Influenza-like Illness Incidence Is Not Reduced by Influenza Vaccination in a Cohort of Older Adults, Despite Effectively Reducing Laboratory-Confirmed Influenza Virus Infections.

Background: Data on the relative contribution of influenza virus and other respiratory pathogens to respiratory infections in community-dwelling older adults (≥60 years) are needed. Methods: A prospective observational cohort study was performed in the Netherlands during 2 winters. Nasopharyngeal and oropharyngeal swabs were collected during influenza-like illness (ILI) episodes and from controls. Viruses and bacteria were identified by multiplex ligation-dependent probe amplification assay and conventional bacterial culture. Results: The ILI incidence in the consecutive seasons was 7.2% and 11.6%, and influenza virus caused 18.9% and 34.2% of ILI episodes. Potential pathogen were detected in 80% of the ILI events with influenza virus, coronaviruses, rhinoviruses, human metapneumovirus, respiratory syncytial virus, parainfluenza viruses, and Haemophilus influenzae being the most common. Influenza vaccination reduced influenza virus infection by 73% (95% confidence interval [CI], 26%-90%) and 51% (95% CI, 7%-74%) in ILI patients. However, ILI incidence was similar between vaccinated (7.6% and 10.8%) and nonvaccinated (4.2% and 11.4%) participants in 2011-2012 and 2012-2013, respectively (P > .05). Conclusions: Influenza virus is a frequent pathogen in older adults with ILI. Vaccination reduces the number of influenza virus infections but not the overall number of ILI episodes: other pathogens fill the gap. We suggest the existence of a pool of individuals with high susceptibility to respiratory infections. Clinical Trials Registration: NTR3386.

Immunogenicity, Safety and Reactogenicity of a Booster Dose of the 10-Valent Pneumococcal Nontypeable H. influenzae Protein D Conjugate Vaccine Coadministered With DTPa-IPV-Hib in Dutch Children: A Randomized Controlled Trial.

BACKGROUND: Immune responses and safety profiles may be affected when vaccines are coadministered. We evaluated the immunogenicity, safety and reactogenicity of a booster dose of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D-conjugate (PHiD-CV; Synflorix GSK Vaccines) and DTPa-IPV-Hib (Pediacel Sanofi Pasteur MSD) when coadministered. METHODS: We performed booster assessment in a randomized controlled trial in the Netherlands. Of 780 enrolled healthy infants, 774 toddlers participated in the booster phase and received (1:1:1) (1) PHiD-CV + DTPa-HBV-IPV/Hib (Infanrix hexa, GSK Vaccines), (2) PHiD-CV + DTPa-IPV-Hib, or (3) 7-valent pneumococcal conjugate vaccine (7vCRM, Prevenar/Prevnar, Pfizer, Inc.) + DTPa-IPV-Hib at 2, 3, 4 and 11-13 months old. Blood samples were taken postprimary, prebooster, 1 and 12 months postbooster. RESULTS: Antipneumococcal antibody responses were comparable between both PHiD-CV groups, except for serotype 18C (conjugated to tetanus toxoid). Anti-18C antibody geometric mean concentrations (GMCs) were higher when coadministered with DTPa-HBV-IPV/Hib. For each vaccine serotype, the percentages of children with antibody concentration ≥ 0.20 µg/mL were within the same ranges between PHiD-CV groups (93.8%-100%). The same was observed for the percentages of participants with opsonophagocytic activity titer ≥ 8 (90.9%-100%). When comparing both DTPa-IPV-Hib groups, postbooster antidiphtheria antibody GMCs were higher when coadministered with 7vCRM, while antitetanus and antipolyribosyl-ribitol phosphate antibody GMCs were higher with PHiD-CV coadministration. Regardless, antibody levels to these antigens were well above thresholds. Safety and reactogenicity profiles were comparable between groups. CONCLUSIONS: Coadministration of a booster dose of PHiD-CV and DTPa-IPV-Hib was immunogenic and well tolerated.

Dysbiosis of upper respiratory tract microbiota in elderly pneumonia patients.

Bacterial pneumonia is a major cause of morbidity and mortality in elderly. We hypothesize that dysbiosis between regular residents of the upper respiratory tract (URT) microbiome, that is balance between commensals and potential pathogens, is involved in pathogen overgrowth and consequently disease. We compared oropharyngeal microbiota of elderly pneumonia patients (n=100) with healthy elderly (n=91) by 16S-rRNA-based sequencing and verified our findings in young adult pneumonia patients (n=27) and young healthy adults (n=187). Microbiota profiles differed significantly between elderly pneumonia patients and healthy elderly (PERMANOVA, P<0.0005). Highly similar differences were observed between microbiota profiles of young adult pneumonia patients and their healthy controls. Clustering resulted in 11 (sub)clusters including 95% (386/405) of samples. We observed three microbiota profiles strongly associated with pneumonia (P<0.05) and either dominated by lactobacilli (n=11), Rothia (n=51) or Streptococcus (pseudo)pneumoniae (n=42). In contrast, three other microbiota clusters (in total n=183) were correlated with health (P<0.05) and were all characterized by more diverse profiles containing higher abundances of especially Prevotella melaninogenica, Veillonella and Leptotrichia. For the remaining clusters (n=99), the association with health or disease was less clear. A decision tree model based on the relative abundance of five bacterial community members in URT microbiota showed high specificity of 95% and sensitivity of 84% (89% and 73%, respectively, after cross-validation) for differentiating pneumonia patients from healthy individuals. These results suggest that pneumonia in elderly and young adults is associated with dysbiosis of the URT microbiome with bacterial overgrowth of single species and absence of distinct anaerobic bacteria. Whether the observed microbiome changes are a cause or a consequence of the development of pneumonia or merely coincide with disease status remains a question for future research.

Early respiratory microbiota composition determines bacterial succession patterns and respiratory health in children.

RATIONALE: Many bacterial pathogens causing respiratory infections in children are common residents of the respiratory tract. Insight into bacterial colonization patterns and microbiota stability at a young age might elucidate healthy or susceptible conditions for development of respiratory disease. OBJECTIVES: To study bacterial succession of the respiratory microbiota in the first 2 years of life and its relation to respiratory health characteristics. METHODS: Upper respiratory microbiota profiles of 60 healthy children at the ages of 1.5, 6, 12, and 24 months were characterized by 16S-based pyrosequencing. We determined consecutive microbiota profiles by machine-learning algorithms and validated the findings cross-sectionally in an additional cohort of 140 children per age group. MEASUREMENTS AND MAIN RESULTS: Overall, we identified eight distinct microbiota profiles in the upper respiratory tract of healthy infants. Profiles could already be identified at 1.5 months of age and were associated with microbiota stability and change over the first 2 years of life. More stable patterns were marked by early presence and high abundance of Moraxella and Corynebacterium/Dolosigranulum and were positively associated with breastfeeding in the first period of life and with lower rates of parental-reported respiratory infections in the consecutive periods. Less stable profiles were marked by high abundance of Haemophilus or Streptococcus. CONCLUSIONS: These findings provide novel insights into microbial succession in the respiratory tract in infancy and link early-life profiles to microbiota stability and respiratory health characteristics. New prospective studies should elucidate potential implications of our findings for early diagnosis and prevention of respiratory infections. Clinical trial registered with www.clinicaltrials.gov (NCT00189020).

The impact of breastfeeding on nasopharyngeal microbial communities in infants.

RATIONALE: Breastfeeding elicits significant protection against respiratory tract infections in infancy. Modulation of respiratory microbiota might be part of the natural mechanisms of protection against respiratory diseases induced by breastfeeding. OBJECTIVES: To study the association between breastfeeding and nasopharyngeal microbial communities, including all cultivable and noncultivable bacteria. METHODS: In this observational study, we analyzed the microbiota of infants that had received exclusive breastfeeding (n = 101) and exclusive formula feeding (n = 101) at age 6 weeks and 6 months by 16S-based GS-FLX-titanium-pyrosequencing. MEASUREMENTS AND MAIN RESULTS: At 6 weeks of age the overall bacterial community composition was significantly different between breastfed and formula-fed children (nonmetric multidimensional scaling, P = 0.001). Breastfed children showed increased presence and abundance of the lactic acid bacterium Dolosigranulum (relative effect size [RES], 2.61; P = 0.005) and Corynebacterium (RES, 1.98; P = 0.039) and decreased abundance of Staphylococcus (RES, 0.48; P 0.03) and anaerobic bacteria, such as Prevotella (RES, 0.25; P < 0.001) and Veillonella (RES, 0.33; P < 0.001). Predominance (>50% of the microbial profile) of Corynebacterium and Dolosigranulum was observed in 45 (44.6%) breastfed infants compared with 19 (18.8%) formula-fed infants (relative risk, 2.37; P = 0.006). Dolosigranulum abundance was inversely associated with consecutive symptoms of wheezing and number of mild respiratory tract infections experienced. At 6 months of age associations between breastfeeding and nasopharyngeal microbiota composition had disappeared. CONCLUSIONS: Our data suggest a strong association between breastfeeding and microbial community composition in the upper respiratory tract of 6-week-old infants. Observed differences in microbial community profile may contribute to the protective effect of breastfeeding on respiratory infections and wheezing in early infancy. Clinical trial registered with www.clinicaltrials.gov (NCT 00189020).

Seven-valent pneumococcal conjugate vaccine and nasopharyngeal microbiota in healthy children.

Seven-valent pneumococcal conjugate vaccine (PCV-7) is effective against vaccine serotype disease and carriage. Nevertheless, shifts in colonization and disease toward nonvaccine serotypes and other potential pathogens have been described. To understand the extent of these shifts, we analyzed nasopharyngeal microbial profiles of 97 PCV-7-vaccinated infants and 103 control infants participating in a randomized controlled trial in the Netherlands. PCV-7 immunization resulted in a temporary shift in microbial community composition and increased bacterial diversity. Immunization also resulted in decreased presence of the pneumococcal vaccine serotype and an increase in the relative abundance and presence of nonpneumococcal streptococci and anaerobic bacteria. Furthermore, the abundance of Haemophilus and Staphylococcus bacteria in vaccinees was increased over that in controls. This study illustrates the much broader effect of vaccination with PCV-7 on the microbial community than currently assumed, and highlights the need for careful monitoring when implementing vaccines directed against common colonizers.

Immunogenicity of 13-valent pneumococcal conjugate vaccine administered according to 4 different primary immunization schedules in infants: a randomized clinical trial.

IMPORTANCE: Immunization schedules with pneumococcal conjugate vaccine (PCV) differ among countries regarding the number of doses, age at vaccinations, and interval between doses. OBJECTIVE: To assess the optimal primary vaccination schedule by comparing immunogenicity of 13-valent PCV (PCV13) in 4 different immunization schedules. DESIGN, SETTING, AND PARTICIPANTS: An open-label, parallel-group, randomized clinical trial of healthy term infants in a general community in The Netherlands conducted between June 30, 2010, and January 25, 2011, with 99% follow-up until age 12 months. INTERVENTIONS: Infants (N = 400) were randomly assigned (1:1:1:1) to receive PCV13 either at ages 2, 4, and 6 months (2-4-6); at ages 3 and 5 months (3-5); at ages 2, 3, and 4 months (2-3-4); or at ages 2 and 4 months (2-4), with a booster dose at age 11.5 months. MAIN OUTCOMES AND MEASURES: Primary outcome measure was antibody geometric mean concentrations (GMCs) against PCV13-included serotypes 1 month after the booster dose measured by multiplex immunoassay. Secondary outcomes included GMCs measured 1 month after the primary series, at 8 months of age, and before the booster. RESULTS: The primary outcome, GMCs at 1 month after the booster dose, was not significantly different between schedules for 70 of 78 comparisons. The 2-4-6 schedule was superior to the 2-3-4 schedule for serotypes 18C (10.2 µg/mL [95% CI, 8.2-12.7] vs 6.5 µg/mL [95% CI, 5.4-7.8]) and 23F (10.9 µg/mL [95% CI, 9.0-13.3] vs 7.3 µg/mL [95% CI, 5.8-9.2]) and superior to the 2-4 schedule for serotypes 6B (8.5 µg/mL [95% CI, 7.1-10.2] vs 5.1 µg/mL [95% CI 3.8-6.7]), 18C (6.6 µg/mL [95% CI, 5.7-7.7]), and 23F (7.2 µg/mL [95% CI, 5.9-8.8]). For serotype 1, the 3-5 schedule (11.7 µg/mL [95% CI, 9.6-14.3]) was superior to the other schedules. Geometric mean concentrations for all 13 serotypes ranged between 1.6 and 19.9 µg/mL. Secondary outcomes demonstrated differences 1 month after the primary series. The 2-4-6 schedule was superior compared with the 3-5, 2-3-4, and 2-4 schedules for 3, 9, and 11 serotypes, respectively. Differences between schedules persisted until the booster dose. CONCLUSIONS AND RELEVANCE: The use of 4 different PCV13 immunization schedules in healthy term infants resulted in no statistically significant differences in antibody levels after the booster dose for almost all serotypes. The choice of PCV schedule will require a balance between the need for early protection and maintaining protection between the primary series and the booster. TRIAL REGISTRATION: trialregister.nl Identifier: NTR2316.

Perceived discomfort levels in healthy children participating in vaccine research.

When assessing the risks of a research protocol, review boards need to consider not only the possible harms but also the expected discomfort levels caused by the various study procedures. However, data on how children experience various study procedures are scarce. This study assessed perceived discomfort levels in 671 healthy children aged 0-2 years under-going vaccinations, venipunctures, and nasopharyngeal swab taking. In half of the study participants, venipunctures caused a moderate or high level of discomfort (49%). Corresponding figures for nasopharyngeal swabbing and vaccinations were 28% and 12%, respectively. Within the reported age group, increasing age was related with higher discomfort levels. In a majority of cases for all study procedures, the perceived levels of discomfort met the parents' expectations.

Superiority of trans-oral over trans-nasal sampling in detecting Streptococcus pneumoniae colonization in adults.

The human nasopharynx is the main reservoir for Streptococcus pneumoniae. We applied conventional and molecular methods to determine the prevalence of S. pneumoniae nasopharyngeal colonization in adults. Paired trans-orally and trans-nasally obtained nasopharyngeal samples from 268 parents of 24-month-old children were assessed for pneumococcal presence. Parents were classified as colonized when live pneumococci were recovered from either sample cultured on medium selective for S. pneumoniae. Of the 52 (19%) colonized parents 49 (18%) were culture-positive in trans-nasal and 10 (4%) in trans-oral samples. Bacterial growth was harvested from these cultures, DNA isolated and tested by quantitative-PCR (qPCR) targeting lytA and piaA genes specific for S. pneumoniae. A sample was considered positive if signals for both genes were detected. Altogether 105 (39%) individuals were classified as positive for pneumococcus by qPCR including 50 (19%) in trans-nasal and 94 (35%) in trans-oral settings. Although significantly more trans-nasal compared to trans-oral samples were culture-positive for S. pneumoniae at the primary diagnostic step (p<0.001) the opposite was observed in qPCR results (p<0.001). To confirm the presence of live pneumococcus in samples positive by qPCR but negative at the initial diagnostic step, we serially-diluted cell harvests, re-cultured and carefully examined for S. pneumoniae presence. Live pneumococci were recovered from an additional 43 parents including 42 positive in trans-oral and 4 in trans-nasal samples increasing the number of individuals culture- and qPCR-positive to 93 (35%) and positive by either of two methods to 107 (40%). There were significantly more trans-oral than trans-nasal samples positive for pneumococcus by both culture and qPCR (n = 71; 27%; vs. n = 50; 19%; p<0.05). Our data suggest that pneumococcal colonization is more common in adults than previously estimated and point towards the superiority of a trans-oral over a trans-nasal approach when testing adults for colonization with S. pneumoniae.

Effects of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D-conjugate vaccine on nasopharyngeal bacterial colonization in young children: a randomized controlled trial.

BACKGROUND: This study evaluated the effects of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) on nasopharyngeal bacterial colonization compared with the 7-valent pneumococcal conjugate vaccine (7vCRM) in young children. METHODS: A randomized controlled trial in the Netherlands, initiated 2 years after 7vCRM introduction, was conducted between 1 April 2008 and 1 December 2010. Infants (N = 780) received either PHiD-CV or 7vCRM (2:1) at 2, 3, 4, and 11-13 months of age. Nasopharyngeal samples taken at 5, 11, 14, 18, and 24 months of age were cultured to detect Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus. Polymerase chain reaction assays quantified H. influenzae and S. pneumoniae and confirmed H. influenzae as nontypeable (NTHi). Primary outcome measure was vaccine efficacy (VE) against NTHi colonization. RESULTS: In both groups, NTHi colonization increased with age from 33% in 5-month-olds to 65% in 24-month-olds. Three months postbooster, VE against colonization was 0.5% (95% confidence interval [CI], -21.8% to 18.4%) and VE against acquisition 10.9% (95% CI, -31.3% to 38.9%). At each sampling moment, no differences between groups in either NTHi prevalence or H. influenzae density were detected. Streptococcus pneumoniae (range, 39%-57%), M. catarrhalis (range, 63%--69%), and S. aureus (range, 9%-30%) colonization patterns were similar between groups. CONCLUSIONS: PHiD-CV had no differential effect on nasopharyngeal NTHi colonization or H. influenzae density in healthy Dutch children up to 2 years of age, implying that herd effects for NTHi are not to be expected. Other bacterial colonization patterns were also similar.

Alternative sampling methods for detecting bacterial pathogens in children with upper respiratory tract infections.

Nasopharyngeal sampling is used for detecting bacteria commonly involved in upper respiratory tract infections, but it requires training and may not always be well tolerated. We sampled children (n = 66) of ages 0 to 4 years, with rhinorrhea, by using a nasopharyngeal swab, a nasal swab, and nose blowing/wiping into a paper tissue. Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus were cultured at similar rates across methods with high concordance (80 to 97%), indicating that they are reliably detected by alternative means.

Salivary immune responses to the 7-valent pneumococcal conjugate vaccine in the first 2 years of life.

BACKGROUND: The CRM197-conjugated 7-valent pneumococcal vaccine (PCV7) is protective against vaccine serotype disease and nasopharyngeal carriage. Data on PCV7-induced mucosal antibodies in relation to systemic or natural anticapsular antibodies are scarce. METHODS: In a randomized controlled setting, children received PCV7 at age 2 and 4 months (2-dose group), at age 2, 4 and 11 months (2+1-dose group) or no PCV7 (control group). From 188 children paired saliva samples were collected at 12 and 24 months of age. From a subgroup of 15 immunized children also serum samples were collected. IgG and IgA antibody-levels were measured by multiplex immunoassay. RESULTS: At 12 months, both vaccine groups showed higher serum and saliva IgG-levels against vaccine serotypes compared with controls which sustained until 24 months for most serotypes. Salivary IgG-levels were 10-20-fold lower compared to serum IgG, however, serum and saliva IgG-levels were highly correlated. Serum and salivary IgA-levels were higher in both vaccine groups at 12 months compared with controls, except for serotype 19F. Higher salivary IgA levels remained present for most serotypes in the 2+1-dose group until 24 months, but not in the 2-dose group. Salivary IgA more than IgG, increased after documented carriage of serotypes 6B, 19F and 23F In contrast to IgG, salivary IgA-levels were comparable with serum, suggesting local IgA-production. CONCLUSIONS: PCV7 vaccination results in significant increases in salivary IgG and IgA-levels, which are more pronounced for IgG when compared to controls. In contrast, salivary anticapsular IgA-levels seemed to respond more to natural boosting. Salivary IgG and IgA-levels correlate well with systemic antibodies, suggesting saliva might be useful as potential future surveillance tool.

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.

Nasopharyngeal colonization elicits antibody responses to staphylococcal and pneumococcal proteins that are not associated with a reduced risk of subsequent carriage.

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.

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.

Lower immunoglobulin G antibody responses to pneumococcal conjugate vaccination at the age of 2 years after previous nasopharyngeal carriage of Streptococcus pneumoniae.

OBJECTIVE: To determine whether nasopharyngeal pneumococcal carriage with serotypes 6B, 19F, or 23F interferes with immunoglobulin G (IgG) antibody responses to vaccination with 7-valent pneumococcal conjugate vaccine (PCV7) at the age 24 months. STUDY DESIGN: Blood samples were collected before and after a PCV7 challenge vaccination at age 24 months from subsets of children participating in a randomized controlled trial. Children previously had received two doses of PCV7 at 2 and 4 months, two plus one doses of PCV7 at 2, 4, and 11 months, or no dosage until 24 months. Nasopharyngeal swabs were cultured at for Streptococcus pneumoniae at age 6 weeks and at 6, 12, 18, and 24 months. IgG responses were determined with enzyme immunoassay. RESULTS: Lower IgG responses against serotypes 6B, 19F, and 23F were observed on PCV7 challenge vaccination at 24 months in children who had received earlier PCV7 vaccinations and had been found positive for homologous carriage compared with non-carriers of these serotypes. Lower non-homologous IgG responses were observed after the PCV7 challenge at 24 months against serotype 6B after earlier 19F carriage and against serotype 19F after earlier 23F carriage compared with children who had not been positive for carriage of these serotypes. CONCLUSIONS: Pneumococcal colonization with serotypes 6B, 19F, and 23F is associated with diminished immune responses against these serotypes on PCV7 vaccination at 2 years of age. Underlying mechanisms deserve further investigation.

Nontypeable Haemophilus influenzae invasive disease in The Netherlands: a retrospective surveillance study 2001-2008.

BACKGROUND: Nontypeable (unencapsulated) strains of Haemophilus influenzae (ntHi) are usually involved in respiratory tract infections and otitis media but may also cause invasive disease. The epidemiology, the course of disease, and the outcome of ntHi invasive disease are not well established. For prevention, risk groups that might benefit from vaccination have to be defined. METHODS: All patients with ntHi invasive disease confirmed by culture of samples collected by the Netherlands Reference Laboratory for Bacterial Meningitis from 41 sentinel hospitals and representative of ∼45% of all Dutch hospitalized ntHi case patients over the period from 2001 through 2008 were included in the study. Data on clinical presentation, course of disease, and outcome as well as patient characteristics and comorbidity were retrospectively retrieved from hospital records. RESULTS: Clinical presentations of 396 cases included mainly invasive pneumonia (190 cases [48%]) and bacteremia without a clinical focus (75 cases [19%]). Comorbidities were present in 327 [83%] and immunodeficiency in 173 [44%] of all cases. The overall case fatality rate within the first month after diagnosis was 12% and the lowest (2%) was among patients aged 5-54 years. The highest extrapolated age-specific incidence rates occurred within the first 6 weeks of life (19.0 cases per 100,000 persons), concerning mostly prematurely born infants with bacteremia within 24 h after birth, and in the first year of life (5.6 cases per 100,000 persons). The highest rate in adults was among elderly patients aged >65 years (2.2 cases per 100,000 persons). CONCLUSIONS: This study provides a detailed overview of invasive ntHi disease cases in the Netherlands. Risk groups are prematurely born infants, elderly patients aged >65 years, and immunocompromised patients.

Impact of infant and preschool pertussis vaccinations on memory B-cell responses in children at 4 years of age.

Whooping cough, caused by Bordetella pertussis, is reemerging in the vaccinated population. Antibody levels to pertussis antigens wane rapidly after both whole-cell (wP) and acellular pertussis (aP) vaccination and protection may largely depend on long-term B- and T-cell immunity. We studied the effect of wP and aP infant priming at 2, 3, 4 and 11 months according to the Dutch immunization program on pertussis-specific memory B-cell responses before and after a booster vaccination with either a high- or low-pertussis dose vaccine at 4 years of age. Purified B-cells were characterized by FACS-analysis and after polyclonal stimulation, memory B-cells were detected by ELISPOT-assays specific for pertussis toxin, filamentous haemagglutinin and pertactin. Before and after the booster, higher memory B-cell responses were measured in aP primed children compared with wP primed children. In contrast with antibody levels, no dose-effect was observed on the numbers of memory B-cell responses. In aP primed children a fifth high-dose aP vaccination tended to induce even lower memory B-cell responses than a low-dose aP booster. In both wP and aP primed children, the number of memory B-cells increased after the booster and correlated with the pertussis-specific antibody concentrations and observed affinity maturation. This study indicates that aP vaccinations in the first year of life induce higher pertussis-specific memory B-cell responses in children 4 years of age compared with Dutch wP primary vaccinations. Since infant aP vaccinations have improved protection against whooping cough in children despite waning antibody levels, this suggests that an enhanced memory B-cell pool induction may have an important role in protection. However, the pertussis-dose of the preschool booster needs to be considered depending on the vaccine used for priming to optimize long-term protection against whooping cough.

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 child's 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 (n = 336), 2+1-doses (336) or no dose (n = 333) 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%; p = 0.019). 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.