Antibiotic resistance of Helicobacter pylori: a cross-sectional study in consecutive patients, and relation to ethnicity.

OBJECTIVES: To assess primary antibiotic resistance in a given population and relate the results to ethnicity. MATERIALS AND METHODS: Consecutive cultures were tested for antibiotic susceptibility with the Etest. Three populations were studied separately: ethnic Dutch people, patients of Turkish descent, and patients originating from Africa and the Middle East. RESULTS: Over a period of 5.5 years, 976 (32%) biopsy specimens from 3010 patients were positive for Helicobacter pylori. Metronidazole and clarithromycin resistance were present in 25.8% and 4.8% of the strains, respectively. The number of metronidazole-resistant strains showed a gradual decrease, while clarithromycin resistance showed a slight increase during the study period. Antimicrobial resistance in patients of Turkish descent and in those originating from Africa or the Middle East was significantly higher than in ethnic Dutch people, 35% and 9.1% versus 21% and 2.9%, respectively (P = 0.003 and P = 0.002). CONCLUSION: It is important to take ethnicity into account when studying antibiotic resistance. The numbers of metronidazole- and clarithromycin-resistant strains can vary considerably between people of different ethnic origin living in the same region.

[Haemophilus influenzae type a as the causative agent of meningitis in an infant]. / Haemophilus influenzae type a als verwekker van meningitis bij een zuigeling.

A 6-month-old girl had been ill with a cold for several days and was increasingly drowsy. She had been fully vaccinated against Haemophilus influenzae type b and had meningitis due to H. influenzae type a. She made a complete recovery after treatment with ceftriaxone and amoxicillin. In the Netherlands, vaccination with the conjugated H. influenzae type b vaccine was started in 1993 and since then invasive infections caused by H. influenzae type b have almost disappeared. Vaccination may suppress carriership of H. influenzae type b. However, vaccination does not elicit cross-protective antibodies against other serotypes of H. influenzae. H. influenzae non-type b may profit from the vaccination state, resulting in a higher carrier rate and an increased incidence of invasive infections. In the Netherlands, H. influenzae type a as the causative agent of an invasive infection has been recorded for the first time since registration started in 1975 and since then five such cases have been reported. In the literature, 45 cases of infection with H. influenzae type a have been described up until now.

Molecular characterisation of 10 Dutch properdin type I deficient families: mutation analysis and X-inactivation studies.

Properdin type I deficiency is characterised by complete absence of extracellular properdin, a positive regulator of the alternative pathway of complement activation. Properdin deficiency is associated with increased susceptibility to severe meningococcal disease. We have identified the genetic defect in 10 Dutch families. Six different mutations and one sequence polymorphism in the properdin gene were found. All amino acid substitutions were limited to conserved amino acids in exons 7 and 8 in contrast to the premature stops that were found in other exons. The missense mutations may alter the protein conformation in such a way that properdin will not be secreted and therefore catabolised intracellularly. The decreased properdin levels found in some healthy females carrying one mutated properdin gene were studied for X-inactivation. Most carriers with extreme low or high properdin levels showed preferential X-inactivation for the normal or mutated X chromosome, respectively. We observed some exceptions, suggesting additional regulation of properdin excretion apart from X-inactivation.

The role of Fcgamma receptor polymorphisms and C3 in the immune defence against Neisseria meningitidis in complement-deficient individuals.

Individuals with either a late (C5-9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti-meningococcal defence in both LCCD persons and properdin-deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcgammaRIIa (CD32) and FcgammaRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcgammaRIIa and FcgammaRIIIb allotypes was assessed in 15 LCCD and in 15 properdin-deficient patients with/without previous meningococcal disease, we found the combination of FcgammaRIIa-R/R131 with FcgammaRIIIb-NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13.9, Fisher's test P = 0.036). No such relation was observed in the properdin-deficient patients. The importance of FcgammaRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcgammaRIIa-R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0.05) less than PMN from FcgammaRIIa-H/H131 donors. When properdin-deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0.001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0.6568, P = 0. 01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcgammaR allotypes. Properdin-deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.

Development of antibodies against tetravalent meningococcal polysaccharides in revaccinated complement-deficient patients.

Individuals deficient in C3 or a late complement component are susceptible to recurrent meningococcal infections. Since they experience meningococcal episodes mostly with uncommon meningococcal serogroups, vaccination with a tetravalent vaccine containing A, C, Y and W135 polysaccharides has been suggested. We vaccinated a cohort of two C3 and 17 late complement component-deficient (LCCD) patients, revaccinated them 7 years later and investigated the development of their IgG antibodies to the capsular polysaccharides of the meningococcal vaccine. Seven years after the first vaccination levels of IgG antibodies declined compared with the levels present at 6 months after the first vaccination, but were still at least four times higher than before vaccination. Levels of antibodies to Y polysaccharide in serum of complement-deficient patients were rather low but they did not differ significantly from those in serum of healthy non-related controls (P = 0.07). Three months after the second vaccination IgG antibodies against all polysaccharides increased, exceeding those measured at 6 months after the first vaccination. In the 8 years of observation after the first vaccination two new meningococcal infections with strains related to the vaccine (serogroup Y strains) occurred in two patients, 3.5 and 5 years after the first vaccination. Our findings show that high IgG antibody levels against the tetravalent meningococcal polysaccharide vaccine were reached after revaccination of two C3 and 17 LCCD individuals 7 years after the first vaccination. Whether revaccination should be required within a period shorter than 7 years is discussed, since two vaccinees developed meningococcal disease to vaccine serogroup Y.

Assessment of complement deficiency in patients with meningococcal disease in The Netherlands.

The frequency of complement deficiency in 176 of 7,732 patients with meningococcal disease in the Netherlands from 1959 through 1992 was assessed. Complement deficiency was found in six patients (3%): 3 (7%) of the patients with Neisseria meningitidis serogroup C disease, 1 (2%) of the patients with N. meningitidis serogroup A disease, and 2 (33%) of the patients with infections due to uncommon serogroups and nongroupable strains of N. meningitidis. Of 91 additional patients with meningococcal infections due to uncommon serogroups, 33% also had complement deficiency. Thirty-four of the 36 complement-deficient patients with meningococcal disease who were from 33 families were 5 years of age or older. Twenty-six additional complement-deficient relatives were found. Screening individuals with meningococcal disease due to uncommon serogroups who were 5 years of age or older identified 30 of the 33 complement-deficient families. Only 27% of the complement-deficient relatives had had meningococcal disease. This risk was lower for relatives with properdin deficiency (18%) than for those deficient in the late component of complement (38%). Therefore, pedigree studies are warranted for identifying those complement-deficient persons who require vaccination for meningococcal disease.

[The bactericidal action of human neutrophils on meningococci in vitro]. / Bakteritsidnoe deistvie neitrofilov cheloveka na meningokokki in vitro.

32 Russian patients with late complement component deficiency (LCCD) were immunize with tetravalent meningococcal polysaccharide vaccine (A + C + W135 + Y). Their immune response and infectious morbidity rate were followed for 6 years and the partial protective efficacy of vaccination was demonstrated. As the antibody-mediated complement-induced bactericidal activity of plasma was completely absent in persons with LCCD, the bactericidal action of human neutrophils on meningococci of groups A, W135 and B was studied under the conditions of incubation with serum samples collected from persons with LCCD before and after vaccination. In LCCD serum alone the exponential growth of meningococci was observed, while the addition of human neutrophils resulted in the essential inhibition of the growth of meningococci (up to their complete elimination). The proportion of serum samples stimulating the elimination of group A and W 135 meningococci by neutrophils was almost 40% of the serum samples collected before vaccination and significantly increased among the serum samples collected after vaccination (up to 84%) or revaccination (up to 90%). At the same time the capacity of an individual serum sample to promote the bactericidal effect of neutrophils against meningococci correlated with its content of specific anti-polysaccharide IgG and IgM antibodies, as well as antibodies to the inner core of lipopolysaccharide. The interaction of neutrophils with meningococci was significantly inhibited after incubation in heat-inactivated serum, suggesting that this interaction was partly mediated along the following path: the binding of IgM and IgG antibodies with bacteria--the activation of complement and the deposition of C3 complement on bacteria--the binding of meningococci with CR3 receptors of neutrophils.

Protection against meningococcal serogroup ACYW disease in complement-deficient individuals vaccinated with the tetravalent meningococcal capsular polysaccharide vaccine.

Individuals with properdin, C3 or late complement component deficiency (LCCD) frequently develop meningococcal disease. Vaccination of these persons has been recommended, although reports on efficacy are scarce and not conclusive. We immunized 53 complement-deficient persons, of whom 19 had properdin deficiency, seven a C3 deficiency syndrome and 27 had LCCD with the tetravalent (ACYW) meningococcal capsular polysaccharide vaccine. Serological studies were performed in 43 of them. As controls 25 non-complement-deficient relatives of the complement-deficient vaccinees and 21 healthy non-related controls were vaccinated. Post-vaccination, complement-deficient individuals and controls developed a significant immunoglobulin-specific antibody response to capsular polysaccharides group A, C, Y, W135, but a great individual variation was noticed. Also, the proportion of vaccinees of the various vaccinated groups with a significant increase in bactericidal titre (assayed with heterologous complement) was similar. Opsonization of meningococci A and W135 with sera of the 20 LCCD individuals yielded in 11 (55%) and eight (40%) sera a significant increase of phagocytic activity after vaccination, respectively. Despite vaccination, four complement-deficient patients experienced six episodes of meningococcal disease in the 6 years post-vaccination. Four episodes were due to serogroup B, not included in the vaccine. Despite good response to serogroup Y upon vaccination, disease due to serogroup Y occurred in two C8beta-deficient patients, 3.5 and 5 years post-vaccination. These results support the recommendation to vaccinate complement-deficient individuals and to revaccinate them every 3 years.

Characterization of Neisseria meningitidis strains causing disease in complement-deficient and complement-sufficient patients.

Serotyping and serosubtyping of meningococci showed no difference between isolates from 44 complement-deficient persons and from 50 complement-sufficient persons with meningococcal disease. Multilocus enzyme electrophoretic typing of the meningococci revealed 54 electrophoretic types that were equally distributed among isolates from complement-deficient and complement-sufficient patients. Analysis of strains isolated from eight complement-deficient persons with 11 recurrences of meningococcal disease showed that one strain was identical to the strain previously isolated from the same individual. Our results indicate that there are no differences between the clonal distributions of strains infecting complement-deficient and complement-sufficient patients. Most recurrences were infections caused by different strains.

Meningococcal disease and polymorphism of FcgammaRIIa (CD32) in late complement component-deficient individuals.

Late complement component-deficient (LCCD) individuals lack plasma bactericidal activity and are highly susceptible to meningococcal disease. Phagocytosis plays a significant role in immune defence against meningococci and involves FcgammaRIIa (CD32) on leucocytes. Two allotypic forms are currently recognized: FcgammaRIIa-R131 and RIIa-H131. Neutrophils with the IIa-H/H131 allotype are more effective in phagocytosis than IIa-R/R131. We studied the distributions of IIa-R131 and IIa-H131 allotypes among 29 Russian LCCD patients who had suffered from recurrent episodes of meningococcal disease. The distribution of IIa-R/R131 to heterozygous IIa-R/H131 to homozygous IIa-H/H131 genotypes was 0.14:0.29:0.57 for LCCD patients who developed the first episode of disease before 10 years of age. The distribution was 0.21:0.64:0.14 for patients who experienced meningococcal disease above the age of 10 years (chi2 = 6, P < 0.05, odds ratio for IIa H/H131 versus R/R131 = 8). Meningococcal disease had a 'grave' course in 14 of 31 disease episodes in patients with IIa-R/R131 and IIa-R/H131 allotypes, in contrast to 1 of 18 episodes in patients with IIa-H/H131 allotype (chi2 = 7, P < 0.01, odds ratio = 14). We conclude that IIa-H/H131 individuals appear to have a higher acquired antibody-mediated phagocytosis-dependent resistance to meningococcal disease above the age of 10 years. Additionally, effective CD32-mediated phagocytosis may restrict the severity of meningococcal disease in LCCD patients with IIa-H/H131 phenotype.

The effect of mannan-binding lectin on opsonophagocytosis of Neisseria meningitidis.

Mannan-binding lectin (MBL), an acute phase protein with a structure and a function very similar to that of C1q, is known to act as an opsonin binding to a number of microorganisms. In order to investigate the effect of MBL on the phagocytic killing of meningococci, a serogroup B meningococcal strain (H44/76) and its unencapsulated variant v24, as well as a serogroup A meningococcal strain were opsonized with MBL (purified from normal human plasma at the State Serum Institute, Denmark) and used in a phagocytic killing assay at a density of 7 x 10(3) CFU/ml. Polymorphonuclear cells (PMNs) from one healthy donor were isolated by density gradient centrifugation over Percoll and added to the system (7 x 10(6) cells/ml). In a first set of experiments without addition of serum or complement, no influence of MBL was observed on the killing of any of these strains. Addition of MBL to non-opsonized bacteria of the serogroup A strain did not result in enhanced killing either; on the contrary, the growth of this strain increased significantly when a high MBL concentration (40 micrograms/ml) was used in the presence of PMNs. Further investigations were performed using sera of five individuals with late complement component deficiency (LCCD) and a concomitant MBL deficiency, vaccinated with a tetra-valent (ACYW135) meningococcal capsular polysaccharide vaccine. Pre- and post-vaccination sera (50% final concentration) were tested against a group A strain opsonized or not with MBL. In only one patient was there a moderate increase of killing of the opsonized bacteria after vaccination compared to pre-vaccination serum. Our results suggest that MBL may not play a significant role in the opsonophagocytosis of meningococci, irrespective of its binding to unencapsulated and serogroup A strains.

Carrier detection by microsatellite haplotyping in 10 properdin type 1-deficient families.

Properdin deficiency carrier identification is relevant, because properdin-deficient persons have an increased risk of contracting meningococcal disease. Vaccination against meningococcal disease at a young age may provide protection. Accurate detection of this deficiency is needed. Microsatellite haplotyping with the PFCI and PFC2 markers closely linked to the properdin gene locus at Xp11.3-Xp11.23 may offer an easy and accurate identification of carriers of the properdin deficiency gene. The chance to study 91 relatives belonging to 10 families with complete (type 1) properdin deficiency offered a unique opportunity to assess whether properdin type 1 deficiency is associated with a distinct microsatellite haplotype. Haplotyping with the closely linked PFC1 and 2 markers yielded five different haplotypes, which did not support the concept of a founder effect. Among the 28 women carriers, two had normal properdin levels and in five the PFC1,2 polymorphism was not informative owing to homozygosity. Extending the microsatellite haplotyping with three additional markers (DXS1126, DXS426 and DXS7) yielded informative haplotypes in all meioses. We concluded that microsatellite haplo-typing using five markers in close proximity to the properdin gene locus is an accurate method of detecting carriers of the properdin deficiency gene and of properdin-deficient persons within a family at a young age.

Heterozygous and homozygous factor H deficiency states in a Dutch family.

Factor H, a 150-kD protein, is an important down-regulating protein of the alternative pathway of the complement system. Presently, only 15 persons, representing seven families, have been described with homozygous factor H deficiency. Deficiency of this protein, inherited as an autosomal recessive trait and resulting in uncontrolled breakdown of C3, results in depletion of components of the alternative pathway (factor B, properdin) and of the terminal pathway (C5), and is associated with the onset of bacterial infections, glomerulonephritis and systemic lupus erythematosus (SLE). The proband of the family in this study suffered from subacute cutaneous lupus erythematosus and had had meningococcal meningitis due to serogroup X. She had a complete factor H deficiency at the protein level as determined by Western blotting. Among 21 relatives of the proband studied, encompassing three generations, 10 had low factor H levels, including the two children of the proband, indicating a heterozygous factor H deficiency state. In serum samples of the proband and 11 relatives prospectively studied, a strong correlation of factor H levels with C3, C3 haemolytic activity, factor B and properdin levels (P < 0.0001) was found. Alternative pathway protein levels were significantly lower (Mann-Whitney test; Z values 3.6-2.7) in sera from the four heterozygous relatives studied than in sera from the seven non-deficient relatives. In addition, a defect of the 37/42-kD H-related protein was found in the proband and two of 21 relatives, compared with four of 40 controls. A defect of the 24/29-kD H-related protein was present in one of 21 relatives studied and in none of the 40 controls.

Comparison of commercial diagnostic tests for identification of serogroup antigens of Neisseria meningitidis.

In the study that is described the sensitivities and specificities of three commercial tests and the standard Reference Laboratory test, used since 1961, to identify Neisseria meningitidis serogroups were compared. The tests marketed by Difco, Murex/Wellcome, and Sanofi/Pasteur showed overall sensitivities of 92, 95, and 100%, respectively, and specificities of 67, 88, and 82%, respectively. When limited to the common serogroups A, B, and C, the three tests yielded sensitivities of 93, 97, and 100%, respectively, and specificities of 98, 100, and 98%, respectively. However, determination of the uncommon serogroups X, W-135, Y, Z, and 29E with these tests is either unreliable or not possible.

Fulminant meningococcal septic shock in a boy with combined inherited properdin and protein C deficiency.

A 7-year-old patient with fulminant septic shock due to Neisseria meningitidis of the uncommon serogroup Y developed extensive gangrene of the limbs. Multiple amputations were necessary and a pulmonary embolism occurred within 2 days post-operatively. Complement and haemostatic system studies, done after recovery, showed a complete absence of properdin antigen and a low protein C antigen and activity level in plasma. Defective haemolytic activity in gel by the alternative pathway of complement activation could be restored with purified properdin, indicating a properdin deficiency type 1. Protein C antigen level as well as activity were in agreement with a protein C deficiency type I. The polymerase chain reaction (PCR) product of exon five of the protein C gene showed a substitution of 72Gly by Arg. Both deficiencies were traced among relatives of the patient. Serum of the father of the patient's mother was also properdin-deficient. Microsatellite haplotyping of the X-chromosome of the patient and his relatives showed that a distinct haplotype cosegregated with the properdin deficiency (Lodscore 2.25; four informative meioses). The protein C type I deficiency was present in the patient's mother and her mother and cosegregated with the mutation found. So far as is known, this is the first patient described with combined inherited properdin deficiency and protein C deficiency.