Elske J. M. van Gils

Variability and Diversity of Nasopharyngeal Microbiota in Children: A Metagenomic Analysis

The nasopharynx is the ecological niche for many commensal bacteria and for potential respiratory or invasive pathogens like Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Disturbance of a balanced nasopharyngeal (NP) microbiome might be involved in the onset of symptomatic infections with these pathogens, which occurs primarily in fall and winter. It is unknown whether seasonal infection patterns are associated with concomitant changes in NP microbiota. As young children are generally prone to respiratory and invasive infections, we characterized the NP microbiota of 96 healthy children by barcoded pyrosequencing of the V5–V6 hypervariable region of the 16S-rRNA gene, and compared microbiota composition between children sampled in winter/fall with children sampled in spring. The approximately 1000000 sequences generated represented 13 taxonomic phyla and approximately 250 species-level phyla types (OTUs). The 5 most predominant phyla were Proteobacteria (64%), Firmicutes (21%), Bacteroidetes (11%), Actinobacteria (3%) and Fusobacteria (1,4%) with Moraxella, Haemophilus, Streptococcus, Flavobacteria, Dolosigranulum, Corynebacterium and Neisseria as predominant genera. The inter-individual variability was that high that on OTU level a core microbiome could not be defined. Microbiota profiles varied strongly with season, with in fall/winter a predominance of Proteobacteria (relative abundance (% of all sequences): 75% versus 51% in spring) and Fusobacteria (absolute abundance (% of children): 14% versus 2% in spring), and in spring a predominance of Bacteroidetes (relative abundance: 19% versus 3% in fall/winter, absolute abundance: 91% versus 54% in fall/winter), and Firmicutes. The latter increase is mainly due to (Brevi)bacillus and Lactobacillus species (absolute abundance: 96% versus 10% in fall/winter) which are like Bacteroidetes species generally related to healthy ecosystems. The observed seasonal effects could not be attributed to recent antibiotics or viral co-infection. The NP microbiota of young children is highly diverse and appears different between seasons. These differences seem independent of antibiotic use or viral co-infection.

Effect of Reduced-Dose Schedules With 7-Valent Pneumococcal Conjugate Vaccine on Nasopharyngeal Pneumococcal Carriage in Children: A Randomized Controlled Trial

CONTEXTnThe effects of reduced-dose schedules of 7-valent pneumococcal conjugate vaccine (PCV-7) on pneumococcal carriage in children are largely unknown, although highly relevant in the context of subsequent herd effects.nnnOBJECTIVEnTo examine the effects of a 2-dose and 2 + 1-dose PCV-7 schedule on nasopharyngeal pneumococcal carriage in young children compared with controls.nnnDESIGN, SETTING, AND PATIENTSnA randomized controlled trial of nasopharyngeal carriage of Streptococcus pneumoniae enrolling 1003 healthy newborns and 1 of their parents in a general community in The Netherlands, with follow-up to age 24 months and conducted between July 7, 2005, and February 14, 2008.nnnINTERVENTIONnInfants were randomly assigned to receive 2 doses of PCV-7 at 2 and 4 months; 2 + 1 doses of PCV-7 at 2, 4, and 11 months; or no dosage (control group).nnnMAIN OUTCOME MEASUREnVaccine serotype pneumococcal carriage rates in infants in the second year of life.nnnRESULTSnAt 12 months, vaccine serotype pneumococcal carriage was significantly decreased after both PCV-7 schedules, with vaccine serotype pneumococcal carriage rates of 25% (95% confidence interval [CI], 20%-30%) and 20% (95% CI, 16%-25%) in the 2-dose and 2 + 1-dose schedule groups, respectively, vs 38% (95% CI, 33%-44%) in the control group (both P < .001). At 18 months, in the 2 + 1-dose schedule group, vaccine serotype pneumococcal carriage had further decreased to 16% (95% CI, 12%-20%) and, at 24 months, to 14% (95% CI, 11%-18%; both P < .001); whereas in the 2-dose schedule group, vaccine serotype pneumococcal carriage had remained stable at 18 months (24%; 95% CI, 20%-29%), but at 24 months had further decreased to 15% (95% CI, 11%-19%; both P < .001). In the control group, vaccine serotype pneumococcal carriage remained around 36% to 38% until 24 months.nnnCONCLUSIONnCompared with no pneumococcal vaccination, a 2 + 1-dose and 2-dose schedule of PCV-7 resulted in significant reductions of vaccine serotype pneumococcal carriage in the second year of life.nnnTRIAL REGISTRATIONnclinicaltrials.gov Identifier: NCT00189020.

Associations between Pathogens in the Upper Respiratory Tract of Young Children: Interplay between Viruses and Bacteria

Background High rates of potentially pathogenic bacteria and respiratory viruses can be detected in the upper respiratory tract of healthy children. Investigating presence of and associations between these pathogens in healthy individuals is still a rather unexplored field of research, but may have implications for interpreting findings during disease. Methodology/Principal Findings We selected 986 nasopharyngeal samples from 433 6- to 24-month-old healthy children that had participated in a randomized controlled trial. We determined the presence of 20 common respiratory viruses using real-time PCR. Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus were identified by conventional culture methods. Information on risk factors was obtained by questionnaires. We performed multivariate logistic regression analyses followed by partial correlation analysis to identify the overall pattern of associations. S. pneumoniae colonization was positively associated with the presence of H. influenzae (adjusted odds ratio 1.60, 95% confidence interval 1.18–2.16), M. catarrhalis (1.78, 1.29–2.47), human rhinoviruses (1.63, 1.19–2.22) and enteroviruses (1.97, 1.26–3.10), and negatively associated with S. aureus presence (0.59, 0.35–0.98). H. influenzae was positively associated with human rhinoviruses (1.63, 1.22–2.18) and respiratory syncytial viruses (2.78, 1.06–7.28). M. catarrhalis colonization was positively associated with coronaviruses (1.99, 1.01–3.93) and adenoviruses (3.69, 1.29–10.56), and negatively with S. aureus carriage (0.42, 0.25–0.69). We observed a strong positive association between S. aureus and influenza viruses (4.87, 1.59–14.89). In addition, human rhinoviruses and enteroviruses were positively correlated (2.40, 1.66–3.47), as were enteroviruses and human bocavirus, WU polyomavirus, parainfluenza viruses, and human parechovirus. A negative association was observed between human rhinoviruses and coronaviruses. Conclusions/Significance Our data revealed high viral and bacterial prevalence rates and distinct bacterial-bacterial, viral-bacterial and viral-viral associations in healthy children, hinting towards the complexity and potential dynamics of microbial communities in the upper respiratory tract. This warrants careful consideration when associating microbial presence with specific respiratory diseases.

Long-Term Effects of Pneumococcal Conjugate Vaccine on Nasopharyngeal Carriage of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis

Background Shifts in pneumococcal serotypes following introduction of 7-valent pneumococcal conjugate vaccine (PCV-7) may alter the presence of other bacterial pathogens co-inhabiting the same nasopharyngeal niche. Methodology/Principal Findings Nasopharyngeal prevalence rates of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis were investigated before, 3 and 4.5 years after introduction of PCV-7 in the national immunisation program in children at 11 and 24 months of age, and parents of 24-month-old children (n≈330/group) using conventional culture methods. Despite a virtual disappearance of PCV-7 serotypes over time, similar overall pneumococcal rates were observed in all age groups, except for a significant reduction in the 11-month-old group (adjusted Odds Ratio after 4.5 years 0.48, 95% Confidence Interval 0.34–0.67). Before, 3 and 4.5 years after PCV-7 implementation, prevalence rates of S. aureus were 5%, 9% and 14% at 11 months of age (3.59, 1.90–6.79) and 20%, 32% and 34% in parents (1.96, 1.36–2.83), but remained similar at 24 months of age, respectively. Prevalence rates of H. influenzae were 46%, 65% and 65% at 11 months (2.22, 1.58–3.13), 52%, 73% and 76% at 24 months of age (2.68, 1.88–3.82) and 23%, 30% and 40% in parents (2.26, 1.58–3.33), respectively. No consistent changes in M. catarrhalis carriage rates were observed over time. Conclusions/Significance In addition to large shifts in pneumococcal serotypes, persistently higher nasopharyngeal prevalence rates of S. aureus and H. influenzae were observed among young children and their parents after PCV-7 implementation. These findings may have implications for disease incidence and antibiotic treatment in the post-PCV era.

Carriage of Streptococcus pneumoniae 3 years after start of vaccination program, the Netherlands.

To evaluate the effectiveness of the 7-valent pneumococcal conjugate vaccine (PCV7) program, we conducted a cross-sectional observational study on nasopharyngeal carriage of Streptococcus pneumoniae 3 years after implementation of the program in the Netherlands. We compared pneumococcal serotypes in 329 prebooster 11-month-old children, 330 fully vaccinated 24-month-old children, and 324 parents with age-matched pre-PCV7 (unvaccinated) controls (ages 12 and 24 months, n = 319 and n = 321, respectively) and 296 of their parents. PCV7 serotype prevalences before and after PCV7 implementation, respectively, were 38% and 8% among 11-month-old children, 36% and 4% among 24-month-old children, and 8% and 1% among parents. Non-PCV7 serotype prevalences were 29% and 39% among 11-month-old children, 30% and 45% among 24-month-old children, and 8% and 15% among parents, respectively; serotypes 11A and 19A were most frequently isolated. PCV7 serotypes were largely replaced by non-PCV7 serotypes. Disappearance of PCV7 serotypes in parents suggests strong transmission reduction through vaccination.

Effect of Seven-Valent Pneumococcal Conjugate Vaccine on Staphylococcus aureus Colonisation in a Randomised Controlled Trial

Background Heptavalent pneumococcal conjugate vaccine (PCV7) shifts nasopharyngeal colonisation with vaccine serotype pneumococci towards nonvaccine serotypes. Because of the reported negative association of vaccine serotype pneumococci and Staphylococcus aureus in the nasopharynx, we explored the effect of PCV7 on nasopharyngeal colonisation with S. aureus in children and parents. Methodology/Principal Findings This study was part of a randomised controlled trial on the effect of PCV7 on pneumococcal carriage, enrolling healthy newborns who were randomly assigned (1∶1∶1) to receive PCV7 (1) at 2 and 4 months of age (2) at 2, 4 and 11 months or (3) no PCV7 (controls). Nasopharyngeal colonisation of S. aureus was a planned secondary outcome. Nasopharyngeal swabs were obtained from all children over a 2-year period with 6-months interval and from one parent at the childs age of 12 and 24 months and cultured for Streptococcus pneumoniae and S. aureus. Between July 2005 and February 2006, 1005 children were enrolled and received either 2-doses of PCV7 (nu200a=u200a336), 2+1-doses (336) or no dose (nu200a=u200a333) before PCV7 implementation in the Dutch national immunization program. S. aureus colonisation had doubled in children in the 2+1-dose group at 12 months of age compared with unvaccinated controls (10.1% versus 5.0%; pu200a=u200a0.019). A negative association for co-colonisation of S. pneumoniae and S. aureus was observed for both vaccine serotype (adjusted odds ratio (aOR) 0.53, 95% confidence interval (CI) 0.38–0.74) and nonvaccine serotype pneumococci (aOR 0.67, 95% CI 0.52–0.88). Conclusions/Significance PCV7 induces a temporary increase in S. aureus colonisation in children around 12 months of age after a 2+1-dose PCV7 schedule. The potential clinical consequences are unknown and monitoring is warranted. Trial Registration ClinicalTrials.gov NCT00189020

Pneumococcal Conjugate Vaccination and Nasopharyngeal Acquisition of Pneumococcal Serotype 19A Strains

CONTEXTnThe rapid increase in multiresistant serotype 19A as a cause of invasive and respiratory pneumococcal disease has been associated in time with the widespread implementation of 7-valent pneumococcal conjugate vaccination (PCV-7) in several countries. Because spontaneous fluctuations in time and antibiotic selective pressure may have induced this serotype 19A increase, controlled studies are needed to assess the role of PCV-7.nnnOBJECTIVEnTo examine the association of PCV-7 vaccination and nasopharyngeal acquisition of serotype 19A pneumococci, their clonal distribution, and antibiotic susceptibility.nnnDESIGN, SETTING, AND PATIENTSnPost hoc per-protocol completers analysis as part of a randomized controlled trial of nasopharyngeal Streptococcus pneumoniae carriage enrolling 1003 healthy newborns with follow-up to the age of 24 months in The Netherlands, which has low antibiotic resistance rates. The study was conducted before widespread PCV-7 implementation in infants, between July 7, 2005, and February 14, 2008. Nasopharyngeal swabs were obtained at the age of 6 weeks and at 6, 12, 18, and 24 months.nnnINTERVENTIONnInfants were randomly assigned to receive 2 doses of PCV-7 at 2 and 4 months; 2 + 1 doses of PCV-7 at 2, 4, and 11 months; or no dosage (unvaccinated control group).nnnMAIN OUTCOME MEASUREnCumulative proportion of children with nasopharyngeal acquisition of a new serotype 19A strain from 6 through 24 months of age.nnnRESULTSnNine hundred forty-eight children completed the study. Fifty-four nasopharyngeal serotype 19A carriage isolates from 318 in the 2-dose group, 66 isolates from 327 in the 2 + 1-dose group, and 33 isolates from 303 in the unvaccinated were collected from 6 weeks through 24 months. The cumulative proportion who tested positive for new nasopharyngeal serotype 19A acquisition from 6 through 24 months of age was significantly higher in those having received the 2 + 1-dose PCV-7 schedule (16.2%; 95% confidence interval [CI], 12.6%-20.6%) vs those who were unvaccinated (9.2%; 95% CI, 6.5%-13.0%; relative risk [RR], 1.75; 95% CI, 1.14-2.70) but not after a 2-dose schedule (13.2%; 95% CI, 9.9%-17.4%; RR, 1.43; 95% CI, 0.91-2.25). There were 28 different sequence types identified, including 6 new types. The proportion of children with new 19A acquisition who had used antibiotics in the last 6 months (18.7%) did not differ among groups. Five isolates were penicillin-intermediate susceptible and another 3 were nonsusceptible to erythromycin and azithromycin, all in the vaccine groups.nnnCONCLUSIONnA 2 + 1-dose PCV-7 schedule was associated with an increase in serotype 19A nasopharyngeal acquisition compared with unvaccinated controls.nnnTRIAL REGISTRATIONnclinicaltrials.gov Identifier: NCT00189020.

Effect of 7-valent pneumococcal conjugate vaccine on nasopharyngeal carriage with Haemophilus influenzae and Moraxella catarrhalis in a randomized controlled trial

Seven-valent CRM197-conjugated pneumococcal conjugate vaccine (PCV7(CRM197)) reduces both vaccine serotype nasopharyngeal colonization and vaccine serotype acute otitis media by 50-60%. However, overall pneumococcal carriage and impact on otitis media are partly offset by concomitant increase of nonvaccine serotypes. We investigated in a randomized controlled trial the impact of 2-doses and 2+1-doses of PCV7(CRM197) on carriage of Streptococcus pneumoniae and of other nasopharyngeal commensals and well-known otitis media pathogens, Haemophilus influenzae and Moraxella catarrhalis, in children. Nasopharyngeal swabs were collected at the age of 6 weeks and at 6, 12, 18 and 24 months. We observed high carriage rates up to 68% for S. pneumoniae, 71% for H. influenzae and 68% for M. catarrhalis at the age of 18 months. Reduced dose (CRM197) schedules induced a slight reduction in overall pneumococcal carriage but no increases in the presence of H. influenzae and M. catarrhalis.

Nasopharyngeal Colonization Elicits Antibody Responses to Staphylococcal and Pneumococcal Proteins That Are Not Associated with a Reduced Risk of Subsequent Carriage

ABSTRACT Knowledge of the immunological correlates of Staphylococcus aureus and Streptococcus pneumoniae colonization is required for the search for future protein vaccines. We evaluated natural antibody levels against pneumococcal and staphylococcal proteins in relation to previous bacterial colonization with both pathogens. In a randomized controlled trial, nasopharyngeal samples were obtained from children at 1.5, 6, 12, 18, and 24 months and cultured for S. aureus and S. pneumoniae. Approximately 50% of the children were PCV7 vaccinated. Serum IgG against 18 pneumococcal and 40 staphylococcal proteins was semiquantified by Luminex technology from 111 12 month olds and 158 24 month olds. Previous culture-proven S. aureus colonization was associated with higher IgG levels against 6/40 staphylococcal proteins (ClfB, ClfA, Efb, CHIPS, LukD, and LukF [P ≤ 0.001]) compared to noncarriers. Previous pneumococcal colonization was associated with increased IgG levels against 12/18 pneumococcal proteins compared to noncarriers (P ≤ 0.003). Increasing age was associated with higher levels of antibodies to most pneumococcal proteins and lower levels of antibodies to over half the staphylococcal proteins, reflecting natural colonization dynamics. Anti-S. pneumoniae and anti-S. aureus protein antibodies at the age of 12 months were not negatively correlated with subsequent colonization with the homologous species in the following year and did not differ between PCV7-vaccinated and nonvaccinated children. Colonization with S. aureus and S. pneumoniae induces serum IgG against many proteins, predominantly proteins with immune-modulating functions, irrespective of PCV7 vaccination. None of them appeared to be protective against new acquisition with both pathogens, possibly due to the polymorphic nature of those proteins in the circulating bacterial population.

Comparability of antibody response to a booster dose of 7-valent pneumococcal conjugate vaccine in infants primed with either 2 or 3 doses

In this cohort study we compared IgG antibody levels between infants immunized with 7-valent CRM197-conjugated pneumococcal vaccine (PCV-7) at 2, 4 and 11 months and at 2, 3, 4 and 11 months of age, as measured by double adsorption ELISA. Pre- and post-booster levels following the 2+1- and 3+1-dose schedule were comparable for 5 out of 7 serotypes except for serotypes 6B and 19F. The proportion of children reaching post-booster antibody thresholds were comparable except for 6B (>or=1.0 microg/ml and >or=5.0 microg/ml) and 19F (>or=5.0 microg/ml). Surveillance studies are warranted for vaccine impact on 6B and 19F disease cases after reduced-dose PCV-7 schedules.