Lotte Lambertsen

Rapid Pneumococcal Evolution in Response to Clinical Interventions

Streptococcus pneumonia evades vaccines and drugs by high levels of recombination and rapid adaptation. Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain23F-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.

Pneumococcal Serotypes and Mortality following Invasive Pneumococcal Disease: A Population-Based Cohort Study

Analyzing population-based data collected over 30 years in more than 18,000 patients with invasive pneumococcal infection, Zitta Harboe and colleagues find specific pneumococcal serotypes to be associated with increased mortality.

Serotype IX, a Proposed New Streptococcus agalactiae Serotype

ABSTRACT We identified three isolates of Streptococcus agalactiae (group B streptococcus [GBS]), of human origin, which failed to react with antisera against any of the nine known GBS serotypes. Polyclonal rabbit antisera raised against these isolates and standard GBS typing sera were used in capillary precipitation and Ouchterlony tests to compare the strains with known GBS serotype reference strains. All three previously nontypeable isolates reacted with all three new antisera, producing lines of identity in the Ouchterlony test. Weak cross-reactions with antisera against several GBS serotypes were observed but were removed by absorption with corresponding antigens. The new antisera were used to test 227 GBS isolates that had been nontypeable or difficult to type using standard antisera. Of these, five reacted with the new antisera. These results suggested that all eight isolates belong to the previously unrecognized GBS serotype. They were tested by Western blotting for the Cα and Cβ proteins and by PCR to identify molecular serotypes and surface protein antigen genes. Two segments of the cps gene cluster (3′ end of cpsE-cpsF and 5′ end of cpsG, approximately 700 bp; 3′ end of cpsH and 5′ end of cpsM, approximately 560 bp) were sequenced. All eight isolates expressed Cα, and seven expressing the Cβ protein and the corresponding genes, bca and bac, respectively, were identified. They all share the same, unique partial cps sequence. These results indicate that these eight isolates represent a new S. agalactiae serotype, which we propose should be designated serotype IX.

Temporal Trends in Invasive Pneumococcal Disease and Pneumococcal Serotypes over 7 Decades

Background. Pneumococcal infections have historically played a major role in terms of morbidity and mortality. We explored historical trends of invasive pneumococcal disease (IPD) and pneumococcal serotypes in a population exposed to limited antibiotic selective pressure and conjugate pneumococcal vaccination (PCV). Methods. Retrospective cohort study based on nationwide laboratory surveillance data on IPD collected uninterruptedly in Denmark during 1938-2007. Changes in the reported incidence and trends of pneumococcal serotypes were explored using nonlinear regression analysis. Results. There were 25,502 IPD cases included in our study. The median incidence of IPD increased from 2.8 cases per 100,000 population (interquartile range [IQR], 1.5-2.6) during the first 4 decades to 15.7 cases per 100,000 population (IQR, 7-20.4) during the 1980s and 1990s, mainly attributed to an increase in the number of bacteremia cases. The incidence of meningitis remained relatively stable, with a median of 1.3 cases per 100,000 population (IQR, 0.9-1.6). The proportions of serotypes/groups 4 and 9 increased; the proportion of serotype 18C decreased; the proportions of serotypes 6, 7F, 14, and 23F remained stable; and serotype 2 nearly disappeared. Before the 1960s, serotypes 1, 2, 3, and 5 presented peaks every 2-3 years, becoming less frequent during the 1970s with peaks every 7-10 years. Between 20% and 90% of IPD in children <5 years were caused by PCV serotypes during the last 4 decades. Cases of IPD caused by serotype 19A increased before introduction of PCV. Between 1993 and 2007, the level of resistance to macrolides and β-lactams was ≤6%. Conclusions. The epidemiology of IPD and single serotypes has constantly changed over the past 7 decades. PCV serotypes appeared to dominate the pneumococcal population.

Pneumococcal Capsular Switching: A Historical Perspective

Background.Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching. Methods.Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 were analyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events. Results.We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately 19.0 kb to ≥58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a. Conclusions.Capsular switching has been a regular occurrence among pneumococcal populations throughout the past 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution.

Target Gene Sequencing To Characterize the Penicillin G Susceptibility of Neisseria meningitidis

ABSTRACT Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the PenI phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA ). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

Early effectiveness of heptavalent conjugate pneumococcal vaccination on invasive pneumococcal disease after the introduction in the Danish Childhood Immunization Programme.

We evaluated the effectiveness of the heptavalent pneumococcal conjugate vaccine (PCV7) on invasive pneumococcal disease (IPD) 1 year after PCV7s introduction in the childhood immunization programme through a nationwide cohort study based on laboratory surveillance data. There was a decline in the overall incidence of IPD from 19.4 to 17.1 cases per 100,000 population (incidence rate ratios (IRR) 0.87; 95% confidence interval (CI) [0.81-0.96]), and of meningitis from 1.56 to 1.16 (IRR 0.74; 95% CI [0.57-0.97]) comparing pre-PCV7 (years 2000-2007) and PCV7 (year 2008) periods. In children <2 years, the incidence decreased from 54 to 23 cases per 100,000 (IRR 0.43; 95% CI [0.29-0.62]) and for vaccine-serotypes from 36.7 to 7.7 (IRR 0.20; 95% CI [0.09-0.38]). The incidence of IPD declined approximately 10% (IRR 0.90; 95% CI [0.84-0.97]) in patients aged >or=2 years. The case fatality was 17% in both periods. The administration of PCV7 was followed by a marked decline in the incidence of IPD in both vaccinated and non-vaccinated individuals.

Impact of pneumococcal vaccination in Denmark during the first 3 years after PCV introduction in the childhood immunization programme.

BACKGROUND AND AIMS The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced in Denmark in October 2007 in a 2+1 schedule with a catch-up programme for children up to 17 months of age. To assess the impact of PCV we evaluated on the whole population: (1) direct and indirect effects on incidence of invasive pneumococcal disease (IPD), (2) changes in pneumococcal serotype distribution and (3) IPD related mortality. METHODS We compared disease incidence in pre-PCV (years 2000-2007) and PCV periods (years 2008-2010) based on national surveillance data. RESULTS In children aged 0-5 years the overall incidence of IPD decreased from 26.7 to 16.3 cases per 100,000 (IRR 0.58; 95% Confidence Interval (CI) [0.48-0.69]) and case fatality declined from 1.8% (12 deaths) in the eight-year pre-PCV period to 0% (no deaths) in the three-year PCV period. In the whole population the overall incidence of IPD and of IPD caused by vaccine serotypes declined significantly from 19.5 to 17.7 and from 7.7 to 3.8 cases per 100,000 persons comparing the two periods. The incidence of IPD due to non-vaccine serotypes (NVT-IPD) increased significantly from 11.8 to 13.9 cases per 100,000 in the whole population (incidence rate ratio 1.18; 95% CI [1.12-1.24]) with predominance of the serotypes 1.7F and 19A. CONCLUSIONS We report a marked decline in incidence in IPD in both vaccinated and non-vaccinated age groups and a minor but statistically significant increase in incidence of IPD due to NVTs in both vaccinated and non-vaccinated groups with predominance of serotypes covered by higher valence pneumococcal conjugate vaccines.

Variation at the capsule locus, cps, of mistyped and non-typable Streptococcus pneumoniae isolates.

The capsule polysaccharide locus (cps) is the site of the capsule biosynthesis gene cluster in encapsulated Streptococcus pneumoniae. A set of pneumococcal samples and non-pneumococcal streptococci from Denmark, the Gambia, the Netherlands, Thailand, the UK and the USA were sequenced at the cps locus to elucidate serologically mistyped or non-typable isolates. We identified a novel serotype 33B/33C mosaic capsule cluster and previously unseen serotype 22F capsule genes, disrupted and deleted cps clusters, the presence of aliB and nspA genes that are unrelated to capsule production, and similar genes in the non-pneumococcal samples. These data provide greater understanding of diversity at a locus which is crucial to the antigenic diversity of the pathogen and current vaccine strategies.

International External Quality Assurance for Laboratory Identification and Typing of Streptococcus agalactiae (Group B Streptococci)

ABSTRACT We report the results from the first international multicenter external quality assessment (EQA) studies for molecular and serological typing of group B streptococcus (GBS) strains as part of DEVANI (Design of a Vaccine against Neonatal Infections), a pan-European program. A questionnaire-based surveillance was undertaken among eight laboratories participating in DEVANI and six laboratories not participating in DEVANI from 13 countries in order to assess their current microbiological procedures for GBS screening, diagnosis, and typing. GBS strains from three EQA distributions were characterized using molecular and serological methods based on GBS capsular polysaccharide typing. Participants were asked to test the first distribution using their current serotyping and genotyping methods. The Strep-B-Latex agglutination method was the most widely used method, with a typeability value of >90%. A multiplex PCR assay for GBS capsular gene typing was also used by 2 of 14 centers, which achieved a typeability value of 93%; this assay detected only 9 of 10 GBS capsular polysaccharide genes. From the second and third EQA studies, standardized protocols were prepared for serological and molecular typing of GBS strains based on the Strep-B-Latex agglutination method and a novel multiplex PCR assay that detected all 10 GBS capsular types (Ia to IX). These standardized protocols are being used by many European laboratories, and as the use of these methods increases, it is imperative to continuously improve and assess laboratory performance and offer training to any laboratories that have technical difficulties.