Multiple-Locus Variable Number Tandem Repeat Analysis for Streptococcus pneumoniae: Comparison with PFGE and MLST

In the era of pneumococcal conjugate vaccines, surveillance of pneumococcal disease and carriage remains of utmost importance as important changes may occur in the population. To monitor these alterations reliable genotyping methods are required for large-scale applications. We introduced a high throughput multiple-locus variable number tandem repeat analysis (MLVA) and compared this method with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLVA described here is based on 8 BOX loci that are amplified in two multiplex PCRs. The labeled PCR products are sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The composite of the number of repeats of the BOX loci makes up a numerical profile that is used for identification and clustering. In this study, MLVA was performed on 263 carriage isolates that were previously characterized by MLST and PFGE. MLVA, MLST and PFGE (cut-off of 80%) yielded 164, 120, and 87 types, respectively. The three typing methods had Simpson's diversity indices of 98.5% or higher. Congruence between MLST and MLVA was high. The Wallace of MLVA to MLST was 0.874, meaning that if two strains had the same MLVA type they had an 88% chance of having the same MLST type. Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%. For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup. Overall, MLVA is a promising genotyping method that is easy to perform and a relatively cheap alternative to PFGE and MLST. In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in the Netherlands before the introduction of the 7-valent conjugate vaccine

Population Structure of Invasive Streptococcus pneumoniae in the Netherlands in the Pre-Vaccination Era Assessed by MLVA and Capsular Sequence Typing

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA)

Pneumococcal population in the era of vaccination: changes in composition and the relation to clinical outcomes

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 serotype

Invasive pneumococcal disease: Clinical outcomes and patient characteristics 2-6 years after introduction of 7-valent pneumococcal conjugate vaccine compared to the pre-vaccine period, the Netherlands

Implementation of 7-valent pneumococcal conjugate vaccine (PCV7) in the Dutch national immunization program for infants led to a shift from vaccine to non-vaccine serotypes in invasive pneumococcal disease (IPD) in all age groups. We studied the impact of the serotype shift on clinical syndromes and outcomes. Pneumococcal isolates from hospitalized IPD patients obtained from nine sentinel microbiology laboratories, covering 25% of the Dutch population, were serotyped. Clinical syndromes, outcomes and patient characteristics in the post-PCV7 (2008-2012) period were compared with the pre-PCV7 period (2004-2006). Serotype specific propensity of the association with empyema, meningitis and death was calculated. Invasive pneumonia incidence significantly decreased in children <5 years and elderly ≥65 years, but increased in 5-64 years old from 4.92 to 5.58 cases/100.000/year (RR 1.13 95% CI 0.99-1.29). Empyema incidence significantly increased in elderly 65 years and older from 0.61 to 2.60 cases/100.000/year (RR 4.28 95% CI 1.97-9.33), mainly due to serotype 1. The incidence of meningitis only declined significantly in children <5 years. IPD case-fatality decreased in children <5 years from 5% to 3%, in 5-64 years old from 9% to 7% and in elderly ≥65 years significantly from 22% to 17%, due to lower case-fatality rates for most emerging non-PCV7 serotypes. An increase in empyema incidence was observed in persons ≥65 years old in the post-PCV7 era, mainly due to the emergence of serotype 1, although overall IPD case-fatality decreased. Extended conjugate vaccines that target serotype 1 or serotypes with high case-fatality may offer further reduction of pneumococcal disease burde

Invasive Pneumococcal Disease 3 Years after Introduction of 10-Valent Pneumococcal Conjugate Vaccine, the Netherlands

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

Immunogenicity of 13-Valent Pneumococcal Conjugate Vaccine Administered According to 4 Different Primary Immunization Schedules in Infants A Randomized Clinical Trial

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

Dynamics and Determinants of Pneumococcal Antibodies Specific against 13 Vaccine Serotypes in the Pre-Vaccination Era

<div><p>Introduction</p><p>Introduction of pneumococcal conjugate vaccines (PCVs) for infants decreased overall invasive pneumococcal disease (IPD), while non-vaccine serotype IPD increased. To fully understand this serotype replacement, knowledge about serotype dynamics in the pre-vaccine era is needed. In addition to IPD surveillance and carriage studies, the serotype replacement can be investigated by serosurveillance studies. The current study compared the results of two Dutch serosurveillance studies conducted in 1995–1996 (PIENTER1) and 2006–2007 (PIENTER2).</p><p>Methods</p><p>Participants in these studies donated a blood sample and completed a questionnaire. Pneumococcal antibodies of serotypes included in PCV13 were measured with a fluorescent-bead based multiplex immunoassay. Geometric mean antibody concentrations (GMCs) and determinants of pneumococcal antibody levels were investigated.</p><p>Results</p><p>GMCs were higher in PIENTER2 for serotypes 1, 6A, 6B, 9V, 18C, 19F and 23F and lower for 3 and 5. Age, day care attendance, household size, vaccination coverage, and urbanisation rate were associated with pneumococcal antibodies in children. Education level, ethnicity, age, low vaccination coverage sample, urbanisation rate, and asthma/COPD were associated with pneumococcal antibodies in elderly. The determinants significantly associated with pneumococcal IgG were slightly different for the elderly in PIENTER1 compared to the elderly in PIENTER2.</p><p>Conclusion</p><p>Although most of the serotype antibody levels remained stable, some of the serotype-specific antibody levels varied during the pre-vaccine era, indicating that exposure of certain serotypes changes without interference of PCVs.</p></div

CT assignment per serotype included in the snapshot.

1<p>Frequency of the CT.</p>2<p>Frequency of the serotype.</p>3<p>Number of mutations with the major CT of the particular serotype.</p>4<p>Silent mutation.</p>5<p>Non-silent mutation.</p>6<p>Number of mutations with the <i>wzh</i> gene segment published by Bentley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020390#pone.0020390-Bentley1" target="_blank">[21]</a>.</p>7<p>Also perfect match with <i>wzh</i> sequences of 6B isolates AY359459, AY359455, AY359449.</p

Oligonucleotide primer sequences used in this study.

1<p>Underlined sequences represent the M13-tail.</p>2<p>Sequences with accession number CR931632 to CR931722 were used for primer design.</p