albeit limited, has been observed (Black et al., 2002; Eskola et al., 2001). At nasopharyngeal level, however, replacement of vaccine-type pneumococci with non-vaccine serotypes, as a result of vaccination, has also been observed (Dagan et al., 2002; Mbelle et al., 1999; Veenhoven et al., 2003). In addition, replacement of mucosal disease, i.e. (recurrent) acute otitis media, has been found (Eskola et al., 2001; Veenhoven et al., 2003). What effect this serotype replacement has on invasive disease remains unclear, although it has been demonstrated that several non-vaccine serotypes clearly have high potential for causing invasive disease (Brueggemann et al., 2003). Because few data are available on the age-related incidence and serotype distribution of S. pneumoniae among healthy children, a cross-sectional study was performed in the summer of 2002 among 3200 healthy children aged 1–19 years, in which the prevalence and determinants of pneumococcal carriage were studied (Bogaert et al., 2003). That study showed a significant age-related colonization rate, with a peak incidence of 55 % at 3 years of age, followed by a gradual decline to a stable colonization rate of 10 %, which was reached after the age of 10 years. Moreover, a significant age-related serotype distribution was noticed, with a primary peak of 7-valent conjugate vaccine (7vPCV) serotypes early in life, followed by a secondary peak with non-vaccine serotypes (Bogaert et al., 2003).
In this study, we investigated the molecular epidemiological dynamics and resistance profiles of the pneumococcal strains collected during the latter study in order to obtain detailed insight into the occurrence and age-related distribution of pneumococcal genotypes and resistance profiles.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The study was approved by the Medical Ethics Review Board of Erasmus MC, Rotterdam, The Netherlands.
Cultures.
Nasopharyngeal samples were obtained with rayon-tipped dacron pernasal swabs (Copan, Italy), transported in Amies transport medium to the medical microbiology laboratory, and plated within 6 h of sampling. The swabs were plated on gentamicin blood agar for the isolation of S. pneumoniae. Identification of S. pneumoniae isolates was performed by standard methods, as described by Lenette et al. (1985). The pneumococcal isolates were serotyped by the capsular swelling method (Quellung reaction), using commercially available antisera (Statens Serum Institute, Copenhagen, Denmark). Susceptibility testing was performed by the disk-diffusion method. Resistance was defined by measuring the zone diameters for the respective antibiotics, as defined by the National Committee for Clinical Laboratory Standards (NCCLS) (2002). Strains showing reduced susceptibility to oxacillin were additionally tested for penicillin and cefotaxime resistance by the Etest (AB Biodisk). Multidrug resistance was defined as resistance to three or more classes of antimicrobial agents.
Restriction fragment end-labelling (RFEL) typing.
Pneumococcal strain typing by RFEL was done as described by van Steenbergen et al. (1995), and adapted by Hermans et al. (1995). Briefly, purified pneumococcal DNA was digested by the restriction enzyme EcoRI. The DNA restriction fragments were end-labelled at 72 °C with [
-32P]dATP using DNA polymerase (Goldstar; Eurogentec). After the radiolabelled fragments were denatured, and separated electrophoretically on a 6 % polyacrylamide sequencing gel containing 8 M urea, the gel was transferred onto filter paper, vacuum dried (HBI), and exposed for variable times at room temperature to ECL hyperfilm (Amersham Laboratories).
Computer-assisted analysis of DNA band patterns.
RFEL autoradiographs were converted to images (Agfa Arcus II; Agfa Gevaert), and analysed by computer (Windows version Bionumerics; Applied Maths). DNA fragments were analysed as described previously (Sluijter et al., 1998). For evaluation of the genetic relatedness of the isolates, we used the following definitions: isolates of a particular RFEL type are 100 % identical by RFEL analysis (Centers for Disease Control Prevention, 1997); an RFEL cluster represents a group of RFEL types that differ in only one band (approx. >95 % genetic relatedness) (Black et al., 2002).
International comparison.
The genotypes were compared with an international collection of pneumococcal isolates representing 751 distinct RFEL types originating from 17 different countries in America, Europe, Africa and Asia (Bogaert et al., 2002, 2005; Hermans et al., 1997; Overweg et al., 2000), in which the first 26 international pandemic clones, as described by the Pneumococcal Molecular Epidemiological Network (PMEN) in 2002, are present (McGee et al., 2001).
Multilocus sequence typing (MLST).
The genotypes of the 24 largest clusters (n>4) were verified by MLST analysis, and one, two or three isolates per cluster were analysed. For this purpose, a fully automated method for MLST was used, as described by Jefferies et al. (2003). The MLST types were compared with the global PMEN database (http://www.pneumo.com/physician/pmen/pmen_history.asp).
Data analysis.
P values for differences were calculated with the 
2 test, using GraphPad Prism version 3.00 for Windows (GraphPad Software).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The age-related distribution of the most prevalent serotypes is depicted in Fig. 1. The 7vPCV serotypes, as well as the cross-protective serotypes, invariably showed peak incidences at the age of 1–2 years, whereas the incidence of the majority of the non-vaccine serotypes peaked at an older age.
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, all genetic clusters are represented in a genetic dendrogram, which includes the serotype distribution, age distribution, and number of isolates representing each cluster. In contrast to the age-related serotype distribution, the dendrogram suggests no additional correlation between age and genetic profile, except for cluster I, which showed a mean age of 11 years, compared with 6 years for the entire pneumococcal population.
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, we list the nine largest genetic clusters, each representing more than ten isolates. The largest cluster, cluster V, consisted of 45 strains of serotype 6B (Veenhoven et al., 2003), 6A (Butler et al., 1999) and 19F (Black et al., 2002), representing five genotypes. No homology was observed with any of the PMEN clones. The second and third largest clusters, cluster VIII (18 strains) and cluster I (17 strains), represented two and three genotypes, respectively, and harboured mainly serotype 3. Only one major cluster, cluster IX, representing serotypes 9V and 19F (13 strains), showed close homology (>95 %) with an international clone, which was Spain9V-3. MLST analysis showed that cluster IX and Spain9V-3 had five out of seven alleles in common. In contrast to the original PMEN clone, all Dutch isolates were fully susceptible to penicillin, tetracycline, erythromycin and cotrimoxazole. The remaining clusters, i.e. cluster II (13 strains), cluster III (10 strains), cluster IV (13 strains), cluster VI (10 strains) and cluster VII (12 strains), represented predominantly the serotypes 14, 15, 23F, 23F and 23F, respectively. Six of the nine major clusters consisted of 7vPCV serotypes, whereas only three clusters represented the non-vaccine serotypes 3 and 15.
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). This method confirmed the presence of an additional PMEN clone, namely England14-9. The remaining clusters showed sequence types (STs) differing from the international multidrug-resistant clones; however, most clusters matched STs present in the international database.
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gives the number of strains, resistance level, and the mean ages of the children carrying these isolates. No significant difference was observed for the mean ages of the children carrying resistant isolates (5·4 years) versus children carrying susceptible isolates (5·9 years), or for the individual resistance profiles.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In our cohort, the largest genetic cluster comprised mainly serotype 6A and 6B isolates. These isolates were fully susceptible to penicillin, tetracycline, erythromycin and cotrimoxazole. In addition, comparison of the genotypes of this cluster showed no close homology with any of the known serotype 6B clones from the PMEN database (McGee et al., 2001). Of the genotypes observed in our collection, only five showed close homology with five distinct PMEN clones (McGee et al., 2001). These data indicate that the international drug-resistant clones do not significantly contribute to colonization and spread of pneumococci among children in The Netherlands. The data confirm the observations of recent studies performed in The Netherlands among day-care-centre attendees, and children suffering from recurrent acute otitis media (Bogaert et al., 2001, 2005).
Recently, we observed an age-related pneumococcal serotype distribution within our study population (Bogaert et al., 2003). Pneumococcal 7vPCV serotypes showed an initial peak incidence of 30 % at the age of 1 year, after which a decline was observed to an incidence of 2–3 % after the age of 8 years. For non-vaccine serotypes, an initial increase in incidence was observed to 27 % at the age of 4 years, followed by a decline, which stabilized after the age of 15 years at 4 % (Bogaert et al., 2003). This was mainly caused by the predominance of a limited number of predominant non-vaccine serotypes after the age of 3 years, i.e. serotypes 3, 8, 10 and 11. Children under 2 years of age show the highest risk for pneumococcal infections, and since these children carry mainly 7vPCV serotype strains, a difference in virulence between 7vPCV and non-vaccine serotypes might exist. Therefore, we investigated the age-related distribution of genotypes. In contrast to the predominant serotype 3 cluster I with a mean age of 11 years, no age-related genetic clustering was observed. This observation supports the analyses of Brueggemann and Sandgren who reported that pneumococcal serotype and host factors, such as age, might be more important than genetic background in the progression of colonization to disease (Crook & Spratt, 1998; Sandgren et al., 2004).
When evaluating the nine largest clusters, each harbouring 10 or more isolates, we observed that six of these clusters represented the conjugate vaccine serotypes 6B, 14 and 23F, whereas the remaining three clusters consisted of the non-vaccine serotypes 3, 8 and 15. These data suggest that besides 7vPCV serotype pneumococci, non-vaccine pneumococcal serotypes are also able to spread at high frequency within the community. These findings are in accordance with several vaccination studies, in which replacement of 7vPCV serotypes with non-vaccine pneumococcal serotype carriage was found in the nasopharynx after vaccination with a pneumococcal conjugate vaccine (Dagan et al., 2002; Mbelle et al., 1999; Veenhoven et al., 2003). However, the question remains whether these non-vaccine serotype strains are as pathogenic as their 7vPCV-serotype counterparts. Several studies have shown that replacement of 7vPCV serotypes with non-vaccine serotype pneumococci can cause mucosal disease (Eskola et al., 2001; Veenhoven, 2003); with respect to invasive disease, replacement has not yet been observed. However, Brueggemann et al. (2003) have demonstrated epidemiological evidence for the high invasive capacity of certain non-vaccine serotypes.
In 18·1 % of the strains studied, susceptibility testing showed resistance to at least one of the most commonly used antimicrobial drugs. Resistance to a single drug was observed most often (13·6 %), and multidrug resistance was found in only 1·9 % of the isolates. Most commonly, we observed resistance to cotrimoxazole (12·9 %), which is a first-line antibiotic used commonly in children in The Netherlands. Finally, we found antibiotic resistance to be associated with 7vPCV-serotype pneumococci, which is in line with previous studies (Bogaert et al., 2002; Dobay et al., 2003; Watanabe et al., 2003).
We have established that drug resistance, with respect to pneumococcal carriage and disease, is still of minor concern in The Netherlands; however, a strict policy with respect to antibiotic prescription is still required.
In conclusion, pneumococcal colonization isolates from healthy Dutch children represent a heterogeneous genetic population of mostly susceptible strains, which display a high tendency to spread horizontally, irrespective of the age of the colonized children.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 August 2005;
revised 27 October 2005;
accepted 7 November 2005.
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5 out of 7 alleles showed homology with Spain9V-3.