点击显示 收起
Department of Infectious, Parasitic and Immune-Mediated Diseases and National Centre of Epidemiology Surveillance and Health Promotion, Istituto Superiore di Sanità, Rome, Italy
Health Protection Agency Haemophilus Reference Unit, Respiratory and Systemic Infection Laboratory, Centre for Infections, London, United Kingdom
Most invasive Haemophilus influenzae type b strains possess a duplication of the capsulation locus. Further amplification resulting in as many as 5 copies has been described. To verify whether amplification is involved in vaccine failure, the number of copies of the locus was determined by Southern blotting in 90 strains from children with true vaccine failure (TVF) between 1993 and 1999 and in 139 strains from unvaccinated children (50 collected between 1993 and 1999 and 89 collected between 1991 and 1992, before routine immunization was introduced). A significantly greater proportion of strains from TVFs contained multiple copies, compared with strains from control children (24% vs. 10%; P = .0379), which suggests that amplification of the capb locus may be a contributory factor in vaccine failure. The presence of multiple-copy strains was associated with disease other than meningitis.
The introduction of conjugate vaccines against Haemophilus influenzae type b (Hib) has resulted in a dramatic reduction in invasive Hib disease in young children worldwide [1]. Routine immunization with conjugate Hib vaccine was introduced in the United Kingdom and the Republic of Ireland in October 1992. Hib vaccine was offered at age 2, 3, and 4 months in the United Kingdom and at age 2, 4, and 6 months in the Republic of Ireland. No booster dose was offered during the second year of life in either country. Despite the effectiveness of the vaccine, rare cases of invasive disease caused by Hib strains in previously immunized children have been reported [2]. Recently, an increase in the incidence of invasive Hib disease has been observed in the United Kingdom, predominantly in immunized children [3]. Although predisposing host risk factors and the use of less immunogenic vaccines have been considered [4, 5], the possibility that particular virulent traits of the bacterium may contribute to vaccine failure cannot be ruled out.
The Hib polysaccharide capsule, a polymer of ribose ribitol phosphate (PRP), is an important virulence determinant of the microorganism [6]. The genes involved in Hib capsule expression are found within the capsulation (cap) locus, an 18-kb DNA segment of the chromosome [7]. Most invasive Hib strains contain a partial duplication of the cap locus that consists of 1 intact copy of the locus and a second copy with a 1.2-kb deletion within the bexA gene (which is necessary for polysaccharide export) and the IS1016 insertion element that flanks the locus. The duplicate arrangement has been supposed to serve as a template for further amplifications of capb gene sequences [7]. Up to 5 copies of the repeat have been detected in invasive strains, and the relationship between the production of polysaccharide capsule and the number of copies of the locus has been demonstrated [8]. The presence of multiple copies (>2 repeats) has been shown to be associated with decreased susceptibility of the bacterium to complement-mediated lysis and decreased complement-mediated opsonization, which suggests that amplification is used by Hib strains to increase resistance to complement-dependent host defense mechanisms [9].
In the present study, we investigated whether amplification of the capb locus might play a role in cases of invasive Hib infection in previously immunized children. To this aim, the number of copies of the capb locus was determined in Hib strains isolated from children with true vaccine failure (TVF) and from control children of the same age class in the United Kingdom and the Republic of Ireland.
MATERIALS AND METHODS
A total of 229 Hib strains isolated from children <60 months old with invasive disease in the United Kingdom and the Republic of Ireland were analyzed. Cases were detected through population-based national surveillance of invasive H. influenzae disease conducted in both countries during the study period. A case was defined as the isolation of H. influenzae from normally sterile site, combined with a clinical picture compatible with invasive H. influenzae disease, regardless of vaccination status. Both surveillance systems involved sending strains to a reference laboratory (in the United Kingdom, Haemophilus Reference Unit, Oxford; in the Republic of Ireland, Microbiology Department, Waterford General Hospital), where strains were identified as Hib by serotyping and polymerase chain reaction (PCR) capsular genotyping. TVF was defined as invasive Hib disease occurring either >2 weeks after the administration of a single dose of vaccine to a child >1 year old or >1 week after the administration of at least 2 doses to an infant 1 year old [4]. Of the 229 Hib strains analyzed in the present study, 90 were isolated from children with TVF between 1993 and 1999, 50 were from unvaccinated children during the same years (the 19931999 control group), and 89 were from unvaccinated children (±1 month old vs. the TVF group) during the years 19911992, before routine immunization was introduced (the 19911992 control group).
The strains had been passaged 3 times before study. The Hib strain Eagan was also included in the study as a reference strain [10]. Because KpnI and SmaI restriction sites flank the cap locus of the encapsulated H. influenzae strains [8], the number of copies of the capb locus was determined by Southern-blot analysis based on the size of the fragments after digestion of the chromosome with these enzymes and by use of the 480-bp amplicon as a probe (capsule type bspecific) obtained by PCR [10] of the reference Hib strain Eagan. Genomic DNA agarose plugs for pulsed-field gel electrophoresis (PFGE) were prepared as described elsewhere [11], starting from overnight broth cultures inoculated with at least 10 colonies. After digestion of the plugs with KpnI and SmaI enzymes (20 U of each; New England Biolabs), restriction fragments were separated by PFGE, transferred to a nylon membrane, hybridized with the probe, and visualized by autoradiograph. The KpnI/SmaI fragment for a 2-copy strain was expected to be 45 kb, because it includes 2 repeats of the locus (18 + 17 kb) plus additional segments (10 kb) upstream and downstream of the cap region [8]. Three-, 4-, and 5-copy strains featured KpnI/SmaI fragments of increased size, with additional DNA in 18-kb increments for each further copy (63, 81, and 99 kb, respectively).
Statistical analysis was performed by use of Epi Info for Windows (version 3.3; Centers for Disease Control and Prevention) and StatXact (version 6.2; Cytel Software). Medians were compared by use of the Mann-Whitney U test. Proportions were compared by use of the 2 test or, when appropriate, Fisher's exact test. A stratified analysis was conducted to evaluate the potential confounding effect of geographical location (United Kingdom or Republic of Ireland), age (12 or >12 months), and site of isolation of the strain (blood or cerebrospinal fluid ).
The Cochran-Armitage exact test for linear trend was used to compare the proportion of strains with multiple copies observed in unvaccinated children over time (19911992, 19931994, 19951996, 19971998, and 1999) and the proportion of strains by number of capb locus copies (i.e., 1 copy or 2, 3, 4, and 5 copies) in the TVF group versus unvaccinated children.
RESULTS
The mean and median ages of the children belonging to 3 groups were 23.7 and 22.4 months (range, 3.858.5 months) for the TVF group, 14.0 and 9.2 months (range, 1.458.8 months; P < .0001 vs. the TVF group and the 19911992 control group) for the 19931999 control group, and 23.0 and 21.5 months (range, 3.559.5 months) for the 19911992 control group.
All Hib strains analyzed showed the presence of hybridizing signals by Southern blotting (figure 1). Most strains (187/229), including strain Eagan, exhibited hybridization bands at the expected position for a 2-copy arrangement of the capb locus (45 kb). Of the remaining 42 strains, 6 showed hybridizing bands at <30 kb, which suggests that they harbored a single copy of the locus, whereas 36 showed bands at the expected positions for 3-copy (23 strains), 4-copy (10 strains), or 5-copy (3 strains) arrangements, respectively (table 1). In most strains harboring multiple copies of the locus, multiple bands representing different numbers of the repeats were simultaneously visible (figure 1). The proportion of strains that contained multiple copies was significantly higher in the TVF group (22/90 [24.4%]) than in the 19931999 control group (5/50 [10.0%]) (odds ratio [OR], 2.9 [95% confidence interval {CI}, 1.038.25]; P = .0379) (table 1).
The percentage of multiple-copy strains in unvaccinated children did not vary by time period, being 10% in both the 19911992 and 19931999 (9/89 vs. 5/50; OR, 1.0 [95% CI, 0.323.21]; P = .9831) control groups. Moreover, the proportion of multiple-copy strains isolated from both groups of unvaccinated children during the years 19911999 did not show any significant variation over time (P = 1.0000, Cochran-Armitage exact test for trend).
When the proportion of multiple-copy strains from TVFs was compared with that found in strains from all 139 unvaccinated children, irrespective of the year of isolation, again a significantly greater proportion of TVF strains contained multiple copies (22/90 strains from the TVF group vs. 14/139 from unvaccinated children; OR, 2.9 [95% CI, 1.396.01]; P = .0035). The OR of multiple-copy strains in the TVF group, compared with that found in unvaccinated children, increased along with the number of capb locus copies (3 vs. 2 copies, OR, 2.39 [95% CI, 0.995.74]; P = .0462; 4 vs. 2 copies, OR, 4.14 [95% CI, 1.2313.93]; P = .0169). This dose-response effect was confirmed by the Cochran-Armitage exact test for trend (P = .0119).
No significant difference in mean age at the time of disease onset by copy number of the capb locus was found (TVF group, 23.3 months for 1 or 2 copies and 25.1 months for multiple-copy strains, P = .6122; 19931999 control group, 14.3 months for 1 or 2 copies and 12.0 months for multiple-copy strains, P = .9227; 19911992 control group, 23.7 months for 1 or 2 copies and 16.3 months for multiple-copy strains, P = .1359). When the proportion of multiple-copy strains from the TVF group was compared with that found in strains from all unvaccinated children, the stratified analysis by age group, geographical location, and site of isolation of the strain did not reveal any confounding effect due to these variables.
Clinical presentation data were available for all but 8 children in the 19911992 control group. The proportion of children presenting with meningitis was similar in all groups: 55 in the TVF group (61.1%), 30 in the 19931999 control group (60.0%), and 56 in the 19911992 control group (69.1%) (P = .4509). In all groups, there was no significant difference in age at onset of disease between children with meningitis and those with other diagnoses (TVF group, 22.4 months for meningitis and 25.9 months for other diagnosis, P = .3825; 19931999 control group, 11.6 months for meningitis and 17.7 months for other diagnosis, P = .0684; 19911992 control group, 21.3 months for meningitis and 26.7 months for other diagnosis, P = .0819).
Meningitis was more frequently associated with 1- or 2-copy strains than were other clinical presentations in all 3 groups of patients (TVF group, 46 cases of meningitis due to 1- or 2-copy strains/55 cases of meningitis vs. 22 other clinical presentation due to 1- or 2 copy-strains/35 other clinical presentations, OR, 0.33 [95% CI, 0.120.89], P = .0253; 19931999 control group, 29/30 vs. 16/20; OR, 0.14 [95% CI, 0.011.34], P = .0759; 19911992 control group, 53/56 vs. 20/25; OR, 0.23 [95% CI, 0.051.04], P = .0551). When all 221 cases with known clinical presentation were considered, irrespective of vaccination status and year of isolation, the proportion of meningitis due to 1- or 2-copy strains was 90.8% (128/141), compared with 72.5% (58/80) of other clinical presentations (OR, 0.27 [95% CI, 0.130.57]; P = .0003). The ORs adjusted for vaccination status, age group, geographical location, and site of isolation were, respectively, 0.27 (95% CI, 0.120.58), 0.28 (95% CI, 0.130.58), 0.27 (95% CI, 0.130.57), and 0.21 (95% CI, 0.070.65).
Finally, clinical outcome was known for 193 of 229 cases. Overall, 5 children died (2.6%); the case-fatality ratio was not significantly associated with the number of copies of the capb locus (4/160 1- or 2-copy strains vs. 1/33 multiple-copy strains; P = 1.0000).
DISCUSSION
In the present study, we determined the number of copies of the capb locus in 229 invasive Hib strains isolated from children who had or had not previously received conjugate Hib vaccine, and we found that the number of copies of the capb locus ranged from 1 to 5. Considering the number of copies by group of children, we observed a significantly greater proportion of multiple-copy strains from patients with TVF, compared with unvaccinated children during the same time period (19931999 control group). Actually, because of the obvious difficulty in collecting invasive strains from unvaccinated children after routine Hib immunization was introduced, children in the 19931999 control group were less numerous than those in the TVF group and also were not of exactly the same age. Because no variation in the proportion of multiple-copy strains occurred during 19911999, we included all strains from the 19911992 control group in the analysis, and the results further confirmed that multiple-copy strains were more frequent in the TVF group than in the control groups. Of note, as a result of the increased size of the control sample, the statistical significance of the difference strongly increased, compared with that found for the TVF group versus the 19931999 control group. Overall, the probability of being infected with a multiple-copy strain was 3 times higher in children with TVF than in unvaccinated children. Interestingly, when strains were stratified into categories on the basis of numbers of copies of the capb locus, ORs increased with increasing number of copies, which suggests a dose-response effect. Stratified analysis did not reveal any confounding effect due to age, geographic location, or site of isolation.
Cases of invasive Hib disease in vaccinated children have been related to associated medical conditions of the host or to both quantitative and qualitative defects in specific anti-Hib antibody production or, recently, to the use of less immunogenic combined vaccines that contain the acellular pertussis component [4, 5]. Several other factorssuch as changes in the immunization schedule, the inability of the conjugate vaccine to induce pathogen-specific helper T cells, and a decline in natural boosting due to reduced circulation of Hib as result of vaccinationhave been suggested to contribute to the recent increase in incidence of invasive Hib disease within the vaccinated population in the United Kingdom [12, 13]. Given the importance of factors related to both host and vaccine, the results of the present study suggest that a particular virulent trait of the microorganismamplification of the capb locusmay also play a role in vaccine failures, at least in some cases. It may be supposed that strains with an amplified capb locus are more capable of overcoming host defense and invading the bloodstream of immunized patients (especially those with a suboptimal anti-PRP antibody concentration), in whom the presence of both serum factors and antibodies to capsule polysaccharide may play a role in maintenance of the amplified state. This is in agreement with previous studies, which suggest that having multiple copies of the capb locus might be a means of evading the immune response in blood [9]. Because the amplified state is unstable in vitro and bacteria in this state are virtually impossible to maintain as a pure culture [8], our strains harboring multiple copies often showed, in Southern blotting, a mixed population that contained different numbers of the capb locus at the same time.
It is noteworthy that, in the present study, the clinical presentation of meningitis was more frequently associated with 1- or 2-copy strains than with multiple-copy strains in all the groups of children. This result does not seem to be related to differences in age groups, site of isolation (CSF or blood), vaccination status, or geographic location, because no confounding effect due to those variables was revealed by our analysis. Recent studies of the invasion of the meninges by extracellular pathogens have shown that this occurs as consequence of both a high degree of bacteremia and the pathogen's ability to directly interact with the blood-brain barrier endothelial cells [14, 15]. In Neisseria meningitidis infection, the presence of type IV pili and other bacterial adhesins seems to be so essential for the interaction of virulent capsulated bacteria with the cellular component of the blood-brain barrier that nonpiliated capsulated strains are unable to interact efficiently with host cells [14]. Therefore, a possible explanation for our results is that, although the presence of a thicker capsule on the cell surface of multiple-copy strains is an advantage in terms of survival in the bloodstream, other surface components may be involved in the direct interaction between bacteria and brain endothelial cells. In this case, a thicker capsule could even be a disadvantage, in that it may interfere with this interaction. Further studies are needed to address this point.
To our knowledge, this is the first study to have investigated the number of copies of the capb locus in invasive Hib strains isolated from vaccine recipients and nonrecipients after the introduction of large-scale vaccination. The results show that strains containing amplified capb sequences were quite common among our Hib strains and were significantly more frequent among strains from children with TVF than among those from unvaccinated children, which suggests that the amplification of the capb locus may contribute to failure. Even if the actual role of the number of copies of capb in TVF has to be further investigated, these data illustrate the need to closely monitor Hib strains from children with TVF to verify whether, after 1999, selection of Hib strains harboring multiple copies of the capb locus has occurred.
Acknowledgments
We are grateful to Ann Moloney (Waterford Regional Hospital, Waterford) and Mary Cafferkey (Irish Meningococcal and Meningitis Reference Laboratory, Children's Hospital, Temple Street, Dublin), for providing strains from the Republic of Ireland.
References
1. Wenger JD, Booy R, Heath PT, Moxon ER. Epidemiological impact of conjugate vaccines on invasive disease caused by Haemophilus influenzae type b. In: Levine MM, Woodrow GC, Kaper JB, Cobon GS, eds. New generation vaccines. 2nd ed. New York: Marcell Dekker, 1997:489502. First citation in article
2. Booy R, Heath PT, Slack MPE, Begg N, Moxon ER. Vaccine failures after primary immunization with Haemophilus influenzae type-b conjugate vaccine without booster. Lancet 1997; 349:1197202. First citation in article
3. Ramsay ME, McVernon J, Andrews NJ, Heath PT, Slack MP. Estimating Haemophilus influenzae type b vaccine effectiveness in England and Wales by use of the screening method. J Infect Dis 2003; 188:4815. First citation in article
4. Heath PT, Booy R, Griffiths H, et al. Clinical and immunological risk factors associated with Haemophilus influenzae type b conjugate vaccine failure in childhood. Clin Infect Dis 2000; 31:97380. First citation in article
5. McVernon J, Andrews N, Slack MPE, Ramsay ME. Risk of vaccine failure after Haemophilus influenzae type b (Hib) combination vaccines with acellular pertussis. Lancet 2003; 361:15213. First citation in article
6. Moxon ER, Kroll JS. The role of bacterial polysaccharide capsules as virulence factors. In: Jann K, Jann B, eds. Bacterial capsules. Vol 150. Berlin: Springer-Verlag, 1990:6585. First citation in article
7. Kroll JS, Loynds BM, Moxon ER. The Haemophilus influenzae capsulation gene cluster: a compound transposon. Mol Microbiol 1991; 5:154960. First citation in article
8. Corn PG, Anders J, Takala AK, Kyhty H, Hoiseth SK. Genes involved in Haemophilus influenzae type b capsule expression are frequently amplified. J Infect Dis 1993; 167:35664. First citation in article
9. Noel GJ, Brittingham A, Granado AA. Mosser DM. Effect of amplification of the cap b locus on complement-mediated bacteriolysis and opsonization of type b Haemophilus influenzae. Infect Immun 1996; 64:476975. First citation in article
10. Falla TJ, Crook DWM, Brophy LN, Maskell D, Kroll JS, Moxon ER. PCR for capsular typing of Haemophilus influenzae. J Clin Microbiol 1994; 32:23826. First citation in article
11. Cerquetti M, Ciofi degli Atti ML, Cardines R, et al. Invasive type e Haemophilus influenzae disease in Italy. Emerg Infect Dis 2003; 9:25861. First citation in article
12. McVernon J, Mitchison NA, Moxon ER. T helper cells and efficacy of Haemophilus influenzae type b conjugate vaccination. Lancet Infect Dis 2004; 4:403. First citation in article
13. Kelly DF, Moxon ER, Pollard AJ. Haemophilus influenzae type b conjugate vaccines. Immunology 2004; 113:16374. First citation in article
14. Nassif X, Bourdoulous S, Eugène E, Couraud PO. How do the extracellular pathogens cross the blood-brain barrier Trends Microbiol 2002; 10:22732. First citation in article
15. Kim KS. Strategy of Escherichia coli for crossing the blood-brain barrier. J Infect Dis 2002; 186(Suppl 2):S2204. First citation in article