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.

Neisseria meningitidis strains from patients with invasive meningococcal disease differ in stimulation of cytokine production.

Cytokine production was determined in vitro after stimulation with three different Neisseria meningitidis (NM) strains. Virulent NM B strain isolated from a patient with mild course of invasive meningococcal disease (IMD) elicited higher cytokine production than NM B and NM C hypervirulent strains isolated from patients with moderate and fatal course of IMD, respectively. Endotoxin concentration after in vitro stimulation correlated with cytokine production: the highest endotoxin levels were observed with virulent NM B strain. Serum cytokines and endotoxin levels showed an opposite trend. These results suggest that inflammatory response during IMD is predominantly influenced by host factors.

Dynamics of PCR-based diagnosis in patients with invasive meningococcal disease.

Invasive meningococcal disease continues to be a life-threatening condition and rapid diagnosis is important for the administration of appropriate treatment. This study focused on the use of PCR for the diagnosis of meningococcal aetiology and the dynamics of PCR-based diagnosis over time in various biological samples. Sixty cerebrospinal fluid (CSF) and 144 serum samples collected during the first week of hospitalisation from 37 patients with laboratory-confirmed invasive meningococcal disease were investigated. Overall, 91.9% of CSF samples and 45.9% of serum samples were PCR-positive, while culture of CSF and blood was positive for only 35% and 39% samples, respectively. Positive PCR results were obtained until day 7 with CSF and until day 5 with serum. It is therefore recommended that samples for molecular diagnosis should be collected early in the course of suspected invasive meningococcal disease.

[Molecular epidemiology of invasive meningococcal disease in the Czech Republic]. / Molekulární epidemiologie invazivního meningokokového onemocneni v Ceské republice.

The study objective is to present results of clonal analysis of Neisseria meningitidis isolates from invasive meningococcal disease in the Czech Republic in 1993-2005. The method of multilocus sequence typing (MLST) revealed genetic heterogeneity of serogroup B isolates and homogeneity of serogroup C isolates. Three clonal sequence type complexes (STC-18, STC-32 and STC-41/44) prevailed among serogroup B isolates and almost a quarter of isolates have ST unclassifiable into clonal complexes. STC-11 showed clear predominance among serogroup C isolates. Hypervirulent clonal complex STC-11 prevailed in 1993-2004 while another clonal complex, STC-41/44, became more widespread for the first time in 2005. MLST analysis showed differences between the meningococcal population in the Czech Republic and those in western European countries and elsewhere. The results of clonal analysis of Neisseria meningitidis isolates from invasive meningococcal disease provide background information for evaluation of the adequacy of recommended measures in the focus of the disease and for updating the recommended vaccination strategy in the Czech Republic.

Invasive meningococcal disease and latex agglutination test--is it still beneficial for diagnosis?

We showed current clinical usefulness of the latex agglutination (LA) test for confirmation of meningococcal etiology on 32 cerebrospinal fluid, 77 serum and 93 urine samples collected during the first week of hospitalization from 19 patients with laboratory confirmed invasive meningococcal disease. The positivity of the LA test in cerebrospinal fluid was 47%, in serum 42% and in urine 24%, while the PCR of cerebrospinal fluid and serum was positive in 95 and 47% cases, respectively. The latest positivity of the LA test was detected on day 2 in cerebrospinal fluid, on day 3 in serum and on day 4 in urine. In the group of patients who had received antibiotic therapy we found nonsignificant reduction of LA positivity and also statistically significant reduction of culture positivity in CSF (p = 0.04); the PCR positivity changed minimally. In blood samples, nonsignificant reduction of culture positivity and no difference in LA and PCR positivity was found. We did not find any statistically significant relationship between test results and clinical forms. The LA test can be therefore considered to be an auxiliary diagnostic method, rapid and easily practicable but less sensitive than PCR. It can be recommended especially for local laboratories where PCR is not available and the patient already received antibiotics before admission to the hospital.

Interactions of invasive and noninvasive strains of Neisseria meningitidis with monkey epithelial cells, mouse monocytes and human macrophages.

Adherence and phagocytosis of invasive and noninvasive Neisseria meningitidis strains was investigated using light, fluorescence and electron microscopy. Invasive strains were isolated from the cerebrospinal fluid and/or blood of the patients with invasive meningococcal disease and noninvasive strains from the nasopharynx and/or larynx of healthy carriers. Adherence/endocytosis was studied on monkey kidney cells (the LLC-MK2 cell line) and phagocytosis on mouse monocytes and human macrophages (the P388D1 and U-937 cell lines, respectively). Although invasive and noninvasive meningococci isolated in the same cluster showed identical genotype and phenotype markers, they were found to interact differently with epithelial cells as well as with monocytes/macrophages. Invasive isolates displayed higher adherence to the surface of LLC-MK2 cells compared to noninvasive ones. Phagocytosis by P388D1 cells of noninvasive strains was effective and the bacteria were damaged by cytolysis. In contrast, invasive bacteria frequently persisted in "coiling" vacuoles and in effect could destroy the host cell. This is the first demonstration of coiling phagocytosis induced by meningococci. Efficiency of phagocytosis by U-937 cells was significantly higher for the noninvasive than invasive strains. Different behaviour of invasive and noninvasive strains of N. meningitidis observed during 4 hours of interactions with epithelial cells and monocytes/macrophages reflects well the higher pathogenic potential of invasive bacteria.

[Characterization of Haemophilus influenzae strains using multilocus sequencing]. / Charakterizace kmenu Haemophilus influenzae metodou multilokusové sekvenacní typizace.

First results of multilocus sequence typing (MLST) of Haemophilus influenzae strains are presented. MLST of 28 H. influenzae strains isolated from patients with invasive diseases in the Czech Republic is indicative of clonal homogeneity of these strains: 22 out of 26 H. influenzae b strains tested were of the same sequence type, ST-6. Four strains were of two sequence types newly described in this study: ST-83 (3 strains) and ST-84 (1 strain). Two nontypeable H. influenzae strains were assigned to sequence types other than ST-6: ST-3 and ST-85 newly described in this study. First MLST results show ST-6 to be typical of H. influenzae b isolated from patients with invasive diseases in the Czech Republic. The sequence types newly described in this study, i.e. ST-83, ST-84 and ST-85, were submitted to the worldwide H. influenzae MLST database (http://haemophilus.mlst.net).

[Study of long-term and multiple carriage of Neisseria meningitidis in a healthy population using molecular biology methods]. / Studie dlouhodobosti a mnohocetnosti nosicství Neisseria meningitidis ve zdravé populaci molekulárními biologickými metodami.

The aim of the study was to investigate the long-term duration and multiple carriage of Neisseria meningitidis in a healthy population. In this prospective study, 206 students at the age of 15 to 19 years were monitored from October 2002 to March 2003. Nasopharyngeal and laryngeal swabs were sampled in one-month intervals and cultured on a selective medium. All colonies detected in primary culture were saved for the study, a maximum of 20 colonies in cases of massive growth. A total of 1,242 isolates were obtained, all of them being examined by a molecular biology method RAPD (randomly amplified polymorphic DNA analysis). By other methods (determination of phenotype by slide agglutination and whole-cell ELISA test, the determination of sequence type, ST by multilocus sequence typing, testing of susceptibility to antibiotics) were always tested the first isolates from individual carriers and isolates from the same carrier with different RAPD characteristics, being 35 altogether. There were thirty three carriers of N. meningitidis detected among the 206 students (16%). The carriage of N. meningitidis was of long-term duration. The study of strains of N. meningitidis with molecular biology methods made it clear that the carrier population of meningococci is heterogeneous. The population of carrier meningococci was of clonal character and mostly non-virulent ST-complexes were detected. Multiple carriage of different clones of N. meningitidis is rare and usually of short-term duration. The colonization of upper respiratory tract by a single clone of N. meningitidis does not protect from colonization by other clone.

Development of the epidemiological situation in invasive meningococcal disease in the Czech Republic caused by emerging Neisseria meningitidis clone ET-15/37.

Meningococcal clone ET-15/37, which appeared as a new one in the Czech Republic in 1993, caused an emergency epidemiological and clinical situation in invasive meningococcal disease, characterized by a high fatality rate (20%) compared to the "normal" fatality rate due to "non ET-15/37" strains. Morbidity rate increased since the first year of the new clone occurrence and reached the peak in 1995. This clone has spread all over the country and investigation of the epidemiological markers of Neisseria meningitidis allowed to quickly recognize the emergency situation and subsequently to provide a targeted vaccination with A + C polysaccharide meningococcal vaccine which prevented the spread of the disease caused by Neisseria meningitidis C. The most frequent phenotype of ET-15/37 clone was C:2a:P1.2(P1.5) and its percentage achieved 80% of group C Neisseria meningitidis strains tested. This antigenic shift of Neisseria meningitidis was associated with the age shift in invasive meningococcal disease morbidity: teenagers started to be the most affected age group and later age group of 1-4 olds followed with high morbidity rates. In 1995 B variant of ET-15/37 clone, B:2a:P1.2(P1.5), appeared, causing a high fatality rate, too. Some data are indicative of a possible decrease in the invasive meningococcal disease incidence in the Czech Republic; nevertheless, the active surveillance and detailed investigation of meningococci have to be continued. After four years following the vaccination and chemoprophylaxis strategy recommended in the Guidelines, set up by the National Reference Laboratory for Meningococcal Infections in 1993, it is possible to conclude, that there have been practically no secondary cases of invasive meningococcal disease in the Czech Republic.

Multilocus sequence typing of Neisseria meningitidis directly from cerebrospinal fluid.

MLST typing of Neisseria meningitidis directly from clinical material was introduced in the National Reference Laboratory for Meningococcal Infections in Prague. Four cerebrospinal fluid samples were obtained from patients with suspected meningococcal invasive disease. In all samples, all classical laboratory methods gave negative results and the only positive method was PCR, which revealed Neisseria meningitidis group C (two specimens) and group B (two specimens), respectively. MLST performed directly from cerebrospinal fluid revealed that the strains causing the two group C infections were of sequence type (ST) 11, while the two group B infections were characterized as ST-32 and ST-33 respectively. Multi-locus sequence typing of meningococci directly from clinical material offers the opportunity to improve further the surveillance of meningococcal disease and has now been introduced into the routine portfolio of tests employed at the national reference laboratory of the Czech Republic.

Neisseria meningitidis RTX protein FrpC induces high levels of serum antibodies during invasive disease: polymorphism of frpC alleles and purification of recombinant FrpC.

The Neisseria meningitidis FAM20 strain secretes two proteins of unknown function, FrpA and FrpC, which contain typical RTX domains found in cytotoxins from other gram-negative pathogens. To evaluate whether the Frp proteins could be involved in meningococcal virulence, 65 isolates of all serogroups were screened by PCR for the presence of both frp genes. The frpA allele was, however, poorly conserved. A single strain harbored an frpA allele of the previously described size, while large insertions were detected in the frpA loci of 22 isolates (34%), and the 42 remaining isolates (65%) did not contain frpA at all. In contrast, frpC alleles, albeit of variable length, were detected in all invasive and most carrier strains. This suggests that meningococci may produce a family of FrpC proteins of various molecular masses. High levels of both immunoglobulin G (IgG) and IgA class antibodies recognizing recombinant FrpC were indeed detected in convalescent-phase sera of most patients at 2 and 4 to 5 weeks after the first symptoms of meningococcal disease. These results show that FrpC-like proteins are produced and may play a role in invasive meningococcal infections.

Carried meningococci in the Czech Republic: a diverse recombining population.

Population and evolutionary analyses of pathogenic bacteria are frequently hindered by sampling strategies that concentrate on isolates from patients with invasive disease. This is especially so for the gram-negative diplococcus Neisseria meningitidis, a cause of septicemia and meningitis worldwide. Meningococcal isolate collections almost exclusively comprise organisms originating from patients with invasive meningococcal disease, although this bacterium is a commensal inhabitant of the human nasopharynx and very rarely causes pathological effects. In the present study, molecular biology-based techniques were used to establish the genetic relationships of 156 meningococci isolated from healthy young adults in the Czech Republic during 1993. None of the individuals sampled had known links to patients with invasive disease. Multilocus sequence typing (MLST) showed that the bacterial population was highly diverse, comprising 71 different sequence types (STs) which were assigned to 34 distinct complexes or lineages. Three previously identified hyperinvasive lineages were present: 26 isolates (17%) belonged to the ST-41 complex (lineage 3); 4 (2.6%) belonged to the ST-11 (electrophoretic type [ET-37]) complex, and 1 (0.6%) belonged to the ST-32 (ET-5) complex. The data were consistent with the view that most nucleotide sequence diversity resulted from the reassortment of alleles by horizontal genetic exchange.