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In the Netherlands, the national immunization program includes 7-valent pneumococcal conjugate vaccine (PCV7) for all newborns born after April 1, 2006. We compared the incidence of invasive pneumococcal disease (IPD) and patient and disease characteristics before PCV7 introduction (June 2004-June 2006) with those after PCV7 introduction (June 2008-June 2010). Culture-confirmed IPD cases were identified by 9 sentinel laboratories covering ≈25% of the Dutch population. Significant declines in overall IPD incidence were observed in children <2 (60%) and in persons >65 (13%) years of age. A trend toward gradual increases in non-PCV7 serotype IPD infections was observed in all age groups; the largest increases were among persons 50-64 (37%) and >65 (25%) years of age. In adults, the proportion of immunocompromised persons increased among IPD patients. Overall, deaths from IPD decreased from 16% to 12% because of a lower case-fatality rate for persons with non-PCV7 serotype IPD. [ABSTRACT FROM AUTHOR]

SUMMARYThe burden of respiratory infections is mainly seen in primary healthcare. To evaluate the potential impact of new preventive strategies against respiratory infections, such as the implementation of pneumococcal conjugate vaccines for infants in 2006 in The Netherlands, we conducted a baseline retrospective cohort study of electronic primary-care patient records to assess consultation rates, comorbidities and antibiotic prescription rates for respiratory infections in primary care. We found that between 1995 and 2005, overall registered consultation rates for lower respiratory tract infections had increased by 42·4%, upper respiratory infections declined by 4·9%, and otitis media remained unchanged. Concomitantly, there was a steady rise in overall comorbidity (75·7%) and antibiotic prescription rates (67·7%). Since Dutch primary-care rates for respiratory infections changed considerably between 1995 and 2005, these changes must be taken into account to properly evaluate the effect of population-based preventive strategies on primary-care utilization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the Netherlands, the 7-valent pneumococcal conjugate vaccine (PCV-7) was implemented in a 3+1-dose schedule in the national immunization program for infants born after April 1, 2006. To assess the vaccine's effectiveness, we compared disease incidence before and after vaccine implementation (June 2004-June 2006 and June 2006-June 2008, respectively). We serotyped 2,552 invasive pneumococcal isolates from throughout the Netherlands, covering 25% of the country's population. Clinical characteristics were extracted from hospital records. After June 2006, vaccine-serotype invasive pneumococcal disease (IPD) decreased 90% (95% confidence interval [CI] 68%-97%) in children age eligible for PCV-7; simultaneously, however, non-vaccine-serotype IPD increased by 71% (not significant), resulting in a 44% total net IPD reduction (95% CI 7%-66%). IPD rates did not change for other age groups. In the Netherlands, PCV-7 offered high protection against vaccine-serotype IPD in vaccinated children, but increases of non-vaccine-serotype IPD reduced net vaccine benefits. [ABSTRACT FROM AUTHOR]

Few studies have examined the relationship between viral activity and bacterial invasive disease, considering both influenza virus and respiratory syncytial virus (RSV). This study aimed to assess the potential relationship between invasive pneumococcal disease (IPD), meningococcal disease (MD), and influenza virus and RSV activity in The Netherlands. Correlations were determined between population-based data on IPD and MD during 1997-2003 and influenza virus and RSV surveillance data. Incidence rate ratios of disease during periods of high influenza virus and RSV activity over the peri-seasonal and summer baseline periods were calculated. The analyses comprised 7266 and 3072 cases of IPD and MD. When data from all seasons were included, the occurrence of pneumococcal bacteraemia and MD correlated significantly with influenza virus and RSV activity both in children and adults. Periods of increased influenza virus and RSV activity showed higher rates of pneumococcal bacteraemia in older children and adults than the peri-season period. Rates of MD in children were also higher during periods of increased influenza virus activity; the same appeared true for MD in older children during periods of increased RSV activity. Although no causal relationship may be inferred from these data, they support a role for influenza virus and RSV in the pathogenesis of IPD and MD. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To determine whether vaccinations aggravate the course of autoimmune diseases such as juvenile idiopathic arthritis (JIA) and whether the immune response to vaccinations may be hampered by immunosuppressive therapy for the underlying disease. METHODS: In this multicenter cohort study, 234 patients with JIA (ages 1-19 years) were vaccinated with meningococcal serogroup C (MenC) conjugate to protect against serogroup C disease (caused by Neisseria meningitidis). Patients were followed up for disease activity for 1 year, from 6 months before until 6 months after vaccination. IgG antibody titers against MenC polysaccharide and the tetanus carrier protein were determined by enzyme-linked immunosorbent assay and toxin binding inhibition assay, respectively. A serum bactericidal assay was performed to determine the function of the anti-MenC antibodies. RESULTS: No change in values for any of the 6 components of the core set criteria for juvenile arthritis disease activity was seen after MenC vaccination. Moreover, no increase in the frequency of disease relapse was detected. Mean anti-MenC IgG concentrations in JIA patients rose significantly within 6-12 weeks after vaccination. Of 157 patients tested, 153 were able to mount anti-MenC IgG serum levels >2 micro g/ml, including patients receiving highly immunosuppressive medication. The 4 patients with a lower anti-MenC antibody response displayed sufficient bactericidal activity despite receiving highly immunosuppressive medication. CONCLUSION: The MenC conjugate vaccine does not aggravate JIA disease activity or increase relapse frequency and results in adequate antibody levels, even in patients receiving highly immunosuppressive medication. Therefore, patients with JIA can be vaccinated safely and effectively with the MenC conjugate. [ABSTRACT FROM AUTHOR]

Background: Immunization with meningococcal ACWY conjugate vaccine induces protective antibodies against invasive meningococcal disease (IMD) caused by serogroups A, C, W and Y. We studied MenACWY-TT vaccine immunogenicity in adolescents with a heterogenous group of primary and secondary immune deficiency including patients with systemic lupus erythematosus, mixed connective tissue disease, vasculitis, uveitis, 22Q11 syndrome, sickle cell disease, and patients who underwent stem cell transplantation for bone marrow failure., Findings: We enrolled 69 individuals aged 14-18 years diagnosed with a primary or secondary immune deficiency in a prospective observational cohort study. All patients received a single dose of MenACWY-TT vaccine during the catch-up campaign 2018-19 because of the IMD-W outbreak in the Netherlands. Capsular polysaccharide-specific (PS) IgG concentrations against MenACWY were measured before and 3-6, 12, and 24 months after vaccination. Overall, geometric mean concentrations (GMCs) of MenACWY-PS-specific IgG were lower in patients compared to data from healthy, aged-matched controls (n = 75) reaching significance at 12 months postvaccination for serogroup A and W (adjusted GMC ratios 0.26 [95% CI: 0.15-0.47] and 0.22 [95% CI: 0.10-0.49], respectively). No serious adverse events were reported by study participants., Conclusions: The MenACWY conjugate vaccine was less immunogenic in adolescent patients with primary or secondary immunodeficiency compared to healthy controls, urging the need for further surveillance of these patients and supporting considerations for booster MenACWY conjugate vaccinations in these patient groups., (© 2023. The Author(s).)

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules 1-5 . Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo 6 . To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the Kras LSL-G12D/+ Trp53 fl/fl mouse model (KP/K b Strep) 7 . This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8 + T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance 8 . Beyond cancer, the K b Strep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: While physicians are often confronted with immunoglobulin A (IgA) deficiency in children with recurrent infections, the clinical relevance of this finding is unclear. Large-scale studies examining the significance of IgA deficiency in children are hampered by differences in techniques for measuring IgA and the physiological increase of IgA with age. Both result in a variety of reference values used for diagnosing IgA deficiency. We propose a new laboratory-independent method to accurately compare IgA measurements in children of varying ages., Methods: We present a method to standardise IgA values for age and laboratory differences. We applied this method to a multicentre case-control study of children under the age of seven suffering from recurrent respiratory tract infections (rRTI, cases) and children who had IgA measured as part of coeliac disease screening (controls). We defined IgA deficiency as serum IgA measurements < 2.5% for age-specific reference values., Results: We developed reference values for IgA for seven age groups and five different laboratory assays. Using these reference values, IgA measurements from 417 cases and 224 controls were standardised to compare groups. In children aged 2 years and older, IgA deficiency was observed in 2.9% (7/242) of cases and 0% (0/189) of controls ( P  = 0.02)., Conclusion: We present a method to compare IgA values in cohorts that vary in age and laboratory assay. This way, we showed that IgA deficiency was more prevalent in children with rRTI compared with controls. This implicates that IgA deficiency may be a clinically relevant condition, even in young children., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Background: Prior to introducing pneumococcal conjugate vaccines (PCVs), Streptococcus pneumoniae was most commonly isolated from the middle ear fluid of children with acute otitis media (AOM). Reducing nasopharyngeal colonisation of this bacterium by PCVs may lead to a decline in AOM. The effects of PCVs deserve ongoing monitoring since studies from the post-PCV era report a shift in causative otopathogens towards non-vaccine serotypes and other bacteria. This updated Cochrane Review was first published in 2002 and updated in 2004, 2009, 2014, and 2019., Objectives: To assess the effect of PCVs in preventing AOM in children up to 12 years of age., Search Methods: We searched CENTRAL, MEDLINE, Embase, CINAHL, LILACS, Web of Science, and two trials registers, ClinicalTrials.gov and WHO ICTRP, to 11 June 2020., Selection Criteria: Randomised controlled trials of PCV versus placebo or control vaccine., Data Collection and Analysis: We used the standard methodological procedures expected by Cochrane. The primary outcomes were frequency of all-cause AOM and adverse effects. Secondary outcomes included frequency of pneumococcal AOM and frequency of recurrent AOM (defined as three or more AOM episodes in six months or four or more in one year). We used GRADE to assess the certainty of the evidence., Main Results: We included 15 publications of 11 trials (60,733 children, range 74 to 37,868 per trial) of 7- to 11-valent PCVs versus control vaccines (meningococcus type C vaccine in three trials, and hepatitis A or B vaccine in eight trials). We included one additional publication of a previously included trial for this 2020 update. We did not find any relevant trials with the newer 13-valent PCV. Most studies were funded by pharmaceutical companies. Overall, risk of bias was low. In seven trials (59,415 children), PCVs were administered in early infancy, whilst four trials (1318 children) included children aged one year and over who were either healthy or had a history of respiratory illness. There was considerable clinical heterogeneity across studies, therefore we reported results from individual studies. PCV administered in early infancy PCV7 The licenced 7-valent PCV with CRM197 as carrier protein (CRM197-PCV7) was associated with a 6% (95% confidence interval (CI) -4% to 16%; 1 trial; 1662 children) and 6% (95% CI 4% to 9%; 1 trial; 37,868 children) relative risk reduction (RRR) in low-risk infants (moderate-certainty evidence), but was not associated with a reduction in all-cause AOM in high-risk infants (RRR -5%, 95% CI -25% to 12%). PCV7 with the outer membrane protein complex of Neisseria meningitidis serogroup B as carrier protein (OMPC-PCV7) was not associated with a reduction in all-cause AOM (RRR -1%, 95% CI -12% to 10%; 1 trial; 1666 children; low-certainty evidence). CRM197-PCV7 and OMPC-PCV7 were associated with 20% (95% CI 7% to 31%) and 25% (95% CI 11% to 37%) RRR in pneumococcal AOM, respectively (2 trials; 3328 children; high-certainty evidence), and CRM197-PCV7 with 9% (95% CI -12% to 27%) and 10% (95% CI 7% to 13%) RRR in recurrent AOM (2 trials; 39,530 children; moderate-certainty evidence). PHiD-CV10/11 The effect of a licenced 10-valent PCV conjugated to protein D, a surface lipoprotein of Haemophilus influenzae, (PHiD-CV10) on all-cause AOM in healthy infants varied from 6% (95% CI -6% to 17%; 1 trial; 5095 children) to 15% (95% CI -1% to 28%; 1 trial; 7359 children) RRR (low-certainty evidence). PHiD-CV11 was associated with 34% (95% CI 21% to 44%) RRR in all-cause AOM (1 trial; 4968 children; moderate-certainty evidence). PHiD-CV10 and PHiD-CV11 were associated with 53% (95% CI 16% to 74%) and 52% (95% CI 37% to 63%) RRR in pneumococcal AOM (2 trials; 12,327 children; high-certainty evidence), and PHiD-CV11 with 56% (95% CI -2% to 80%) RRR in recurrent AOM (1 trial; 4968 children; low-certainty evidence). PCV administered at a later age PCV7 We found no evidence of a beneficial effect on all-cause AOM of administering CRM197-PCV7 in children aged 1 to 7 years with a history of respiratory illness or frequent AOM (2 trials; 457 children; moderate-certainty evidence) and CRM197-PCV7 combined with a trivalent influenza vaccine in children aged 18 to 72 months with a history of respiratory tract infections (1 trial; 597 children; moderate-certainty evidence). CRM197-PCV9 In 1 trial including 264 healthy daycare attendees aged 1 to 3 years, CRM197-PCV9 was associated with 17% (95% CI -2% to 33%) RRR in parent-reported all-cause otitis media (very low-certainty evidence). Adverse events Nine trials reported on adverse effects (77,389 children; high-certainty evidence). Mild local reactions and fever were common in both groups, and occurred more frequently in PCV than in control vaccine groups: redness (< 2.5 cm): 5% to 20% versus 0% to 16%; swelling (< 2.5 cm): 5% to 12% versus 0% to 8%; and fever (< 39 °C): 15% to 44% versus 8% to 25%. More severe redness (> 2.5 cm), swelling (> 2.5 cm), and fever (> 39 °C) occurred less frequently (0% to 0.9%, 0.1% to 1.3%, and 0.4% to 2.5%, respectively) in children receiving PCV, and did not differ significantly between PCV and control vaccine groups. Pain or tenderness, or both, was reported more frequently in PCV than in control vaccine groups: 3% to 38% versus 0% to 8%. Serious adverse events judged to be causally related to vaccination were rare and did not differ significantly between groups, and no fatal serious adverse event judged causally related to vaccination was reported., Authors' Conclusions: Administration of the licenced CRM197-PCV7 and PHiD-CV10 during early infancy is associated with large relative risk reductions in pneumococcal AOM. However, the effects of these vaccines on all-cause AOM is far more uncertain based on low- to moderate-certainty evidence. We found no evidence of a beneficial effect on all-cause AOM of administering PCVs in high-risk infants, after early infancy, and in older children with a history of respiratory illness. Compared to control vaccines, PCVs were associated with an increase in mild local reactions (redness, swelling), fever, and pain and/or tenderness. There was no evidence of a difference in more severe local reactions, fever, or serious adverse events judged to be causally related to vaccination., (Copyright © 2020 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.)

Background: Prior to introducing pneumococcal conjugate vaccines (PCVs), Streptococcus pneumoniae was most commonly isolated from middle ear fluid of children with acute otitis media (AOM). Reducing nasopharyngeal colonisation of this bacterium by PCVs may lead to a decline in AOM. The effects of PCVs deserve ongoing monitoring since studies from the post-PCV era report a shift in causative otopathogens towards non-vaccine serotypes and other bacteria. This updated Cochrane Review was first published in 2002 and updated in 2004, 2009, and 2014. The review title was changed (to include the population, i.e. children) for this update., Objectives: To assess the effect of PCVs in preventing AOM in children up to 12 years of age., Search Methods: We searched CENTRAL, MEDLINE, Embase, CINAHL, LILACS, Web of Science, and trials registers (ClinicalTrials.gov and WHO ICTRP) to 29 March 2019., Selection Criteria: Randomised controlled trials of PCV versus placebo or control vaccine., Data Collection and Analysis: We used the standard methodological procedures expected by Cochrane. The primary outcomes were frequency of all-cause AOM and adverse effects. Secondary outcomes included frequency of pneumococcal AOM and frequency of recurrent AOM (defined as three or more AOM episodes in six months or four or more in one year). We used GRADE to assess the quality of the evidence., Main Results: We included 14 publications of 11 trials (60,733 children, range 74 to 37,868 per trial) of 7- to 11-valent PCVs versus control vaccines (meningococcus type C vaccine in three trials, and hepatitis A or B vaccine in eight trials). We included two additional trials for this update. We did not find any relevant trials with the newer 13-valent PCV. Most studies were funded by pharmaceutical companies. Overall, risk of bias was low. In seven trials (59,415 children) PCVs were administered in early infancy, while four trials (1318 children) included children aged one year and over who were either healthy or had a history of respiratory illness. There was considerable clinical heterogeneity across studies, therefore we did not perform meta-analyses.Adverse eventsNine trials reported on adverse effects (77,389 children; high-quality evidence). Mild local reactions and fever were common in both groups, and occurred more frequently in PCV than in control vaccine groups: redness (< 2.5 cm): 5% to 20% versus 0% to 16%; swelling (< 2.5 cm): 5% to 12% versus 0% to 8%; and fever (< 39 °C): 15% to 44% versus 8% to 25%. More severe redness (> 2.5 cm), swelling (> 2.5 cm), and fever (> 39 °C) occurred less frequently (0% to 0.9%, 0.1% to 1.3%, and 0.4% to 2.5%, respectively in children receiving PCV) and did not differ significantly between PCV and control vaccine groups. Pain or tenderness, or both was reported more frequently in PCV than in control vaccine groups: 3% to 38% versus 0% to 8%. Serious adverse events judged causally related to vaccination were rare and did not differ significantly between groups, and no fatal serious adverse event judged causally related to vaccination was reported.PCV administered in early infancyPCV7The effect of a licenced 7-valent PCV with CRM197 as carrier protein (CRM197-PCV7) on all-cause AOM varied from -5% (95% confidence interval (CI) -25% to 12%) relative risk reduction (RRR) in high-risk infants (1 trial; 944 children; moderate-quality evidence) to 6% (95% CI -4% to 16%; 1 trial; 1662 children) and 6% (95% CI 4% to 9%; 1 trial; 37,868 children) RRR in low-risk infants (high-quality evidence). PCV7 with the outer membrane protein complex of Neisseria meningitidis serogroup B as carrier protein (OMPC-PCV7), was not associated with a reduction in all-cause AOM (RRR -1%, 95% CI -12% to 10%; 1 trial; 1666 children; high-quality evidence).CRM197-PCV7 and OMPC-PCV7 were associated with 20% (95% CI 7% to 31%) and 25% (95% CI 11% to 37%) RRR in pneumococcal AOM, respectively (2 trials; 3328 children; high-quality evidence) and CRM197-PCV7 with 9% (95% CI -12% to 27%) to 10% (95% CI 7% to 13%) RRR in recurrent AOM (2 trials; 39,530 children; high-quality evidence).PHiD-CV10/11The effect of a licenced 10-valent PCV conjugated to protein D, a surface lipoprotein of Haemophilus influenzae, (PHiD-CV10) on all-cause AOM varied from 6% (95% CI -6% to 17%; 1 trial; 5095 children) to 15% (95% CI -1% to 28%; 1 trial; 7359 children) RRR in healthy infants (moderate-quality evidence). PHiD-CV11 was associated with 34% (95% CI 21% to 44%) RRR in all-cause AOM (1 trial; 4968 children; high-quality evidence).PHiD-CV10 and PHiD-CV11 were associated with 53% (95% CI 16% to 74%) and 52% (95% CI 37% to 63%) RRR in pneumococcal AOM (2 trials; 12,327 children; high-quality evidence) and PHiD-CV11 with 56% (95% CI -2% to 80%) RRR in recurrent AOM (1 trial; 4968 children; moderate-quality evidence).PCV administered at later agePCV7We found no evidence of a beneficial effect on all-cause AOM of administering CRM197-PCV7 in children aged 1 to 7 years with a history of respiratory illness or frequent AOM (2 trials; 457 children; high-quality evidence) and CRM197-PCV7 combined with a trivalent influenza vaccine in children aged 18 to 72 months with a history of respiratory tract infections (1 trial; 597 children; high-quality evidence).CRM197-PCV9In 1 trial including 264 healthy day-care attendees aged 1 to 3 years, CRM197-PCV9 was associated with 17% (95% CI -2% to 33%) RRR in parent-reported all-cause OM (low-quality evidence)., Authors' Conclusions: Administration of the licenced CRM197-PCV7 and PHiD-CV10 during early infancy is associated with large relative risk reductions in pneumococcal AOM. However, the effects of these vaccines on all-cause AOM is far more uncertain. We found no evidence of a beneficial effect on all-cause AOM of administering PCVs in high-risk infants, after early infancy (i.e. in children one year and above), and in older children with a history of respiratory illness. Compared to control vaccines, PCVs were associated with an increase in mild local reactions (redness, swelling), fever, and pain and/or tenderness. We found no evidence of a difference in more severe local reactions, fever, or serious adverse events judged causally related to vaccination.

Fed-batch processes are commonly used tools in high cell density cultivation of Escherichia coli . However, the setup of a fed-batch process can be challenging, especially when working with genetically modified strains. This work deals with the design of a specific strategy for the leucine auxotrophic E. coli strain K12 ER2507 that is of interest for arabinose-inducible gene expression systems due to its ara-14 mutation. To set up this process the optimum yield coefficient of biomass from leucine was determined by use of a design of experiments tool. Unfortunately, the yield coefficient is negatively influenced by both leucine and glucose concentrations. Furthermore, an inhibitory effect of leucine reduced the specific growth rate even at low concentration. Under consideration of these problems, an improved high cell density cultivation (iHCDC) for E. coli K12 ER2507 was established. The start medium was set up without any C-source to omit a batch phase with high glucose concentration and substrate feeding was initiated directly upon inoculation. Due to the poor solubility of leucine in feed medium, the amino acid was dissolved in the base. With the improved high cell density cultivation process a final cell density of more than 90 g/L was obtained reproducibly., (© 2017 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

We provide a tutorial on differential item functioning (DIF) analysis, an analytic method useful for identifying potentially biased items in assessments. After explaining a number of methodological approaches, we test for gender bias in two scenarios that demonstrate why DIF analysis is crucial for developing assessments, particularly because simply comparing two groups' total scores can lead to incorrect conclusions about test fairness. First, a significant difference between groups on total scores can exist even when items are not biased, as we illustrate with data collected during the validation of the Homeostasis Concept Inventory. Second, item bias can exist even when the two groups have exactly the same distribution of total scores, as we illustrate with a simulated data set. We also present a brief overview of how DIF analysis has been used in the biology education literature to illustrate the way DIF items need to be reevaluated by content experts to determine whether they should be revised or removed from the assessment. Finally, we conclude by arguing that DIF analysis should be used routinely to evaluate items in developing conceptual assessments. These steps will ensure more equitable-and therefore more valid-scores from conceptual assessments., (© 2017 P. Martinková et al. CBE—Life Sciences Education © 2017 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Objective: We examined associations between observed neighborhood conditions (good/adverse) and psychosocial outcomes (stress, depressive symptoms, resilience, and sense of control) among middle-aged and older African Americans., Methods: The sample included 455 middle-aged and older African Americans examined in Wave 10 of the African American Health (AAH) study. Linear regression was adjusted for attrition, self-selection into neighborhoods, and potential confounders, and stratified by the duration at current address (<5 vs ≥5 years) because of its hypothesized role as an effect modifier., Results: Among individuals who lived at their current address for ≥5 years, residing in neighborhoods with adverse versus good conditions was associated with significantly less stress (standardized β = -0.18; P = .002) and depressive symptoms (standardized β = -0.12; P = .048). Among those who lived at their current address for <5 years, residing in neighborhoods with adverse versus good conditions was not significantly associated with stress (standardized β = 0.18; P = .305) or depressive symptoms (standardized β = 0.36; P = .080)., Conclusion: Neighborhood conditions appear to have significant, complex associations with psychosocial factors among middle-aged and older African Americans. This holds important policy implications, especially since adverse neighborhood conditions may still result in adverse physical health outcomes in individuals with >5 years at current residence despite being associated with better psychosocial outcomes.

Nasopharyngeal and oropharyngeal samples are commonly used to direct therapy for lower respiratory tract infections in non-expectorating infants with cystic fibrosis (CF).We aimed to investigate the concordance between the bacterial community compositions of 25 sets of nasopharyngeal, oropharyngeal and bronchoalveolar lavage (BAL) samples from 17 infants with CF aged ∼5 months (n=13) and ∼12 months (n=12) using conventional culturing and 16S-rRNA sequencing.Clustering analyses demonstrated that BAL microbiota profiles were in general characterised by a mixture of oral and nasopharyngeal bacteria, including commensals like Streptococcus , Neisseria , Veillonella and Rothia spp. and potential pathogens like Staphylococcus aureus , Haemophilus influenzae and Moraxella spp. Within each individual, however, the degree of concordance differed between microbiota of both upper respiratory tract niches and the corresponding BAL.The inconsistent intra-individual concordance between microbiota of the upper and lower respiratory niches suggests that the lungs of infants with CF may have their own microbiome that seems seeded by, but is not identical to, the upper respiratory tract microbiome., (Copyright ©ERS 2017.)

Implementation of pneumococcal conjugate vaccines in the Netherlands (PCV7 in 2006 and PCV10 in 2011) for infants caused a shift in serotypes in invasive pneumococcal disease (IPD). We explored sex differences in serotype-specific IPD incidence before and after vaccine introduction. Incidences in the pre-PCV7 (June 2004-May 2006), post-PCV7 (June 2008-May 2011) and post-PCV10 period (June 2013-May 2015), stratified by age, were compared. Incidence was higher in men for all age groups (overall in men: 16.7, 15.5 and 14.4/100,000 and women: 15.4, 13.6 and 13.9/100,000 pre-PCV7, post-PCV7 and post-PCV10, respectively), except for 20-39 year-olds after PCV7 and 40-64 year-olds after PCV10 introduction. After PCV7 and PCV10 introduction, the overall IPD incidence decreased in men aged 20-39 years (from 5.3 pre-PCV7 to 4.7 and 2.6/100,000 post-PCV7 and post-PCV10, respectively), whereas it showed a temporary increase in women (from 3.9/100,000 pre-PCV7 to 5.0/100,000 post-PCV7 and back to 4.0/100,000 post-PCV10) due to replacement disease. PCV10 herd effects were observed throughout, but in women older than 40 years, a significant increase in non-PCV10 serotype offset a decrease in overall IPD incidence. Ongoing surveillance of IPD incidence by sex is important to evaluate the long-term effects of PCV implementation., (This article is copyright of The Authors, 2017.)

Since the early 1990s, the Netherlands has experienced several large measles epidemics, in 1992-94, 1999-2000 and in 2013-14. These outbreaks mainly affected orthodox Protestants, a geographically clustered population with overall lower measles-mumps-rubella first dose (MMR-1) vaccination coverage (60%) than the rest of the country (> 95%). In the 2013-14 epidemic described here, which occurred between 27 May 2013 and 12 March 2014, 2,700 cases were reported. Several control measures were implemented including MMR vaccination for 6-14-month-olds and recommendations to reduce the risk in healthcare workers. The vast majority of reported cases were unvaccinated (94%, n = 2,539), mostly for religious reasons (84%, n = 2,135). The median age in the epidemic was 10 years, 4 years older than in the previous epidemic in 1999-2000. A likely explanation is that the inter-epidemic interval before the 2013-2014 epidemic was longer than the interval before the 1999-2000 epidemic. The size of the unvaccinated orthodox Protestant community is insufficient to allow endemic transmission of measles in the Netherlands. However, large epidemics are expected in the future, which is likely to interfere with measles elimination in the Netherlands and elsewhere., (This article is copyright of The Authors, 2017.)

Capturing the complexity and waning patterns of co-occurring immunoglobulin (Ig) responses after clinical B. pertussis infection may help understand how the human host gradually loses protection against whooping cough. We applied bi-exponential modelling to characterise and compare B. pertussis specific serological dynamics in a comprehensive database of IgG, IgG subclass and IgA responses to Ptx, FHA, Prn, Fim2/3 and OMV antigens of (ex-) symptomatic pertussis cases across all age groups. The decay model revealed that antigen type and age group were major factors determining differences in levels and kinetics of Ig (sub) classes. IgG-Ptx waned fastest in all age groups, while IgA to Ptx, FHA, Prn and Fim2/3 decreased fast in the younger but remained high in older (ex-) cases, indicating an age-effect. While IgG1 was the main IgG subclass in response to most antigens, IgG2 and IgG3 dominated the anti-OMV response. Moreover, vaccination history plays an important role in post-infection Ig responses, demonstrated by low responsiveness to Fim2/3 in unvaccinated elderly and by elevated IgG4 responses to multiple antigens only in children primed with acellular pertussis vaccine (aP). This work highlights the complexity of the immune response to this re-emerging pathogen and factors determining its Ig quantity and quality.

Rationale: Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections and hospitalizations in infants worldwide. Known risk factors, however, incompletely explain the variability of RSV disease severity, especially among healthy children. We postulate that the severity of RSV infection is influenced by modulation of the host immune response by the local bacterial ecosystem., Objectives: To assess whether specific nasopharyngeal microbiota (clusters) are associated with distinct host transcriptome profiles and disease severity in children less than 2 years of age with RSV infection., Methods: We characterized the nasopharyngeal microbiota profiles of young children with mild and severe RSV disease and healthy children by 16S-rRNA sequencing. In parallel, using multivariable models, we analyzed whole-blood transcriptome profiles to study the relationship between microbial community composition, the RSV-induced host transcriptional response, and clinical disease severity., Measurements and Main Results: We identified five nasopharyngeal microbiota clusters characterized by enrichment of either Haemophilus influenzae, Streptococcus, Corynebacterium, Moraxella, or Staphylococcus aureus. RSV infection and RSV hospitalization were positively associated with H. influenzae and Streptococcus and negatively associated with S. aureus abundance, independent of age. Children with RSV showed overexpression of IFN-related genes, independent of the microbiota cluster. In addition, transcriptome profiles of children with RSV infection and H. influenzae- and Streptococcus-dominated microbiota were characterized by greater overexpression of genes linked to Toll-like receptor and by neutrophil and macrophage activation and signaling., Conclusions: Our data suggest that interactions between RSV and nasopharyngeal microbiota might modulate the host immune response, potentially affecting clinical disease severity.

The earliest studies in the late 19th century on Streptococcus pneumoniae (S. pneumoniae) carriage used saliva as the primary specimen. However, interest in saliva declined after the sensitive mouse inoculation method was replaced by conventional culture, which made isolation of pneumococci from the highly polymicrobial oral cavity virtually impossible. Here, we tested the feasibility of using dried saliva spots (DSS) for studies on pneumococcal carriage. Saliva samples from children and pneumococcus-spiked saliva samples from healthy adults were applied to paper, dried, and stored, with and without desiccant, at temperatures ranging from -20 to 37 °C for up to 35 days. DNA extracted from DSS was tested with quantitative-PCR (qPCR) specifically for S. pneumoniae. When processed immediately after drying, the quantity of pneumococcal DNA detected in spiked DSS from adults matched the levels in freshly spiked raw saliva. Furthermore, pneumococcal DNA was stable in DSS stored with desiccant for up to one month over a broad range of temperatures. There were no differences in the results when spiking saliva with varied pneumococcal strains. The collection of saliva can be a particularly useful in surveillance studies conducted in remote settings, as it does not require trained personnel, and DSS are resilient to various transportation conditions.

Rationale: Cystic fibrosis (CF) is characterized by early structural lung disease caused by pulmonary infections. The nasopharynx of infants is a major ecological reservoir of potential respiratory pathogens., Objectives: To investigate the development of nasopharyngeal microbiota profiles in infants with CF compared with those of healthy control subjects during the first 6 months of life., Methods: We conducted a prospective cohort study, from the time of diagnosis onward, in which we collected questionnaires and 324 nasopharynx samples from 20 infants with CF and 45 age-matched healthy control subjects. Microbiota profiles were characterized by 16S ribosomal RNA-based sequencing., Measurements and Main Results: We observed significant differences in microbial community composition (P < 0.0002 by permutational multivariate analysis of variance) and development between groups. In infants with CF, early Staphylococcus aureus and, to a lesser extent, Corynebacterium spp. and Moraxella spp. dominance were followed by a switch to Streptococcus mitis predominance after 3 months of age. In control subjects, Moraxella spp. enrichment occurred throughout the first 6 months of life. In a multivariate analysis, S. aureus, S. mitis, Corynebacterium accolens, and bacilli were significantly more abundant in infants with CF, whereas Moraxella spp., Corynebacterium pseudodiphtericum and Corynebacterium propinquum and Haemophilus influenzae were significantly more abundant in control subjects, after correction for age, antibiotic use, and respiratory symptoms. Antibiotic use was independently associated with increased colonization of gram-negative bacteria such as Burkholderia spp. and members of the Enterobacteriaceae bacteria family and reduced colonization of potential beneficial commensals., Conclusions: From diagnosis onward, we observed distinct patterns of nasopharyngeal microbiota development in infants with CF under 6 months of age compared with control subjects and a marked effect of antibiotic therapy leading toward a gram-negative microbial composition.

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.

Background: Vaccination of infants with pneumococcal conjugate vaccines (PCV) has resulted in major shifts in circulating serotypes., Aim: To investigate the impact of PCV7 on the clonal composition of the pneumococcal population, and the relation of clonal lineages and clinical outcome., Materials & Methods: By using multiple-locus variable number of tandem repeat analysis, we assessed the pneumococcal populations before (n = 1154), 2-3 years after (n = 1190) and 4-6 years after (n = 1244) the introduction of PCV7 in The Netherlands., Results: We found statistically significant shifts in clonal lineages within serotypes 1 and 12F based on multiple-locus variable number of tandem repeat analysis results after the implementation of PCV7. Within serotype 12F, the increasing clonal lineage was significantly more associated with pneumonia., Conclusion: Shifts in clonal lineages within serotypes could impact the outcomes of pneumococcal disease and fill the niche of the vaccine serotypes.

The Community-Acquired Pneumonia Immunization Trial in Adults (CAPiTA) demonstrated the efficacy of 13-valent pneumococcal conjugate vaccine (PCV13) in preventing vaccine-type community-acquired pneumonia and vaccine-type invasive pneumococcal disease in elderly subjects. We examined the cost-effectiveness of PCV13 vaccination in the Netherlands. Using a Markov-type model, incremental cost-effectiveness ratios (ICER) of PCV13 vaccination in different age- and risk-groups for pneumococcal disease were evaluated using a societal perspective. Estimates of quality-adjusted life-years (QALYs), costs, vaccine efficacy and epidemiological data were based on the CAPiTA study and other prospective studies. The base-case was PCV13 vaccination of adults aged 65-74 years compared to no vaccination, assuming no net indirect effects in base-case due to paediatric 10-valent pneumococcal conjugate vaccine use. Analyses for age- and risk-group specific vaccination strategies and for different levels of hypothetical herd effects from a paediatric PCV programme were also conducted. The ICER for base-case was €8650 per QALY (95% CI 5750-17,100). Vaccination of high-risk individuals aged 65-74 years was cost-saving and extension to medium-risk individuals aged 65-74 years yielded an ICER of €2900. Further extension to include medium- and high-risk individuals aged ≥18 years yielded an ICER of €3100.PCV13 vaccination is highly cost-effective in the Netherlands. The transferability of our results to other countries depends upon vaccination strategies already implemented in those countries., (Copyright ©ERS 2015.)

Three years after a 7-valent pneumococcal conjugate vaccine was replaced by a 10-valent pneumococcal conjugate vaccine in the Netherlands, we observed a decrease in incidence of invasive pneumococcal disease caused by Streptococcus pneumoniae serotypes 1, 5, and 7F. Our data do not support or exclude cross-protection against serotype 19A.

Background: Antibody levels wane rapidly after Meningococcal serogroup C conjugate (MenCC) vaccination in young children, rendering the need for an adolescent booster dose. It is not clear whether circulating memory B cells are associated with persistence of MenC-specific antibody levels., Methods: Measurement of MenC-specific IgG and IgA memory B cells and levels of serum and salivary MenC-specific IgG and IgA in healthy 10-, 12- and 15-year-olds prior to and one month and one year after a MenCC booster vaccination. All participants had received a primary MenCC vaccination nine years earlier., Results: The number of circulating MenC-specific IgG memory B cells prior to booster was low and not predictive for MenC-specific IgG responses in serum or saliva post-booster, whereas the number of MenC-specific IgA memory B cells pre-booster positively correlated with MenC-specific IgA levels in saliva post-booster (R = 0.5, P<0.05). The booster induced a clear increase in the number of MenC-specific IgG and IgA memory B cells. The number of MenC-PS-specific IgG memory B cells at 1 month post-booster was highest in the 12-year-olds. The number of MenC-specific memory B cells at one month post-booster showed no correlation with the rate of MenC-specific antibody decay throughout the first year post-booster., Conclusions: Circulating MenC-specific IgA memory B cells correlate with IgA responses in saliva, whereas circulating MenC-specific IgG memory B cells are not predictive for MenC-specific IgG responses in serum or saliva. Our results are suggestive for age-dependent differences in pre-existing memory against MenC.

Respiratory tract infections are a major global health concern, accounting for high morbidity and mortality, especially in young children and elderly individuals. Traditionally, highly common bacterial respiratory tract infections, including otitis media and pneumonia, were thought to be caused by a limited number of pathogens including Streptococcus pneumoniae and Haemophilus influenzae. However, these pathogens are also frequently observed commensal residents of the upper respiratory tract (URT) and form-together with harmless commensal bacteria, viruses and fungi-intricate ecological networks, collectively known as the 'microbiome'. Analogous to the gut microbiome, the respiratory microbiome at equilibrium is thought to be beneficial to the host by priming the immune system and providing colonization resistance, while an imbalanced ecosystem might predispose to bacterial overgrowth and development of respiratory infections. We postulate that specific ecological perturbations of the bacterial communities in the URT can occur in response to various lifestyle or environmental effectors, leading to diminished colonization resistance, loss of containment of newly acquired or resident pathogens, preluding bacterial overgrowth, ultimately resulting in local or systemic bacterial infections. Here, we review the current body of literature regarding niche-specific upper respiratory microbiota profiles within human hosts and the changes occurring within these profiles that are associated with respiratory infections., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Incidence of pneumococcal disease is disproportionally high in infants and elderly. Nasopharyngeal colonisation by Streptococcus pneumoniae is considered a prerequisite for disease but unlike in children, carriage in elderly is rarely detected. Here, we tested for S. pneumoniae in nasopharyngeal and saliva samples collected from community-dwelling elderly with influenza-like-illness (ILI). Trans-nasal nasopharyngeal, trans-oral nasopharyngeal and saliva samples (n = 270 per sample type) were collected during winter/spring 2011/2012 from 135 persons aged 60-89 at onset of ILI and 7-9 weeks later following recovery. After samples were tested for pneumococci by conventional culture, all plate growth was collected. DNA extracted from plate harvests was tested by quantitative-PCRs (qPCR) specific for S. pneumoniae and serotypes included in the 13-valent pneumococcal conjugated vaccine (PCV13). Pneumococci were cultured from 14 of 135 (10%) elderly with none of the sampled niches showing superiority in carriage detection. With 76/270 (28%) saliva, 31/270 (11%) trans-oral and 13/270 (5%) trans-nasal samples positive by qPCR, saliva was superior to nasopharyngeal swabs (p<0.001) in qPCR-based carriage detection. Overall, from all methods used in the study, 65 of 135 (48%) elderly carried pneumococci at least once and 26 (19%) at both study time points. The difference between carriage prevalence at ILI (n = 49 or 36%) versus recovery (n = 42 or 31%) was not significant (p = 0.38). At least 23 of 91 (25%) carriage events in 19 of 65 (29%) carriers were associated with PCV13-serotypes. We detected a large reservoir of pneumococci in saliva of elderly, with PCV13-serotype distribution closely resembling the contemporary carriage of serotypes reported in the Netherlands for PCV-vaccinated infants.

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).

Analysis of the Dutch national invasive pneumococcal disease (IPD) surveillance data by sex reveals an increase in the incidence of serotype-1 disease in young female adults in The Netherlands after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the national immunization schedule. This has led to an overall increase in IPD in women aged 20-45 years, which was not observed in men of the same age. No other differences in serotype shifts possibly induced by the introduction of PCV7 were observed between the sexes in this age group. Serotype 1 is a naturally fluctuating serotype in Europe and it has been associated with disease in young healthy adults before. It remains uncertain whether or not there is an association between the observed increase in serotype-1 disease in young female adults and the implementation of PCV7 in The Netherlands.

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).

While nasopharyngeal sampling is the gold standard for the detection of Streptococcus pneumoniae carriage, historically seen, saliva sampling also seems highly sensitive for pneumococcal detection. We investigated S. pneumoniae carriage in saliva from fifty schoolchildren by conventional and molecular methods. Saliva was first culture-enriched for pneumococci, after which, DNA was extracted from all bacterial growth and tested by quantitative-PCR (qPCR) for pneumococcus-specific genes lytA and piaA. Next, serotype composition of the samples was determined by serotype-specific qPCRs, conventional-PCRs (cPCR) and sequencing of cPCR amplicons. Although only 2 (4%) of 50 samples were positive by conventional diagnostic culture, 44 (88%) were positive for pneumococci by qPCR. In total, we detected the presence of at least 81 pneumococcal strains representing 20 serotypes in samples from 44 carriers with 23 carriers (52%) positive for multiple (up to 6) serotypes. The number of serotypes detected per sample correlated with pneumococcal abundance. This study shows that saliva could be used as a tool for future pneumococcal surveillance studies. Furthermore, high rates of pneumococcal carriage and co-carriage of multiple pneumococcal strains together with a large number of serotypes in circulation suggests a ubiquitous presence of S. pneumoniae in saliva of school-aged children. Our results also suggest that factors promoting pneumococcal carriage within individual hosts may weaken competitive interactions between S. pneumoniae strains.

Background: Daycare attendance is an established risk factor for upper respiratory tract infections (URTI) and acute otitis media (AOM). Whether this results in higher use of healthcare resources during childhood remains unknown. We aim to assess the effect of first year daycare attendance on the timing and use of healthcare resources for URTI and AOM episodes during early childhood., Methods: In the Wheezing-Illnesses-STudy-LEidsche-Rijn birth cohort, 2,217 children were prospectively followed up to age six years. Children were categorized according to first-year daycare attendance (yes versus no) and age at entry when applicable (age 0 to 2 months, 3 to 5 months and 6 to 12 months). Information on general practitioner (GP) diagnosed URTI and AOM, GP consultations, antibiotic prescriptions and specialist referral was collected from medical records. Daycare attendance was recorded by monthly questionnaires during the first year of life., Results: First-year daycare attendees and non-attendees had similar total six-year rates of GP-diagnosed URTI and AOM episodes (59/100 child-years, 95% confidence interval 57 to 61 versus 56/100 child-years, 53 to 59). Daycare attendees had more GP-diagnosed URTI and AOM episodes before the age of one year and fewer beyond the age of four years than non-attendees (Pinteraction <0.001). Daycare attendees had higher total six-year rates for GP consultation (adjusted rate ratio 1.15, 1.00 to 1.31) and higher risk for specialist referrals (hazard ratio: 1.43, 1.01 to 2.03). The number of antibiotic prescriptions in the first six years of life was only significantly increased among children who entered daycare between six to twelve months of age (rate ratio 1.32, 1.04 to 1.67). This subgroup of child-care attendees also had the highest overall URTI and AOM incidence rates, GP consultation rates and risk for specialist referral., Conclusions: Children who enter daycare in the first year of life, have URTI and AOM at an earlier age, leading to higher use of healthcare resources compared to non-attendees, especially when entering daycare between six to twelve months. These findings emphasize the need for improved prevention strategies in daycare facilities to lower infection rates at the early ages.

Background: Meningococcal serogroup C (MenC) specific antibody levels decline rapidly after a single primary MenC conjugate (MenCC) vaccination in preschool children. A second MenCC vaccination during (pre)adolescence might attain longer lasting individual and herd protection. We aimed to establish an appropriate age for a (pre)adolescent MenCC booster vaccination., Methods: A phase-IV trial with healthy 10-year-olds (n = 91), 12-year-olds (n = 91) and 15-year-olds (n = 86) who were primed with a MenCC vaccine nine years earlier. All participants received a booster vaccination with the same vaccine. Serum bactericidal antibody assay titers (SBA, using baby rabbit complement), MenC-polysaccharide (MenC-PS) specific IgG, IgG subclass and avidity and tetanus-specific IgG levels were measured prior to (T0) and 1 month (T1) and 1 year (T2) after the booster. An SBA titer ≥8 was the correlate of protection., Results: 258 (96.3%) participants completed all three study visits. At T0, 19% of the 10-year-olds still had an SBA titer ≥8, compared to 34% of the 12-year-olds (P = 0.057) and 45% of the 15-year-olds (P<0.001). All participants developed high SBA titers (GMTs>30,000 in all age groups) and MenC-PS specific IgG levels at T1. IgG levels mainly consisted of IgG1, but the contribution of IgG2 increased with age. At T2, 100% of participants still had an SBA titer ≥8, but the 15-year-olds showed the highest protective antibody levels and the lowest decay., Conclusion: Nine years after primary MenCC vaccination adolescents develop high protective antibody levels in response to a booster and are still sufficiently protected one year later. Our results suggest that persistence of individual--and herd--protection increases with the age at which an adolescent booster is administered., Trial Registration: EU Clinical Trials Database 2011-000375-13 Dutch Trial Register NTR3521.

Background: Acute otitis media (AOM) is a very common respiratory infection in early infancy and childhood. The marginal benefits of antibiotics for AOM in low-risk populations in general, the increasing problem of bacterial resistance to antibiotics and the huge estimated direct and indirect annual costs associated with otitis media (OM) have prompted a search for effective vaccines to prevent AOM., Objectives: To assess the effect of pneumococcal conjugate vaccines (PCVs) in preventing AOM in children up to 12 years of age., Search Methods: We searched CENTRAL (2013, Issue 11), MEDLINE (1995 to November week 3, 2013), EMBASE (1995 to December 2013), CINAHL (2007 to December 2013), LILACS (2007 to December 2013) and Web of Science (2007 to December 2013)., Selection Criteria: Randomised controlled trials (RCTs) of PCVs to prevent AOM in children aged 12 years or younger, with a follow-up of at least six months after vaccination., Data Collection and Analysis: Two review authors independently assessed trial quality and extracted data., Main Results: We included 11 publications of nine RCTs (n = 48,426 children, range 74 to 37,868 per study) of 7- to 11-valent PCV (with different carrier proteins). Five trials (n = 47,108) included infants, while four trials (n = 1318) included children aged one to seven years that were either healthy (one study, n = 264) or had a previous history of upper respiratory tract infection (URTI), including AOM. We judged the methodological quality of the included studies to be moderate to high. There was considerable clinical diversity between studies in terms of study population, type of conjugate vaccine and outcome measures. We therefore refrained from pooling the results.In three studies, the 7-valent PCV with CRM197 as carrier protein (CRM197-PCV7) administered during early infancy was associated with a relative risk reduction (RRR) of all-cause AOM ranging from -5% in high-risk children (95% confidence interval (CI) -25% to 12%) to 7% in low-risk children (95% CI 4% to 9%). Another 7-valent PCV with the outer membrane protein complex of Neisseria meningitidis (N. meningitidis) serogroup B as carrier protein, administered in infancy, did not reduce overall AOM episodes, while a precursor 11-valent PCV with Haemophilus influenzae (H. influenzae) protein D as carrier protein was associated with a RRR of all-cause AOM episodes of 34% (95% CI 21% to 44%).A 9-valent PCV (with CRM197 carrier protein) administered in healthy toddlers was associated with a RRR of (parent-reported) OM episodes of 17% (95% CI -2% to 33%). CRM197-PCV7 followed by 23-valent pneumococcal polysaccharide vaccination administered after infancy in older children with a history of AOM showed no beneficial effect on first occurrence and later AOM episodes. In a study in older children with a previously diagnosed respiratory tract infection, performed during the influenza season, a trivalent influenza vaccine combined with placebo (TIV/placebo) led to fewer all-cause AOM episodes than vaccination with TIV and PCV7 (TIV/PCV7) when compared to hepatitis B vaccination and placebo (HBV/placebo) (RRR 71%, 95% CI 30% to 88% versus RRR 57%, 95% CI 6% to 80%, respectively) indicating that CRM197-PCV7 after infancy may even have negative effects on AOM., Authors' Conclusions: Based on current evidence of the effects of PCVs for preventing AOM, the licensed 7-valent CRM197-PCV7 has modest beneficial effects in healthy infants with a low baseline risk of AOM. Administering PCV7 in high-risk infants, after early infancy and in older children with a history of AOM, appears to have no benefit in preventing further episodes. Currently, several RCTs with different (newly licensed, multivalent) PCVs administered during early infancy are ongoing to establish their effects on AOM. Results of these studies may provide a better understanding of the role of the newly licensed, multivalent PCVs in preventing AOM. Also the impact on AOM of the carrier protein D, as used in certain pneumococcal vaccines, needs to be further established.

The upper respiratory tract (URT) is a distinct microbial niche of low-density bacterial communities and, also, a portal of entry for many potential pathogens, including Streptococcus pneumoniae. Thus far, animal models have been used to study the dynamics of and interactions between limited numbers of different species in the URT. Here, we applied a deep sequencing approach to explore, for the first time, the impact of S. pneumoniae acquisition on URT microbiota in a mouse model, as well as potential age-dependent effects. Young-adult and elderly mice were inoculated intranasally with S. pneumoniae, and nasal lavage samples were collected for up to 28 days postcolonization. Bacterial DNA extracted from lavage samples was subjected to barcoded pyrosequencing of the V5-to-V7 hypervariable region of the small-subunit rRNA gene. We observed highly diverse microbial profiles, with the presence overall of 15 phyla and approximately 645 operational taxonomic units (OTUs). We noted differences in the composition of microbiota between young and elderly mice, with a significantly higher abundance of Bacteroidetes in the young mice. The introduction of S. pneumoniae into the URT led to a temporary dominance of pneumococci in the microbiota of all mice, accompanied by a significant decrease in microbial diversity. As mice gradually cleared the colonization, the diversity returned to baseline levels. Diversification was accompanied by an early expansion of Bacteroidetes, Staphylococcus spp., and Lachnospiraceae. Moreover, the Bacteroidetes expansion was significantly greater in young-adult than in elderly mice. In conclusion, we observed differences in URT microbiota composition between naive young-adult and elderly mice that were associated with differences in pneumococcal clearance over time.

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.

Streptococcus pneumoniae is a frequent asymptomatic colonizer of the nasopharyngeal niche and only occasionally progresses toward infection. The burden of pneumococcal disease is particularly high in the elderly, and the mechanisms behind this increased susceptibility are poorly understood. Here we used a mouse model of pneumococcal carriage to study immunosenescence in the upper respiratory tract (URT). Nasal mucosa-associated lymphoid tissue (NALT) showed increased expression of Toll-like receptor 1, interleukin-1β, NLRp3 inflammasome, and CCL2 in naive elderly compared to young animals. This suggests an increased proinflammatory expression profile in the NALT of aged mice at baseline. Simultaneously, we observed a more tolerogenic profile in respiratory epithelia of naive elderly compared to young adult mice with upregulation of the NF-κβ pathway inhibitor peroxisome proliferator-activated receptor gamma (PPARγ). After nasal instillation of pneumococci, pneumococcal colonization was prolonged in elderly mice compared to in young adults. The delay in clearance was associated with absent or delayed upregulation of a proinflammatory mediator(s) in the NALT, diminished influx of macrophages into the URT niche, and absent downregulation of PPARγ in respiratory epithelium, accompanied by diminished expression of cathelicidin (CRAMP) at the site of colonization. These findings suggest that unresponsiveness to pneumococcal challenge due to altered mucosal immune regulation is the key to increased susceptibility to disease in the elderly.

Pneumococcal conjugate vaccines (PCVs) have been introduced worldwide. However, few developing countries have high-quality surveillance systems available for monitoring vaccine impact. We evaluated whether data from nasopharyngeal carriage studies can be used to accurately monitor post-PCV changes in the incidence of invasive pneumococcal disease (IPD) among children under 5 years of age. For various dates during 1991-2010, data on nasopharyngeal pneumococcal carriage and on IPD before and after administration of 7-valent PCV (PCV7) were available from England and Wales, the Netherlands, the Navajo and White Mountain Apache American Indian populations, and the US states of Massachusetts and Alaska. We estimated the change in carriage prevalence for each serotype in each study and then either calculated the average change (inverse variance-weighted) among vaccine and nonvaccine serotypes (model 1) or used mixed-effects models to estimate the change for each serotype individually, pooling serotype data within or between studies (models 2 and 3). We then multiplied these values by the proportion of IPD caused by each serotype during the pre-PCV7 period to obtain an estimate of post-PCV7 disease incidence. Model 1 accurately captured overall changes in IPD incidence following PCV7 introduction for most studies, while the more detailed models, models 2 and 3, were less accurate. Carriage data can be used in this simple model to estimate post-PCV changes in IPD incidence.

Neutrophil recruitment is essential in clearing pneumococcal infections. The first step in neutrophil extravasation involves the interaction between P-selectin on activated endothelium and P-Selectin Glycoprotein 1 (PSGL-1) on neutrophils. Here, we identify pneumococcal Zinc metalloproteinase C as a potent inhibitor of PSGL-1. ZmpC degrades the N-terminal domain of PSGL-1, thereby disrupting the initial rolling of neutrophils on activated human umbilical vein endothelial cells. Furthermore, mice infected with wild-type strain in the model of pneumococcal pneumonia showed lower lungs neutrophil infiltration compare to animals infected with ZmpC mutant. In addition, we confirmed the association of zmpC with serotype 8 and 11A and found it to be associated with serotype 33F as well. In conclusion, wereport PSGL-1 as a novel target for ZmpC and show that ZmpC inhibits neutrophil extravasation during pneumococcal pneumonia., (© 2013 John Wiley & Sons Ltd.)

This study investigated long-term cellular and humoral immunity against pertussis after booster vaccination of 4-year-old children who had been vaccinated at 2, 3, 4, and 11 months of age with either whole-cell pertussis (wP) or acellular pertussis (aP) vaccine. Immune responses were evaluated until 2 years after the preschool booster aP vaccination. In a cross-sectional study (registered trial no. ISRCTN65428640), blood samples were taken from wP- and aP-primed children prebooster and 1 month and 2 years postbooster. Pertussis vaccine antigen-specific IgG levels, antibody avidities, and IgG subclasses, as well as T-cell cytokine levels, were measured by fluorescent bead-based multiplex immunoassays. The numbers of pertussis-specific memory B cells and gamma interferon (IFN-γ)-producing T cells were quantified by enzyme-linked immunosorbent spot assays. Even 2 years after booster vaccination, memory B cells were still present and higher levels of pertussis-specific antibodies than prebooster were found in aP-primed children and, to a lesser degree, also in wP-primed children. The antibodies consisted mainly of the IgG1 subclass but also showed an increased IgG4 portion, primarily in the aP-primed children. The antibody avidity indices for pertussis toxin and pertactin in aP-primed children were already high prebooster and remained stable at 2 years, whereas those in wP-primed children increased. All measured prebooster T-cell responses in aP-primed children were already high and remained at similar levels or even decreased during the 2 years after booster vaccination, whereas those in wP-primed children increased. Since the Dutch wP vaccine has been replaced by aP vaccines, the induction of B-cell and T-cell memory immune responses has been enhanced, but antibody levels still wane after five aP vaccinations. Based on these long-term immune responses, the Dutch pertussis vaccination schedule can be optimized, and we discuss here several options.

We investigated the effects of our Bio-ITEST teacher professional development model and bioinformatics curricula on cognitive traits (awareness, engagement, self-efficacy, and relevance) in high school teachers and students that are known to accompany a developing interest in science, technology, engineering, and mathematics (STEM) careers. The program included best practices in adult education and diverse resources to empower teachers to integrate STEM career information into their classrooms. The introductory unit, Using Bioinformatics: Genetic Testing, uses bioinformatics to teach basic concepts in genetics and molecular biology, and the advanced unit, Using Bioinformatics: Genetic Research, utilizes bioinformatics to study evolution and support student research with DNA barcoding. Pre-post surveys demonstrated significant growth (n = 24) among teachers in their preparation to teach the curricula and infuse career awareness into their classes, and these gains were sustained through the end of the academic year. Introductory unit students (n = 289) showed significant gains in awareness, relevance, and self-efficacy. While these students did not show significant gains in engagement, advanced unit students (n = 41) showed gains in all four cognitive areas. Lessons learned during Bio-ITEST are explored in the context of recommendations for other programs that wish to increase student interest in STEM careers.

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.

Respiratory infectious diseases are mainly caused by viruses or bacteria that often interact with one another. Although their presence is a prerequisite for subsequent infections, viruses and bacteria may be present in the nasopharynx without causing any respiratory symptoms. The upper respiratory tract hosts a vast range of commensals and potential pathogenic bacteria, which form a complex microbial community. This community is assumed to be constantly subject to synergistic and competitive interspecies interactions. Disturbances in the equilibrium, for instance due to the acquisition of new bacteria or viruses, may lead to overgrowth and invasion. A better understanding of the dynamics between commensals and pathogens in the upper respiratory tract may provide better insight into the pathogenesis of respiratory diseases. Here we review the current knowledge regarding specific bacterial-bacterial and viral-bacterial interactions that occur in the upper respiratory niche, and discuss mechanisms by which these interactions might be mediated. Finally, we propose a theoretical model to summarize and illustrate these mechanisms.

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.

Immunization with acellular pertussis vaccine (aP) induces higher specific antibody levels and fewer adverse reactions than does immunization with the whole-cell vaccine (wP). However, antibody levels in infants induced by both types of pertussis vaccines wane already after 1 year. Therefore, long-term T-cell responses upon vaccination might play a role in protection against pertussis. In a cross-sectional study (ISRCTN65428640), we investigated T-helper (Th) cell immune responses in wP- or aP-vaccinated children before and after an aP low-dose or high-dose preschool booster at 4 years of age in The Netherlands. T cells were stimulated with pertussis vaccine antigens. The numbers of gamma interferon-producing cells and Th1, Th2, Th17, and interleukin-10 (IL-10) cytokine concentrations were determined. In addition, pertussis-specific IgE levels were measured in plasma. Children being vaccinated with aP vaccinations at 2, 3, 4, and 11 months of age still showed higher pertussis-specific T-cell responses at 4 years of age than did wP-vaccinated children. These T-cell responses failed to show a typical increase in cytokine production after a fifth aP vaccination but remained high after a low-dose booster and seemed to decline even after a high-dose booster. Importantly, elevated IgE levels were induced after this booster vaccination. In contrast, wP-vaccinated children had only low prebooster T-cell responses, and these children showed a clear postbooster T-cell memory response even after a low-dose booster vaccine. Four high-dose aP vaccinations in infancy induce high T-cell responses still present even 3 years after vaccination and enhanced IgE responses after preschool booster vaccination. Therefore, studies of changes in vaccine dosage, timing of pertussis (booster) vaccinations, and the possible association with local side effects are necessary.