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Spijkerman J.S.,University Utrecht | Spijkerman J.S.,Spaarne Hospital | van Gils E.J.M.,University Utrecht | van Gils E.J.M.,Spaarne Hospital | And 6 more authors.
Emerging Infectious Diseases | Year: 2011

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-monthold children, and 8% and 1% among parents. Non-PCV7 serotype prevalences were 29% and 39% among 11-monthold 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.


Van Gils E.J.M.,University Utrecht | Van Gils E.J.M.,Spaarne Hospital Hoofddorp | Veenhoven R.H.,Spaarne Hospital Hoofddorp | Hak E.,University Utrecht | And 10 more authors.
JAMA - Journal of the American Medical Association | Year: 2010

Context: The rapid increase in multiresistant serotype19Aas a cause of invasive and respiratory pneumococcal disease has been associated in time with the widespread implementation of 7-valent pneumococcal conjugate vaccination (PCV-7) in several countries. Because spontaneous fluctuations in time and antibiotic selective pressuremayhave induced this serotype 19A increase, controlled studies are needed to assess the role of PCV-7. Objective: To examine the association of PCV-7 vaccination and nasopharyngeal acquisition of serotype 19A pneumococci, their clonal distribution, and antibiotic susceptibility. Design, Setting, and Patients: Post hoc per-protocol completer's analysis as part of a randomized controlled trial of nasopharyngeal Streptococcus pneumoniae carriage enrolling 1003 healthy newborns with follow-up to the age of 24 months in the Netherlands, which has low antibiotic resistance rates. The study was conducted before widespread PCV-7 implementation in infants, between July 7, 2005, and February 14, 2008. Nasopharyngeal swabs were obtained at the age of 6 weeks and at 6, 12, 18, and 24 months. Intervention: Infants were randomly assigned to receive 2 doses of PCV-7 at 2 and 4 months; 2 + 1 doses of PCV-7 at 2, 4, and 11 months; or no dosage (unvaccinated control group). Main Outcome Measure: Cumulative proportion of children with nasopharyngeal acquisition of a new serotype 19A strain from 6 through 24 months of age. Results: Nine hundred forty-eight children completed the study. Fifty-four nasopharyngeal serotype 19A carriage isolates from 318 in the 2-dose group, 66 isolates from 327 in the 2 + 1-dose group, and 33 isolates from 303 in the unvaccinated were collected from 6 weeks through 24 months. The cumulative proportion who tested positive for new nasopharyngeal serotype 19A acquisition from 6 through 24 months of age was significantly higher in those having received the 2 + 1-dose PCV-7 schedule (16.2%;95% confidence interval [CI], 12.6%-20.6%) vs those who were unvaccinated (9.2%; 95% CI, 6.5%-13.0%; relative risk [RR], 1.75;95%CI, 1.14-2.70) but not after a 2-dose schedule (13.2%; 95% CI, 9.9%-17.4%; RR, 1.43; 95% CI, 0.91-2.25). There were 28 different sequence types identified, including 6 new types. The proportion of children with new 19A acquisition who had used antibiotics in the last 6 months (18.7%) did not differ among groups. Five isolates were penicillin-intermediate susceptible and another 3 were nonsusceptible to erythromycin and azithromycin, all in the vaccine groups. Conclusion: A 2 + 1-dose PCV-7 schedule was associated with an increase in serotype 19A nasopharyngeal acquisition compared with unvaccinated controls. Trial Registration: clinicaltrials.gov Identifier: NCT00189020. ©2010 American Medical Association. All rights reserved.


Spijkerman J.,University Utrecht | Spijkerman J.,Spaarne Hospital | Prevaes S.M.P.J.,University Utrecht | Prevaes S.M.P.J.,Spaarne Hospital | And 8 more authors.
PLoS ONE | Year: 2012

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. © 2012 Spijkerman et al.


Bogaert D.,University Utrecht | Keijser B.,TNO | Huse S.,Josephine Bay Paul Center for Comparative Molecular Biology and Evolution | Rossen J.,St Elisabeth Hospital | And 6 more authors.
PLoS ONE | Year: 2011

The nasopharynx is the ecological niche for many commensal bacteria and for potential respiratory or invasive pathogens like Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. Disturbance of a balanced nasopharyngeal (NP) microbiome might be involved in the onset of symptomatic infections with these pathogens, which occurs primarily in fall and winter. It is unknown whether seasonal infection patterns are associated with concomitant changes in NP microbiota. As young children are generally prone to respiratory and invasive infections, we characterized the NP microbiota of 96 healthy children by barcoded pyrosequencing of the V5-V6 hypervariable region of the 16S-rRNA gene, and compared microbiota composition between children sampled in winter/fall with children sampled in spring. The approximately 1000000 sequences generated represented 13 taxonomic phyla and approximately 250 species-level phyla types (OTUs). The 5 most predominant phyla were Proteobacteria (64%), Firmicutes (21%), Bacteroidetes (11%), Actinobacteria (3%) and Fusobacteria (1,4%) with Moraxella, Haemophilus, Streptococcus, Flavobacteria, Dolosigranulum, Corynebacterium and Neisseria as predominant genera. The inter-individual variability was that high that on OTU level a core microbiome could not be defined. Microbiota profiles varied strongly with season, with in fall/winter a predominance of Proteobacteria (relative abundance (% of all sequences): 75% versus 51% in spring) and Fusobacteria (absolute abundance (% of children): 14% versus 2% in spring), and in spring a predominance of Bacteroidetes (relative abundance: 19% versus 3% in fall/winter, absolute abundance: 91% versus 54% in fall/winter), and Firmicutes. The latter increase is mainly due to (Brevi)bacillus and Lactobacillus species (absolute abundance: 96% versus 10% in fall/winter) which are like Bacteroidetes species generally related to healthy ecosystems. The observed seasonal effects could not be attributed to recent antibiotics or viral co-infection. The NP microbiota of young children is highly diverse and appears different between seasons. These differences seem independent of antibiotic use or viral co-infection. © 2011 Bogaert et al.


Bruin J.P.,Regional Laboratory of Public Health | Kostrzewa M.,Bruker | Van Der Ende A.,University of Amsterdam | Badoux P.,Regional Laboratory of Public Health | And 3 more authors.
European Journal of Clinical Microbiology and Infectious Diseases | Year: 2014

Generally accepted laboratory methods that have been used for decades do not reliably distinguish between H. influenzae and H. haemolyticus isolates. H. haemolyticus strains are often incorrectly identified as nontypeable Haemophilus influenzae (NTHi). To distinguish H. influenzae from H. haemolyticus we have created a new database on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) bio-typer 2 and compared the results with routine determination of Haemophilus (growth requirement for X and V factor), and multilocus sequence typing (MLST). In total we have tested 277 isolates, 244 H. influenzae and 33 H. haemolyticus. Using MLST as the gold standard, the agreement of MALDI-TOF MS was 99.6 %. MALDI-TOF MS allows reliable and rapid discrimination between H. influenzae and H. haemolyticus. © 2013 Springer-Verlag Berlin Heidelberg.


Bruin J.P.,Regional Laboratory of Public Health | Diederen B.M.W.,Regional Laboratory of Public Health | IJzerman E.P.F.,Regional Laboratory of Public Health | Den Boer J.W.,Regional Laboratory of Public Health | Mouton J.W.,Radboud University Nijmegen
Diagnostic Microbiology and Infectious Disease | Year: 2013

Objectives: Routine use of disk diffusion tests for detecting antibiotic resistance in Legionella pneumophila has not been described. The goal of this study was to determine the correlation of MIC values and inhibition zone diameter (MDcorr) in clinical L. pneumophila isolates. Methods: Inhibition zone diameter of 183 L. pneumophila clinical isolates were determined for ten antimicrobials. Disk diffusion results were correlated with MICs as determined earlier with E-tests. Results: Overall the correlation of MIC values and inhibition zone diameters (MDcorr) of the tested antimicrobials is good, and all antimicrobials showed a WT distribution. Of the tested fluoroquinolones levofloxacin showed the best MDcorr. All macrolides showed a wide MIC distribution and good MDcorr. The MDcorr for cefotaxim, doxycycline and tigecycline was good, while for rifampicin and moxifloxacin, they were not. Conclusion: Overall good correlation between MIC value and disk inhibition zone were found for the fluoroquinolones, macrolides and cefotaxim. © 2013 Elsevier Inc..


Bruin J.P.,Regional Laboratory of Public Health | Ijzerman E.P.F.,Regional Laboratory of Public Health | den Boer J.W.,Regional Laboratory of Public Health | Mouton J.W.,Radboud University Nijmegen | And 2 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2012

Objectives: The purpose of this study was to establish wild-type (WT) distributions and determine the epidemiological cut-off values (ECOFF) in clinical L. pneumophila serogroup 1 isolates for 10 antimicrobials commonly used for the treatment of Legionella infections using a method feasible in a routine clinical laboratory. Methods: MICs of 183 clinical L. pneumophila serogroup 1 isolates, collected as part of an outbreak detection program, were tested using E-test methodology on buffered charcoal yeast extract agar supplemented with -ketoglutarate (BCYE-). The MICs were read after 2 days of incubation at 35 C with increased humidity and without CO 2. ECOFFs were determined according to EUCAST methodology and expressed as WT X mg/L. Results: All antimicrobials showed a WT distribution, although the width varied from 2 two-fold dilutions to 8 dilutions, depending on antibiotic class. The ECOFFs determined were 1.0 mg/L for ciprofloxacin, 0.50 mg/L for levofloxacin, 1.0 mg/L for moxifloxacin, 1.0 mg/L for erythromycin, 1.0 mg/L for azithromycin, 0.50 mg/L for clarithromycin, 1.0 mg/L for cefotaxime, 0.032 mg/L for rifampicin, 16 mg/L for tigecycline, and 8 mg/L for doxycycline. Conclusion: All isolates were inhibited by low concentrations of the fluoroquinolones and macrolides tested, with somewhat higher MICs for the fluoroquinolones. Rifampicin was found to be the most active against L. pneumophila isolates in vitro. These data can be used as a reference for the detection of resistance in clinical L. pneumophila isolates and as a setting of clinical breakpoints. © 2012 Elsevier Inc.


Bruin J.P.,Regional Laboratory of Public Health | Peeters M.F.,St Elisabeth Hospital | Ijzerman E.P.F.,Regional Laboratory of Public Health | Diederen B.M.W.,Regional Laboratory of Public Health
European Journal of Clinical Microbiology and Infectious Diseases | Year: 2010

We evaluated a new immunochromatographic assay (Legionella V-TesT, Coris BioConcept, Gembloux, Belgium) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Test devices were read at various time points to determine the optimum incubation time regarding performance. The results were compared with those obtained with the BinaxNOW urinary antigen test. The sensitivity and specificity were 82.2% and 98.6%, respectively, for the Legionella V-TesT and 83.9% and 100%, respectively, for the BinaxNOW urinary antigen test after 15 min of incubation. When tests were examined after 60 min, the sensitivity for both tests increased to 91.5%. © 2010 Springer-Verlag.


Bruin J.P.,Regional Laboratory of Public Health | Diederen B.M.W.,Regional Laboratory of Public Health
European Journal of Clinical Microbiology and Infectious Diseases | Year: 2013

We evaluated the ability of a new antigen test (TRU Legionella® assay, Meridian Bioscience, Cincinnati, OH, USA) to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained with the Binax NOW® urinary antigen test (Binax, Portland, ME, USA). The sensitivities and specificities were 73 % [95 % confidence interval (CI), 65 to 81 %] and 100 %, respectively, for the Meridian TRU Legionella test and 77 % (95 % CI, 68 to 84 %) and 100 %, respectively, for the Binax NOW urinary antigen test. The sensitivity of the Meridian TRU Legionella test increased to 81 % if tests were re-examined after 60 min of incubation. Prolonged incubation did not affect the specificity. We conclude that both assays evaluated have similar performance characteristics and are suitable for the rapid diagnosis of Legionnaires' disease. © 2012 Springer-Verlag.


Boers S.A.,Regional Laboratory of Public Health | van der Reijden W.A.,Regional Laboratory of Public Health | Jansen R.,Regional Laboratory of Public Health
PLoS ONE | Year: 2012

Multilocus sequence typing (MLST) is a widely used system for typing microorganisms by sequence analysis of housekeeping genes. The main advantage of MLST in comparison to other typing techniques is the unambiguity and transferability of sequence data. However, a main disadvantage is the high cost of DNA sequencing. Here we introduce a high-throughput MLST (HiMLST) method that employs next-generation sequencing (NGS) technology (Roche 454), to generate large quantities of high-quality MLST data at low costs. The HiMLST protocol consists of two steps. In the first step MLST target genes are amplified by PCR in multi-well plates. During this PCR the amplicons of each bacterial isolate are provided with a unique DNA barcode, the multiplex identifier (MID). In the second step all amplicons are pooled and sequenced in a single NGS-run. The MLST profile of each individual isolate can be retrieved easily using its unique MID. With HiMLST we have profiled 575 isolates of Legionella pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae in mixed species HiMLST experiments. In conclusion, the introduction of HiMLST paves the way for a broad employment of the MLST as a high-quality and cost-effective method for typing microbial species. © 2012 Boers et al.

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