Streptococcus pneumoniae serotypes 8 and 22F causing invasive pneumococcal disease in the Czech Republic in 2014-2020: whole genome sequencing (WGS) analysis. / Streptococcus pneumoniae sérotypu 8 a 22F pusobící invazivní pneumokokové onemocnení v Ceské republice v letech 2014­2020: analýza metodou sekvenace celého genomu (WGS).

AIM: An analysis is presented of whole genome data of Streptococcus pneumoniae serotypes 8 and 22F isolated in the Czech Republic from invasive pneumococcal disease (IPD) in 2014-2020. New multivalent pneumococcal conjugate vaccines (PCVs) are effective against these serotypes. Recently, serotypes 8 and 22F have been among the leading causes of IPD in the Czech Republic. S. pneumoniae isolates from the Czech Republic were compared with those of the same serotypes recovered in other countries in the same period and available in the international database PubMLST. MATERIAL AND METHODS: Isolates from IPD of serotypes 8 (22 isolates) and 22F (21 isolates) recovered in the Czech Republic in 2014-2020 were subjected to whole genome sequencing (WGS). The genomes were analysed and compared using the international database PubMLST. RESULTS: Most of the studied Czech serotype 8 isolates belong to two main subpopulations. The first subpopulation, dominated by ST-53 isolates, is part of a highly abundant group of genetically close European and non-European isolates that are clearly separated on the phylogenetic network. The second subpopulation of Czech serotype 8 isolates (dominated by ST-404) is more genetically variable and forms a separate lineage on the global phylogenetic network, with no other European isolates. Czech isolates of serotype 22F are a homogeneous population with a clear predominance of ST-433, which belongs to a genetically close European population. CONCLUSION: The analysis of WGS data of IPD isolates of serotypes 8 and 22F provided a detailed insight into the genetic relationships between the Czech populations of these serotypes. It also allowed comparison of the Czech populations with the matched populations from other European and non-European countries. The obtained results add to the body of knowledge about the spread of genetic lineages causing IPD in the Czech Republic in the post-vaccination period and provide a basis for considering whether the use of the new multivalent PCVs in the Czech Republic would be beneficial.

Comparison of invasive and non-invasive isolates of Neisseria meningitidis by whole genome sequencing, Czech Republic, 2005-2021.

AIM: Whole genome sequencing (WGS) analysis of candidate virulence genes of epidemiologically and/or clinically related invasive and non-invasive isolates of Neisseria meningitidis from 2005-2021. MATERIAL AND METHODS: Seventy-nine isolates were selected for analysis from three different categories: cases of invasive meningococcal disease (IMD) and their healthy contacts, different clinical specimens from the same IMD case, and different clinical specimens from the same IMD case and their healthy contacts. WGS was used to analyse sequence variability in candidate N. meningitidis virulence factor genes, with more than 250 loci studied. RESULTS: The frequency of sequence changes in the candidate N. meningitidis virulence factor genes of invasive and non-invasive isolates varied widely. The highest level of variability was observed in the pilus genes, especially pilE and pglA. Our study detected variability in the opacity protein A (opaA) gene in more than half of the isolates analysed, with the frequency of opaA gene changes reaching almost 70% in MenC isolates. Higher frequency of changes were also observed in the genes for capsule production, especially in those of the D+D' capsular region. CONCLUSIONS: The results obtained support the hypothesis that serogroup-specific genetic mechanisms are also involved in the pathogenicity of N. meningitidis. These data add to the body of knowledge necessary for the development of new effective IMD vaccines.

Analysis of meningococcal vaccine uptake in patients with invasive meningococcal disease, Czech Republic, 2006-2022.

In 2006-2022, 958 cases of invasive meningococcal disease (IMD) were reported to the surveillance programme in the Czech Republic, of which 21 (2.19%) had a history of vaccination with one of the meningococcal vaccines. Data analysis shows that these vaccines provide a very good protection against IMD. It was found that vaccinated patients with IMD either were not vaccinated against the causative serogroup and/or did not receive a booster dose. The results of this analysis show the benefit of both vaccines available in the Czech Republic: recombinant vaccine containing meningococcal serogroup B antigens (MenB vaccine) and tetravalent conjugate vaccine containing antigens of four meningococcal serogroups A, C, W, Y (A, C, W, Y conjugate vaccine). The results also show the benefit of meningococcal vaccine booster doses and the need for giving MenB vaccine to young children as early as possible.

Analysis of epidemiological and molecular data from invasive meningococcal disease surveillance in the Czech Republic, 1993-2020.

AIM: An analysis is presented of epidemiological and molecular data from invasive meningococcal disease (IMD) surveillance in the Czech Republic (CR) for 1993-2020, comparing trends in four seven-year periods: 1993-1999, 2000-2006, 2007-2013, and 2014-2020. MATERIAL AND METHODS: IMD surveillance data are generated by linking National Reference Laboratory for Meningococcal Diseases data and epidemiological data routinely reported to the infectious diseases information systems, with duplicate data removal. Whole genome sequencing (WGS) was used for analysis of selected isolates from IMD cases. In this study, WGS data are analysed on 323 isolates recovered from IMD cases in the Czech Republic between 1993-2020. RESULTS: Over the entire study period 1993-2020, 2,674 cases were recorded in the IMD surveillance programme, of which 272 were fatal. In the first seven-year period, the highest incidence rate of 2.2/100,000 population was reported in 1995, a gradual decline from 0.8 to 0.6/100,000 was observed in the third period, and in the last period, this decline continued until 2020, achieving a low of 0.2/100,000. In all four study periods, the age group 0-11 months was the most affected, followed by 1-4-year-olds and 15-19-year-olds. Serogroup B caused the highest number of cases (43.6%), followed by serogroups C (34.9%), Y (1.8%), and W (1.5%). Serogroup X was only found in three cases. The overall case fatality rate in the entire study period was 10.2%, with no decline seen in the four periods. The highest case fatality rate was associated with serogroup Y (14.6%), followed by serogroups W (12.5%), C (12.0%), and B (8.1%). In terms of age, the highest case fatality rate was observed in people aged 65+ (24.7%). The WGS data for 323 IMD isolates from 1993-2020 showed the highest representation of eight clonal complexes: cc11, cc44/41, cc32, cc267, cc23, cc18, cc35, and cc865. Isolates of serogroup C, cc11, from the last study period form two genetically distinct populations with distinct phenotypes that are genetically distant from the lineage of cc11 isolates from the first two periods. The study population included a unique Czech subpopulation of serogroup W isolates (ST-3342, cc865), recorded only in the last two periods (2007-2020), whose position in the phylogenetic network supports the theory that the serogroup W population in the Czech Republic originated from serogroup B isolates as a result of serogroup switch (capsule switch). Clonal complexes cc41/44, cc32, cc267, cc18, and cc35 are predominantly composed of serogroup B isolates, while cc23 includes exclusively serogroup Y isolates. CONCLUSIONS: The analysis of surveillance data over a 28-year period confirms that the vaccination strategy currently used in the Czech Republic, i.e., vaccination of young children and adolescents with a combination of MenB vaccine and quadrivalent conjugate ACWY vaccine, appears to be the most appropriate in the context of the long-term epidemiological situation of IMD in the CR.

Detailed molecular characterization of Neisseria meningitidis isolates by whole genome sequencing (WGS), Czech Republic, 2010-2019.

AIM: Presentation of the first results of the analysis of Neisseria meningitidis isolates from invasive meningococcal disease and from clinically and/or epidemiologically linked cases from 2010-2019. Whole genome sequencing (WGS) was used for the study. MATERIAL AND METHODS: The study set included 59 isolates of N. meningitidis from 2010-2019. Serogrouping was done by conventional serological methods and confirmed by RT-PCR. WGS was used for detailed molecular characterization, covering not only basic genes but also ribosomal and capsular genes, antibiotic resistance gene penA, and outer membrane protein gene porA. RESULTS: WGS analysis of N. meningitidis isolates resulted in a detailed molecular characterization. In a large part of the genes analysed, new mutated allelic variants were found. They were submitted to the PubMLST database and subsequently annotated by the curator. All 59 study isolates were assigned to BAST types, characterized by a unique combination of allelic variants of N. meningitidis B vaccine (MenB vaccine) antigen genes. Overall, 32 different BAST types were identified, and 10 isolates either carried an unknown combination of BAST loci or a new allelic variant in some of the BAST loci. Furthermore, the MenDeVAR index, which provides information on the functional effect of MenB vaccines on a given isolate, was determined. CONCLUSIONS: The results obtained add to the body of knowledge of the transmission of invasive and non-invasive strains of N. meningitidis in the population. The WGS analysis provided detailed data on the coverage of these strains by new MenB vaccines.

Population analysis of Streptococcus pneumoniae serotype 19A by whole genome sequencing in the Czech Republic and in Europe after serotype 19A inclusion in pneumococcal conjugate vaccine.

AIM: To present the results of whole genome sequencing (WGS) analysis of Streptococcus pneumoniae serotype 19A and to compare them with the respective data from Europe. The vaccine serotype 19A is widely distributed in the Czech Republic. MATERIAL AND METHODS: WGS was used in this study as the most powerful available method for detailed characterization of S. pneumoniae. Nineteen Czech isolates of S. pneumoniae 19A were analysed and compared with 415 European isolates included in the PubMLST database. RESULTS: S. pneumoniae serotype 19A causes all types of pathogen - host interaction, from carriage to noninvasive and invasive pneumococcal disease (IPD). In 2010 - 2019, 3872 cases of IPD were reported within the surveillance programme in the Czech Republic, with 323 of these caused by serotype 19A. WGS data of the Czech serotype 19A isolates show a numerous and genetically related subpopulation of three sequencing types: ST-199, ST-416, and ST-3017. Within this subpopulation, the largest is the cluster of nine ST-199 isolates. High relatedness of ST-199 isolates is also confirmed by the fact that all but one isolate, 117/2019 (novel rST- -137805), share the same ribosome sequencing profile - rST-11365. Outside the above-mentioned subpopulation, there are only four isolates that form three separate genetic lines of serotype 19A. A highly similar situation is observed across European countries, where about half of all serotype 19A isolates form a genetically closely related subpopulation (ST-199, ST-416, ST-450, ST-667, ST-3017, and ST-10360) while isolates which are not part of this subpopulation represent a large number of unrelated genetic lines. CONCLUSIONS: The study has shown a mostly homogeneous population of S. pneumoniae serotype 19A to circulate in the post-vaccination era in both the Czech Republic and Europe, with some unrelated isolates located outside this population.

Implementation and use of whole genome sequencing (WGS) in the surveillance of invasive pneumococcal disease, Czech Republic, 2017-2019.

AIM: In order to improve the surveillance of invasive pneumococcal disease (IPD), the National Reference Laboratory (NRL) for Streptococcal Infections implemented whole genome sequencing (WGS) of Streptococcus pneumoniae. This article reports the first WGS data on S. pneumoniae isolates in the Czech Republic. MATERIAL AND METHODS: Thirty-five isolates of S. pneumoniae from IPD recovered in 2017-2019 were selected for WGS. These were serotypes 4, 8, 9V, 19A, and 22F, which were determined by the Quellung reaction in combination with endpoint multiplex PCR (mPCR). Multilocus sequence typing (MLST) is routinely used for more detailed analysis termed sequence typing. The selected isolates were analysed by WGS on the Illumina MiSeq platform. The sequences obtained were processed using the Velvet de novo Assembler software. The assembled genomes were uploaded into the PubMLST database, using the BIGSdb platform, and then scanned automatically and molecularly characterized. The isolates were compared at three resolution levels: seven MLST genes, 53 ribosomal genes (rMLST), and 1420 genes (all loci). The all loci scheme covers MLST genes, ribosomal genes, and core genome MLST genes (cgMLST). These are all currently defined genes of S. pneumoniae available in the PubMLST database. Distance matrices based on the number and variability of all loci analysed were generated automatically using the Genome Comparator tool. Phylogenetic networks were created and edited with the SplitsTree4 package, using the NeighborNet algorithm. The final graphics were edited with the Inkscape software. RESULTS: Based on an overall view of the phylogenetic networks, it can be concluded that the genetic lines within each of S. pneumoniae serotypes 4, 8, 9V, 19A, and 22F are highly unrelated, to the same extent as if the isolates were of different serotypes. S. pneumoniae isolates of the same serotype, whether or not of the same sequence type, can be described, based on the results, as a non-homogeneous group with a number of unrelated genetic clusters that share genes assigning them to a specific serotype. WGS has also shown its discriminatory power, allowing the assignment of isolates of the same serotype and sequence type to different genetic clusters. CONCLUSION: Of the methods used so far in the Czech Republic, WGS allows the most detailed characterization of S. pneumoniae isolates. It is highly desirable to integrate it in the molecular surveillance of IPD in the Czech Republic, similarly to other countries in Europe and in the world.

Surveillance of invasive meningococcal disease based on whole genome sequencing (WGS), Czech Republic, 2015.

AIM: To test the potential of whole genome sequencing (WGS) for molecular surveillance of invasive meningococcal disease in the Czech Republic. To check the success of the new method in the identification of gene and protein variants and to compare the outcomes between WGS and conventional sequencing methods. MATERIAL AND METHODS: WGS was carried out in a set of 20 N. meningitidis isolates from invasive meningococcal disease cases in the Czech Republic in 2015. WGS was performed using the Illumina MiSeq platform. The WGS data were processed by the Velvet de novo Assembler software, and the resultant genome contigs were submitted to the Neisseria PubMLST web database containing allelic and genomic data on strains of the genus Neisseria. The genomes were analysed and compared using the BIGSdb Genome Comparator, which is part of the PubMLST database. WGS data were compared at several levels of resolution: MLST (Multi Locus Sequence Typing), rMLST (ribosomal MLST), cgMLST (core genome MLST), and "all loci", i.e. all genes of N. meningitidis defined in the PubMLST database by 6 November 2017 (3028 loci). The WGS method was used to characterise in detail the genes of antigens involved in vaccines against N. meningitidis B. RESULTS: The new WGS method provided detailed characteristics of N. meningitidis isolates, which improved the results obtained previously by conventional sequencing methods. High quality WGS data made it possible to identify novel alleles and novel sequence types that could not be recognized by conventional sequencing methods. The analysis of genetic diversity confirmed closer relatedness between isolates belonging to the same clonal complex. The most accurate information on genetic diversity of isolates was obtained by the comparison of WGS data at the cgMLST and "all loci" levels. Distant relatedness of three clonal complexes (cc32, cc35, and cc269) was found. WGS data also provided more accurate information on the coverage of isolates by MenB vaccines in comparison with conventional sequencing data. CONCLUSIONS: The WGS method showed a higher discrimination potential and allowed a more accurate determination of genetic characteristics of N. meningitidis. The integration of the WGS method in routine molecular surveillance of invasive meningococcal disease in the Czech Republic is desirable.