Publications (13)60.66 Total impact
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Article: Different cytokine production and toll-like receptor expression induced by heat-killed invasive and carrier strains of Neisseria meningitidis.
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ABSTRACT: Neisseria meningitidis may cause severe invasive disease. The carriage state of the pathogen is common, and the reasons underlying why the infection becomes invasive are not fully understood. The aim of this study was to compare the differences between invasive and carrier strains in the activation of innate immunity. The monocyte expression of TLR2, TLR4, CD14, and HLA-DR, cytokine production, and the granulocyte oxidative burst were analyzed after in vitro stimulation by heat-killed invasive (n = 14) and carrier (n = 9) strains of N. meningitidis. The expression of the cell surface markers in monocytes, the oxidative burst, and cytokine concentrations were measured using flow cytometry. Carrier strains stimulated a higher production of inflammatory cytokines and oxidative burst in granulocytes than invasive strains (all p < 0.001), whereas invasive strains significantly up-regulated TLR2, TLR4 (p < 0.001), and CD14 (p < 0.01) expression on monocytes. Conversely, the monocyte expression of HLA-DR was higher after the stimulation by carrier strains (p < 0.05) in comparison to invasive strains. The LPS inhibitor polymyxin B abolished the differences between the strains. Our findings indicate different immunostimulatory potencies of invasive strains of N. meningitidis compared with carrier strains.Apmis 03/2013; · 1.99 Impact Factor -
Article: Predicted strain coverage of a meningococcal multicomponent vaccine (4CMenB) in Europe: a qualitative and quantitative assessment.
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ABSTRACT: BACKGROUND: A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe. METHODS: We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain. FINDINGS: 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63-90, range of point estimates 73-87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries. INTERPRETATION: MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future. FUNDING: Novartis Vaccines and Diagnostics.The Lancet Infectious Diseases 02/2013; · 17.39 Impact Factor -
Article: Capsule null locus meningococci: typing of antigens used in an investigational multicomponent meningococcus serogroup B vaccine.
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ABSTRACT: The investigational multicomponent meningococcus serogroup B vaccine (4CMenB) targets the antigenetically variable population of serogroup B meningococci. Forty-one strains of capsule null locus (cnl) meningococci, which are frequent among healthy carriers, were selected from nine sequence types (ST), which belong to four clonal complexes (cc), and three countries. They were antigen sequence typed and analyzed for antigen expression to predict whether these strains harbor the genes and express the four vaccine antigens of 4CMenB as measured by the meningococcal antigen typing system (MATS). The PorA variant used in the vaccine was not found. The nadA gene was absent in all but one strain, which did not express the antigen in vitro. Only strains of clonal complex ST-198 harbored a factor H binding protein (FHBP) allele of the cross-reactive variant 1 family which is included in the vaccine. All these strains expressed the antigen. Five variants of the Neisserial heparin binding antigen (NHBA) gene were identified. Expression of NHBA was observed in all strains with highest levels in ST-198 cc and ST-845. The data suggest a potential impact of 4CMenB immunization at least on cnl meningococci of the ST-198 cc and ST-845.Vaccine 11/2011; 30(2):155-60. · 3.77 Impact Factor -
Article: Serotype-specific invasive disease potential of Streptococcus pneumoniae in Czech children.
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ABSTRACT: To estimate the invasive disease potential of Streptococcus pneumoniae serotypes, invasive isolates (n=138) were compared with nasopharyngeal isolates (n=153) from children under 6 years of age in the Czech Republic. Odds ratios (ORs) based on a comparison of the distribution of S. pneumoniae serotypes amongst invasive and carriage isolates were calculated for individual serotypes and 172 strains were characterized using multilocus sequence typing. The ORs of serotypes 9V and 14 were significantly greater than 1, suggesting an association with invasive disease, while serotypes 6A and 23F were significantly associated with carriage (ORs less than 1). A single predominant clone with high invasive disease potential was found in each of the 9V, 7F, 14 and 1 serotypes while carriage-associated serotypes were highly heterogeneous.Journal of Medical Microbiology 09/2010; 59(Pt 9):1079-83. · 2.50 Impact Factor -
Article: Multicenter study for defining the breakpoint for rifampin resistance in Neisseria meningitidis by rpoB sequencing.
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ABSTRACT: Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (n = 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of > or = 0.25 mg/liter (n = 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of < 0.25 mg/liter were also examined (n = 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of > 1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of < or = 1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of < or = 1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database (http://pubmlst.org/neisseria/). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.Antimicrobial Agents and Chemotherapy 09/2010; 54(9):3651-8. · 4.84 Impact Factor -
Article: Multidrug-resistant epidemic clones among bloodstream isolates of Pseudomonas aeruginosa in the Czech Republic.
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ABSTRACT: To determine whether the high proportion of antimicrobial resistance among hospital isolates of Pseudomonas aeruginosa in the Czech Republic is associated with the spread of multidrug-resistant clones, we investigated 108 bloodstream isolates collected prospectively in 2007. The isolates originated from 48 hospitals in 36 cities and were serotyped, tested for susceptibility to 10 anti-Pseudomonas agents and studied by multilocus sequence typing, macrorestriction analysis and class 1 integron typing. Forty-five isolates were fully susceptible, while 14 and 49 isolates were resistant to 1-2 and 3-9 agents, respectively. A total of 42 multilocus sequence types (ST) were identified, of which ST235 (serotype O11), ST175 (O4) and ST132 (O6) included 19, 16 and 5 isolates, respectively. These three STs encompassed 40 (82%) of 49 isolates resistant to more than two agents and originated from 29 hospitals in 22 cities. Isolates of the same ST had highly similar macrorestriction patterns. Twelve ST235 isolates harbored an integron variable region with the gene cassette array of aacA7-aadA6-orfD, while 15 ST175 isolates shared a region with the aadB-aadA13 array and all ST132 isolates carried a region with aacA4. A carbapenemase-encoding gene (bla(IMP-7)) was detected in a single strain (ST357). In conclusion, the multidrug resistance of Czech P. aeruginosa bloodstream isolates in 2007 was predominantly associated with three epidemic clones, one of which belongs to international clonal complex CC235.Research in Microbiology 02/2010; 161(3):234-42. · 2.76 Impact Factor -
Article: Clonal distribution of invasive pneumococci, Czech Republic, 1996-2003.
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ABSTRACT: We conducted surveillance on invasive pneumococci isolated from adults in the Czech Republic during 1996-2003. The 7 most prevalent serotypes were characterized. Coverage with the 7-valent pneumococcal conjugate vaccine was low. Our observations confirm that detection methods may have modified the expected effect of this vaccine.Emerging Infectious Diseases 02/2010; 16(2):287-9. · 6.79 Impact Factor -
Article: Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria meningitidis.
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ABSTRACT: Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.The Journal of Infectious Diseases 07/2009; 200(3):379-89. · 6.41 Impact Factor -
Article: Target gene sequencing to characterize the penicillin G susceptibility of Neisseria meningitidis.
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ABSTRACT: Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, Pen(I)) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3' half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the Pen(I) phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.Antimicrobial Agents and Chemotherapy 09/2007; 51(8):2784-92. · 4.84 Impact Factor -
Article: Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway.
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ABSTRACT: The distribution of serogroups and multilocus sequence types (STs) in collections of disease-associated and carried meningococci from the period 1991 to 2000 in three European countries (the Czech Republic, Greece, and Norway) was investigated. A total of 314 patient isolates and 353 isolates from asymptomatic carriers were characterized. The frequency distributions of serogroups and clone complexes differed among countries and between disease and carrier isolate collections. Highly significant differentiation was seen at each housekeeping locus. A marked positive association of serogroup C with disease was evidenced. The ST-11 complex was strongly positively associated with disease; associations for other clone complexes were weaker. The genetic diversity of the clone complexes differed. A single ST dominated the ST-11 clone complex, while the ST-41/44 complex exhibited greater levels of diversity. These data robustly demonstrated differences in the distribution of meningococcal genotypes in disease and carrier isolates and among countries. Further, they indicated that differences in genotype diversity and pathogenicity exist between meningococcal clone complexes.Journal of Clinical Microbiology 12/2004; 42(11):5146-53. · 4.15 Impact Factor -
Article: Microevolution through DNA exchange among strains of Neisseria meningitidis isolated during an outbreak in the Czech Republic
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ABSTRACT: Neisseria meningitidis is a highly variable bacterium. Indeed, N. meningitidis is naturally competent for transformation, and horizontal DNA exchange between strains may lead to mosaic genetic loci in N. meningitidis. We studied such an exchange in nature during an epidemic provoked by N. meningitidis. This epidemic started in the Czech Republic in 1993 and the original epidemic clone was shown to have the antigenic formula (serogroup:serotype:serosubtype) C:2a:P1.2,5. We analysed 145 meningococcal strains isolated in the Czech Republic between 1993 and 1997 using serological and genetic typing methods (multilocus enzyme electrophoresis and polymorphism of pilA and pilD genes). This analysis showed that genetic exchange between epidemic and endemic strains had occurred. Exchanges involved mostly surface- exposed structures such as the capsule, giving rise to new meningococcal variants. The expansion of these variants should be kept under close surveillance.RésuméMicroévolution à travers un échange d'ADN entre des souches de Neisseria meningitidis isolées au cours d'une épidémie en République tchèque. Neisseria meningitidis est capable d'une grande variabilité. Cela résulte de sa compétence naturelle pour la transformation. Ainsi, des échanges horizontaux d'ADN entre différentes souches de N. meningitidis conduisent à des structures mosaïques pour un certain nombre de gènes. Nous avons étudié ce type d'échange lors d'une épidémie provoquée en République tchèque par N. meningitidis. L'épidémie démarra en 1993 et le clone épidémique avait la formule antigénique suivante : (sérogroupe:sérotype:sous-type) C:2a:P1.2,5. Nous avons analysé 145 souches isolées en République tchèque entre 1993 et 1997. Les méthodes de typages sérologiques et génétiques montrèrent qu'il y avait eu des échanges génétiques entre souches épidémiques et endémiques de N. meningitidis impliquant les gènes correspondant aux structures de surface les plus exposées, telle la capsule. De nouveaux variants sont ainsi apparus et leur développement doit rester soumis à une surveillance active.Research in Microbiology 06/1999; · 2.76 Impact Factor -
Article: Reactivity of the new monoclonal antibody ‘22’ with meningococcal strains isolated from patients and carriers in Greece
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ABSTRACT: Previous studies found that the majority of Neisseria meningitidis isolates from either patients or carriers in Greece do not react with the monoclonal antibodies used at present in the whole-cell ELISA (WCE) for determination of serotype and subtype antigens. A new monoclonal antibody designated ‘22’ produced by the National Meningococcal Reference Laboratory in the Czech Republic was assessed in the whole-cell ELISA with 257 non-typable meningococcal strains from both patients (52) and carriers (205). The carrier strains included 34 non-typable isolates from two immigrant populations: ethnic Greeks who have immigrated from Russia since 1989 (19/75) and Kurdish refugees (15/34). Approximately 10% of the meningococcal strains isolated from patients and 11.7% of the carrier strains reacted with the reagent. Although the majority of meningococcal isolates from resident Greeks were not typable with the antibody, 11/19 (57.9%) of the carrier strains from Russian immigrants and 4/15 (20%) of those from the Kurdish refugees reacted with the new reagent.FEMS Immunology & Medical Microbiology 08/1997; 19(1):1 - 5. · 2.44 Impact Factor -
Article: The presence of pilus and clade type among invasive pneumococci recovered in the Czech Republic.
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ABSTRACT: total of 223 invasive isolates of serotypes 4 (55), 6B (20), 14(55), 9V (46), and 19F (47) recovered from 1996 to 2003 were studied. Serotyping was performed by the Quellung reaction and all isolates were analyzed by MLST. MICs of penicillin, erythromycin, clindamycin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole were determined by broth microdilution method. The presence of pilus was performed by PCR of the sortase B, C, and D genes which are a structural part of the rlrA pathogenicity islet. Confirmation of pneumococcal pilus absence was done by amplification of the entire pilus islet with primers PFL-up and PFL-dn and comparison of the obtained PCR product with R6 DNA as non-piliated control. For pilus positive strains, the clade type (I, II, and III) was determined using PCR. The pilus was found in all serotypes, although the proportion of pilus positive isolates among serotypes varied from 1.7% (1) in serotype 14 to 100% of piliated isolates in serotype 6B, Figure 1. Out of 88 pilus positive isolates, 25% (22) were penicillin non-susceptible (MIC of penicillin > 0.06 mg/l). The ST's were homogenous for presence of the pilus, Table 1. The pilus has been found in all isolates of serotypes 9V, 6B, and 4 representing Spain 9V -3 (ST156), Poland 6B -20 (ST315), and Sweden 4 -38 (ST205) clones, respectively. Among piliated isolates, 59 (67%) were defined as clade type I, 17 (19.3%) as clade type II, and 12 (13.6%) as clade type III, Figure 2. The clade I was found in serotypes 4 (ST205, 247, and 2342), serotype 14 (ST143), 9V (ST156 and 162), and serotype 19F (ST380); clade II in serotype 6B (ST176 and 1770); clade III in serotypes 4 (ST2341) and 6B (ST315). During the study period, the prevalence of serotype 4 in invasive isolates increased from 1.7% in 1996 to 11.1% in 2003, the analogical pattern was also seen in serotype 9V (from 3.3% to 5.3%), Figure 3. The expresion of the pilus could constitute a selective advantage in competition with other serotypes and could contribute to the increase of serotype 4 among invasive pneumococcal isolates and the expansion of Spain 9V -3 clone among PNSP isolates. Streptococcus pneumoniae is major cause of bacterial meningitis and community acquired pneumonia among children and adults. Although there is at least 91 different serotypes, only limited number of them demonstrated higher frequency in invasive pneumococcal disease. The effective spread of given serotypes represented by a few invasive clones was not fully elucidated yet and number of virulence factors has been suggested to influence the success of particular clones. One of the identified virulence factor is pneumococcal adhesive pilus encoded by rlrA islet, which can be organised into 3 clades. The aim of the study was to determine the prevalence of the pilus and a clade type among pilus positive isolates in a collection of invasive strains of serotypes 4, 6B, 9V, 14, and 19F.
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1997
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Státní Zdravotní Ústav
Praha, Hlavni mesto Praha, Czech Republic
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