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Journal of Clinical Microbiology, June 2007, p. 1753-1758, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02134-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic and Antigenic Analysis of Invasive Serogroup Y Neisseria meningitidis Isolates Collected from 1999 to 2003 in Canada ^,

Raymond S. W. Tsang,^1* Averil M. Henderson,¹ Marissa L. Cameron,¹ Shaun D. Tyler,² Shari Tyson,² Dennis K. S. Law,¹ Jan Stoltz,¹ and Wendell D. Zollinger³

Laboratory for Vaccine Preventable Bacterial Diseases,¹ DNA Core Facility, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada,² Department of Bacterial Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland³

Received 17 October 2006/ Returned for modification 8 January 2007/ Accepted 9 April 2007

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One hundred forty serogroup Y Neisseria meningitidis isolates recovered from patients with invasive meningococcal disease (IMD) in Canada from 1999 to 2003 were analyzed by genetic and serological methods. Seventy-four isolates (52.9%) belonged to serotype 2c, and most have serosubtype antigen P1.5,2 (37 isolates, 26%) or P1.5 (31 isolates, 22%). Forty-eight isolates (34.3%) belonged to serotype 14 and have serosubtype antigen P1.5,2 (13 isolates, 9%) or P1.5 (7 isolates, 5%) or were nonserosubtypeable (27 isolates, 19%). Thirteen isolates (9.3%) were nonserotypeable. Multilocus sequence typing identified two unrelated clonal populations of serogroup Y meningococci causing invasive disease in Canada: ST-23 and ST-167 clonal complexes. Almost all ST-167-related isolates were typed as 2c:P1.5, while strains of the ST-23 clonal complex were either serotype 14 or 2c but with the serosubtype antigen P1.5,2. In contrast to previous reports that patients with serogroup Y disease are usually older, 26% of the Canadian serogroup Y cases were found in the 10-to-19-year-old age group and another 11% were in the 20-to-39-year-old age group.

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Neisseria meningitidis, the causative agent of invasive meningococcal disease (IMD), is a significant human pathogen. IMD occurs worldwide and can carry a case fatality rate of over 10% (26, 38). In a small number of colonized subjects, the bacteria gain entrance into the bloodstream from the upper respiratory tract to cause systemic diseases such as meningococcemia, meningitis, pneumonia, and other clinical manifestations (18, 26, 39). When these gram-negative diplococci are recovered from invasive disease cases, they are usually covered in a polysaccharide capsule. This capsule is the basis for serogroup differentiation and is the target for vaccine development. Currently 13 serogroups are recognized, and 5 of these (A, B, C, Y, and W135) are responsible for the majority of disease cases (20). Purified capsular polysaccharide vaccines have been used to protect against N. meningitidis serogroups A, C, Y, and W135 (2). Diphtheria or tetanus toxoid-conjugated A and C polysaccharide vaccines have also been developed (9), and more recently a conjugated tetravalent vaccine against A, C, Y, and W135 has been licensed in the United States and Canada (21, 23). No safe and effective vaccine against all strains of serogroup B meningococci has been developed due to the poor immunogenicity of the serogroup B capsule, which shares a common molecular structure with the carbohydrate component of the neural cell adhesion molecule (n-CAM) in humans (11, 13).

Serological classification of N. meningitidis isolates into serotypes and serosubtypes is based on detection of specific epitopes on the outer membrane proteins, PorB and PorA, respectively (15). Thus, strains of N. meningitidis can be described by their serogroup, serotype, and serosubtype antigens, for example, C:2a:P1.5,2 (12, 15).

Serological characterization of the serotype and serosubtype antigens is being replaced and supplemented by DNA sequencing of their genes and additional genes (34). Other genetic techniques used to study the population biology of N. meningitidis and to compare the genetic relatedness of strains include multilocus sequence typing (MLST) (17) and pulsed-field gel electrophoresis (30).

Invasive meningococcal disease has certain unique epidemiological features. For example, disease incidence fluctuates, with periods of increased activity every 10 to 15 years (16, 35). Also different hyperinvasive lineages or clones of meningococci cause disease with unique epidemiology (5). Furthermore, there are geographical differences in the serogroup distribution of N. meningitidis strains causing IMD. For instance, in Canada (32) and the United States (22, 25) serogroup Y meningococci have been responsible for a noticeable proportion of all IMD cases. This is in contrast to most European countries (8), where serogroup Y N. meningitidis causes only a small percentage of all IMD and is often found in healthy carriers (7, 40).

In order to have a better understanding of serogroup Y meningococcal disease in Canada, we characterized invasive serogroup Y isolates received at the Public Health Agency of Canada's National Microbiology Laboratory (NML) over a 5-year period from 1999 to 2003. This report describes the antigenic and genetic characteristics of invasive serogroup Y N. meningitidis in Canada and compares the data with those reported in literature from other countries. While strains that belong to the sequence type (ST) 23 (ST-23) clonal complex were responsible for a significant percentage of all invasive serogroup Y disease in Canada, an unrelated clonal group of the ST-167 complex was also found to be a common cause of invasive serogroup Y disease.

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Bacterial strains and serological analysis of surface antigens. Isolates of N. meningitidis from IMD patients were submitted by provincial and territorial public health laboratories in Canada to the NML for characterization as part of our national surveillance activity for this disease (32). Patient information, including age, sex, source of specimen, and province of origin, was obtained from specimen requisition forms. Serogroup determination by standard bacterial agglutination test (24) was performed with rabbit antisera against all 13 different serogroups (A, B, C, D, H, I, K, L, W135, X, Y, Z, and 29E). Isolates that were autoagglutinable or nongroupable were tested by an indirect whole-cell enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies to the common serogroups of B, C, Y, and W135 (33). Serotyping and serosubtyping of meningococci were done by an indirect whole-cell ELISA (1) using monoclonal antibodies against the serotype antigens 1, 2a, 2b, 4, 14, and 15 and serosubtype antigens P1.1, P1.2, P1.4, P1.5, P1.6, P1.7, P1.9, P1.10, P1.12, P1.13, P1.14, P1.15, and P1.16 (Rijksinstituut voor Volksgezondheid en Milieu, National Institute of Public Health, Bilthoven, The Netherlands). In addition, the serotype antigen 2c was tested with monoclonal antibodies as previously described (32).

Genetic characterization of meningococci. Genogrouping was done by PCR and PCR-ELISA methods described by Borrow et al. (3, 4). Partial sequences of the porA and porB genes that cover the hypervariable regions (VRs) were determined by methods described by Sacchi et al. (27, 28).

PorA variable regions, VR1 and VR2, are described using nomenclature according to the Neisseria PorA website (http://neisseria.org/nm/typing/pora) and that proposed by Sacchi et al. (28). MLST was performed as described by Maiden et al. (17), and isolates were assigned sequence types and clonal complex designations according to the Neisseria MLST website (http://pubmlst.org/neisseria/). Relationships between allelic profiles and STs were analyzed for patterns of evolutionary descent using the eBURST algorithm (10).

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Serogroup distribution for N. meningitidis causing IMD in Canada. From 1999 to 2003, a total of 1,007 N. meningitidis isolates recovered from patients with invasive diseases (only one isolate per patient was included) were analyzed at the NML. The majority of the isolates belonged to the four most common serogroups: B, C, Y, and W135 (Fig. 1). Only three isolates belonged to other serogroups (one isolate each of serogroups 29E, X, and Z).

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FIG. 1. Serogroup distribution of meningococcal isolates (N. meningitidis strains) from invasive meningococcal disease cases in Canada from 1999 to 2003.

A total of 140 serogroup Y isolates from individual IMD cases were analyzed during this 5-year period. The number of serogroup Y isolates received per year ranged from 17 isolates (10.1%) in 1999 to a high of 38 isolates (25.3%) in 2003. A total of 60 isolates (42.9%) were from the province of Ontario, but the numbers and percentages of serogroup Y meningococci among all invasive meningococcal isolates in that province ranged from a low of 8 isolates (11.4%) in 1999 to a high of 15 isolates (30.6%) in 2003. One hundred thirteen of the serogroup Y isolates (80.7%) came from three provinces: British Columbia, Ontario, and Quebec.

Seventy-two cases (51.4%) of invasive serogroup Y meningococcal disease were found in females, 67 cases (47.9%) were found in males, and the information for one case was not available. Most of the 140 invasive serogroup Y isolates were recovered from blood (113 isolates or 80.7%) or cerebrospinal fluid (CSF; 22 isolates or 15.7%) cultures. There were also three isolates (2.1%) recovered from synovial fluids and one from a thyroid cyst. In one case, both the patient's CSF and blood specimens grew an identical serogroup Y meningococcus.

Serotype and serosubtype antigens of invasive serogroup Y N. meningitidis in Canada. Seventy-four (52.9%) of the invasive serogroup Y isolates were identified as serotype 2c, 48 isolates (34.3%) as serotype 14, 3 isolates (2.1%) as serotype 15, 1 isolate as serotype 1, and 1 isolate as serotype 2a. Thirteen isolates (9.3%) did not react with the panel of serotyping monoclonal antibodies and were therefore deemed nonserotypeable (NT). The porB gene VR sequences of all 13 NT isolates were determined (see Table S1 in the supplemental material). Three NT isolates had PorB VR1 to -4 identical to strains identified as serotype 19,14; two isolates showed identity to serotype 2c, and one isolate had PorB VR1 to -4 identical to serotype 4,7 (27). The remaining seven isolates had PorB VRs that displayed mosaic patterns, showing changes that suggested either point mutations or recombination events (see Table S1 in the supplemental material).

Fifty-five (39.3%) of the 140 invasive serogroup Y isolates had the serosubtype antigen P1.5,2; 45 isolates (32.1%) had the P1.5 antigen, and 34 isolates (24.3%) were found to be nonserosubtypeable (P1.-). There were four isolates (2.9%) with the serosubtype antigen P1.16 and one isolate (0.7%) each of serosubtypes P1.2 and P1.6. DNA sequencing of the porA genes revealed that most of the P1.5,2 isolates had PorA VR types 5-1 (5a) for VR1 and 2-2 (2c) for VR2, while those of the P1.5 phenotype were characterized by the PorA VR types 5a for VR1 and 10-1 (10a) or 10-4 (10d) for VR2. The porA gene sequences for all 34 P1.- isolates were also analyzed. Nineteen of the P1.- isolates (55.9%) had PorA VR types 5-2 (5c) for VR1 and 10a for VR2; 8 isolates (23.5%) had PorA VR types 5c for VR1 and 10-2 (10b) for VR2; and the remaining 7 strains all had different PorA VR combinations, each occurring only once (see Table S2 in the supplemental material).

There were 15 different serotype-serosubtype antigen combinations, and they were presented in Table 1 together with MLST STs of the isolates showing these different antigenic formulas. The most common antigenic combinations encountered were Y:2c:P1.5,2 (26.4%), Y:2c:P1.5 (22.1%), Y:14:P1.- (19.3%), and Y:14:P1.5,2 (9.3%). The remaining 11 combinations each made up no more than 5% of all serogroup Y isolates, but together they combined to account for 22.9%.

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TABLE 1. Distribution of invasive serogroup Y meningococci in Canada from 1999 to 2003 by year of isolation, province of origin, antigenic formula, and MLST STs

Age distribution of serogroup Y IMD cases. Of the 137 cases with known age information, the distributions of their ages were described in Table 2.

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TABLE 2. Age distribution of serogroup Y invasive meningococcal disease cases in Canada from 1999 to 2003

Genotypes of invasive serogroup Y meningococci. MLST analysis of these serogroup Y isolates identified two unrelated clonal groups. Ninety-two isolates (65.7%) belonged to the ST-23 clonal complex, and, within this clonal complex, there were 65 isolates identified as ST-23, 10 isolates as ST-1625, and another 9 isolates as ST-569. The remaining eight isolates were divided among five other STs (Table 3).

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TABLE 3. ST allelic profiles of invasive serogroup Y meningococci belonging to ST-23 and ST-167 clonal complexes in Canada from 1999 to 2003

Thirty-eight isolates belonged to another clonal group, the ST-167 clonal complex, unrelated to ST-23 at each of the seven housekeeping gene alleles. This clonal group was made up of isolates displaying nine different STs, but the most common were ST-3923 (19 isolates), ST-3980 (6 isolates), and ST-1624 (6 isolates) (Table 3).

Besides these two clonal groups, to which 93% of the invasive serogroup Y meningococci belonged, there were nine other STs identified, and these were mostly represented by single isolates. Their characteristics are described in Table 4. eBURST analysis of STs and their allelic profiles produced by MLST of the 140 serogroup Y N. meningitidis identified two mutually exclusive groups of related genotypes. The founding genotype in group 1 was ST-1624, with three single-locus variants (SLVs), ST-3923, ST-3949, and ST-572, and one double-locus variant, ST-3980. In group 2, ST-23 was determined to be the founding genotype and four SLVs were identified: ST-5733, ST-1625, ST-3704, and ST-5730.

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TABLE 4. Canadian invasive serogroup Y meningococci from 1999 to 2003 belonging to an ST unrelated to ST-23 and ST-167 clonal complexes

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In Canada, IMD has been a notifiable disease since 1924 (35; Public Health Agency of Canada, notifiable disease online [http://dsol-smed.phac-aspc.gc.ca/dsol-smed/ndis/index_e.html]). Surveillance is carried out in all provinces and territories by local public health officials and the provincial and territorial public health laboratories. Isolates from IMD cases are submitted to the NML for serogroup confirmation and further antigenic and genetic analysis. The Division of Immunization and Respiratory Diseases, Center for Infectious Disease Prevention and Control, links case-by-case epidemiological data from provinces and territories with laboratory data provided by the NML and publishes the case information in the Canadian Communicable Disease Report (CCDR). Recent CCDR reports indicated 18, 25, 30, 39, and 41 invasive serogroup Y cases were found in years 1999 to 2003, respectively, with a combined total of 153 serogroup Y IMD cases (29, 37). For comparison, NML received a total of 140 invasive serogroup Y isolates during the same period of time, suggesting a recovery rate of about 91.5%. These reports also indicated that the proportion of IMD isolates belonging to serogroup Y showed an increasing trend from 8.4% in 1999 up to 21.5% in 2003. Although it is difficult to know for sure if our collection of isolates is a true representation of all invasive serogroup Y strains from disease cases in Canada, there is no indication to suggest any bias in our sample collection. The number of serogroup Y cases reported by provinces to the national notifiable disease system is very similar to the number of invasive serogroup Y isolates received at the NML (Table 1); therefore, our sample represents the majority of all invasive serogroup Y isolates found in Canada.

Patients contracting serogroup Y meningococcal disease have been previously reported to be older and often have chronic underlying medical conditions (18). In our study 37 cases (26.4%) were found in the 10- to 19-year-old age group and 16 cases (11.4%) in the 20- to 39-year-old age group (Table 2). The proportion of cases in the 10- to 19-year-old age group increased from 2 cases (11.8%) in 1999 to a peak of 12 cases (41.4%) in 2001 and subsequently declined to 10 cases (26.3%) by 2003. Therefore, it is important to follow this trend to determine if there is a true shift in the age group of patients with serogroup Y disease.

The invasive serogroup Y isolates were divided by serotype antigens into two major types, specifically, serotype 2c (74 isolates or 52.9%) and serotype 14 (48 isolates or 34.3%) (Table 1). The geographical distribution of these two serotypes varied across Canada. Over 50% of the serotype 2c isolates appeared in British Columbia and Ontario, e.g., 38 isolates were from Ontario and 13 isolates were from British Columbia versus only 9 serotype 2c isolates in Québec. In contrast, serotype 14 was found predominantly in Québec (17 isolates), Ontario (16 isolates), and British Columbia (8 isolates).

Harrison et al. described two PorB profiles among the invasive serogroup Y isolates studied in Maryland (14). Isolates from the early 1990s had the class 2 PorB allele 2-55, whereas isolates from the late 1990s possessed the class 3 PorB allele 3-36. The class 2 PorB allele 2-55 has a VR2 (loop V) peptide sequence identical to that of serotype 2c, and the class 3 PorB allele 3-36 has a VR4 (loop VII) peptide sequence identical to that of serotype 14 (27). This suggests that the two major serotypes (2c and 14) observed in Canada were also found in the United States. However, instead of geographical variations of these two serotypes, Harrison et al. reported a temporal shift that occurred in Maryland. A temporal shift was not observed for the serogroup Y strains in Canada.

MLST divided the 140 invasive serogroup Y isolates into two genetic populations: ST-23 and ST-167 clonal complexes. Usually a clonal complex is named after the most commonly encountered ST in a group of related STs. However, for the serogroup Y strains in Canada that belonged to the ST-167 clonal complex, there has been only 1 strain identified as ST-167 while 19 isolates of ST-3923 were found (Table 3). Whether this finding is unique for our locality will require further studies of isolates from other countries.

In this study there was an apparent association between the antigens expressed by a strain and its ST. For example, most isolates within the ST-167 clonal complex were identified as serotype 2c with serosubtype antigen P1.5. In contrast, isolates within the ST-23 clonal complex were either serotype 14 or serotype 2c, with the serosubtype antigen P1.2,5. Association of antigens and genotypes is well established in many other clonal groups such as the ET-37 complex (36). According to the N. meningitidis MLST website (accessed on 8 April 2007), serogroup Y isolates accounted for only 42% of those identified as belonging to the ST-167 clonal complex. Furthermore, 69% of these ST-167 clonal complex isolates were from carriers and only 25% were recovered from invasive-disease cases. In this regard, it is of interest that a single case of serogroup Y meningococcal disease in Ireland was caused by a strain genotyped as ST-167 and serotyped as NT:P1.5 (19).

Several reports have described many serogroup Y meningococci belonging to clonal complex ST-23. In Taiwan, serogroup Y first emerged in 2001 and caused more disease than serogroups A and C combined (6). In that study, 11 of 13 serogroup Y isolates examined were ST-23. Recently Harrison et al. found that 94% of invasive serogroup Y disease cases in Maryland were due to isolates of the ST-23 clonal complex (14). Of the 96 serogroup Y isolates that belonged to the ST-23 clonal complex, 39 isolates (40.6%) were ST-23, 23 isolates (24.0%) were ST-1622, 17 isolates (17.7%) were ST-1625, and 9 isolates (9.4%) were ST-1621. While ST-23 serogroup Y meningococci have been found to be associated with invasive disease in Asia and the United States, in Europe they appeared to be isolated more frequently from healthy carriers than from patients with invasive disease (7, 40).

In Canada, besides ST-23, two other members of this clonal complex, ST-1625 (10 cases) and ST-569 (9 cases), were also found to be associated with invasive diseases. ST-1625 was detected by Harrison et al. in the Maryland study; however, ST-569 was not (14). Studies in Japan (31), Taiwan (6), and Europe (7, 40) did not find these two genotypes to be associated with serogroup Y meningococci or with serogroup Y disease. When we analyzed the ages of patients suffering from invasive serogroup Y disease due to the ST-23, ST-1625, or ST-569 clones, the median ages of patients were 41, 16, and 18 years, respectively (data not shown). This may suggest some potential differences in the epidemiology of serogroup Y disease due to the different STs within the ST-23 clonal complex.

The limitation of our study is the lack of clinical data from these 140 invasive serogroup Y cases to match with the antigenic and genotypic information of the isolates. Nevertheless, this report serves to provide some baseline information on the types of invasive serogroup Y meningococci in Canada, which will serve for future references as well as for comparison with data from other countries. For future surveillance of serogroup Y disease, a unified epidemiological and laboratory surveillance system which collects relevant clinical data with matching microbiological characteristics of the causative strains would allow researchers a better understanding of the epidemiology of this disease as well as the behavior of the bacteria.

 
We acknowledge the contributions of the directors and staff of provincial and territorial public health laboratories for supporting the national meningococcal disease surveillance program by submitting meningococcal disease isolates for antigenic and genetic analysis.

This study made use of the Neisseria Multi Locus Sequence Typing website (http://pubmlst.org/neisseria/) developed by Keith Jolley and Man-Suen Chan and sited at the University of Oxford; the development of this site has been funded by the Wellcome Trust and European Union. We acknowledge the Health Canada's Genomics R&D Fund for providing funding for this work.

 
* Corresponding author. Mailing address: Vaccine Preventable Bacterial Diseases, National Microbiology Laboratory, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2. Phone: (204) 789-6020. Fax: (204) 789-2018. E-mail: Raymond_Tsang{at}phac-aspc.gc.ca

Published ahead of print on 18 April 2007.

Supplemental material for this article may be found at http://jcm.asm.org/.

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Journal of Clinical Microbiology, June 2007, p. 1753-1758, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02134-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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