Prevalence of the M1UK sublineage among emm1 Streptococcus pyogenes invasive strains isolated in the Czech Republic from December 2022 to May 2023.

AIMS: Since December 2022, an increase in invasive disease caused by Streptococcus pyogenes has been observed in the Czech Republic, with a shift in the clinical presentation and age of patients. Unlike in previous years, invasive disease is more common in children and adolescents under 18 years of age and in previously healthy middle-aged adults. An increase has been noticed in the number of S. pyogenes isolates from primarily sterile sites such as haemoculture, cerebrospinal fluid, pleural effusion fluid, joint fluid, and postmortem specimens. Routine emm gene typing revealed emm1 to be the predominant emm type of S. pyogenes. Between January 2023 and July 2023, 46% of all S. pyogenes isolates from invasive cases were assigned to the emm1 type. The globally spread M1UK sublineage is characterized by differences in the expression of seven genes, including the streptococcal pyrogenic toxin A (speA) gene, compared to historical emm1 iGAS strains. The aim of this study is to determine whether the more toxigenic M1UK sublineage is associated with the increase in invasive disease in the Czech Republic. METHODS: Whole genome sequencing of 41 S. pyogenes isolates from patients with invasive disease recovered in the Czech Republic in 2018 and 2019 and from December 2022 to May 2023 was performed using the MiSeq instrument (Illumina). Bioinformatics analysis was performed using freely available online tools the Bacterial and Viral Bioinformatics Resource Center. RESULTS: Based on whole genome sequencing data of 41 emm1 isolates of S. pyogenes from patients with invasive infectious disease recovered in 2018 and 2019 and from December 2022 to May 2023, the M1UK sublineage was found to be predominant from December 2022 to May 2023. CONCLUSION: The reason for the spread of the M1UK sublineage in the Czech Republic late in 2022 and in the first half of 2023 is not entirely clear, but it may be related to reduced immunity due to limited GAS transmission during lockdowns, especially in children. Another factor that may have contributed to the high incidence of invasive infectious diseases is the seasonal circulation of respiratory viruses.

Streptococcus pneumoniae serotypes 8 and 22F causing invasive pneumococcal disease in the Czech Republic in 2014-2020: whole genome sequencing (WGS) analysis.

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.

Posibilities for use of whole genome sequencing (WGS) for the analysis of Streptococcus pneumoniae isolates.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive coccus causing both non-invasive and invasive infectious diseases. Pneumococcal diseases are vaccine preventable. Invasive pneumococcal diseases (IPD) meeting the international case definition are reported nationally and internationally and are subject to surveillance programmes in many countries, including the Czech Republic. An important part of IPD surveillance is the monitoring of causative serotypes and their frequency over time and in relation to ongoing vaccination programmes. In the world and in the Czech Republic, whole genome sequencing (WGS) is increasingly used for pneumococci, which allows for serotyping from sequencing data, precise analysis of their genetic relationships, and the study of genes present in their genome. Whole-genome sequencing enables the generation of reliable and internationally comparable data that can be easily shared. Sequencing data are analysed using bioinformatics tools that require knowledge in the field of natural sciences with an emphasis on genetics and expertise in bioinformatics. This publication presents some options for pneumococcal analysis, i.e., serotyping, multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), whole genome MLST (wgMLST), single nucleotide polymorphism (SNP) analysis, assignment to Global Pneumococcal Sequence Cluster (GPSC), and identification of virulence genes and antibiotic resistance genes. The WGS strategies and applications for Europe and WGS implementation in practice are presented. WGS analysis of pneumococci allows for improved IPD surveillance, thanks to molecular serotyping, more detailed typing, generation of internationally comparable data, and improved evaluation of the effectiveness of vaccination programmes.

Detection of superantigens in Streptococcus pyogenes isolates based on whole genome sequencing data.

Streptococcus pyogenes causes a variety of human diseases ranging from uncomplicated respiratory tract and skin infections to severe invasive diseases possibly involving toxic shock syndrome. Besides the emm gene-encoded M protein, important virulence factors are pyrogenic exotoxins, referred to as superantigens. The National Reference Laboratory for Streptococcal Infections has newly introduced bioinformatics tools for processing S. pyogenes whole genome sequencing data. Using the SRST2 software and BV-BRC platform, WGS data of 10 S. pyogenes isolates from patients with invasive disease were analysed, and emm type, sequence type, and superantigen encoding gene profiles were determined. The Unicycler assembly pipeline with the SPAdes de novo assembler was used to assemble genome sequences from short reads.

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.

Monitoring changes in invasive disease caused by Haemophilus influenzae in the Czech Republic between 1999 and 2020.

AIM: To assess the trends and changes in the incidence of invasive disease caused by Haemophilus influenzae in the Czech Republic (CR) between 1999 and 2020 with regard to the introduction of childhood vaccination against H. influenzae serotype b (Hib) in 2001. Characterization of strains by multilocus sequence typing (MLST) and search for correlations between serotypes, sequence types, and patient groups or clinical manifestations of the disease. MATERIAL AND METHODS: A total of 623 invasive H. influenzae strains from surveillance of invasive Haemophilus disease in the Czech Republic were analysed. All strains were biotyped based on phenotypic characteristics and serotyped using slide agglutination with specific a-f antisera. Three hundred and eighty-three strains from the collection of the National Reference Laboratory for Haemophilus Infections (NRL HEM) originating from surveillance in the CR were analysed by MLST and assigned to sequence types (ST). For analyses, the dataset was supplemented with five strains from the PubMLST database of serotypes rarely or not at all found in the CR. Similarity calculations based on MLST and strain (serotype, biotype, ST) and patient (diagnosis, sex, age) data were performed in BioNumerics 7.6. RESULTS: After the introduction of Hib vaccination in 2001, a dramatic decline of more than 90% was observed in invasive Hib disease over the following years. Between 1999 and 2020, a total of 623 cases of invasive disease caused by H. influenzae were recorded in the CR, with about 20 cases reported annually in recent years. At present, the dominant agents causing Haemophilus invasive disease in the CR are non-enveloped strains (HiNT) followed by strains of Hif and Hie serotypes. The most common manifestation of Haemophilus invasive disease in the pre-vaccination era was meningitis, while now it is sepsis. Sequence types of 383 strains from the NRL HEM collection originating from surveillance in the CR were analysed. The results showed high clonality of the encapsulated strains and diversity of HiNT strains, which is consistent with the results of others. Strain similarity analysis showed no demonstrable relationships between patient age or clinical manifestation and serotype and ST. CONCLUSION: In invasive Haemophilus disease, there has been a dramatic change as a result of Hib vaccination after 2001, with a reduction of cases caused by Hib from tens to units annually. In the last decade, the situation in the CR has been stable with no significant changes in the number of cases or in the representation of causative serotypes and is in line with the reports from other EU countries. In order to monitor further developments, it is desirable that the NRL HEM should continue the surveillance of invasive disease caused by H. influenzae, including molecular biological characteristics of strains. MLST allows the characterisation of strains based on allelic variants of selected housekeeping genes, but it does not allow the association of specific H. influenzae sequence types with patient age, sex or clinical manifestations. In the future, whole genome sequencing could be a useful tool for determining the correlation between the disease and specific strains.

Molecular genotyping of Streptococcus agalactiae isolates with non-typeable serotype, Czech Republic, 2008­2020

The NRL for Streptococcal Infections performed serotyping of 1038 isolates of Streptococcus agalactiae from 972 patients by the latex agglutination method in 2008­2020. Forty-three isolates (4.4%) whose serotyping failed were classified as non-typeable. The aim of the present study was to determine the genotype of these non-typeable isolates using multiplex polymerase chain reaction (mPCR). Genotyping was successful in the entire set of 43 non-typeable isolates. The most common genotype was V (41.9%), followed by Ia (20.9%). The isolates serotyped by latex agglutination were predominantly assigned to serotype III (29.2%) and V (26.2%). Complete data were obtained on the prevalence of S. agalactiae serotypes/genotypes in the Czech Republic in 2008­2020. Monitoring the serotype and genotype distribution of the pathogen is a prerequisite for the introduction of a potential vaccine against S. agalactiae into clinical practice.

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.

Invasive pneumococcal diseases in adults admitted to the Na Bulovce Hospital: Serotype replacement after the implementation of general childhood pneumococcal vaccination.

OBJECTIVE: The aim of this study was to analyse epidemiological and clinical characteristics of invasive pneumococcal disease (IPD) in adults before and after the introduction of the general childhood conjugate pneumococcal vaccination programme in the Czech Republic. MATERIAL AND METHODS: The retrospective observational sentinel study included adults with IPD admitted to the Na Bulovce Hospital in Prague from 1/2000 through 12/2019. A case of IPD was defined as isolation of Streptococcus pneumoniae from a primarily sterile site. RESULTS: A total of 304 IPD cases were diagnosed during the study period, with a male to female ratio of 1.49:1 and age median of 58 years (IQR 43-73). The most prevalent clinical forms were bacteraemic pneumonia (185 cases; 60.9%) and purulent meningitis (90; 29.6%). A total of 157/293 patients (53.6%) required intensive care, and the case fatality rate was 25.3% (n = 77). The serotype was determined in 292 (96.0%) isolates, the most prevalent being serotypes 3 (38; 12.5%), 4 (28; 9.2%), 7F (24; 7.9%), 8 (21; 6.9%), and 1 (18; 5.9%). Both clinical and epidemiological characteristics of IPD caused by the most prevalent serotypes differed considerably. Patients diagnosed with serotype 3 were older, more frequently required intensive care, and showed higher mortality. The proportion of IPD caused by non-PCV13 serotypes increased from 28.8% (19/66) in 2000-2005 to 54.8% (40/70) in 2015-2019 (p = 0.001). CONCLUSION: The study demonstrated that invasive diseases caused by the most prevalent pneumococcal serotypes differ in their epidemiological and clinical characteristics and case fatality rate. During the study period, there was a significant increase in IPD caused by non-PCV 13 serotypes, limiting the effect of vaccination in adults.

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.

Clonal characterisation of Streptococcus pneumoniae strains using MLST and MLVA - Can MLVA improve the characterisation?

AIM: To determine clonal characteristics of Streptococcus pneumoniae (S. pneumoniae) strains causing invasive pneumococcal disease (IPD) in the Czech Republic (CR) in 2017. Clonal assignment of strains was performed in the National Reference Laboratory for Streptococcal Infections (NRL) by the routinely used method, multilocus sequence typing (MLST), and a newly introduced method, multiple-locus variable number tandem repeat analysis (MLVA). MATERIAL AND METHOD: The study strains were 87 isolates of S. pneumoniae selected from those referred to the NRL within the IPD surveillance programme from all over the CR in 2017. The study set covers S. pneumoniae isolates of both pneumococcal 13-valent conjugate vaccine serotypes (1, 4, and 9V) and non-vaccine serotypes (8, 9N, and 22F) widely spread in the CR. The study methods were MLST, the standard method used worldwide for the characterisation of pneumococcal isolates based on sequencing of a set of gene regions, and MLVA, which allows to characterise isolates based on the number of tandem repeats in intergenic regions. RESULTS: MLST revealed and confirmed a high level of clonal homogeneity of S. pneumoniae isolates of serotypes 1, 9N, 9V, and 22F and a considerable genetic variability of serotype 4 and 8 isolates. There was a general correlation between the MLST and MLVA clonal complex assignments. In comparison with MLST, MLVA has superior clonal discriminatory power. Isolates with the newly determined MLVA profiles should be assigned to new MLVA types (MT). Nevertheless, the new web support of the MLVA scheme for S. pneumoniae is less relevant as it does not provide services comparable to those available from the web support for MLST characterisation. CONCLUSIONS: MLST continues to be the standard method for clonal characterisation of S. pneumoniae isolates from IPD for the purposes of both national and international surveillance. MLST characteristics of isolates are helpful in the study of clonal variability conducted by both national and transnational public health protection authorities. MLVA is not routinely used but can serve as a complementary method for rapid identification of clonal relatedness between isolates, e.g. those from local outbreaks. It is more suitable for the detection of emergence and spread of a virulent clonal variant.

Surveillance of antibiotic resistance of Streptococcus pneumoniae in the Czech Republic, respiratory study results, 2010-2017.

AIM: The purpose of the surveillance performed from October to December in 2010-2017 was to monitor the trends in the susceptibility to beta-lactam and macrolide antibiotics in Streptococcus pneumoniae isolates from respiratory tract infections in the Czech Republic. MATERIAL AND METHODS: Between 42 and 55 laboratories participated in the study every year. Consecutive non-duplicate pneumococcal isolates from relevant microbiological specimens from patients with community-acquired bacterial respiratory tract infection were sequentially included in the study. Laboratories recorded qualitative results of penicillin and erythromycin susceptibility testing; susceptibility to antibiotics was determined by the disk diffusion method. Penicillin non-susceptible and/or erythromycin resistant isolates were referred to the National Reference Laboratory for Antibiotics, where the minimum inhibitory concentration of each antibiotic was tested using the broth microdilution method, and their serotyping was performed in the National Reference Laboratory for Streptococcal Infections. Twenty-six isolates from 2017 were analysed by the multilocus sequence typing method. RESULTS: In total, 7 491 pneumococcal strains were examined, of which 53.7% (4 023) were from the upper respiratory tract and 47.7% (3 573) from children under 15 years of age. Non-susceptibility to penicillin decreased from 2.6% in 2010 to 1.2% in 2017, while resistance to erythromycin increased from 7.4% to 9.7% over the same period. Penicillin non-susceptible isolates were mostly of serotypes 19A, 19F, and 15A. Macrolide resistant but penicillin susceptible isolates were predominantly represented by serotypes 19A and 3. The presence of the Taiwan19F-14 clone was confirmed in penicillin non-susceptible isolates by MLST, and the most frequently identified sequence type (ST) in macrolide resistant isolates was ST416 classified into the Netherlands15B-37 clone. CONCLUSIONS: The respiratory study of antibiotic resistance in S. pneumoniae confirmed the decreasing trend of resistance to penicillin but revealed a growing resistance to macrolide antibiotics in the Czech Republic. The results of our study confirm that antibiotic resistance in the vaccination era is associated primarily with the non-vaccine serotypes, and the clonal expansion of macrolide resistant serotype 19A was apparently supported by the growing prescription of macrolide antibiotics.

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.

Molecular characterization of Streptococcus pneumoniae isolates recovered from cases of pneumococcal vaccine failure in children under five years of age in the Czech Republic in 2012-2014.

OBJECTIVE: Molecular characterization of Streptococcus pneumoniae isolates recovered in the Czech Republic from previously vaccinated patients diagnosed with invasive pneumococcal disease caused by vaccine serotypes in 2012-2014. The target group were children under five years of age. MATERIAL AND METHODS: S. pneumoniae strains are referred to the National Reference Laboratory for Streptococcal Infections (NRL) from all over the Czech Republic. Pneumococcal vaccine failure was identified based on the international definition. The NRL routinely performs identification and typing of isolates using both conventional and molecular methods. To characterize in more detail eight isolates from cases of pneumococcal vaccine failure, multilocus sequence typing (MLST) was used. RESULTS: In 2012-2014, eight S. pneumoniae strains were recovered from children under five years of age diagnosed with vaccine failure. The strains were assigned to serotypes 1 (n = 4), 14 (n = 2), and 3 (n = 2). Serotype 1 isolates were of one sequence type, ST306, which is typical for serotype 1. Similarly, serotype 14 was exclusively of ST124, which is often linked to serotype 14 in the Czech Republic, although in other geographical areas, other STs are often found. Serotype 3 is highly heterogeneous, which is also reflected in multiple STs found in it. We identified a clonal complex comprising ST505 and ST124. ST124 is for the first time reported in S. pneumoniae of serotype 3 in the world. CONCLUSION: Results are presented of a pilot project conducted in the Czech Republic and focused on molecular characterization of S. pneumoniae strains recovered from cases of pneumococcal vaccine failure in children under five years of age. The data obtained are helpful in improving the surveillance of IPD in the Czech Republic.

[The possibilities for the implementation of whole genome sequencing (WGS) of Neisseria meningitidis in molecular surveillance of invasive meningococcal disease in the Czech Republic]. / Moznosti implementace sekvenace celého genomu (WGS) Neisseria meningitidis do molekulární surveillance invazivního meningokokového onemocnení v Ceské republice.

The implementation of the surveillance of invasive meningococcal disease is recommended worldwide. The Whole Genome Sequencing (WGS) method increasingly comes to the fore, which provides the possibilities for further detailed characterization of Neisseria meningitidis and makes it possible to integrate all conventional sequencing approaches into one method. Six N. meningitidis isolates from 2013 and 2015, characterized previously by Sanger amplicon sequencing, were selected to be studied by the novel WGS method. WGS data analysis has confirmed the accuracy of this method in determining epidemiological markers. The aim of this communication is to point out the possibility for the implementation of WGS into molecular surveillance of invasive meningococcal disease in the Czech Republic. The National Reference Laboratory for Meningococcal Infections (NRL/MEN) will continue to use WGS for molecular characterization of selected isolates of N. meningitidis and for the improvement of molecular surveillance of invasive meningococcal disease in the country.

[Clonal analysis of the meningococcal populations from invasive disease and healthy carriers isolated in the Czech Republic in 1971-2014 (May)]. / Klonální analýza populace meningokoku z invazivního onemocnení a od zdravých nosicu izolovaných v Ceské republice v období 1971--2014 (kveten).

OBJECTIVE: To present the results of clonal analysis of the meningococcal populations isolated from invasive disease and healthy carriers in the Czech Republic over four decades. MATERIAL AND METHODS: A total of 2179 isolates of Neisseria meningitidis from 1971-2014 (May) were studied: 1093 isolates from patients with invasive meningococcal disease (IMD) and 1086 isolates from healthy carriers. All study isolates were analysed by multilocus sequence typing (MLST), one of the major methods used in molecular epidemiology of IMD. RESULTS: More than 94 % of N. meningitidis isolates from IMD were assigned to serogroups B or C. The strains of the leading serogroup B were genetically highly heterogeneous: 1093 isolates were assigned to 25 clonal complexes. Similarly, the strains of the second leading serogroup C appeared genetically heterogeneous and were classified into 19 clonal complexes. The third leading serogroup Y of IMD isolates showed an opposite tendency and appeared highly homogeneous, with only three clonal complexes being detected. Over 75% of the predominant clonal complexes of IMD isolates of both serogroup Y (cc23) and serogroup C (cc11) were classified as hypervirulent and, as such, posed the highest risk to the host population. Over 80% of IMD isolates of serogroup B were assigned to hypervirulent clonal complexes (cc32, cc41/44, cc18, cc269, and cc11). Of 1086 N. meningitidis isolates from healthy carriers, 41.4% were non-serogroupable, i.e. designated N. meningitidis NG. Classification of these isolates into clonal complexes was highly heterogeneous. In total, 28 clonal complexes were identified of which only a minority were hypervirulent. CONCLUSIONS: The analysis of MLST data on strains collected over four decades revealed that the population of N. meningitidis strains involved in IMD differ genetically from N. meningitidis strains isolated from healthy carriers. These results are relevant to both the optimal use of preventive measures in a focus of IMD and to the development of an effective meningococcal vaccine and vaccination strategy guidelines.

[Real-time PCR detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae DNA in clinical specimens]. / Detekce DNA Neisseria meningitidis, Haemophilus influenzae a Streptococcus pneumoniae v klinickém materiálu metodou real-time PCR.

The study aim was to implement a molecular real-time polymerase chain reaction (PCR) assay recommended by the CDC (Centers for Disease Control and Prevention) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical (culture negative) specimens from patients with suspected invasive bacterial disease. Clinical specimens are referred to the National Reference Laboratory (NRL) for Meningococcal Infections, Unit for Airborne Bacterial Infections, Centre for Epidemiology and Microbiology, National Institute of Public Health from various regions of the Czech Republic. Clinical specimens are, in particular, cerebrospinal fluid, anti-coagulated blood or serum and, exceptionally, post-mortem specimens. The NRL has implemented molecular diagnosis of these bacterial pathogens involved in meningitis and sepsis from clinical specimens since 1999. The first diagnostic method was semi-nested PCR followed by electrophoretic analysis. In 2014, a molecular qualitative real-time PCR assay was implemented.

Predicted strain coverage of a new protein-based meningococcal vaccine in the Czech Republic.

BACKGROUND: Recent data indicate that Neisseria meningitidis B strains cause about 70% of invasive meningococcal disease (IMD) cases in Europe and the availability of a vaccine effective against N. meningitidis B is desirable. A new protein-based MenB vaccine was licensed for use in Europe in January 2013. Meningococcal antigen typing system (MATS) was developed to predict strain coverage of this vaccine. Reports have recently been published for a European consortium, including aggregated data for the Czech Republic. The aim of this paper is to provide a detailed breakdown of MATS results for the Czech N. meningitidis B isolates. MATERIALS AND METHODS: One hundred and eight N. meningitidis B isolates from IMD collected in the Czech Republic during 2007-2010 were selected. MATS analysis was done according to the method previously published. RESULTS: Based on MATS analysis, the overall estimate of strain coverage of the new MenB vaccine for a panel of 108 Czech N. meningitidis B strains is 74% (95% CI: 59-87%). Thirty-nine strains (36%) are predicted to be covered by a single antigen and 41 strains (38%) by more than one antigen. For 28 strains (26%), no antigen coverage was found. CONCLUSIONS: MATS analysis showed that the new protein-based MenB vaccine could protect against a substantial proportion of IMD caused by N. meningitidis B in the Czech Republic. Continued detailed surveillance of IMD will be essential if the MenB vaccine is introduced to the country.

[Sequencing analysis of the antigens included in the four-component vaccine against serogroup B meningococcus in Czech isolates of Neisseria meningitidis from 2007-2013]. / Sekvenacní analýza antigenu zarazených v ctyrkomponentní vakcíne proti meningokoku B v ceských izolátech Neisseria meningitidis v období 2007-2013.

OBJECTIVE: Study of the antigens included in the newly registered four-component vaccine against meningococcus B (MenB vaccine) produced by the reverse vaccinology method and assessment of the potential of the vaccine for use in the Czech Republic. MATERIAL AND METHODS: Czech isolates of Neisseria meningitidis were screened for four antigens: fHbp (factor H binding protein), NHBA (Neisseria heparin binding antigen), NadA (neisserial adhesin A), and PorA P1.4 outer membrane protein. A total of 304 N. meningitidis isolates from 2007-2013 were included in the study: 262 isolates from invasive meningococcal disease (IMD) (203 serogroup B isolates and 59 non-B isolates) and 42 isolates from healthy carriers. RESULTS: The gene encoding the fHbp peptide was detected in all study isolates from both IMD cases and healthy carriers. The two types of isolates differed in the distribution of fHbp variants. The fHbp1 variant prevailed in the IMD isolates (both B and non-B) while the fHbp2 variant was expressed more often in the carrier isolates. The presence of the nhba gene encoding the NHBA peptide was revealed in all study isolates from both IMD cases and healthy carriers. The serogroup B isolates from IMD cases differed from the non-B isolates from IMD cases and from the carrier isolates in the distribution of NHBA variants. The presence of the nadA gene encoding the NadA peptide was only found in 26.6% of serogroup B isolates from IMD cases in comparison to 40.7% of non-B isolates from IMD cases. As few as 4.8% of isolates from healthy carriers harboured the nadA gene. The PorA P1.4 protein included in the new MenB vaccine was only detected in two serogroup B isolates from IMD cases (of the total of 262 serogroup B and non-B isolates from IMD cases) and in none of the isolates from healthy carriers. Isolates from both B and non-B IMD cases were positive most often for the combination of the antigens NHBA + fHbp1, followed by the NHBA antigen alone and then by the combination NHBA + fHbp1 + NadA-1+2/3. Isolates from healthy carriers showed a different antigen distribution pattern: the NHBA antigen alone was the most widespread, followed by the combination NHBA + fHbp1. CONCLUSIONS: The antigens included in the four-component MenB vaccine were revealed by sequencing in a large proportion of the Czech isolates of N. menin-gitidis from both IMD cases and healthy carriers. This four-component vaccine registered in Europe since January 2013 has proven suitable for use in the Czech Republic.

[Clinical and microbiological characteristics of severe infections caused by group A streptococci]. / Klinická a mikrobiologická charakteristika závazných infekcii sposobených streptokokmi skupiny A.

AIM OF THE STUDY: Analysis of data on the occurrence of severe infections caused by group A streptococci, emm typing of the isolated strains, and survey of resistance to the selected antibiotics. MATERIAL AND METHODS: In 2012 and 2013, 43 S. pyogenes strains were isolated from severe infections. The isolates were most often recovered from wounds and abscesses (36 patients), followed by blood culture (5 patients), sputum (1 patient), and autopsy samples (1 patient). Antimicrobial susceptibility to selected antibiotics ( penicillin, erythromycin, clindamycin, spiramycin, and tetracycline) was tested by the disk-diffusion method and the minimal inhibitory concentrations (MICs) were determined. RESULTS: In 2012 and 2013, an increase in severe streptococcal infections was observed, with 27 and 16 cases reported, respectively. Resistance to macrolides was detected in 18.5% and 12.5% of isolates, respectively, and resistance to tetracycline in 25.9% and 20% of isolates, respectively. The isolated S. pyogenes strains were most often assigned to emm type 1. Risk factors for streptococcal infection were analyzed, with injury being most often identified as a risk factor- in seven and two patients, respectively. The patients were most frequently treated by beta-lactam antibiotics (penicillin and ampicillin). In 2012, two cases were fatal. CONCLUSION: This study reports cases of invasive group A streptococcal (GAS) infection as seen in clinical practice. An increased occurrence of S. pyogenes was observed in the hospital, with the isolates exhibiting elevated resistance to erythromycin and tetracycline. It is necessary to monitor the trends of antimicrobial resistance and the distribution of emm types among group A streptococcal isolates. Cooperation of the laboratory and clinical professionals in the prevention and therapy of streptococcal infections, invasive in particular, is crucial for risk reduction.