Influence of Two Vaccination Campaigns on Genetic Diversity of Invasive Neisseria meningitidis Isolates in Northern Spain (1997–2008)

BACKGROUND: Neisseria meningitidis diversifies rapidly, due to its high recombination rates. The aim of this study was to analyze the possible impact of two vaccination campaigns (a once-off A/C polysaccharide vaccination campaign in people aged 18 months to 20 years old in 1997, and a meningococcal C conjugate vaccination campaign in children aged < or = 6 years old from 2000 to 2008) on diversification of the population of invasive isolates obtained between 1997 and 2008. All of the 461 available isolates were included (2, 319, 123, 11 and 6 belonging to serogroups A, B, C, Y and W-135, respectively). METHODOLOGY/PRINCIPAL FINDINGS: The isolates were analyzed for diversity using multilocus sequence typing, eBURST and the S.T.A.R.T.2 program. One hundred and seven sequence types (ST) and 20 clonal complexes were obtained. Five different STs (ST11, ST8, ST33, ST1163 and ST3496) included 56.4% of the isolates. With the exception of ST11, all other STs were associated with a specific serogroup. Epidemic circulation of serogroup C ST8 isolates was detected in 1997-1998, as well as epidemic circulation of ST11 isolates (serogroups B and C) in 2002-2004. The epidemic behavior of serogroup B ST11 (ST11_B:2a:P1.5) was similar, although with lesser intensity, to that of ST11 of serogroup C. Although clonality increased during epidemic years, the overall diversity of the meningococcal population did not increase throughout the 12 years of the study. CONCLUSION: The overall diversity of the meningococcal population, measured by the frequency of STs and clonal complexes, numbers of alleles, polymorphic sites, and index of association, remained relatively constant throughout the study period, contradicting previous findings by other researchers

Molecular epidemiological characterisation of carried Neisseria meningitidis isolates in Scotland, 1974 - 2004 and a comparison with an invasive meningococcal disease strain collection

Neisseria meningitidis is an important cause of meningitis and septicaemia worldwide. Invasive meningococcal disease (IMD) is cyclical and varies by age group, being more common in children, especially those under 5 years. However, IMD is a rare outcome relative to asymptomatic carriage of the organism by the host and disease-causing isolates represent the tip of the iceberg in terms of the overall meningococcal population biology. The emphasis of previous research into N. meningitidis has rested firmly on the study of IMD isolates. In more recent times, however, there has been a more conscious effort to re-dress this balance to aid in our understanding of the relationship between carriage and disease. The Scottish Haemophilus, Legionella, Meningococcus and Pneumococcus Reference Laboratory (SHLMPRL) have accrued an extensive isolate archive dating back to the 1960s. Only recently with the expansion of molecular techniques has the SHLMPRL begun to investigate this valuable resource. For around a decade or more the SHLMPRL have routinely used genotyping techniques to characterise all IMD isolates it receives. However, these methods have hitherto not been employed to investigate isolates of the archive not obtained from cases of IMD. This study sought to characterise a collection of carried meningococci assembled from the isolate archive of the SHLMPRL that was obtained during the 31-year period, 1974 – 2004. Multi-locus sequence typing (MLST), porA Variable Region (VR) subtyping and a panel of genogrouping PCRs was used to characterise 791 carriage isolates. Temporal analyses of the data revealed how the presence of individual serogroups, clonal complexes, Sequence Types (STs), PorA subtypes and individual strain types in carried meningococci had changed in Scotland. Furthermore, these data were used in a comparison with a previously characterised collection of IMD isolates obtained during the same 31-year period to investigate the association of individual serogroups, clonal complexes, STs, PorA subtypes and individual strain types either with a carriage phenotype or with invasive disease. Nongroupable isolates [Odds Ratio: OR 17.66; 95% Confidence Interval: CI (12.71 to 24.54)] and those of serogroups W135 [OR 2.49; 95% CI (1.72 to 3.60)], Y [OR 6.26; 95% CI (4.18 to 9.39)], X [OR 3.13; 95% CI (1.20 to 8.14)], Z [OR 131.89; 95% CI (18.00 to 960.76)] and 29E [OR 21.30; 95% CI (4.76 to 95.36)] were significantly associated with a carriage phenotype. In contrast, serogroups A [OR 3.64; 95% CI (1.96 to 6.76)], B [OR 2.65; 95% CI (2.25 to 3.12)] and C [OR 1.92; 95% CI (1.58 to 2.33)] were significantly associated with invasive disease. The carriage strain collection reported herein was also observed to be highly diverse with the majority of STs identified only once. This diversity observed within the carriage strain collection [0.981; 95% CI (0.955, 1.006)] was significantly greater than the diversity within an IMD strain collection [0.938; 95%CI (0.934, 0.942)] from the same period. Temporal changes in the most prevalent clonal complexes (ccs) were observed throughout the 31-year period with increases in cc22, cc41/44 and cc269 and decreases in cc1, cc5, cc8, cc11, cc32, cc35, cc37, cc254, cc334 and cc364. Furthermore, for several ccs a significant association with a carriage phenotype (cc22, cc23, cc35, cc92, cc167, cc174, cc212, cc213, cc254, cc461, cc750, cc1157 and meningococci unassigned to a clonal complex) or with invasive disease (cc1, cc8, cc11, cc32, cc41-44 and cc269) was observed. Four lineages were identified amongst capsule null locus-containing meningococci, two of which, cc53 and cc1117, contained a unique allele (cnl-8) that was distinct from isolates of these lineages reported elsewhere. Despite significant associations at the level of cc, distinct differences in those associations were apparent for individual STs within a given clonal complex; most notably the significant association of ST41 and both ST43 and ST44 with invasive disease and a carriage phenotype, respectively. A feature of the carriage strain collection was the concentration of cc8 isolates during the period 1984 – 1986 and the high proportion of isolates obtained from individuals resident in Lanarkshire at a time when an episode of increased disease was experienced within that region. In this study, cc8 was found to be significantly associated with invasive disease. Furthermore, whilst ST8 was also significantly associated with invasive disease the most common strain type within cc8, C:8:8:5,2,36-2, [OR 1.68; 95% CI (1.25, 2.27)] was however, significantly associated with a carriage phenotype. The strain types B:213:22,14,36 [OR 2.38 (95% CI 1.40, 4.07)] and B:43:19,15-1,36 [OR 2.88 (95% CI 1.42, 5.88)] were also significantly associated with a carriage phenotype. Due to the heterogeneity of PorA subtypes in meningococci in Scotland the potential coverage by experimental or licensed PorA-based OMV vaccines would be limited. Therefore the introduction of monovalent or multivalent PorA-based vaccines in Scotland may be of little benefit. Improved strain coverage as a whole, not just against those of serogroup B, may require the addition of other vaccine antigens. Several other vaccine targets, including factor H-binding protein, are currently under investigation to improve coverage. Surveillance of these antigens and of the different lineages and serogroups in carriage and IMD isolates is essential to accurately monitor the effects that future vaccines will have on the meningococcus. We must remain vigilant despite a downward trend in cases of meningococcal disease in more industrialised countries

Independent evolution of the core and accessory gene sets in the genus Neisseria: insights gained from the genome of Neisseria lactamica isolate 020-06

<p>Abstract</p> <p>Background</p> <p>The genus <it>Neisseria </it>contains two important yet very different pathogens, <it>N. meningitidis </it>and <it>N. gonorrhoeae</it>, in addition to non-pathogenic species, of which <it>N. lactamica </it>is the best characterized. Genomic comparisons of these three bacteria will provide insights into the mechanisms and evolution of pathogenesis in this group of organisms, which are applicable to understanding these processes more generally.</p> <p>Results</p> <p>Non-pathogenic <it>N. lactamica </it>exhibits very similar population structure and levels of diversity to the meningococcus, whilst gonococci are essentially recent descendents of a single clone. All three species share a common core gene set estimated to comprise around 1190 CDSs, corresponding to about 60% of the genome. However, some of the nucleotide sequence diversity within this core genome is particular to each group, indicating that cross-species recombination is rare in this shared core gene set. Other than the meningococcal <it>cps </it>region, which encodes the polysaccharide capsule, relatively few members of the large accessory gene pool are exclusive to one species group, and cross-species recombination within this accessory genome is frequent.</p> <p>Conclusion</p> <p>The three <it>Neisseria </it>species groups represent coherent biological and genetic groupings which appear to be maintained by low rates of inter-species horizontal genetic exchange within the core genome. There is extensive evidence for exchange among positively selected genes and the accessory genome and some evidence of hitch-hiking of housekeeping genes with other loci. It is not possible to define a 'pathogenome' for this group of organisms and the disease causing phenotypes are therefore likely to be complex, polygenic, and different among the various disease-associated phenotypes observed.</p

A molecular epidemiological analysis of meningococcal isolates within Scotland 1972-1998

Neisseria meningitidis is an important cause of meningitis and bacteraemia worldwide and is associated with high case-fatality rates. Meningococcal disease continues to remain a public health issue in Scotland and the rest of Europe. Typing methods are used for epidemiological purposes to investigate outbreaks and the spread of meningococci and to examine the population structure of the organism in order to better understand its variation and evolution. Reference institutes have employed such methods for a number of decades for the diagnosis and detection of meningococci. However, phenotypic methods for serogrouping, serotyping and serosubtyping meningococci, although providing good strain information, can lead to endemic strains appearing identical using these methods when they are in fact quite different. More recently methods have been developed to further characterise bacteria. These methods have included PCR for the detection of meningococcal disease within blood, serogrouping and sequencing of housekeeping genes (MLST) and antigen genes such as PorA. These molecular epidemiological methods were used for the retrospective typing of invasive meningococci in Scotland, 1972-1998, using a fully automated procedure. The results of these were then analysed using statistical packages to examine the population structure of the organism. In total there were 2517 invasive isolates, received by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) from the start of 1972 to the end of 1998. Serogroup distribution changed from year to year during the time period 1972-1998 but serogroups B and C were dominant throughout this period. Serogroup B was the dominant serogroup throughout the seventies and early eighties until serogroup C became dominant during the mid 1980s. This increase in dominance of serogroup C has been found in this study not to be associated with one particular sequence type (ST) but is associated with a number of STs, which include ST-8, ST-11, ST-206 and ST-334. This is in contrast to the increase in serogroup C disease in the 1990s that was due to the ST-11 clonal complex. While there was much diversity in the STs (309 different STs among the 2517 isolates), only ten accounted for 1562 isolates (59.9%). These were ST-11, ST-8, ST-41, ST-153, ST-1, ST-32, ST-33, ST-269, ST-334 and ST-60. There were 177 new STs found during the time period. The STs were further differentiated into 31 clonal complexes, with 57 singleton types. As with the STs, although there was much diversity in the clonal complexes, only seven accounted for 1993 isolates. It was found that with PorA variable region (VR) types there were certain combinations significantly more common than others. There was a strong link with PorA type and ST and more so with clonal complex. This link was evident with the PorA type 5, 2-1, 36-2, which occured in 70 isolates representing the ST-11 complex and in all but two isolates representing ST-11. Similarly PorA type 18-3, 1, 35-1 was associated with 15 isolates belonging to the ST41-44 complex. However, this was not the case with all PorA combinations as the PorA type 19, 15, 36 was associated with 10 different complexes. There was some association between serogroup and PorA VR types. There was strong evidence of certain VR1, 2 and 3 regions being associated with certain serogroups, although this was not definitive. For example, of 192 isolates with PorA type 19, 15, 36, 85.4% were associated with serogroup B. Genosubtyping of the porA gene has been shown to increase the power of differentiation within clonal meningococcal populations. For, example, seven isolates that had the same serogroup, ST, VR1 and VR2 could be differentiated by their VR3 type. Using cluster detection software SaTScan to analyse all isolates, it was found there were 29 clusters in Scotland, from 1972-1998. These clusters included 63 cases, which accounted for 2.5% of all cases. A range of different strains caused the clusters that were identified in this study, some caused by hypervirulent strains. These strain types were responsible for a number of cases throughout the world as well as in Scotland during the period of this study. However it was also shown that there were clusters identified in this study caused by lesser-known strain types that were not responsible for many cases and that appear to be unique to Scotland or the UK. This study is the first to look at the detection of clusters over a time period of 26 years and to identify clusters that would have previously been unidentified due to lack of suitable characterisation techniques. The results in this study indicate that the multivalent preparation produced by the Netherlands Vaccine Institute (Nonavalent vaccine) had the potential, based on the PorA types that it contains, to prevent the majority of serogroup B infection that had occurred in Scotland, from 1972-1998. It also had the potential, although not to the same extent as serogroup B, to protect against other serogroups. For the age groups that would potentially have been the first to be immunised with any vaccine as part of the childhood vaccination programme, the 0-4 years old group, the potential coverage was over 92% which is comparable with the coverage seen with the serogroup C meningococcal conjugate (MCC) vaccine, of approximately 90%

Molecular characterization of non-groupable Neisseria meningitidis causing invasive disease in South Africa

The University of the Witwatersrand, Faculty of Health Sciences In fulfilment of the requirements for the degree of Master of Science in Medicine. 27 October 2017.Background The meningococcal capsule is an important virulence determinant. Unencapsulated meningococci lacking capsule biosynthesis genes and containing the capsule null locus (cnl) are predominantly non-pathogenic. Rare cases of invasive meningococcal disease caused by cnl isolates belonging to sequence types (ST) and clonal complexes (cc) ST-845 (cc845), ST-198 (cc198), ST-192 (cc192) and ST-53 (cc53) have been documented. The clinical significance of these isolates however remains unclear. We identified four invasive cnl meningococci through laboratory-based surveillance in South Africa from 2003 through 2013, which we aimed to characterize using whole genome data. Results One isolate [NG: P1.7-2,30: F1-2: ST-53 (cc53)] contained cnl allele 12, and caused empyema in an adult male with bronchiectasis from tuberculosis, diabetes mellitus and a smoking history. Three isolates were NG: P1.18-11,42-2: FΔ: ST-192 (cc192) and contained cnl allele 2. One patient was an adolescent male with meningitis. The remaining two isolates were from recurrent disease episodes (eight months apart) in a male child with deficiency of the sixth complement component, and with the exception of two single nucleotide polymorphisms, contained identical core genomes. The ST-53 (cc53) isolate possessed alleles for NHBA peptide 191 and fHbp variant 2; whilst the ST-192 (cc192) isolates contained NHBA peptide 704 and fHbp variant 3. All four isolates lacked nadA. Comparison of the South African genomes to 65 additional cnl genomes on the PubMLST Neisseria database (http://pubmlst.org/neisseria/), determined that most putative virulence genes could be found in both invasive and carriage phenotypes. Conclusions Although rare, invasive disease by cnl meningococci may be associated with host immunodeficiency and such patients may benefit from protein-based meningococcal vaccines.LG201

Molecular characterisation of Neisseria meningitidis serogroup B isolates in South Africa, 2002- 2006

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2011Despite being a fulminant pathogen, Neisseria meningitidis (meningococcus) is part of the commensal flora of the human nasopharynx. Globally, five meningococcal serogroups (A, B, C, Y and W135) cause the majority of invasive disease. Most serogroup B cases occur sporadically but may be endemic or epidemic within a geographic region. In South Africa, there are limited data on invasive serogroup B clones and the antigenic diversity of certain meningococcal outer membrane proteins. This study examined the molecular epidemiology of serogroup B meningococci in South Africa from 2002 through 2006. Invasive meningococcal isolates were submitted to a national laboratory-based surveillance system. For this study, serogroup B isolates were characterised by pulsed-field gel electrophoresis (PFGE), PorA, FetA and multilocus sequence (MLST) typing. PorA, FetA and multilocus sequence (MLST) typing were performed on all 2005 isolates (n=58) and randomly selected isolates from other years (n=25). A total of 2144 invasive cases were reported over the study period. Of these, 76% (1627/2144) had viable isolates available for serogrouping and 307 (19%) were serogroup B. Serogroup B cases were reported from across the country however the majority were from the Western Cape province. The highest incidence of serogroup B was in children less than 5 years of age. Isolates displayed a high level of diversity by PFGE. Despite this diversity the majority of serogroup B meningococci collected over the 5-year period could be grouped into several clonal clusters representative of global invasive MLST clonal complexes. Overall, the most predominant MLST clones in South Africa were ST-32/ET-5 and ST-41/44/lineage 3. In addition, at least 19 PorA types and 16 FetA types were determined among selected isolates. Globally invasive serogroup B disease is caused by heterogeneous strains however, prolonged outbreaks in several countries have been due to strains of the ST-32/ET-5 and ST-41/44/lineage 3 clonal complexes. At present, serogroup B disease in South Africa is not dominated by an epidemic clone, however, global clonal complexes ST-32/ET-5 and ST-41/44/lineage 3 are circulating in Western Cape and Gauteng, respectively

The Impact of Nucleotide Sequence Analysis on Meningococcal Vaccine Development and Assessment

Since it became available as a routine tool in biology, the determination and analysis of nucleotide sequences has been applied to the design of vaccines and the investigation of their effectiveness. As vaccination is primarily concerned with the interaction of biological molecules with the immune system, the utility of sequence data is not immediately obvious and, indeed, nucleotide sequence data are most effective when used to complement more conventional immunological approaches. Here, the impact of sequencing on the field of vaccinology will be illustrated with reference to the development and implementation of vaccines against Neisseria meningitidis (the meningococcus) over the 30-year period from the late-1980s to the late-2010s. Nucleotide sequence-based studies have been important in the fight against this aggressive pathogen largely because of its high genetic and antigenic diversity, properties that were only fully appreciated because of sequence-based studies. Five aspects will be considered, the use of sequence data to: (i) discover vaccine antigens; (ii) assess the diversity and distribution of vaccine antigens; (iii) determine the evolutionary and population biology of the organism and their implications for immunization; and (iv) develop molecular approaches to investigate pre- and post-vaccine pathogen populations to assess vaccine impact. One of the great advantages of nucleotide sequence data has been its scalability, which has meant that increasingly large data sets have been available, which has proved invaluable in the investigation of an organism as diverse and enigmatic as the meningococcus

Bacterial Meningitis

This timely collection of expert papers draws attention to the global burden of meningitis and the challenges faced by the WHO’s roadmap to defeat meningitis by 2030. The three main goals of the meningitis roadmap are to eliminate epidemics of bacterial meningitis, reduce cases and deaths from vaccine-preventable bacterial meningitis, and reduce disability and improve quality of life after meningitis of any cause. This book includes a wide range of original research and reviews on epidemiology and vaccination of bacterial meningitis that have direct relevance to advancing the goals of the roadmap