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.

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.

[2 families with meningococcal infection and a hereditary disorder of the 5th component of the complement system]. / Twee families met meningokokkeninfectie en een erfelijke afwijking van de vijfde component van het complementsysteem.

Within a period of six months, a 20-year-old female with a homozygous deficiency of the C5 component of complement developed meningococcal meningitis twice (different serogroups). Additional C5 deficiencies were not found in relatives. Homozygous deficiency of C5 was also present in another family in which a 16-year-old female and an 18-year-old sister suffered from meningococcal meningitis. Some characteristics of meningococcal disease in patients with C5 deficiency differed from meningococcal disease in patients with a normal complement system: meningitis occurred at a relatively advanced age, was associated with serogroups W-135, B and X and recurred in two of three patients.

[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.

[Complement deficiencies and meningococcal disease in The Netherlands]. / Complementdeficiënties en meningokokkenziekte in Nederland.

OBJECTIVE: To determine the prevalence of complement system deficiencies in patients who have survived a Neisseria meningitidis infection. DESIGN: Retrospective. SETTING: Reference laboratory for bacterial meningitis of the University of Amsterdam and the National Institute of Public Health and Environmental Protection. METHOD: Out of the files of the laboratory 187 patients who had experienced a meningococcal infection in the Netherlands between 1959-1990 were selected in two groups according to the infecting bacterial strain: 97 patients with a serogroup X, Y, Z, W135, 29E, or non-groupable strains and 90 patients with an infection due to serogroup A or C. The patients were asked for their cooperation by their family doctor and one of us visited the patients at home to take blood samples. The complement activity was studied with a haemolysis in gel test and with an assay of haemolytic activity in free solution. RESULTS: Complement deficiency was present in 18% of the 187 patients who had experienced a meningococcal infection. The highest prevalence was found in patients older than 10 years who had developed infections due to serogroups X, Y, W135, or non-groupable strains (45%). Of the patients with a serogroup A or C infection, 3% had an complement deficiency. Of the complement deficiencies, 42% concerned a component of the alternative pathway, 12% a deficiency of C3, and 46% a component of the terminal route. The most commonly found deficiencies were properdin deficiency (39%) and C8 deficiency (18%). 30% of the complement deficient patients reported other family members having experienced meningitis. Recurrent meningitis was only observed in patients with terminal route deficiencies. CONCLUSION: We recommend that patients with a meningococcal infection due to serogroups X, Y, W135 or non-groupable strains should be screened for complement deficiency.

[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.

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.

Binding of mannan-binding protein to various bacterial pathogens of meningitis.

Mannan-binding protein (MBP), a calcium-dependent plasma lectin, may play a role in the innate defence against microorganisms. After binding to carbohydrate structures at the bacterial surface, MBP activates the classical pathway of the complement system. To investigate the binding capacity of MBP to various bacteria associated with meningitis, an assay was developed to study the binding of MBP to bacteria grown in a semisynthetic fluid culture medium. Salmonella montevideo (containing a mannose-rich lipopolysaccharide (LPS)), used as a positive control strain, showed binding of radiolabelled MBP at a level of 80% compared with binding of MBP to zymosan. Binding of labelled MBP to Salm. montevideo was time-dependent, temperature-dependent and saturable. The binding was inhibited by unlabelled MBP, by mannose and by N-acetyl-D-glucosamine. Among bacterial pathogens often found to cause meningitis, a wide range of MBP binding capacities could be determined. The encapsulated Neisseria meningitidis (representatives from 11 serogroups other than group A were included: n = 22), N. mucosa (n = 1), Haemophilus influenzae type b (n = 10) and Streptococcus agalactiae (n = 5) had a low MBP binding capacity of 21.7% (95% confidence interval (CI) 3.3-40.1%). Escherichia coli K1 (n = 11), Strep. suis (n = 5), Strep. pneumoniae (n = 10) and N. meningitidis serogroup A (n = 2) showed intermediate MBP binding capacity of 58.4% (95% CI 40.0-76.8%). A third group consisting of non-encapsulated Listeria monocytogenes (n = 11), non-encapsulated H. influenzae (n = 2), non-encapsulated N. meningitidis (n = 2), N. cinera (n = 1) and N. subflava (n = 1) strains had a high MBP binding capacity of 87.5% (95% CI 62.5-112.5%). The majority of encapsulated pathogens causing bacterial meningitis seem to have a rather low MBP binding capacity.

Complement deficiency predisposes for meningitis due to nongroupable meningococci and Neisseria-related bacteria.

Nongroupable meningococci or bacteria related to the genus Neisseria rarely cause meningitis. Complement deficiency has been identified as a major predisposing factor for meningococcal disease. To assess whether patients with meningitis due to such strains have a complement deficiency, we studied 12 persons. Six patients had meningitis due to nongroupable strains of meningococci, and six patients had meningitis due to Moraxella species or Acinetobacter species. Inherited complement component C7 or C8 deficiency was found in two persons who had had meningitis due to nongroupable meningococci, and one C8-deficient person had had meningitis caused by Moraxella osloensis. Hypocomplementemia resulting from CSF drain-associated shunt nephritis was found in one person with meningitis due to Moraxella nonliquefaciens and in one person with meningitis due to Acinetobacter lwoffi. This rather high frequency of inherited or acquired complement deficiencies among patients with meningitis due to nongroupable meningococci, Moraxella species, and Acinetobacter species justifies the recommendation that such patients must be studied for complement deficiency.

C5 deficiency in a patient with primary Sjögren's syndrome.

We report a case of C5 deficiency in combination with Sjögren's syndrome (SS). Our patient presented with polyarthritis and complaints of oral and ocular dryness. In the serum there was a very low titer of total hemolytic complement (CH50) due to a deficiency of the fifth complement component. C5 deficiency is often associated with recurrent life threatening infections, membranous glomerulonephritis, and discoid lupus erythematosus, but it has not been described in association with primary SS.

Complement deficiencies in patients over ten years old with meningococcal disease due to uncommon serogroups.

46 patients in whom meningococcal disease due to serogroups X, Y, Z, W135, or 29E had developed after the age of 10 years were investigated retrospectively for complement deficiency. Complement deficiency was found in half of the patients: properdin deficiency in 9 patients, C3 deficiency syndromes in 5, and homozygous deficiency of a terminal component (C5, C6, C7, or C8) in 9. Meningococcal infections recurred in 5 of the 9 patients with terminal complement component deficiencies but not in the other complement-deficient patients. The findings show that meningococcal disease due to uncommon serogroups is often associated with complement deficiency.

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.

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.

Deficiency of the beta subunit of the eighth component of complement presenting as arthritis and exanthem.

A 13-year-old boy presented with juvenile chronic arthritis of 6 months' duration. Antinuclear antibodies, anti-double-stranded DNA antibodies, and rheumatoid factor were not detected. Western blotting showed a deficiency of the beta subunit of the eighth component of complement. The same deficiency was present in the patient's sister. C8 beta deficiency is usually detected in individuals who survive meningococcal disease. There was no such history in this family. Juvenile chronic arthritis has not previously been described in patients with C8 beta deficiency.

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.

Inherited complement deficiency in children surviving fulminant meningococcal septic shock.

UNLABELLED: We evaluated the complement system in 29 children (mean age: 4.5 years) who survived fulminant meningococcal septic shock. No terminal complement deficiencies were found. One patient, who experienced the most dramatic disease course, had a decreased haemolytic activity in the haemolysis-in-gel test for the alternative pathway. The properdin concentration in serum of this patient was < 0.1 microgram/ml (n = 17.1-27.7 micrograms/ml). Coagulation studies revealed a heterozygeous type I protein C deficiency as well. He was the only patient with a Neisseria meningitidis group Y infection. CONCLUSION: Fulminant meningococcal disease due to uncommon serogroups should lead to screening of the alternative pathway of complement activation.

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.