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.

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.

Volume 38, no. 12, p. 4492-4498, 2000. We described the genetic characterization of 156 Neisseria meningitidis isolates obtained from healthy young adults in the Czech Republic during 1993. Subsequent work has established that a further 61 isolates collected during that year had been stored separately and had been overlooked. These isolates were not a random sample of those collected, as isolates with a phenotype resembling the strain responsible for a disease outbreak that year were overrepresented. All but one of the additional meningococci were isolated from individuals who were 20 to 24 years old, giving a total of 190 isolates from this age group, rather than the 130 isolates originally reported; the other isolate was from the younger cohort (age range, 15 to 19 years).The revised multilocus sequence typing (MLST) data are available at the Neisseria MLST website (http://neisseria.mlst.net/ links.htm). These data show a total of 88 sequence types (STs), which were resolved into 16 clonal complexes (lineages), with the remaining STs not presently assigned to clonal complexes. The six most prevalent clonal complexes were the ST-11 complex (33 isolates . Since the original publication, some minor changes have been made to the assignment and names of the clonal complexes; for example, the ST-41 complex has been renamed the ST-44 complex. The present assignments are available at the PubMLST isolate database website (http://neisseria .mlst.net).The principal conclusions of the paper, that the population was diverse and that this diversity was principally generated by recombination, are unaltered (Table 1; Fig. 1). However, the revised data show that the prevalence of meningococci belonging to the ST-11 (ET-37) complex was almost six times higher than that calculated on the basis of the data from the 156 original isolates and much greater than any previously measured prevalence of this complex among carriage isolates. This affects estimates of the overrepresentation of ST-11 complex meningococci among isolates from invasive disease. Of the 27 meningococcal isolates carried by members of the younger cohort (age range, 15 to 19 years), 3 (11.1%) belonged to the ST-11 complex. As 10 (22.7%) of the 44 cases of invasive disease in this age group were caused by ST-11 complex organisms, this clonal complex was overrepresented approximately twofold among disease-causing meningococci, as originally reported. However, meningococcus carriage data for individuals in the older cohort (age range, 20 to 24 years) indicated that a total of 30 (15.8%) of the 190 isolates belonged to the ST-11 complex, with 2 out of 9 cases of disease having been caused by . This clonal complex was therefore overrepresented by approximately 1.4-fold (not the originally reported 16-fold) among the disease-associated meningococci isolated from this age group. The ST-11 complex meningococci were found in five of the geographical regions sampled, suggesting that the distribution of these meningococci was widespread. At present, it is unclear why the ...

Twenty clinical samples (18 cerebrospinal fluid samples and 2 articular fluid samples) were sent to 11 meningococcus reference centers located in 11 different countries. Ten of these laboratories are participating in the EU-MenNet program (a European Union-funded program) and are members of the European Monitoring Group on Meningococci. The remaining laboratory was located in Burkina Faso. Neisseria meningitidis was sought by detecting several meningococcus-specific genes (crgA, ctrA, 16S rRNA, and porA). The PCRbased nonculture method for the detection of N. meningitidis gave similar results between participants with a mean sensitivity and specificity of 89.7 and 92.7%, respectively. Most of the laboratories also performed genogrouping assays (siaD and mynB/sacC). The performance of genogrouping was more variable between laboratories, with a mean sensitivity of 72.7%. Genogroup B gave the best correlation between participants, as all laboratories routinely perform this PCR. The results for genogroups A and W135 were less similar between the eight participating laboratories that performed these PCRs.

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.

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.

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.

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.