1. Surveillance of meningococcal infections by the National Reference Center (J-M Alonso, M-K Taha et al.)
N. meningitidis is a major agent of meningitis (30% of bacterial meningitis) and of meningococcemias (septicemias) worldwide. Beside neurologic sequellae, purpura fulminans and fatal septic shock can complicate meningococcal infections. Meningococcal infections are potentially epidemic. Their world incidence varies, according to countries, from 1 to 10 per 100,000 inhabitants, with mortality rates reaching up to 10%. In France the incidence was stable at <1/100,000 for the past eleven years, but increased by 21% in 2000. Nevertheless, the cases remain sporadic and no epidemic occurred. The proportion of the various serogroups among the 520 strains recovered from invasive infections is 66% for the prominent serogroup B, followed by the serogroup C (19,2%), the serogroup W135 (7,2) and serogroup Y (2%). Serogroup W135 was of concern in 2000 due to the emergence and spread of an epidemic clone associated with the Hajj. A total of 19 cases, including 4 fatal cases, occurred among pilgrims and their contacts, all due to N.meningitidis W135:2a/P1-2,5. Identical cases were recorded in the UK, Netherlands, Middle East and USA. Molecular typing showed that these strains belonged to the clonal complex ET-37. These results led us to investigate the origin of this clone by DNA sequencing, and particularly the emergence of a new antigenic variant escaping the immunity induced by the A&C vaccine which is obligatory for the Hajj pilgrims since 1987. The epidemic potential of such an antigenic variant, which emerged among immunologically naive populations, highlights the need for careful reevaluation of vaccination strategies.
Forty-five fatal cases of meningococcal infection have been recorded in France in 2000 (mortality rate of 9% compared to 5% in 1999). Fatal cases mostly occurred among children under 5 years and adults over 65.
2. Experimental research.
Our research themes are based on the constant surveillance of the actual status of meningococcal diseases, considering the evolution of the bacterial molecular determinants as well as the criteria of host susceptibility. They are conducted in collaboration with the correspondents of the Reference Center at the national and international levels. Our goals are to improve the diagnostic and typing methods and to provide better knowledge on the pathogenesis of theses diseases.
2.1. Molecular epidemiology of N.meningitidis.
Direct characterization of N.meningitidis from clinical samples (blood, cerebrospinal fluid, etc.) permits its determination by amplification of the regulatory gene crgA, followed by that of the genes siaD, encoding the capsule for serogroups B, C, Y/W135 or the gene mynB encoding the synthesis of capsular polysaccharides of serogroup A. By using these methods, 85 additional cases of meningococcal disease were recorded in 2000 of 269 suspect cases for which culture was negative.
Molecular typing of N.meningitidis is essential for assessing an epidemiological link between cases.
Polymorphic chromosomal loci pilA, pilD, regF, igaA and crgA are amplified by PCR and further analyzed by multilocus DNA fingerprinting. Various alleles are then defined that are specific for each clonal group. This technique, fully correlated with the multilocus enzyme electrophoresis, and completed by the pulsed-field electrophoresis and the multilocus sequence typing, permitted to characterize several geographic foci in France that confirmed an endemic condition due to heterogeneous genotypes.
2.2. The polymorphism of the gene penA and its relationship to diminished susceptibility to penicillin.
N.meningitidis strains with diminished susceptibility to penicillin (MIC*0.125mg/L) were detected since 1994 in France, reaching 30% in 2000. We have studied the polymorphism of the gene penA among 25 clinical strains, including 13 penicillin-susceptible strains (MIC<0.125mg/L) and 12 strains with diminished susceptibility (1 mg/L>MIC*0.125mg/L), of different geographical origin, antigenic formulas, and from different pathologic conditions. We observed that for different genotypes, according to restriction patterns of the genes pilA, pilD, crgA, regF and igaA, strains with MIC of penicillin <0.125mg/L (penS) harbored identical penA allele, whereas strains with MIC >0.125mg/L (penI) harbored altered sequences of penA. Transformation of penS strains into penI was obtained in vitro by using total DNA or PCR-amplified products of altered penA gene. Also mixed culture of both genotypes led to equivalent transformation, suggesting a direct correlation between alterations of penA and the expression of diminished susceptibility to penicillin. Detecting such gene alterations will be essential for penicillin susceptibility testing whenever PCR diagnosis will be used.
2.3. Molecular genetic of virulence of N.meningitidis.
The meningococcal infectious process first involves bacterial adhesion to epithelial target cells. This is mostly due to the pili. The PilC1 protein plays a major role in the pili assembly and the adhesion to the cell. The gene pilC1 harbors a promoter region necessary for the induction of its expression when the bacteria adhere to the cell. A new transcriptional regulator has been identified, the gene crgA, of the lysR family, whose expression is induced by contact with the target cell along with that of pilC1. The gene crgA seems to be involved in adhesion, mostly in switching to intimate adhesion of N.meningitidis to the cell. These adhesion determinants also play an important role in the invasive process of the blood vessels by activating the TNFa in the endothelial cells in the presence of monocytes.