Background
Pneumonia is a frequent cause of childhood deaths in many developing countries and its leading cause is
Streptococcus pneumoniae [
1‐
3]. In The Gambia, there is a high burden of pneumococcal disease in young children in both urban and rural settings [
4‐
9]. During a recently concluded pneumococcal conjugate vaccine trial in The Gambia, the incidence of invasive pneumococcal disease in children who had not received the vaccine was 363 (95% CI 157, 715) per 100 000 person-years in children aged 6 weeks to 5 months and 576 (95%CI 369, 857) per 100,00 person years in those aged 6–11 months [
7]. Similar figures have been reported from rural Kenya in a population that had not received Hib or pneumococcal conjugate vaccines [
10]. The pneumococcus is also a major cause of otitis media, responsible for much morbidity in young children worldwide [
11]. HIV infection increases susceptibility to pneumococcal infection and, as a consequence, the incidence of pneumococcal infection in children and adults is increasing in many parts of sub-Saharan Africa [
12,
13]. Emergence of antibiotic resistant pneumococci has complicated case-management strategies for pneumococcal disease. Therefore, long-term prevention of pneumococcal disease in young children by vaccination is a major international public health priority.
S. pneumoniae is subdivided into 91 different serotypes [
14], all of which are capable of causing invasive disease. Geographic variations in the distribution of pneumococcal serotypes exist and some serotypes are associated selectively with disease in children or adults [
15]. The advent of pneumococcal conjugate vaccines provides a potential way of preventing pneumococcal disease although available vaccines contain only a limited number of serotypes. The results of a recently concluded efficacy trial of a 9-valent pneumococcal conjugate vaccine (Wyeth) in Basse, a typical rural African setting in The Gambia, are highly encouraging as they showed protective efficacies, among children who received three doses of vaccine according to protocol, of 37% against radiological pneumonia, 77% against invasive pneumococcal disease of vaccine serotype, 50% against overall pneumococcal disease, 15% against all-cause hospitalizations and 16% against all-cause mortality [
16]. The licensed 7-valent pneumococcal conjugate vaccine (Prevenar
®) has had a dramatic impact on the incidence of invasive pneumococcal disease in vaccinated infants in the United States and has also protected unvaccinated adults through the induction of herd protection [
17]. However, concerns have been raised about the long terms effects of vaccines containing limited numbers of serotypes on the overall pneumococcal population [
17]. Thus, prior to the planned introduction of Prevenar
R into the routine immunisation programme of The Gambia, we studied 131 but presented data on 127
S. pneumoniae isolates obtained from vaccinated or unvaccinated children (<2 years old) during The Gambian vaccine trial (between August 2000 and April 2004) by BOX-PCR genotyping and MLST to determine the current population structure of pneumococci in the country.
Discussion
One hundred and thirty-two S. pneumoniae isolates were recovered from vaccinated and unvaccinated children aged 2–29 months during the course of a pneumococcal conjugate vaccine trial conducted in The Gambia of which 131 were characterized by serotyping, antibiotic susceptibility, BOX-PCR and MLST and 127 had unique MLST and/or antibiotic susceptibility pattern and were included in this study.
There was no particular association of clones with specific presentations of invasive pneumococcal disease (data not shown) although three out of six (50%) cases of meningitis were caused by pneumococci belonging to serotype 12F with ST989. This result concurs with findings in Northern Ghana [
23] and Niger where serotype 12F ST989 isolates have been detected in patients with meningitis. Serotype 12F ST989 isolates have also been identified in nasopharyngeal carriers in a village in Kenya [
24] and in Qatar [
25]. Similarly, a SLV of ST989, serotype 12B ST2211, was responsible for a case of meningitis in Niger [
25].
In this study, we found twenty-nine different serotypes corresponding to seventy-two MLST types (sequence types). MLST is the definitive, internationally accepted method for monitoring the spread of clones through the pneumococcal population [
20,
21,
26]. Our data showed that serotype and genotype distribution varied considerably in vaccinated and unvaccinated children suggesting a high degree of fluidity in the pneumococcal population dynamics (figure
2).
Although the 9-valent vaccine used in our trial contained glycoproteins for serotypes 1 and 9V, pneumococci of these serotypes caused a small number of cases of invasive disease with no reduction in cases in vaccinated children. For example, 4 vaccinated children (from whom 5 isolates were obtained – Table
1) had invasive disease due to a serotype 1 infection compared with 2 unvaccinated children (from whom 3 isolates were obtained – Table
1). There was no clustering of cases of serotype 1 in either vaccinated or unvaccinated groups over time during the period 2002–2004. The very small number of serotype 1 and 9V cases detected precludes precise estimation of serotype-specific efficacy for these two serotypes but these findings raise the possibility that efficacy was lower against serotypes 1 and 9V than against other vaccine-type serotypes, despite the fact that the vaccine elicited strong immune responses, as measured by ELISA, for all serotypes contained in the 9-valent vaccine, including serotypes 1 and 9V (data not shown). All but one of the seven serotype 1 isolates in our study had the same genotype, ST 618. One of the isolates, from an unvaccinated child, had a novel ST (ST3336) which is a SLV of ST 618. ST 618 and its single and double locus variant have recently been observed to cause meningitis in Ghana, Burkina Faso and Egypt [
25]. Furthermore, ST618 belongs to the same clonal complex (ST 217) of serotype 1 that has caused an outbreak of meningitis in Northern Ghana [
23], indicating a dominant West African clone. The ST217 hypervirulent clonal complex is typical of lineage B serotype 1 pneumococci that are predominant within Africa and Israel [
26]. A lower efficacy for serotype 1 and 9V could be explained by heterogeneity or intraspecies variations among these invasive pneumococci.
Isolates belonging to the same genotype (clone) can express different types of capsular polysaccharide through horizontal recombination of pneumococcal DNA by natural transformation [
20]. This phenomenon, capsular switching, leads to the emergence of a new clone expressing a different capsule [
17]. There was no direct evidence of capsular switching within our isolates. However, comparative analysis of our serotype and MLST results with those published in the MLST database showed that some genotypes found in our isolates express different serotypes in other areas. For example, we found genotype ST63 to be serotype 14 and associated with bacteremia and pneumonia (figure
2) and ST63 serotype 14 is associated with meningitis in Niger [
27]. Elsewhere, ST63 has been found to express serotype 15A, 19A and 23F capsular polysaccharides [
25], suggesting that ST63 has a high propensity for capsular switching. ST912 expressing serotype 9V capsular polysaccharide has previously been isolated from a carrier in The Gambia [
25] but in this study we found ST912 expressing serotype 6A polysaccharide. Similarly, ST2179 expressing serotype 6A capsular polysaccharide has previously been isolated from a Gambian carrier [
25] while in this study we found ST2179 expressing serotype 9V polysaccharide and causing invasive disease, suggesting capsular switching. Similarly, ST2174 expressing a serotype 23F capsule has previously been isolated from a carrier in The Gambia [
25] but in this study we found ST2174 expressing serotype 19A capsular polysaccharide again suggesting capsular switching. This is plausible as cluster analysis from our data shows that serotype 19A and 23F are genetically linked (figure
2). Although our data is suggestive that capsular switching can occur naturally, a large dataset will be required to show the impact of conjugate vaccination on this process. We have recently set up nasopharyngeal pneumococcal carriage studies in 21 villages in The Gambia to address this question [
28].
The use of PCV has the potential to reduce substantially the number of drug-resistant pneumococcal infections [
29]. Antimicrobial resistance in pneumococci is closely linked to serotype and genotype. Although we found no penicillin resistant isolates in this study, there is evidence from The Gambia that isolates of vaccine serotype have significantly higher intermediate resistance to penicillin than those of non-vaccine serotype (p < 0.01) [
28]. Similarly, isolates of 9-valent serotype were less likely to be resistant to tetracycline than those of a non-vaccine serotype (39.0% vs. 52.6%; p < 0.05). In contrast, isolates of non-vaccine serotype were more likely to be resistant to chloramphenicol (p < 0.0001). Interestingly, we identified two serotype 6B isolates with ST385 and ST3323 which were-resistant to three antibiotics; chloramphenicol, cotrimoxazole and tetracycline. ST385 and ST3323 are also SLV and DLV respectively to ST273. ST273 is known to be a major pandemic antimicrobial-resistant clone, Greece 6B-22 [
30,
31] of pneumococci as defined by the pneumococcal molecular epidemiology network.
Acknowledgements
We would like to acknowledge Ishrat Hakeem, Ebou Mbowe, Lamin Juwara, Mark Saaka, Ousman Secka and Pauline Sambou for technical assistance. We also acknowledge the use of the
S. pneumoniae MLST database [
25] which is housed at Imperial College, London.
The Gambia Pneumococcal Vaccine Trial was funded by grants from MRC (UK), the National Institute of Allergy and Infectious Diseases at the USA National Institutes of Health through contract N01-AI-25477; WHO through contract V23/181/127; the Children's Vaccine Program at PATH; and US Agency for International Development
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MA and RA conceived the study and wrote the paper with GE, FC and BMG. MA, HD-A, JO'C and VP performed PCR, sequence reactions, edited and aligned DNA sequences for MLST analysis. EB cultured and identified bacteria isolates from clinical samples. GE, BO, CO, AV, SMAZ and FC participated in field and clinical aspects of the study. TA was involved in statistical analysis. All authors read and approved the final manuscript.