Abstract
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.
Individuals with complement deficiencies have a high risk of contracting meningococcal disease (7, 13) due to defective serum bactericidal activity and/or phagocytosis (6, 18). Meningococcal disease recurs in 39% of the individuals with the late component of complement deficiency (LCCD) (C5 to C9) and in 6% of properdin-deficient persons (5, 6, 10, 12, 17). Whether these recurrences are real reinfections with a new strain or recrudescences is debatable (6, 7).
Standard characterization of meningococcal strains comprises serogrouping, serotyping, and serosubtyping (2). Multilocus enzyme electrophoresis (MEE) of metabolic enzymes is used to investigate the clonal spread of meningococcal strains. Serogroups W135, X, Y, and Z are often isolated from complement-deficient persons with meningococcal disease (8, 13, 17). In previous studies, it was shown that meningococci from complement-deficient and complement-sufficient patients have similar serum sensitivity (14), serotypes, and serosubtypes but that their electrophoretic types (ETs) are different (11).
In this study, we determined the serotypes, serosubtypes, and ETs of 44 first meningococcal isolates from 44 complement-deficient persons and 50 strains isolated from 50 complement-sufficient persons, all of whom were Caucasians. The demographic characteristics of the patients and the serogroups of the strains isolated from the patients are presented in Table 1. Properdin deficiency was present in 15 patients, factor H deficiency was present in 1 patient, C3 nephritic factor (C3NeF) was present in 4 patients, and C3 deficiency was present in 2 patients. An LCCD was present in 21 patients, being C5 deficiency in 4 patients, C6 deficiency in 2 patients, C7 deficiency in 7 patients, and C8 deficiency in 8 patients. Two isolates were obtained from a patient with two episodes of meningococcal disease; the patient had low classical and alternative pathway hemolytic activities in reconvalescent-phase sera, compatible with increased complement consumption. Tests for C3NeF were negative. In total, 11 strains were isolated from eight complement-deficient patients with second or third episodes of meningococcal disease. For comparison, 50 meningococcal isolates of serogroups A, B, C, W135, X, and Y and nongroupable meningococci from patients with meningococcal disease and with normal serum complement hemolytic activity were studied. These isolates were matched with those of the complement-deficient persons for year of isolation and the serogroups isolated. Since four serogroup B, one serogroup W135, one serogroup Z, and five serogroup C isolates were found in the complement-deficient patients with second and third episodes of meningococcal disease, 11 strains of such serogroups isolated from complement-sufficient patients were also studied.
TABLE 1.
Characteristics of patients and distribution of serogroups according to the patient’s complement state
| Characteristic | Complement state
|
|
|---|---|---|
| Deficient | Sufficient | |
| No. of males/no. of females | 13/31 | 22/28 |
| Mean age (yr) at first meningococcal disease | 16.5 | 23.1 |
| Age range (yr) | 1–49 | 0–82 |
| No. of strains of the following serogroup: | ||
| A | 1 | 1 |
| B | 3 | 9 |
| C | 6 | 7 |
| W135 | 16 | 19 |
| X | 3 | 3 |
| Y | 13 | 8 |
| Z | 0 | 1 |
| Nongroupable | 2 | 2 |
| Total no. of strains | 44 | 50 |
Serotyping and subtyping were done by whole-cell enzyme-linked immunosorbent assay with specific monoclonal antibodies (15). MEE based on 14 enzymes was performed as described previously (4, 11), and distinctive multilocus genotypes were designated ETs. The ETs found were also compared with those of 278 serogroup B strains isolated from patients with meningococcal serogroup B disease in The Netherlands during the period from 1958 to 1986 (4, 15, 16). In addition, we assessed whether 11 meningococcal strains isolated during recurrences of meningococcal disease among eight complement-deficient persons differed from those isolated during a previous episode of meningococcal disease.
No significant difference in serotypes and subtypes was found among the isolates recovered from complement-deficient and complement-sufficient patients (Table 2). Of the isolates from complement-deficient patients, 25% were not serotypeable or subtypeable. Among isolates from complement-sufficient patients, 34% were not typeable and 24% were not subtypeable (Table 2). None of the serotypes or serotypes except serotype-serosubtype 2a:P1,2 clustered in a particular serogroup; 60% of serotype-serosubtype 2a:p1,2 strains belonged to serogroup W135. MEE typing of the 105 isolates yielded 54 ETs. The distribution of the ETs in the dendrogram (Fig. 1) is similar for either the 44 first isolates or the 55 isolates from the complement-deficient patients for all meningococcal disease episodes in comparison with the distribution of the ETs for the 50 meningococcal isolates from the complement-sufficient patients. The cluster most frequently found was the ET-37 complex (here represented by ETs 42 through 49), encompassing the 15 first isolates from complement-deficient patients and 18 strains from complement-sufficient patients. No segregation of strains from patients with a specific complement deficiency in a certain ET was observed. The ETs of the strains from complement-deficient patients were distributed throughout the dendrogram when the strains were analyzed with the 278 serogroup B strains isolated from patients during the period from 1958 to 1986 (data not shown).
TABLE 2.
Serotypes and serosubtypes of meningococcal strains causing disease in complement-deficient and complement-sufficient patientsa
| Serotype | Total no. of strains from CD patients/total no. of strains from CS patients | No. of strains of the following P1 subtypes from CD patients/no. of strains from CS patients
|
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 2 | 4 | 5 | 6 | 7 | 10 | 12 | 1,2 | 1,7 | 1,16 | 2,5 | 5,15 | 14,15 | Not subtypeable | ||
| 2a | 14/19 | 9/9 | 1/0 | 2/4 | 2/3 | 0/1 | 0/2 | ||||||||
| 2b | 2/2 | 0/1 | 1/0 | 1/1 | |||||||||||
| 4 | 11/6 | 1/0 | 1/0 | 0/1 | 1/1 | 1/2 | 2/0 | 1/0 | 1/0 | 3/2 | |||||
| 14 | 4/1 | 0/1 | 4/0 | ||||||||||||
| 15 | 2/5 | 0/2 | 2/3 | ||||||||||||
| Not typeable | 11/17 | 1/1 | 3/2 | 4/6 | 0/1 | 0/1 | 1/1 | 2/5 | |||||||
| Total | 44/50 | 11/12 | 1/2 | 4/2 | 4/7 | 1/1 | 4/6 | 2/0 | 1/0 | 0/1 | 0/1 | 4/5 | 0/1 | 1/0 | 11/12 |
CD, complement deficient; CS, complement sufficient.
FIG. 1.
Genetic relationships among 54 ETs of 105 Neisseria meningitidis isolates recovered from complement-deficient (CD) and complement-sufficient (CS) patients in The Netherlands. The dendrogram was generated by the average linkage method of clustering from a matrix of coefficients of genetic distance on the basis of 14 polymorphic enzyme loci. ETs are numbered sequentially from top to bottom. The number of strains from complement-sufficient and complement-deficient patients within a certain ET are indicated, as is the complement deficiency factor (NI, not identified deficiency). Strains from complement-deficient patients with second or third episodes of meningococcal disease are marked with an asterisk.
In total, 11 recurrences of meningococcal disease occurred in four patients with C3 deficiency syndrome and four patients with LCCD (Table 3). The serogroups of the meningococcal isolates causing the second disease episode were different from the serogroups of those causing the first episode except for one isolate from a C3-deficient patient. This patient had her second episode except for one isolate from a C3-deficient patient. This patient had her second episode of meningococcal disease 42 days after her first episode. The serotypes, serosubtypes, and ETs of both isolates were identical, indicating a recrudescence. In the first episode, she presented with meningitis which was successfully treated with 12 × 106 U of intravenous penicillin per day for 10 days. The isolate was cultured from a cerebrospinal fluid specimen. In the second episode she presented with septicemia. Gram staining and culture of the cerebrospinal fluid were negative. Three years later, a third episode of meningitis with sepsis was due to a different strain of the same serogroup. Two other patients had a third episode of meningococcal disease caused by strains distinct from those that caused the previous episodes.
TABLE 3.
Characteristics of N. meningitidis strains from complement-deficient patients with multiple disease episodes and interval between episodesa
| Complement deficiency | First episode
|
Second episode
|
Serogroup:serotype: serosubtype (ET) of third isolate | ||
|---|---|---|---|---|---|
| Serogroup:serotype:serosubtype (ET) of first isolate | Interval (yr) | Serogroup:serotype:serosubtype (ET) of second isolate | Interval (yr) | ||
| C3NeF | B:4:P1.4 (ET-16) | 1.3 | C:4:P1.15 (ET-50) | ||
| NI | C:NT:NT (ET-34) | 2 | W135:NT:NT (ET-18) | ||
| C3 | Y:4:P1.12 (ET-5) | 3 | C:NT:P1.5 (ET-35) | ||
| C3 | B:2a:P1.2 (ET-42) | 0.13 | B:2a:P1.2 (ET-42) | 3 | B:15:P1.4P1.7 (ET-17) |
| C5 | X:4:P1.7 (ET-7) | 0.33 | B:4:NT (ET-8) | ||
| C7 | X:4:P1.1P1.2 (ET-9) | 1 | C:NT:P1.1P1.7 (ET-4) | 1 | Z:nt:P1.2 (ET-1) |
| C7 | B:nt:P1.6 (ET-18) | 5 | C:2a:P1.2P1.5 (ET-46) | ||
| C8 | Y:NT:NT (ET-33) | 0.6 | C:2a:NT (ET-43) | 1.25 | B:4:P1.4 (ET-37) |
NI, not identified; NT, not typeable.
Our findings strongly indicate that the different serotypes and serosubtypes, in combination with their serogroups, were equally distributed among the complement-sufficient and complement-deficient patients. It has been reported that certain ETs are more frequently encountered in patients with disease than in carriers (3). During the period over which strains were studied, meningococcal disease in The Netherlands was caused by strains with a wide variety of ETs, of which a new clonal lineage (lineage III) associated with serogroup B, serotype and serosubtype 4:P1.4, has caused a gradual increase in the incidence of meningococcal disease since 1980 (4, 15, 16). In our study groups, only one strain from a complement-deficient person belonged to this clonal lineage (ET-17). MEE has been used to examine 17 strains isolated from 13 complement-deficient persons and to compare these strains with strains isolated from the general population of Cape Province, South Africa (11). In that study significantly more strains from complement-deficient individuals than strains from complement-sufficient patients were found to belong to one cluster (cluster F). It was concluded that strains from cluster F are probably less virulent and are able to cause disease predominantly in patients with immune deficiency. In our study ET clustering of the strains from the complement-deficient individuals had a pattern similar to the clustering of strains from complement-sufficient individuals and the serogroup B strains isolated from the general population. Cluster F was represented in The Netherlands by ET-28 to ET-30 and encompassed only six isolates: four from complement-sufficient patients, one from a properdin-deficient patient, and one from a patient with C3NeF. Among the strains studied, the cluster of strains that predominated in The Netherlands belonged to the ET-37 complex, which was represented by 33 isolates: 15 first isolates from complement-deficient individuals and 18 isolates from complement-sufficient persons. Thus, 34% of the strains from the patients with complement deficiency belonged to the ET-37 complex, whereas in the study in South Africa, only 1 of 17 isolates (6%) belonged to that clonal complex. Assuming that strains with low levels of virulence more frequently infect complement-deficient persons, this difference suggests that the strains with low levels of virulence circulating in the population in The Netherlands are different from those circulating in South Africa. However, the ET-37 complex includes clones with high levels of virulence that have been shown to cause epidemic disease in several parts of the world (19) when they are of serogroup B or have the C capsular polysaccharide. Therefore, the assumption that less virulent strains attack complement-deficient patients is not confirmed.
Penicillin is the treatment of preference for meningococcal disease (2). Although it is very effective in eliminating the meningococci from the blood and cerebrospinal fluid, nasopharyngeal carriage of meningococci is not eradicated by intravenous treatment with penicillin (1). Most of our patients were treated with penicillin only. The mean duration of meningococcal carriage has been estimated to be 9.6 months (9). The only recurrence with an identical strain, a serogroup B strain, occurred within 6 weeks after a meningitis episode in a C3-deficient patient who was treated with penicillin only. The treatment resulted in a complete disappearance of the meningococci from the cerebrospinal fluid. Whether this relapse was due to incomplete eradication of the meningococcus from the nasopharyngeal carriage site or a reinfection with the same strain upon reentrance into her family after discharge from the hospital is not known. Nevertheless, in patients with C3 deficiency treatment of the patient and close contacts with rifampin or ciprofloxacin (2) for the eradication of nasopharyngeal carriage seems justified.
Acknowledgments
We thank the medical specialists for kindly providing clinical data. Virma Godfried-Barbij, Agaath Arends, Torill Alvestad, Mark Darren Wools, and Ingun Ytterhaug are thanked for excellent technical assistance.
This work was supported by a World Health Organization grant (grant c11/181/23) to D.A.C. and by Praeventiefonds grant 28-1873.
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