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 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.

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.

Corynebacterium sp. isolated from blood culture of a bacteremic patient. Will the assumptions about a new corynebacterium be confirmed?

A case report is presented of a patient with suspected septicaemia from whose blood culture a new strain of Corynebacterium sp. was isolated. Until now, no report of this strain isolated from human clinical materials has been available in the literature. In addition to a brief clinical description of the case, the article also features morphological, biochemical properties as well as antibiogram of the bacterium. It describes also methods used for the identification of this isolate. The aim of the work was to highlight a novel and rare coryneform strain.

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.

[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.

[Multiple-locus variable number tandem repeat analysis of Bordetella pertussis strains collected in the Czech Republic in 1967-2015: spread of a variant adapted to the population with a high vaccination coverage]. / Multiple-locus variable number tandem repeat analýza souboru kmenu Bordetella pertussis v CR z období 1967-2015: rozsírení varianty adaptované na proockovanou populaci.

AIM: To perform multiple-locus variable number tandem repeat analysis (MLVA) of B. pertussis strains from the collection of the National Reference Laboratory for Diphtheria and Pertussis (NRL/DIPE), National Institute of Public Health (NIPH), Prague. The study strains were isolated from clinical specimens collected mostly in the Czech Republic over a nearly 50-year period from 1967 to 2015 (June). The isolates from three periods characterized by different vaccination strategies and trends in pertussis are compared for genetic diversity and distribution of MLVA types (MT). Based on the results obtained, the suitability for use of MLVA in the analysis of epidemic outbreaks of B. pertussis in the Czech Republic is considered. MATERIAL AND METHODS: DNA samples extracted from B. pertussis strains included in the present study were examined by MLVA using the standard protocol. Data were processed by means of the eBURST algorithm and the calculation of the Simpson diversity index (DI) was used for the statistical analysis. Data were analyzed as a whole and also separately for strains from the three periods: 1967-1980, 1990-2007, and 2008-2015 (June). RESULTS: Fourteen different MT were detected in the study strains, with three of them not being reported before. The most common MTs were MT27 and MT29. MT29 was predominant in 1967-1980 while MT27 was the most prevalent in 1990-2007 and 2008-2015 (June). The DI was the lowest (0.49) in 2008-2015 (June), and comparably higher DIs were calculated for the two previous periods (i.e. 0.667 for 1967-1980 and 0.654 for 1990-2007). CONCLUSION: MLVA revealed a decrease in genetic diversity and shifts in MT distribution of B. pertussis strains isolated from clinical specimens in the Czech Republic from 1967 to 2015 (June). These shifts in the Czech Republic can be characterized as a progressive increase in global MTs at the expense of the locally unique ones. The most common MT, similarly to other geographical areas with long-term high vaccination coverage, is MT27. The results of MLVA of 136 B. pertussis strains can provide a background for using this method in molecular epidemiological analysis of smaller groups of strains.

[Antigenic variability of Bordetella pertussis strains isolated in 1967-2010 in the Czech Republic--possible explanation for the rise in cases of pertussis?]. / Antigenní variabilita kmenu Bordetella pertussis izolovaných v letech 1967-2010 v Ceské republice--mozné vysvetlení vzestupu nemocnosti pertusí?

OBJECTIVE: Comparison of antigenic structures of Bordetella pertussis (B. pertussis) strains isolated from 1967 to 2010 in the Czech Republic. MATERIAL AND METHODS: Seventy strains of B. pertussis were referred to the National Reference Laboratory (NRL) for Pertussis and Diphtheria within the surveillance of pertussis from all over the Czech Republic (CR) between 1967 and 2010. To study the strains, the analysis was performed of the genome sequences encoding the surface immunogenic structures--the pertussis toxin S1 subunit gene (ptxA), pertactin gene region 1 (prnA), type 3 fimbriae gene (fim3)--and pertussis toxin promoter (ptxP) responsible for the regulation of the production of pertussis toxin. RESULTS: For the study set of B. pertussis strains, the sequencing analysis revealed changes in all genomic regions studied. The isolates from three periods differ in the allelic profile. In period I (19671978) with the use of whole cell pertussis vaccine (wP), the following two profiles were the most common: ptxP(1), ptxA(2), prnA(1), fim3(1) and ptxP(1), ptxA(1), prnA(3), fim3(1). In period 2 (19902007) with the switch to acellular pertussis vaccine (aP), the most common profile was: ptxP(3), ptxA(1), prnA(2), fim3(2). Period 3 (20082010) with the use of aP was characterized by the predominance of the following two profiles which had never been found in period 1: ptxP(3), ptxA(1), prnA(2), fim3(2) and ptxP(3) ptxA(1), prnA(2), fim3(1). CONCLUSIONS: Sequencing of the genomic regions ptxP, ptxA, prnA, and fim3 of B. pertussis strains isolated in the CR between 1967 and 2010 confirmed changes in the allelic variants of these regions. The incidence of strains carrying the new allelic variants was increasing after 1995 at the expense of those carrying the original variants. The study results can be interpreted as a partial genetic escape of pathogenic strains of B. pertussis beyond the reach of the pertussis vaccines.

[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.

[Enterovirus sequencing as a new approach to the laboratory diagnosis for clinical and epidemiological purposes]. / Sekvenace enteroviru jako nový prístup k laboratorní diagnostice pro klinické i epidemiologické úcely.

OBJECTIVE: Introducing enterovirus sequencing as an advanced approach to classify the viruses isolated according to the novel nomenclature and to characterize isolates in detail. MATERIAL AND METHODS: Seventy-five specimens collected from 64 patients in two hospitals, Liberec Regional Hospital, and Plzen University Hospital, were analyzed. The study patients' age ranged from four to 54 years, with a median of 15 years in males and 16 years in females. In most patients, the reasons for admission were intense headache, fever, vomiting, tiredness, meningeal symptoms, intestinal symptoms (in two patients), and skin symptoms (in one patient). The specimens collected were rectal and throat swabs, cerebrospinal fluid (CSF) and stool specimens. Molecular detection and typing were performed using the RT-PCR method. A segment of the 5´non-coding RNA was selected for typing. Specimens were amplified using single-step PCR with external primers and with the same primers extended to include M13 sequences (Generi-Biotech). The LASERGENE software (DIASTAR) was used in sequence editing, alignment, and quality check. The sequences obtained were checked against the central GenBank sequence database using the BLAST algorithm. RESULTS: The identification of the study isolates resulted in 61 ECHO viruses 30, three coxsackie viruses B1, one coxsackie virus B3, one coxsackie virus A9, one enterovirus 86, one enterovirus 71, Two ECHO viruses 13/coxsackie virus B5, one ECHO virus 7/30/coxsackie virus B4, one coxsackie virus B4/enterovirus B, one enterovirus 87/ECHO virus 30/enterovirus B, and one ECHO virus 3. All viruses isolated, except enterovirus 71 classified into group A, were of group B. CONCLUSION: The enteroviruses were identified unambigously, although the sequencing only targeted a short, conserved segment that showed considerable variability. The sequencing was an effective alternative to enterovirus identification by the neutralisation test and allowed for detailed characterization of the isolates. The predominance of ECHO 30 as the cause of aseptic meningitis is in accordance with the literature data.

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.

Cluster of three cases of invasive meningococcal disease in a preschool facility in West Bohemia, the Czech Republic.

From 1993 to 2009, there was only one cluster of invasive meningococcal disease (IMD) reported in a community of children in the Czech Republic. This exceptional cluster that occurred in a preschool facility is the focus of this report. In response to the announcement of the disease, anti-epidemic precautions were put in place. Neisseria meningitidis isolates were delivered from local laboratories to the National Reference Laboratory for Meningococcal Infections in Prague. Phenotyping was performed there along with multilocus sequence typing. Related factors and microbiological results were analyzed retrospectively. In October 2009, three girls contracted IMD within a period of 1 week in a 42-member group in a preschool facility attached to the elementary school in Starý Plzenec-Sedlec. In relation to three cases of the disease, another 66 people were registered of which 58 underwent a microbiological examination. N. meningitidis was detected in a total of five (8.6 %) people. The National Reference Laboratory for Meningococcal Infections defined the type of the strain to be C: P1.18-1,34-2,38: F1-7: ST-467 (cc269) and penA27. Tests showed the precise identity of all strains obtained from the three sick children and of two strains contracted through contact with the preschool facility. Despite the complete recovery of all patients with no permanent damage, the need for rapid cooperation between clinical sites, diagnostic laboratories, and epidemiologists was confirmed.

Variability in the concentrations of volatile metabolites emitted by genotypically different strains of Pseudomonas aeruginosa.

AIMS: To characterize the volatile metabolites produced by genotypically diverse strains of Pseudomonas aeruginosa in order to evaluate their potential for use as biomarkers of lung infection in noninvasive breath analysis. METHODS AND RESULTS: Volatile organic compounds (VOCs) emitted from 36 clinical strains of Ps. aeruginosa (belonging to different multilocus sequence types) cultured in liquid and on solid media were analysed by gas chromatography mass spectrometry (GC-MS) and selected ion flow tube mass spectrometry (SIFT-MS). Several previously identified VOCs were detected, including ethanol, acetone, 2-butanone, 2-pentanone, isoprene, aminoacetophenone, dimethyl sulphide, dimethyl disulphide, dimethyl trisulphide and methyl thiocyanate. Additionally, significant production of 3-methyl-butanone, acetophenone, methylthioacetate and methyl thiobutanoate was observed for the first time in this study. SIFT-MS quantifications of VOCs showed high variability between genotypically distinct strains. CONCLUSIONS: The data obtained indicate that the production rates of the volatile biomarkers of Ps. aeruginosa vary by two orders of magnitude between different strains cultured under the same conditions. Similar variability was observed for both liquid and solid media. SIGNIFICANCE AND IMPACT OF THE STUDY: Inter-strain genotypic variability strongly influences the concentrations of the volatile biomarkers from Ps. aeruginosa. A group of several biomarkers quantified in real time in exhaled breath may thus provide a more valuable indicator of the course of pulmonary infections compared to a single biomarker.

Multilocus sequence types in Czech neonatal GBS strains from 2004 to 2008.

The first multilocus sequence typing results of Czech neonatal Streptococcus agalactiae isolates are presented in this paper. The aims of the study were to prove if the Czech isolates belong to potential invasive clonal complex CC17 and to further analyse serotype-sequence type relationships. Twelve sequence types detected among 77 isolates were assigned to 5 clonal complexes. Sequence type variability was found for most of our collection serotypes. As many as 83 % of the invasive isolates were covered by as few as 3 sequence types: S17, ST23 and ST19. ST17 was the only sequence type strictly tied to serotype III. "Hyperinvasive" ST17 was identified in 85 % of the cerebrospinal fluid isolates (6 out of 7), but in 32 % of the blood isolates only.

Sequential analysis of biomarkers in cerebrospinal fluid and serum during invasive meningococcal disease.

The aim of the present study was to determine the profile of different inflammatory molecules in serum and cerebrospinal fluid (CSF) during invasive meningococcal disease (IMD). Their relationship with IMD severity was also assessed. A cohort of 12 patients with IMD was investigated. Paired serum and CSF samples were obtained at the time of diagnostic and follow-up lumbar puncture and were examined using Luminex analysis. IMD severity correlated with serum interleukin-6 (IL-6) and interleukin-1 receptor antagonist (IL-1 ra) on admission. Furthermore, the CSF levels of IL-1 beta, IL-1 ra, IL-6, IL-8, macrophage inflammatory protein-1 beta (MIP-1 beta), and monocyte chemoattractant protein-1 (MCP-1) were significantly higher than their respective serum levels. The strongest correlations were found between serum concentrations of IL-1 beta and IL-1 ra, IL-6, IL-8, and MIP-1 beta, whereas the strongest correlations in CSF were found between endotoxin and IL-8, IL-17, MIP-1 beta, and MCP-1. As was expected, the concentrations of inflammatory molecules in both serum and CSF significantly decreased after antibiotic treatment. With regard to kinetics, a severe course of IMD correlated positively with rapid declines of CSF IL-6 and cortisol levels. Sequential multiple analyses revealed patterns of inflammatory responses that were associated with the severity of IMD, as well as with the compartmentalization and kinetics of the immune reaction.

[Study of hypervirulent complexes of Neisseria meningitidis by methods of molecular biology and possibility of their prevention in the Czech Republic by vaccination]. / Studium hypervirulentních komplexu Neisseria meningitidis metodami molekulární biologie a moznosti prevence jejich výskytu v Ceské republice vakcinací.

A brief report is presented of the results of the Project of the Internal Grant Agency of the Ministry of Health of the Czech Republic that received the Medical Research Award 2008 from the Minister of Health of the Czech Republic (http://www.mzcr.cz/Odbornik/Pages/816-udeleni-ceny-ministra-zdravotnictvi-pro-rok-2008.html). The Science Council of the Internal Grant Agency of the Ministry of Health of the Czech Republic selected 5 award winners of 16 candidate projects recommended by the Specialty Commissions of the Internal Grant Agency. The winning project: Study of hypervirulent complexes of Neisseria meningitidis by molecular biological methods and preventive potential of vaccination against these complexes in the Czech Republic.