Abstract
The relationship between carriage and the development of invasive meningococcal disease is not fully understood. We investigated the changes in meningococcal carriage in 892 military recruits in Finland during a nonepidemic period (July 2004 to January 2006) and characterized all of the oropharyngeal meningococcal isolates obtained (n = 215) by using phenotypic (serogrouping and serotyping) and genotypic (porA typing and multilocus sequence typing) methods. For comparison, 84 invasive meningococcal disease strains isolated in Finland between January 2004 and February 2006 were also analyzed. The rate of meningococcal carriage was significantly higher at the end of military service than on arrival (18% versus 2.2%; P < 0.001). Seventy-four percent of serogroupable carriage isolates belonged to serogroup B, and 24% belonged to serogroup Y. Most carriage isolates belonged to the carriage-associated ST-60 clonal complex. However, 21.5% belonged to the hyperinvasive ST-41/44 clonal complex. Isolates belonging to the ST-23 clonal complex were cultured more often from oropharyngeal samples taken during the acute phase of respiratory infection than from samples taken at health examinations at the beginning and end of military service (odds ratio [OR], 6.7; 95% confidence interval [95% CI], 2.7 to 16.4). The ST-32 clonal complex was associated with meningococcal disease (OR, 17.8; 95% CI, 3.8 to 81.2), while the ST-60 clonal complex was associated with carriage (OR, 10.7; 95% CI, 3.3 to 35.2). These findings point to the importance of meningococcal vaccination for military recruits and also to the need for an efficacious vaccine against serogroup B isolates.
INTRODUCTION
Neisseria meningitidis causes both epidemic and endemic life-threatening diseases worldwide, most notably sepsis and bacterial meningitis. It is also part of the normal nasopharyngeal microbiota of healthy persons (25, 34). The rate of asymptomatic carriage varies greatly depending on the population and epidemiological situation in question, ranging between 10% and 35% among young adults in Europe and the United States (6, 10, 34). Carriage is more common in teenagers and young adults than in young children (3), and the highest transmission and carriage rates have been reported for populations of people living in close contact with one another, such as university students or military recruits sharing dormitories (7). The molecular epidemiology of meningococcal carriage and disease development is not fully understood.
Previous phenotypic and genotypic studies have shown that N. meningitidis strains recovered from carriers are genetically more diverse than those isolated from patients with invasive meningococcal disease (IMD) (8, 9). Relatively few genotypes, the “hyperinvasive lineages,” have been responsible for most IMD, while only a small proportion of the strains isolated from carriers generally belong to these hyperinvasive lineages (26). Since most patients with life-threatening invasive disease have not been in contact with other IMD patients, it is assumed that carriers are the major source of the virulent strains that are potential causes of disease. In order to introduce effective IMD prevention policies, including vaccination, more carriage studies are needed to improve our understanding of the spread of N. meningitidis in populations at heightened risk of meningococcal disease, including military recruits.
The incidence of invasive meningococcal disease in Finland (<0.7 case/100,000 inhabitants/year) is currently low. In contrast to the case in many other European countries, with increases in serogroup C disease during the last few decades, there have been no major meningococcal epidemics or outbreaks in Finland since the serogroup A meningococcal epidemic in the 1970s (27) and a smaller serogroup B epidemic involving military recruits in southern Finland in 1995 and 1996 (31). Thus, meningococcal vaccination is currently recommended in Finland only for high-risk groups, including military recruits, who receive a tetravalent serogroup ACYW135 polysaccharide vaccine as a part of their vaccination program when entering service.
The present study aimed to follow changes in meningococcal carriage in military recruits in Finland during a nonepidemic period (July 2004 to January 2006). To investigate the diversity of the carriage isolates, meningococci isolated from oropharyngeal swabs taken at the beginning and end of military service or during the acute phase of respiratory tract infection were subjected to phenotyping (serogrouping and serotyping) and genotyping (porA typing and multilocus sequence typing [MLST]). For comparison, 84 meningococcal strains isolated from IMD patients in Finland in January 2004 to February 2006 were also analyzed.
(Some of the data in this study were presented previously at the Seventeenth International Pathogenic Conference 2010, Banff, Canada [21a].)
MATERIALS AND METHODS
Subjects.
This work was part of a larger CIAS (cold, infections, and asthma) study assessing risk factors for asthma and respiratory infections in Finnish military conscripts from July 2004 to January 2006 (22), in which a total of 892 men from two intake groups entering the Kainuu Brigade in Kajaani, Northern Finland, in July 2004 (420/1,836 men) and January 2005 (472/1,861 men), were enrolled (Fig. 1). These included all 224 men with a diagnosis of asthma in previous health examinations or in the call-up examination and 668 randomly chosen controls without asthma. The service time of the men was 6 (518 men), 9 (55 men), or 12 (245 men) months, according to their military duties; 29 (13%) asthmatic and 45 (6.7%) nonasthmatic men dropped out before completing their military service. The ages of the conscripts ranged from 18.1 to 24.4 years (median, 19.6 years). The study protocol was accepted by the Medical Ethics Committee of Kainuu Central Hospital, Kajaani, Finland. Participation was voluntary, and all participants signed a declaration of informed consent. The recruits were routinely vaccinated with a tetravalent serogroup ACYW135 polysaccharide vaccine at the beginning of their military service, but no antibiotic prophylaxis was given. No cases of invasive meningococcal disease occurred in the Kainuu Brigade during the study period.
Fig 1.
Study design and pharyngeal swab sampling. Oropharyngeal swabs for bacterial culture were collected at the beginning of military service and again at the end of military service, 6, 9, or 12 months after entry into service. In addition, oropharyngeal swabs were taken during the acute phase of every respiratory infection episode that required consultation with a physician.
Oropharyngeal sampling.
Oropharyngeal swabs for bacterial culture were collected in connection with a health examination performed during the first 2 weeks of service and again at the end of military service, 6, 9, or 12 months after entry into service. In addition, oropharyngeal swabs were taken during the acute phase of every respiratory infection episode that required consultation with a physician. The infectious episodes were diagnosed on the basis of symptoms of respiratory infection and clinical findings, as described in detail previously (22). The samples were taken from the posterior wall of the oropharynx and from both tonsils by use of calcium alginate swabs, which were immediately placed into test tubes containing 1 ml of STGG (skim milk, tryptone, glucose, and glycerol) medium (23). The tubes were vortexed and stored at −70°C within 6 h of collection for later analysis. The samples were cultured in batches after an average storage time of 6 months at −70°C.
Culture, isolation, and characterization of carriage isolates.
The oropharyngeal swabs stored in STGG medium were thawed at room temperature for 15 to 30 min, vortexed for about 10 s, and cultured on nonselective chocolate agar plates. The plates were incubated in 5% carbon dioxide at 37°C and examined at 24 and 48 h for the growth of meningococcus-like colonies. A single colony was picked from each plate with suspected meningococcal growth for subculturing prior to species identification by Gram staining, oxidase reaction, and carbohydrate utilization tests. All isolates identified as N. meningitidis were initially characterized with regard to serogroup and serotype by whole-cell enzyme-linked immunosorbent assay (ELISA) as described previously (37). The monoclonal antibodies for phenotyping were purchased from the National Institute for Biological Standards and Control, United Kingdom, with the exception of the serogroup Y-specific monoclonal antibody 1938 (39), a kind gift from Ulrich Vogel and Heike Claus (University of Würzburg, Germany), and the serogroup B-specific monoclonal antibody NmB 735 (16), purchased from Dade Behring Marburg GmbH (Marburg, Germany). Isolates that did not react with any of the serogrouping reagents (A, B, C, Y, and W135) or with the serotyping reagents (P2.2a, P2.2b, P3.1, P3.4, P3.14, P3.15, and P3.21) were defined as nongroupable (NG) or nontypeable (NT), respectively. The isolates were further analyzed by MLST and porA typing (VR1 and VR2) as previously described (20, 26). A combination of serogroup, serotype, and porA type was used to define each meningococcal isolate.
Characterization of invasive disease isolates.
Out of the total of 91 notifications of IMD cases that were referred to the National Infectious Disease Registry (NIDR) at the National Institute for Health and Welfare (THL) between January 2004 and February 2006, 92% (84/91 cases) were culture-confirmed cases for which corresponding isolates were submitted to the National Meningococcal Reference Laboratory at the THL for species confirmation, serogrouping by latex and/or slide agglutination, and serotyping by whole-cell ELISA. DNAs extracted from the culture-confirmed cases were sent to the University of Oxford for MLST, porA typing, and ClonalFrame analysis.
Statistical analysis.
Statistical analyses were performed using SPSS v.17.0 (SPSS Inc., Chicago, IL). The chi-square test or Fisher's exact test, as appropriate, was used for categorical variables. A logistic regression analysis was used to calculate odds ratios (OR), and the results for the carrier strains were adjusted for smoking and intake group. A two-sided P value of <0.05 was considered statistically significant.
ClonalFrame analysis.
ClonalFrame, version 1.1 (12), a statistical tree-building algorithm, was used to infer the clonal relationships of the isolate sets, taking into account homologous recombination that may have been present. ClonalFrame draws inferences by using a Monte Carlo Markov chain and requires an assessment of the convergence and mixing of its results (13); therefore, several independent runs of ClonalFrame (100,000 to 150,000 iterations) were run for each assessment. The results were compared for convergence by using the Gelman and Rubin statistic (17), and the runs were combined for maximum robustness. The convergence was judged satisfactory, and the samples from the runs were combined for maximum robustness. Statistical support for any grouping of isolates was assessed by the proportion of clonal genealogies exhibiting this grouping in the combined sample. This approach was applied independently to each of the carriage and disease isolate sets separately and in a combined isolate tree.
RESULTS
Serogroups, MLST sequence types (STs), and antigen profiles of meningococcal carriage isolates from army recruits.
A total of 193 carriers were identified among the 892 conscripts, and 215 meningococcal carriage isolates were obtained. Twenty of the 215 oropharyngeal isolates (9.5%) were obtained from swabs collected on entry to military service, 151 (70%) were from swabs collected at the end of service, and 44 (20.5%) were from swabs collected during acute respiratory infections (Table 1). Twenty (10.4%) of the 193 carriers were culture positive for N. meningitidis more than once during their military service, while the remaining carriers (173/193 individuals [89.6%]) were culture positive only once. The carriage of N. meningitidis did not differ between asthmatics and nonasthmatics (P > 0.05), and therefore these two groups were analyzed together. Serogroups B, Y, and W135 accounted for 25.6% (55/215 isolates), 8.4% (18/215 isolates), and 0.4% (1/215 isolates) of the isolates, respectively, and 65.6% (141/215 isolates) of the isolates were NG.
Table 1.
Carriage of N. meningitidis by intake group, service time, and time of sampling
| Intake group | Service time (mo) | Carriage rate (no. of carriers/no. of individuals [%]) |
||
|---|---|---|---|---|
| Entry | Sick visits | End of service | ||
| July 2004 | 6 | 1/235 (0.4) | 9/148 (6) | 27/235 (11.5) |
| 9 | 0/22 | 1/18 (5.6) | 5/22 (22.7) | |
| 12 | 1/125 (0.8) | 3/85 (3.2) | 24/125 (19.2) | |
| Dropouts | 0/38 | 2/21 (9.5) | NAa | |
| January 2005 | 6 | 9/283 (3.2) | 18/269 (6.7) | 68/283 (24) |
| 9 | 1/33 (3) | 0/32 | 7/33 (21) | |
| 12 | 8/120 (6.7) | 9/125 (7.2) | 20/120 (17) | |
| Dropouts | 0/36 | 2/20 (10) | NAa | |
| Total | 20/892 (2.2) | 44/718 (6.1) | 151/818 (18.5) | |
NA, not applicable.
Those entering military service, in general, had a low rate of N. meningitidis carriage (Table 1), and this was significantly (P < 0.001) lower for the summer intake group than for the winter intake group (0.5% [2/420 individuals] versus 3.8% [18/472 individuals]). The rate of carriage was significantly (P < 0.001) higher at the end of military service than it had been at arrival, with 14.7% (56/382 individuals) of those in the summer intake group and 21.8% (95/436 individuals) of those in the winter intake group being culture positive for meningococci (P < 0.001 between the two groups). Only five conscripts (0.6%) were culture positive for N. meningitidis at both the beginning and end of their service.
By MLST, 111 different STs were identified among 214 carriage isolates with complete MLST profiles, of which 57.7% (64/111 STs) were new STs. Overall, 93.5% (200/214 isolates) of the isolates fell into 14 previously known clonal complexes (Table 2). The ST-60 clonal complex was the most common, with 60 (28%) isolates and 20 different STs, followed by the ST-41/44 clonal complex, with 46 isolates (21.5%) and 25 STs, and the ST-23 clonal complex, with 25 isolates (12%) and 11 STs. The two most common STs were ST-4146 (ST-60 clonal complex), with 29 isolates (13.6%), and ST-136 (ST-41/44 clonal complex), with 14 isolates (6.5%). Two-thirds of all carriage isolates were nongroupable by traditional serogrouping methods. Of the clonal complexes represented by two or more isolates (this includes the isolates with unassigned STs, grouped as “unassociated”), five (clonal complexes 254, 178, 198, 53, and 32) could not be serogrouped, three (clonal complexes 60, 35, and 213) were partially serogrouped but with limited results, and the remaining four (clonal complexes 41/44, 23, and 269 and the “unassociated” group) had moderate to high success for serogrouping (Table 2). The majority of serogroupable isolates belonged to serogroup B (55/74 isolates), followed by serogroup Y (18/74 isolates), and one isolate was grouped as a serogroup W135 isolate. All serogroup Y isolates belonged to the ST-23 clonal complex, and 71% (39/55 isolates) of the serogroup B isolates belonged to the ST-41/44 clonal complex (Table 2).
Table 2.
Distribution of carriage isolate (n = 215) profiles within clonal complexes
| Clonal complex | No. of isolatesa | No. of STs | Serogroup(s) (% of isolates) | Most common strain profile(s)b (% of isolates) |
|---|---|---|---|---|
| ST-60 | 60 | 20 | NG (98), B (2) | NG:NT:P1.5,2 (93) |
| ST-41/44 | 46 | 25 | B (85), NG (15) | B:15:P1.17,16-3 (28) |
| ST-23 | 25 | 11 | Y (72), NG (28) | Y:14:P1.5-2,10-1 (24), Y:NT:P1.5-1,2-2 (24) |
| ST-254 | 18 | 6 | NG (100) | NG:4:P1.5-1,10-6 (39), NG:4:P1,5-1,10-8 (39) |
| ST-35 | 17 | 10 | NG (76), B (24) | NG:4:P1.22-1,14 (59) |
| Unassociated | 14 | 13 | B (57), NG (43) | Heterogenous |
| ST-178 | 11 | 8 | NG (100) | NG:NT:P1.5,10-44 (18) |
| ST-198 | 9 | 6 | NG (100) | NG:15:P1.18,25-1 (44) |
| ST-53 | 4 | 2 | NG (100) | NG:21:P1.7,30-5 (75) |
| ST-213 | 3 | 3 | NG (67), B (33) | NG:NT:P1.22,14 (33), NG:15:P1.22,14 (33), B:1:P1.12-3,4 (33) |
| ST-269 | 2 | 2 | B (50), NG (50) | NG:15:P1.7-2,13-1 (50), B:1:P1.21,26 (50) |
| ST-32 | 2 | 2 | NG (100) | NG:15:P1.7,16 (100) |
| ST-22 | 1 | 1 | W135 (100) | W135:NT:P1.18-1,3 (100) |
| ST-461 | 1 | 1 | B (100) | B:1:P1.19-35,13-1 (100) |
| ST-103 | 1 | 1 | NG (100) | NG:NT:P1.5-2,10 (100) |
One isolate had an incomplete MLST profile.
Serogroup:serotype:porA type (VR1, VR2).
The clonal complex distribution showed an increase in diversity between the beginning of service and the end of service for each year. When the study commenced in 2004, two clonal complexes were detected at the beginning of service, and by the end of 2004, there were eight clonal complexes plus 2 unassociated STs. A continued increase in clonal complex distribution was also observed in 2005, increasing from 8 to 14 clonal complexes by the end of the study period (2006).
Univariate statistical analysis revealed a strong association of certain clonal complexes with respiratory infection episodes (Table 3). Further examination of this association by logistic regression analysis showed that the ST-23 clonal complex was significantly more common among isolates collected during the acute phase of respiratory infection (OR, 7.1; 95% confidence interval [95% CI], 2.9 to 17.2) than among those collected at the beginning or end of service. A similar but smaller trend was also observed for the ST-41/44 clonal complex, though this association did not reach statistical significance (OR, 1.5; 95% CI, 0.71 to 3.3) when adjusted for intake group, while the opposite trend was observed for the ST-60 clonal complex (Table 3), which was about 2-fold more common among isolates collected at the beginning or end of service than among those collected during respiratory infections. The distribution of clonal complexes did not differ between smokers and nonsmokers, except for the ST-41/44 clonal complex, which appeared to be more frequent, though not statistically significantly so, among smokers than nonsmokers (59.1% versus 40.9%; P > 0.05).
Table 3.
Association of clonal complexes of carried meningococci with respiratory infection episodes
| Clonal complex | % of carried isolates |
P valuea | OR (95% CI)b | ||
|---|---|---|---|---|---|
| On arrival (n = 20) | During acute respiratory infection episodes (n = 44) | At departure (n = 151) | |||
| ST-60 | 35.0 | 16 | 30.5 | 0.045 | 0.4 (0.18–1.01) |
| ST-41/44 | 10.0 | 27.3 | 21.2 | 0.061 | 1.5 (0.71–3.3) |
| Unassociated | 20.0 | 4.5 | 5.3 | ||
| ST-23 | 5.0 | 31.8 | 6.6 | <0.001 | 7.1 (2.9–17.2) |
| ST-254 | 0 | 9.1 | 9.3 | 0.9 | 1.1 (0.4–3.6) |
| ST-35 | 10.0 | 4.5 | 8.6 | 0.5 | 0.5 (0.1–2.2) |
| ST-178 | 5.0 | 0 | 6.6 | ||
By Pearson chi-square test for the comparison of isolates collected from military recruits on arrival or at departure and during acute respiratory infection episodes.
Adjusted for intake group for the comparison of isolates collected from the military recruits on arrival or at departure and during acute respiratory infection episodes.
Eighty-two strain profiles (serogroup:serotype:porA type) were identified among the 215 carriage isolates, with the most common being NG:NT:P1.5,2 (28%), B:15:P1.17,16-3 (7%), and NG:4:P1.22-1,14 (4.7%). Profile NG:NT:P1.5,2 represented 93% of the 60 ST-60 clonal complex isolates. Profile B:15:P1.17,16-3 (28%) predominated among the 46 ST-41/44 clonal complex isolates (Table 2).
ClonalFrame analysis of carriage isolates.
The relationships of the carriage isolates inferred by ClonalFrame analysis using the MLST alleles clustered 94.4% of carriage isolates into 14 previously known clonal complexes (Fig. 2). Twelve isolates were unassigned to a clonal complex and are the only representatives of these STs currently in the PubMLST Neisseria database.
Fig 2.
Seven-locus MLST ClonalFrame tree for carriage isolates. STs unassigned to a clonal complex are represented by open circles with ST numbers, and clonal complexes are shown by colored circles with ST numbers. When present, the central genotype is indicated by a colored square. Blue arrows indicate peripheral MLST profiles with clonal complex association. Green, clonal complex ST-41/44; purple, clonal complex ST-60; teal, clonal complex ST-23; olive, clonal complex ST-198; orange, clonal complex ST-254; pink, clonal complex ST-35; brown, clonal complex ST-53; red, clonal complex ST-178; yellow, clonal complex ST-213; gray, clonal complex ST-269; blue, clonal complex ST-32; black, clonal complex ST-22; mauve, clonal complex ST-461; light blue, clonal complex ST-103.
Four STs did not cluster with their respective clonal complexes. One of three ST-213 clonal complex isolates and one of seven ST-178 clonal complex isolates incongruently grouped together with a single ST-22 clonal complex isolate; a second isolate from the ST-178 clonal complex and one isolate from the ST-41/44 clonal complex did not group with their assigned clonal complexes. In both of these cases, the unresolved grouping was caused by peripheral isolates of each clonal complex, particularly by differences in the aroE and, to a lesser extent, fumC gene fragments. Finer resolution or clarification of these isolate associations would require the addition of more loci or the use of whole genes instead of typing fragments.
Characterization of isolates collected from conscripts with multiple positive oropharyngeal cultures.
Of the 892 conscripts recruited, 18 (2.0%) had two positive oropharyngeal cultures for N. meningitidis during their service, and 2 (0.2%) conscripts had three positive cultures. To determine whether the paired isolates represented the same or different strains, the antigen profiles and sequence types were compared (Table 4). In 10 cases, the porA types of the strains were identical; in 8 of those 10 cases, the clonal complexes were the same, and in 3 of those 8 cases, the sequence types were also the same.
Table 4.
Strain characteristics of isolates collected from conscripts with multiple positive oropharyngeal cultures
| Recruit no. | Samplea | Isolate profileb | Clonal complex | ST | Timec (days) |
|---|---|---|---|---|---|
| 1 | I (during) | NG:4:P1.5-1,10-6 | ST-254 | 254 | 45 |
| II (end) | NG:4:P1.5-1,10-6 | ST-254 | 254 | ||
| 2 | I (during) | Y:NT:P1.5-1,2-2 | ST-23 | 23 | 15, 294 |
| II (during) | Y:NT:P1.5-1,2-2 | ST-23 | 23 | ||
| III (end) | NG:NT:P1.5,2 | ST-60 | 7543 | ||
| 3 | I (entering) | NG:4:P1.22-1,14 | ST-35 | 35 | 27 |
| II (during) | NG:4:P1.22-1,14 | ST-35 | 35 | ||
| 4 | I (during) | NG:15:P1.7,16 | ST-32 | 1249 | 7 |
| II (end) | NG:15:P1.7,16 | ST-32 | 7478 | ||
| 5 | I (entering) | Y:NT:P1.5-1,2-2 | ST-23 | 7518 | 61 |
| II (during) | Y:NT:P1.5-1,2-2 | ST-23 | 3228 | ||
| 6 | I (entering) | NG:21:P1.7,30-5 | ST-53 | 2126 | 159 |
| II (end) | NG:21:P1.7,30-5 | ST-53 | 53 | ||
| 7 | I (during) | NG:NT:P1.5,2 | Unassociated | 7561 | 170 |
| II (end) | NG:NT:P1.5,2 | ST-60 | 4146 | ||
| 8 | I (during) | B:NT:P1.5-1,2-2 | ST-41/44 | 42 | 26 |
| II (end) | B:NT:P1.5-1,2-2 | ST-41/44 | 2136 | ||
| 9 | I (entering) | B:15:P1.17,16-3 | Unassociated | 7559 | 80, 38 |
| II (during) | NG:NT:P1.5,2 | ST-60 | 7536 | ||
| III (during) | NG:NT:P1.5,2 | ST-60 | 4146 | ||
| 10 | I (entering) | B:15:P1.18,25/25-7 | Unassociated | 7491 | 159 |
| II (end) | B:15:P1.18,25/25-7 | ST-60 | 7535 | ||
| 11 | I (during) | B:15:P1.22-1,14 | ST-41/44 | 136 | 139 |
| II (during) | NG:14:P1.5-2,10-1 | ST-23 | 7568 | ||
| 12 | I (during) | Y:14:P1.5-2,10-28 | ST-23 | 23 | 25 |
| II (end) | B:4:P1.7-1,1 | ST-35 | 7487 | ||
| 13 | I (during) | NG:NT:P1.5,2 | ST-60 | 4146 | 52 |
| II (end) | NG:NT:P1.5,10-82 | ST-178 | 7497 | ||
| 14 | I (entering) | NG:NT:P1.5-32,10-44 | ST-178 | 7864 | 165 |
| II (end) | NG:4:P1.5-32,10-44 | ST-178 | 7865 | ||
| 15 | I (entering) | NG:NT:P1.ND,ND | Unassociated | 7915 | 339 |
| II (end) | NG:NT:P1.5,2 | ST-60 | 7541 | ||
| 16 | I (during) | NG:14:P1.5-2,10-28 | ST-23 | 23 | 105 |
| II (end) | NG:NT:P1.5,2 | ST-60 | 7538 | ||
| 17 | I (during) | B:15:P1.17,16-3 | ST-41/44 | 136 | 246 |
| II (end) | NG:NT:P1.5,2 | ST-60 | 7545 | ||
| 18 | I (during) | NG:4:P1.7-2,4 | ST-41/44 | 303 | 238 |
| II (end) | NG:14:P1.19-2,15 | ST-35 | 472 | ||
| 19 | I (during) | B:NT:P1.19,15-1 | ST-41/44 | 2691 | 240 |
| II (end) | NG:NT:P1.22,14 | ST-213 | 7496 | ||
| 20 | I (entering) | B:15:P1.17,16-3 | Unassociated | 7558 | 163 |
| II (end) | NG:15:P1.17,16-3 | ST-41/44 | 136 |
During, during respiratory tract infection; entering, entering military service; end, ending military service.
Serogroup:serotype:porA type (VR1, VR2). ND, not done.
Time between samples I and II or II and III.
Serogroups, MLST sequence types, and antigen profiles of IMD isolates.
Eighty-four (92%) isolates were recovered from 91 IMD cases that occurred in Finland between January 2004 and February 2006. Sixty percent (50/84 isolates) of these isolates were from patients younger than 25 years, and 18% were from adolescents aged 15 to 19 years. Unlike the carriage isolates, all of these disease isolates were serogroupable. Serogroups B, C, and Y were the predominant serogroups, accounting for 81%, 8.3%, and 8.3% of the isolates, respectively.
Sixty-four STs were identified among the 84 disease isolates with complete MLST profiles, of which 50% were new ones. Over one half of the new STs were assigned to 15 previously known clonal complexes, the majority of which have previously been associated with invasive disease (Table 5). The two most common clonal complexes, ST-41/44 and ST-32, accounted for 40.7% of the IMD isolates, compared with 22.4% of the carriage isolates. Univariate statistical analysis revealed a difference in clonal complex distribution between the patient and carrier isolates. By logistical regression analysis, isolates of the ST-32 clonal complex were significantly associated with invasive disease (OR, 17.8; 95% CI, 3.8 to 81.2), while the isolates of the ST-60 clonal complex were associated with carriage (OR, 10.7; 95% CI, 3.3 to 35.2).
Table 5.
Distribution of invasive meningococcal disease isolate (n = 84) profiles within clonal complexes
| Clonal complex | No. of isolatesa | No. of STs | Serogroup(s) (% of isolates) | Most common strain profile(s)b (% of isolates) |
|---|---|---|---|---|
| Unassociated | 27 | 23 | B (89), C (11) | Heterogenous |
| ST-41/44 | 21 | 12 | B (100) | B:4:P1.7-2,4 (42.9) |
| ST-32 | 12 | 9 | B (84), C (8), Y (8) | B:15:P1.7,16-6 (25) |
| ST-23 | 5 | 4 | Y (100) | Y:14:P1.5-2,10-1 (60) |
| ST-22 | 2 | 2 | B (50), W135 (50) | B:NT:P1.18-1,3 (50), W13:NT:P1.18-1,3 (50) |
| ST-269 | 2 | 2 | B (50), C (50) | B:4:P1.18,25/25-7 (50), C:21:P1.12-1,13-1 (50) |
| ST-60 | 2 | 2 | B (50), W135 (50) | B:15:P1.7,16-6 (50), W135:NT:P1.22-1,14 (50) |
| ST-865 | 2 | 1 | B (100) | B:14:P1.7-2,13-2 (100) |
| ST-162 | 1 | 1 | B (100) | B:NT:P1.7-2,4 (100) |
| ST-167 | 1 | 1 | Y (100) | Y:1:P1.5-1,10-1 (100) |
| ST-174 | 1 | 1 | B (100) | B:14:P1.5-1,10-4 (100) |
| ST-334 | 1 | 1 | B (100) | B:2b:P1.7-2,13-1 (100) |
| ST-364 | 1 | 1 | B (100) | B:NT:P1.12-1,13-1 (100) |
| ST-53 | 1 | 1 | B (100) | B:4:P1.22-1,14 (100) |
| ST-549 | 1 | 1 | B (100) | B:4:P1.5-2,10-2 (100) |
| ST-18 | 1 | 1 | B (100) | B:NT:P1.5-1,10-4 (100) |
Three isolates had incomplete MLST profiles.
Serogroup:serotype:porA type (VR1, VR2).
B:4:P1.7-2,4 (11%), B:15:P1.7,16-6 (4.8%), B:2b:P1.7-2,13-1 (4.8%), and Y:14:P1.5-2,10-1 (4.8%) were the most common profiles among the invasive isolates. These four profiles were found more frequently among the invasive strains than among the carrier strains, whereas profiles NG:NT:P1.5,2 and B:15:P1.17,16-3 were more prevalent among the carrier strains. Profile B:4:P1.7-2,4 represented 42.9% of the ST-41/44 clonal complex isolates among the invasive strains, while profile B:15:P1.17,16-3 predominated in the carriage isolates, representing 28% of the isolates of this clonal complex (Tables 2 and 5).
ClonalFrame analysis of disease isolates.
ClonalFrame analysis clustered 66 of the 84 disease isolates into 15 previously known clonal complexes (Fig. 3). Twenty-two isolates are currently unassigned to a clonal complex, and 20 are the only representatives of STs currently in the PubMLST Neisseria database. The two remaining STs have been observed once (each) before: ST-1470 was observed in Canada in 1999 (single isolate), and ST-2003 was observed in the United Kingdom in 2000 (single isolate). The disease isolates split into 2 groups forming 2 centralized tree nodes, and where a clonal complex is represented by more than one ST, a more defined branch occurs, with two exceptions: the clonal complex ST-23 has two branches from a shared node, and one ST-41/44 isolate does not group with the other ST-41/44 clonal complex isolates. The differences are confined primarily to the abcZ, fumC, and gdh alleles and, to a lesser extent, the gdh allele. As pointed out for the carriage data set, these are also peripheral clonal complex STs, and their allele assignment places them within the assigned ST-32 and ST-41/44 clonal complexes, respectively.
Fig 3.
Seven-locus MLST ClonalFrame tree for disease isolates. STs unassigned to a clonal complex are represented by open circles with ST numbers, and clonal complexes are shown by colored circles and ST numbers. When present, the central genotype is indicated by a colored square. The blue arrow indicates a peripheral MLST profile with clonal complex association. Green, clonal complex ST-41/44; teal, clonal complex ST-23; black, clonal complex ST-22; tan, clonal complex ST-162; purple, clonal complex ST-60; yellow, clonal complex ST-334; mauve, clonal complex ST-549; red, clonal complex ST-865; blue, clonal complex ST-32; brown, clonal complex ST-35; gray, clonal complex ST-269; olive, clonal complex ST-18; orange, clonal complex ST-174; pink, clonal complex ST-167; light blue, clonal complex ST-364.
The clonal complex assignments of these peripheral STs, compared against the known ST cohort for each respective clonal complex in the database, show overlapping MLST profiles, such that the STs do not cluster with their assigned clonal complexes as seen in each tree. As in most bacterial identification, there is a gray area where overlapping characteristics exist, making strict confinement of some isolates to a predefined group complicated and, at the very least, occasionally inconsistent. As in the carriage MLST analysis, finer resolution of the disease isolates would require the addition of more typing loci or the use of whole genes.
DISCUSSION
In the present study, the carriage of N. meningitidis increased from an average of 2.2% at the beginning of military service to 18.5% at the end of military service. The carriage rates reported here are somewhat lower than those in previous studies, in which rates varying from <16% to over 70% have been reported among military recruits (1, 15, 28, 30). These previous studies were performed mainly on unvaccinated populations, however. In our study, a tetravalent serogroup ACYW135 polysaccharide vaccine was given to the recruits as a part of the routine vaccination program at the beginning of their military service. This may have explained the low or absent level of carriage of serogroup C, Y, and W135 meningococci during the period studied, although previous studies suggested that the effect of polysaccharide vaccination on carriage is probably short-term (11).
During the present study (2004 to 2006), the annual incidence of IMD in Finland (<0.9 case/100,000 inhabitants) was relatively low compared with that in other European countries (14), and no cases of IMD occurred in the Kainuu Brigade. It is known that the capsule plays a major role in the pathogenesis of meningococcal disease (24), and all of our patient isolates expressed a polysaccharide capsule. In contrast, 65.6% of the carrier isolates were nongroupable by serological means, suggesting that at least some of them may have been nonencapsulated. Both the disease and serogroupable carrier isolates were predominantly serogroup B. While outbreaks and increases in the incidence of serogroup C disease have occurred in other parts of Europe since the late 1990s (40), this serogroup was relatively uncommon among our disease isolates, and none of the recruits carried meningococci expressing the serogroup C capsule in their oropharynx. Serogroup Y, which has increased in the United States (33) and United Kingdom (36) as well as in Scandinavian countries, including Finland (35, 36), during the past decade, accounted for about 8% of the disease isolates and also for about 8% of the carriage isolates, despite vaccination of the recruits against this serogroup at the time of entering military service. This suggests that the tetravalent ACYW135 polysaccharide vaccine does not prevent the carriage of serogroup Y meningococci completely, although it probably provides protection against the development of invasive disease.
Molecular epidemiological studies have demonstrated that in 2000 to 2002, most of the meningococcal disease in Europe was caused by strains belonging to the ST-41/44, ST-11, ST-32, ST-8, and ST-269 clonal complexes (2, 4). In the present study, executed in July 2004 to January 2006, the ST-41/44 clonal complex caused 25% of IMD cases and 21.5% of all carriage cases. Almost one half of the invasive strains (46.9%) belonged to the three hyperinvasive lineages represented by the ST-41/44, ST-32, and ST-23 clonal complexes, whereas in the case of carriage, the ST-60 clonal complex predominated, accounting for 28% of strains. Clonal complexes ST-41/44, ST-32, and ST-23, which have previously been associated with disease, also accounted for 34% of the carriage isolates. As in previous studies, meningococci belonging to the ST-32 clonal complex were associated with invasive disease, while strains belonging to the ST-60, ST-35, and ST-254 clonal complexes were associated with carriage. The ST-23 clonal complex was found in both carrier (11.7%) and invasive (6%) isolates.
We found respiratory infections to be associated with the carriage of isolates belonging to ST-23. In contrast, clonal complex ST-60, which was found to be associated with carriage, was found more frequently at the end of military service than during respiratory infection episodes or when entering service. To our knowledge, this is the first study to show that clonal complexes associated with invasive disease are present in the oropharynx, especially during respiratory infection episodes. This finding is in line with previous epidemiological (21, 38) and experimental (29) studies showing that susceptibility to infection caused by meningococci is markedly increased following viral infections of the respiratory tract.
Previous studies have found an association between particular clonal complexes and certain serogroups (26). In our study, serogroup B was significantly associated with the ST-41/44 clonal complex in both carrier and invasive strains, while the ST-23 clonal complex showed a strong association with serogroup Y in invasive case and carriage isolates. Isolates belonging to the ST-23 clonal complex are also frequently reported for patients with serogroup Y meningococcal disease in the United States (19). It is also worth mentioning that serogroup C meningococci belonging to the ST-11 clonal complex, which has been responsible for most serogroup C invasive disease cases in Europe during the last decade (41), were absent from both our invasive disease and carriage isolates.
We performed oropharyngeal cultures by using calcium alginate swabs which had been placed immediately into test tubes containing STGG medium, which might have led to a lower yield of positive meningococcal cultures than that for direct plating. A recent literature review by Roberts et al. states that meningococcal carriage should be assessed by swabbing the posterior wall of the oropharynx followed by direct plating or storage of the swab in a transport medium for less than 5 h before culturing (32). To our knowledge, however, there are no reports on the use of an STGG transport medium for the storage of meningococcal isolates at −70°C. STGG medium has previously been found to be suitable for the storage of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis and for nasopharyngeal swabs used for the detection of the same bacteria (23). In order to determine if STGG medium is also suitable for the storage of N. meningitidis, we stored the isolates in STGG medium and found over a 12-month period that the isolates survived at −70°C without changes in bacterial densities (in 12 repeated cultures) (our unpublished observations), suggesting that STGG medium is suitable for the storage of N. meningitidis.
Great variation in the duration of meningococcal carriage has been found previously, and sometimes a persistent carriage state may exist for several months (5, 18). Of the 20 conscripts with more than one positive oropharyngeal culture, 8 carried a strain with the same clonal complex and porA type at two different time points. In seven of these eight cases, the period between the two samples was less than 62 days, and in one case, it was over 5 months (159 days).
To conclude, the results reported here show a significant increase in meningococcal carriage during military service. Our results also show a clear difference in the phenotypic and genotypic distribution of meningococci between patient and carrier strains. Furthermore, a significant association between acute upper respiratory tract infection and the oropharyngeal carriage of certain virulent meningococcal clones is indicated. These findings highlight the importance of meningococcal vaccination of military recruits and also the need for an efficacious vaccine against serogroup B isolates.
ACKNOWLEDGMENTS
We thank Eeva Liisa Heikkinen, Elsi Saarenpää, and Leena Saarinen for their technical assistance. We also thank Heike Claus and Ulrich Vogel from the University of Würzburg for providing the serogroup Y-specific monoclonal antibody.
This study was partly funded by the Finnish Defense Forces and the Scientific Advisory Board for Defense.
Footnotes
Published ahead of print 30 November 2011
The authors have paid a fee to allow immediate free access to this article.
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