Downloaded from on October 7, 2019 by ...7 Martin C.J. Maiden 5, Maija Leinonen 4, Helena Käyhty 8, Maija Toropainen 8 8 9 1Institute of Diagnostics, Department of Medi cal Microbiology,

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Genotypic and phenotypic characterization of the carriage and invasive 1

disease isolates of Neisseria meningitidis in Finland 2

3

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Ulla Jounio1,2,3, Annika Saukkoriipi4, Holly B. Bratcher5, Aini Bloigu4, Raija Juvonen6, 5

Sylvi Silvennoinen-Kassinen1, Ari Peitso3, Terttu Harju7, Olli Vainio1,2, Markku Kuusi8, 6

Martin C.J. Maiden5, Maija Leinonen4, Helena Käyhty8, Maija Toropainen8 7

8

1Institute of Diagnostics, Department of Medical Microbiology, University of Oulu, Oulu, 9

Finland. 2Clinical Microbiology Laboratory, Oulu University Hospital, Oulu, Finland. 10

3Finnish Defence Forces, Centre for Military Medicine, Lahti, Finland. 11

4National Institute for Health and Welfare, Oulu, Finland. 5University of Oxford, Oxford, 12

UK. 6Kainuu Central Hospital, Department of Otorhinolaryngology, Kajaani, Finland. 7Oulu 13

University Hospital, Department of Internal Medicine, Oulu, Finland. 8National Institute for 14

Health and Welfare, Helsinki, Finland 15

16

Address for correspondence: Ulla Jounio 17

Department of Medical Microbiology 18

University of Oulu 19

PO Box 5000 20

90014 University of Oulu, Finland 21

E-mail: [email protected] 22

Phone: +358 8 537 5877 23

Fax: +358 8 537 5908 24

Keywords: N. meningitidis, carriage, invasive disease, genotyping, phenotyping, MLST 25

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.05385-11 JCM Accepts, published online ahead of print on 30 November 2011

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Running title: Characterization of carried and invasive meningococci 26

Some of the data have been presented previously at the Seventeenth International Pathogenic 27

Conference 2010, Banff, Canada (Jounio U, Saarinen L, Bratcher HB, Saukkoriipi A, 28

Juvonen R, Vainio O, Maiden M, Leinonen M, Käyhty H, Toropainen M. Meningococcal 29

Carriage in Army Recruits in Finland, 2004-2005) 30

31

ABSTRACT 32

The relationship between carriage and the development of invasive meningococcal disease is 33

not fully understood. We investigated here the changes in meningococcal carriage in 892 34

military recruits in Finland during a non-epidemic period from July 2004 to January 2006 35

and characterized all the oropharyngeal meningococcal isolates obtained (n=215) by 36

phenotypic (serogrouping, serotyping) and genotypic (porA typing and multilocus sequence 37

typing) methods. For comparison, 84 invasive meningococcal disease strains isolated in 38

Finland between January 2004 and February 2006 were also analysed. The rate of 39

meningococcal carriage was significantly higher at the end of military service than on arrival 40

(18% vs. 2.2%, p<0.001). 74% of serogroupable carriage isolates belonged to serogroup B 41

and 24% to serogroup Y. Most carriage isolates belonged to carriage-associated ST-60 42

clonal complex. However, 21.5% belonged to the hyperinvasive ST-41/44 clonal complex. 43

Isolates belonging to the ST-23 clonal complex were more often cultured from 44

oropharyngeal samples taken during the acute phase of respiratory infection than from 45

samples taken at health examinations at the beginning and end of military service (OR, 6.7; 46

95% CI, 2.7 to 16.4). The ST-32 clonal complex was associated with meningococcal disease 47

(OR, 17.8; 95% CI, 3.8 to 81.2), while the ST-60 clonal complex was associated with 48

carriage (OR, 10.7; 95% CI, 3.3 to 35.2). These findings point to the importance of 49


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meningococcal vaccination for military recruits and the need for an efficacious vaccine also 50

against serogroup B isolates. 51

52

Introduction 53

Neisseria meningitidis causes both epidemic and endemic life-threatening diseases worldwide, most 54

notably sepsis and bacterial meningitis. It is also part of the normal nasopharyngeal microbiota of 55

healthy persons (25, 34). The rate of asymptomatic carriage varies greatly depending on the 56

population and epidemiological situation in question, ranging between 10% and 35% among young 57

adults in Europe and the United States (6, 10, 34). Carriage is more common in teenagers and young 58

adults than in young children (3), and the highest transmission and carriage rates have been reported 59

in populations where people live in close contact with one another, such as university students or 60

military recruits sharing dormitories (7). The molecular epidemiology of meningococcal carriage 61

and disease development is not fully understood. 62

63

Previous phenotypic and genotypic studies have shown that N. meningitidis strains recovered from 64

carriers are genetically more diverse than those isolated from patients with invasive disease (IMD) 65

(8, 9). Relatively few genotypes, the “hyper-invasive lineages”, have been responsible for most of 66

the IMD, while only a small proportion of the strains isolated from carriers generally belong to 67

these hyper-invasive lineages (26). Since most patients with life-threatening invasive disease have 68

not been in contact with other IMD patients, it is assumed that carriers are the major source of the 69

virulent strains that are potential causes of disease. In order to introduce effective IMD prevention 70

policies, including vaccination, more carriage studies are needed to improve our understanding of 71

the spread of N. meningitidis in populations who are at a heightened risk of meningococcal disease, 72

including military recruits. 73

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The incidence of invasive meningococcal disease in Finland (<0.7/100,000 inhabitants/year) is 75

currently low. In contrast to many other European countries with increases in serogroup C disease 76

during the last few decades, there have been no major meningococcal epidemics or outbreaks in 77

Finland since the serogroup A meningococcal epidemic in the 1970´s (27) and a smaller serogroup 78

B epidemic involving military recruits in southern Finland in 1995-1996 (31). Thus meningococcal 79

vaccination is currently recommended in Finland only for high-risk groups, including military 80

recruits who receive a tetravalent serogroup ACYW135 polysaccharide vaccine as a part of their 81

vaccination programme when entering service. 82

83

The present study aimed to follow changes in meningococcal carriage in military recruits in Finland 84

during a non-epidemic period from July 2004 to January 2006. To investigate the diversity of the 85

carriage isolates, meningococci isolated from the oropharyngeal swabs taken at the beginning and 86

end of military service or during the acute phase of respiratory tract infection were subjected to 87

phenotyping (serogrouping and serotyping) and genotyping (porA typing and MLST). For 88

comparison, 84 meningococcal strains isolated from IMD patients in Finland in January 2004 to 89

February 2006 we also analyzed. 90

91

MATERIALS AND METHODS 92

Subjects 93

This work was part of a larger CIAS (Cold, Infections and Asthma) study assessing risk factors for 94

asthma and respiratory infections in Finnish military conscripts from July 2004 to January 2006 95

(22), in which a total of 892 men from two intake groups entering the Kainuu Brigade in Kajaani, 96

Northern Finland, in July 2004 (420/1836) and January 2005 (472/1861), were enrolled (Figure 1). 97

These included all 224 men with a diagnosis of asthma in previous health examinations or in the 98

call-up examination, and 668 randomly chosen controls without asthma. The service time of the 99


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men was six (518 men), nine (55 men), or twelve (245 men) months according to their military 100

duties; twenty-nine (13%) asthmatic and forty-five (6.7%) non-asthmatic men dropped out before 101

completing their military service. The ages of the conscripts ranged from 18.1 to 24.4 years (median 102

19.6). The study protocol was accepted by the Medical Ethics Committee of Kainuu Central 103

Hospital, Kajaani, Finland. Participation was voluntary, and all the participants signed a declaration 104

of informed consent. The recruits were routinely vaccinated with a tetravalent serogroup 105

ACYW135 polysaccharide vaccine at the beginning of their military service but no antibiotic 106

prophylaxis was given. No cases of invasive meningococcal disease occurred in the Kainuu Brigade 107

during the study period. 108

109

Oropharyngeal sampling 110

Oropharyngeal swabs for bacterial culture were collected in connection with a health examination 111

performed during the first two weeks of service and again at the end of military service, 6, 9, 12 112

months after entry to service. In addition, oropharyngeal swabs were taken during the acute phase 113

of every respiratory infection episode that required consultation with a physician. The infectious 114

episodes were diagnosed on the basis of symptoms of respiratory infection and clinical findings as 115

described in detail previously (22). The samples were taken from the posterior wall of the 116

oropharynx and both tonsils with calcium alginate swabs that were immediately placed into test 117

tubes containing 1 ml of STGG (skim milk, tryptone, glucose and glycerol) medium (23). The tubes 118

were vortexed and stored at -70ºC within 6 hours of collection for later analysis. The samples were 119

cultured in batches after an average storage time of 6 months at -70ºC. 120

121

Culture, isolation and characterization of carriage isolates 122

The oropharyngeal swabs stored in STGG were thawed at room temperature for 15-30 minutes, 123

vortexed for about 10 seconds and cultured on non-selective chocolate agar plates. The plates were 124


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incubated in 5% carbon dioxide at 37°C and examined at 24 and 48 hours for the growth of 125

meningococci-like colonies. A single colony was picked from each plate with suspected 126

meningococcal growth for subculturing prior to species identification by Gram staining, oxidase 127

reaction and carbohydrate utilization tests. All isolates identified as N. meningitidis were initially 128

characterized for their serogroup and serotype by whole-cell ELISA as described previously (37). 129

The monoclonal antibodies for phenotyping were purchased from the National Institute for 130

Biological Standards and Control, UK, with the exception of the serogroup Y-specific monoclonal 131

antibody 1938 (39), a kind gift from Ulrich Vogel and Heike Claus (University of Würzburg, 132

Germany), and the serogroup B specific monoclonal antibody NmB 735 (16), purchased from Dade 133

Behring Marburg GmbH (Marburg, Germany). The isolates that did not react with any of the 134

serogrouping reagents (A, B, C, Y, and W135) or with the serotyping reagents (P2.2a, P2.2b, P3.1, 135

P3.4, P3.14, P3.15, and P3.21) were defined as non-groupable (NG) and non-typable (NT), 136

respectively. The isolates were further analysed by multilocus sequence typing (MLST) and porA 137

typing (VR1 and VR2) as previously described (20, 26). A combination of serogroup, serotype and 138

porA type was used to define each meningococcal isolate. 139

140

Characterization of invasive disease isolates 141

Out of the total of 91 notifications of IMD cases that were referred to the National Infectious 142

Disease Registry (NIDR) at the National Institute for Health and Welfare (THL) between January 143

2004 and February 2006, 92% (84/91) were from culture confirmed cases of which corresponding 144

isolates were submitted to the National Meningococcal Reference Laboratory at the THL for 145

species confirmation, serogrouping by latex and/or slide agglutination, and serotyping by whole-cell 146

ELISA. DNA extracted from the culture-confirmed cases was sent to the University of Oxford for 147

MLST, porA typing and ClonalFrame analysis. 148

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Statistical analysis 150

The statistical analyses were performed using SPSS v.17.0 (SPSS Inc. Chicago, IL, USA). The Chi-151

square test or Fisher´s exact test, as appropriate, was used for categorical variables. A logistic 152

regression analysis was used for calculating odds ratios and the results for the carrier strains were 153

adjusted for smoking and intake group. A two-sided P-value of < 0.05 was considered statistically 154

significant. 155

156

Clonal Frame Analysis 157

CLONALFRAME version 1.1 (12), a statistical tree building algorithm, was used to infer the clonal 158

relationship of the isolate sets taking into account homologous recombination that may be present. 159

ClonalFrame draws inference using a Monte-Carlo Markov chain, and requires an assessment of the 160

convergence and mixing of its results, (13) therefore, several independent runs of ClonalFrame 161

100,000 to 150,000 iteration, were run for each assessment. The results were compared for 162

convergence using the Gelman and Rubin statistic (17), and the runs were combined for maximum 163

robustness. The convergence was judged satisfactory, and the samples from the runs were combined 164

for maximum robustness. Statistical support for any grouping of isolates was assessed by the 165

proportion of clonal genealogies exhibiting this grouping in the combined sample. This approach 166

was independently applied to each of the carriage and disease isolate sets separately and a combined 167

isolate tree. 168

169

RESULTS 170

Meningococcal carriage in army recruits: serogroups, MLST sequence types, and antigen profiles 171

of carriage isolates 172

A total of 193 carriers were identified in the 892 conscripts, and 215 carried meningococci were 173

isolated. Twenty out of the 215 oropharyngeal isolates (9.5%) were obtained from swabs collected 174


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on entry to military service, 151 (70%) from swabs collected at the end of service and 44 (20.5%) 175

from swabs collected during acute respiratory infections (Table 1). Twenty (10.4%) of the 193 176

carriers were culture positive for N. meningitidis more than once during their military service, while 177

the remaining carriers (173/193; 89.6%) were culture-positive only once. The carriage of N. 178

meningitidis did not differ between the asthmatics and non-asthmatics (P>0.05) and therefore these 179

two groups were analysed together. Serogroups B, Y and W135 accounted for 25.6% (55/215), 180

8.4% (18/215) and 0.4% (1/215) of the isolates, respectively, and 65.6% (141/215) were NG. 181

182

Those entering military service had in general a low rate of N. meningitidis carriage (Table 1), and 183

this was significantly (p<0.001) lower in the summer intake group than in the winter group [0.5% 184

(2/420) vs. 3.8% (18/472)]. The rate of carriage was significantly (p<0.001) higher at the end of 185

military service, than it had been at arrival, with 14.7% (56/382) of the summer intake group and 186

21.8% (95/436) of those in the winter intake group being culture positive for meningococci 187

(p<0.001 between the two groups). Only five conscripts (0.6%) were culture-positive for N. 188

meningitidis at both the beginning and the end of their service. 189

190

By MLST, 111 different sequence types (STs) among 214 carriage isolates with complete MLST 191

profile were identified, of which 57.7% (64/111) were new STs. Overall 93.5% (200/214) of the 192

isolates fell into 14 previously known clonal complexes (Table 2). ST-60 clonal complex was the 193

most common with 60 (28%) isolates and 20 different STs, followed by ST-41/44 clonal complex 194

with 46 isolates (21.5%) and 25 STs and ST-23 clonal complex with 25 isolates (12%) and 11 STs. 195

The two most common STs were ST-4146 (ST-60 clonal complex) with 29 isolates (13.6%) and 196

ST-136 (ST-41/44 clonal complex) with 14 isolates (6.5%). Two-thirds of all carriage isolates were 197

non-groupable by traditional serogrouping methods. Of the clonal complexes represented by two or 198

more isolates (this includes the isolates with unassigned STs grouped as “unassociated”), five 199


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(clonal complexes 254, 178, 198, 53, 32) could not be serogrouped, three (clonal complexes 60, 35, 200

and 213) were partially serogrouped but with limited results, and the remaining four (clonal 201

complexes 41/44, 23, 269 and unassociated) had moderate to high success for serogrouping (Table 202

2). The majority of serogroupable isolates belonged to serogroup B (55/74) followed by serogroup 203

Y (18/74) and one isolate typed as W135. All serogroup Y isolates belonged to ST-23 clonal 204

complex and 71% (39/55) of the serogroup B isolates belonged to ST-41/44 clonal complex (Table 205

2). 206

207

The clonal complex distribution showed an increase in diversity between the beginning of service 208

and the end of service for each year. When the study commenced in 2004 two clonal complexes 209

were detected at the beginning of service and by the end of 2004 there were eight clonal complexes 210

plus 2 unassociated STs. A continued increase in clonal complex distribution was also observed in 211

2005, increasing from eight clonal complexes to fourteen by the end of the study period in 2006. 212

213

Univariate statistical analysis revealed a strong association of certain clonal complexes with 214

respiratory infection episodes (Table 3). Further examination of this association by logistic 215

regression analysis showed that ST-23 clonal complex was significantly more common among 216

isolates collected during the acute phase of respiratory infection (OR, 7.1; 95% CI, 2.9 to 17.2) 217

compared to those collected at the beginning or at the end of the service. A similar but smaller trend 218

was also observed for the ST-41/44 clonal complex though this association did not reach statistical 219

significance (OR, 1.5; 95% CI, 0.71 to 3.3) when adjusted for intake group, while an opposite trend 220

was observed for ST-60 clonal complex (Table 3) that was about 2-fold more common among 221

isolates collected at the beginning or at the end of the service compared to those collected during 222

respiratory infections. The distribution of clonal complexes did not differ between smokers and 223


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non-smokers except for the ST-41/44 clonal complex which appeared to be more frequent, though 224

not statistically significant, among smokers than non-smokers (59.1% vs. 40.9%, p>0.05). 225

226

Eighty-two strain profiles (serogroup: serotype: porA type) were identified among the 215 carriage 227

isolates, the most common being NG:NT:P1.5,2 (28%), B:15:P1.17,16-3 (7%) and NG:4:P1.22-228

1,14 (4.7%). Profile NG:NT:P1.5,2 represented 93% of the 60 ST-60 clonal complex isolates. 229

B:15:P1.17,16-3 (28%) predominated among the 46 ST-41/44 clonal complex isolates (Table 2). 230

231

232

ClonalFrame Analysis of carriage isolates 233

The relationships of the carriage isolates inferred by ClonalFrame analysis using the MLST typing 234

alleles clustered 94.4% of carriage isolates into 14 previously known clonal complexes (Figure 2). 235

Twelve isolates were unassigned to a clonal complex and are the only representative of these STs 236

currently in the PubMLST Neisseria database. 237

238

Four STs did not cluster with their respective clonal complex. One of three ST-213 clonal complex 239

isolates and one of seven ST-178 clonal complex isolates incongruently group together with a single 240

ST-22 clonal complex isolate; a second isolate from the clonal complex ST-178 and one isolate 241

from the ST-41/44 clonal complex did not group with their assigned clonal complexes. In both of 242

these cases the unresolved grouping are caused by peripheral isolates of each clonal complex; in 243

particular differences in the aroE, and to a lesser extent the fumC, gene fragments. Finer resolution 244

or clarification of these isolate associations would require the addition of more loci, or the use of 245

whole genes instead of the typing fragments. 246

247

Characterization of isolates collected from conscripts with multiple positive oropharyngeal cultures 248


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Of the 892 conscripts recruited, 18 (2.0%) had two positive oropharyngeal cultures for N. 249

meningitidis during their service and two (0.2%) conscripts had three. To determine whether the 250

paired isolates represented the same or different strains, the antigen profiles and sequence types 251

were compared (Table 4). In 10 cases the porA type of the strains were identical, in 8 of those 10 252

the clonal complex was the same, and in three of those eight cases the sequence type was also the 253

same. 254

255

Invasive meningococcal disease: serogroups, MLST sequence types, and antigen profiles of disease 256

isolates 257

84 (92%) isolates were recovered from 91 IMD cases that occurred in Finland between January 258

2004 and February 2006. Sixty percent (50/84) of these isolates were from patients younger than 25 259

years and 18% from adolescents aged 15-19 years. Unlike the carriage isolates, all of these disease 260

isolates were serogroupable. B, C and Y were the predominant serogroups, accounting for 81%, 8.3 261

%, and 8.3% of the isolates, respectively. 262

263

Sixty-four STs were identified among the 84 disease isolates with complete MLST profiles, of 264

which 50% were new ones. Over a half of the new STs were assigned to fifteen previously known 265

clonal complexes, the majority of which have been previously associated with invasive disease 266

(Table 5). The two most common clonal complexes, ST-41/44 and ST-32 accounted for 40.7% of 267

the IMD isolates, compared with 22.4% of the carriage isolates. Univariate statistical analysis 268

revealed a difference in the clonal complex distribution between the patient and carrier isolates. By 269

logistical regression analysis, isolates of the ST-32 clonal complex were significantly associated 270

with invasive disease (OR, 17.8; 95% CI, 3.8 to 81.2) while the isolates of the ST-60 clonal 271

complex were associated with carriage (OR, 10.7; 95% CI, 3.3 to 35.2). 272

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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%) 274

were the most common profiles among the invasive isolates. These four profiles were found more 275

frequently among the invasive strains than among the carrier strains, whereas profiles 276

NG:NT:P1.5,2 and B:15:P1.17,16-3 were more prevalent among the carrier strains. Profile 277

B:4:P1.7-2,4 represented 42.9% of the ST-41/44 clonal complex isolates in the invasive strains, 278

while profile B:15:P1.17,16-3 predominated in the carriage isolates, representing 28% of the 279

isolates of this clonal complex (Tables 2 and 5). 280

281

ClonalFrame Analysis of disease isolates 282

ClonalFrame analysis clustered 66 of the 84 disease isolates into fifteen previously known clonal 283

complexes (Figure 3). Twenty-two isolates are currently unassigned to a clonal complex and twenty 284

are the only representative of the STs currently in the PubMLST Neisseria database. The two 285

remaining STs have been observed once before, ST-1470 in Canada in 1999 (single isolate) and ST-286

2003 in the UK in 2000 (single isolate). The disease isolates split into 2 groups forming 2 287

centralized tree nodes and where a clonal complex is represented by more than one ST, a more 288

defined branch occures with two exceptions. The clonal complex ST-23 has two branches from a 289

shared node and one ST-41/44 isolate did not group with the other ST-41/44 clonal complex 290

isolates. The differences are primarily confined to the abcZ, fumC and gdh alleles and to a lesser 291

extent the gdh allele. As pointed out in the carriage data set, these are also peripheral clonal 292

complex STs and their allele assignment places them within the assigned ST-32 and ST-41/44 293

clonal complexes, respectively. 294

295

The clonal complex assignment of these peripheral STs, when compared against the known ST 296

cohort for each respective clonal complex in the database, show an overlapping MLST profile, such 297

that the ST does not cluster with its assigned clonal complex as seen in each tree. As in most 298


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bacterial identification there is a grey area where overlapping characteristics exist, making strict 299

confinement of some isolates to a predefined group complicated and the very least occasionally 300

inconsistent. As in the carriage MLST analysis, finer resolution of the disease isolates would 301

require the addition of more typing loci or the use of whole genes. 302

303

DISCUSSION 304

In the present study, the carriage of N. meningitidis increased from an average of 2.2% at the 305

beginning of the military service to 18.5% at the end of military service. The carriage rates reported 306

here are somewhat lower than in previous studies, where rates varying from less than 16% to over 307

70% have been reported among military recruits (1, 15, 28, 30). These previous studies were mainly 308

performed on unvaccinated populations, however. In our study, a tetravalent serogroup ACYW135 309

polysaccharide vaccine was given to the recruits as a part of the routine vaccination programme at 310

the beginning of their military service. This may have explained the low or absent level carriage of 311

serogroup C, Y, and W135 meningococci during the period studied although previous studies 312

suggest that the effect of polysaccharide vaccination on carriage is probably short term (11) . 313

314

During the present study from 2004-2006, the annual incidence of IMD in Finland (<0.9/100,000 315

inhabitants) was relatively low compared with that in other European countries (14) and no cases of 316

IMD occurred in the Kainuu Brigade. It is known that the capsule plays a major role in the 317

pathogenesis of meningococcal disease (24), and all our patient isolates expressed a polysaccharide 318

capsule. By contrast, 65.6% of the carrier isolates were non-groupable by serological means, 319

suggesting that at least some of them may have been non-encapsulated. Both the disease and the 320

serogroupable carrier isolates were predominantly serogroup B. While outbreaks and increases in 321

the incidence of serogroup C disease have occurred in other parts of the Europe since the late 322

1990´s (40), this serogroup was relatively uncommon among our disease isolates and none of the 323


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recruits carried meningococci expressing serogroup C capsule in their oropharynx. Serogroup Y, 324

which has increased in the USA (33) and UK (36) as well as in Scandinavian countries including 325

Finland (35, 36) during the past decade, accounted for about 8% of the disease isolates and also 326

about 8% of the carriage isolates despite the vaccination of the recruits against this serogroup at the 327

time of entering military service. This suggests that the tetravalent ACYW135 polysaccharide 328

vaccine does not prevent the carriage of serogroup Y meningococci completely although it probably 329

provides protection against the development of invasive disease. 330

331

332

Molecular epidemiological studies have demonstrated that in 2000-2002, most of the 333

meningococcal disease in Europe was caused by strains belonging to the ST-41/44 clonal complex, 334

ST-11 clonal complex, ST-32 clonal complex, ST-8 clonal complex and ST-269 clonal complex (2, 335

4). In the present study executed in July 2004–January 2006, ST-41/44 clonal complex caused 25% 336

of IMD cases and 21.5% of all carriage cases. Almost a half of the invasive strains (46.9%) 337

belonged to the three hyper-invasive lineages represented by the ST-41/44 clonal complex, ST-32 338

clonal complex and ST-23 clonal complex, whereas in the case of carriage the ST-60 clonal 339

complex predominated, accounting for 28%. Clonal complexes ST-41/44, ST-32, and ST-23, 340

which have been previously associated with disease, also accounted for 34% of the carriage. As in 341

previous studies, meningococci belonging to the ST-32 clonal complex were associated with 342

invasive disease, while strains belonging to the clonal complexes ST-60, ST-35 and ST-254 were 343

associated with carriage. ST-23 clonal complex was found in both carrier (11.7%) and invasive 344

(6%) isolates. 345

346

We found respiratory infections to be associated with the carriage of isolates belonging to ST-23. 347

By contrast, clonal complex ST-60, which was found to be associated with carriage, was more 348


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frequent at the end of military service than during respiratory infection episodes or when entering 349

service. To our knowledge, this is the first study to show that clonal complexes associated with 350

invasive disease are present in the oropharynx especially during respiratory infection episodes. This 351

finding is in line with the previous epidemiological (21, 38) and experimental (29) studies showing 352

that susceptibility to infection caused by meningococci is markedly increased following viral 353

infections of the respiratory tract. 354

355

Previous studies have found an association between particular clonal complexes and certain 356

serogroups (26). In our study, serogroup B was significantly associated with the ST-41/44 clonal 357

complex in both carrier and invasive strains, while ST-23 clonal complex showed strong association 358

with serogroup Y in invasive case and carriage isolates. Isolates belonging to the ST-23 clonal 359

complex are also frequently reported in patients with serogroup Y meningococcal disease in the 360

United States (19). It is also worth mentioning that serogroup C meningococci belonging to ST-11 361

clonal complex that has been responsible for most of serogroup C invasive disease cases in Europe 362

during last decade (41), were absent from both our invasive disease and carriage isolates. 363

364

We performed oropharyngeal cultures by using calcium alginate swabs which had been 365

immediately placed into test tubes containing STGG medium, which might have led to a lower yield 366

of positive meningococcal cultures as compared to direct plating. The recent literature reviewed by 367

Roberts et al. states that meningococcal carriage should be assessed by swabbing the posterior wall 368

of the oropharynx followed by direct plating or the storage of the swab in a transport medium for 369

less than 5 hours before culturing (32). To our knowledge, however, there are no reports on the use 370

of an STGG transport medium for the storage of meningococcal isolates at -70ºC. STGG has 371

previously been found to be a suitable medium for the storage of Streptococcus pneumoniae, 372

Haemophilus influenzae and Moraxella catarrhalis and for the nasopharyngeal swabs used for the 373


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detection of the same bacteria (23). In order to determine if STGG is also suitable for the storage of 374

N. meningitidis, we stored the isolates in STGG medium and found over a 12 month period that the 375

isolates survive at -70ºC without changes in bacterial densities (12 repeated culturing, our 376

unpublished observations), suggesting that STGG medium might be is a suitable medium for the 377

storage of N. meningitidis. 378

379

Great variation in the duration of meningococcal carriage has been found previously, and 380

sometimes a persistent carriage state may exist for several months (5, 18). Of the 20 conscripts with 381

more than one positive oropharyngeal culture, eight carried a strain with the same clonal complex 382

and porA type at two different time points. In seven of the eight cases, the time period between the 383

two samples was less than 62 days and in one case over 5 months (159 days). 384

385

To conclude, the results reported here show a significant increase in meningococcal carriage during 386

the military service. Our results also showed a clear difference in the phenotypic and genetic 387

distribution of meningococci between the patient and the carrier strains. Further, a significant 388

association between an acute upper respiratory infection and the oropharyngeal carriage of certain 389

virulent meningococcal clones was indicated. These findings highlight the importance of 390

meningococcal vaccination of military recruits and the need for an efficacious vaccine also against 391

the serogroup B isolates. 392

393

ACKNOWLEDGEMENTS 394

The authors thank Eeva Liisa Heikkinen, Elsi Saarenpää and Leena Saarinen for their technical 395

assistance. We also thank Heike Claus and Ulrich Vogel from the University of Würzburg for 396

providing the serogroup Y-specific monoclonal antibody. This study was partly funded by the 397

Finnish Defence Forces and the Scientific Advisory Board for Defence. 398

399

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524


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7

75

200

751

2793. 29

97.

7528.

7527.

7534.

7525.

7524.

7529.

507.

Clonal ComplexST-549


Clonal C l

756

755.

7544.

60.

7553.

3735.

7530.

532.

03.

2. 750

7505.

7504.

7500.

9.


Clonal Complex

ST-60

ComplexST-865

7333

22.

1828.

7506.

560.

1249

7476.

463.

32.

1295.

Clonal Complex

ST-22

Clonal Complex

7

2692.

23.

7517.

3.2017.

7477.

34.

Clonal Complex

ST-23

ComplexST-32

303.

41.

154.

7337.

7563.639.

7562.

2267.

7501.7502

7862.

1470.

136.

6591.

7508.

7513.7332.

7330.482.

2.

7511.7479.7484.7485.4777

Clonal ComplexST-41/44 Clonal

Clonal Complex

ST-35

779.

7493.

7494.

7322.

7336.

7523.

0.05


ComplexST-369

ST-35



Clonal Complex

ST-18


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Figure 3: Seven Locus MLST Clonal Frame tree of Disease Isolates.STs unassigned to a clonal complex are represented by an open circle and g p p y pST number, clonal complexes are shown by colour and ST number. When present, the central genotype is indicated by a coloured 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 – clonalGreen 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, Grey – 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.


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Kainuu Brigade intake group and

conscripts enrolled

Pharyngeal swab sampling*

Entry to service Sick visits during End of service

Service time

July 2004

1836

6 mo. n=235

235 swabs taken in July 2004

148 swabs taken in July 2004-January

2005

235 swabs taken in January 2005

conscripts enrolled Entry to service Sick visits during service

End of service

1836 men

420 men enrolled


85 swabs taken in July 2004-July 125 swabs taken in

July 2005

9 mo. n=22

12 mo. n=125


18 swabs taken in July 2004-April

2005

22 swabs taken in April 2005

116 asthmatic 304 non-asthmatic

Total number of swabs taken 420 272 382

July 2004

Dropouts n=38


2005 July 2005n 125

21 swabs taken in July 2004-October

2004no swabs

January 2005

1861 men

6 mo. n=283

9 mo. 33

269 swabs taken in January 2005-July

2005


33 swabs taken in J 2005

32 swabs taken in January 2005- 33 swabs taken in

O t b 2005


472 men enrolled

108 asthmatic 364 non-asthmatic

n=33

12 mo. n=120


January 2005 January 2005October 2005 October 2005

20 swabs taken in

125 swabs taken in January 2005-January 2006


Total number of swabs taken 472 446 436

no swabsDropouts n=36


20 swabs taken in January 2005-

March 2005


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Figure 1. Study design and pharyngeal swab sampling

* Oropharyngeal swabs for bacterial culture were collected at the beginning of the military service and again at the end of military service, 6, 9, or 12 months after entry to service. In addition, oropharyngeal swabs were taken during the

h f i i f i i d h i d l iacute phase of every respiratory infection episode that required consultation with a physician.


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7 7 7 2533. 75

18.

7557.

7564.

7567.

23.

2692.

3228.

7565.

7566.

7568.

3638.

7498.

520.

Clonal Complex

Clonal Complex

ST-23

Clonal Complex

ST-22

7538

3412.

4146.

7536.

7537.

7539 .7 5 4 0 .

7 5 4 1 .7 37 8 .

7 4 9 9 .7 3 4 5 .7351.7 3 2 7 .7556.7561.

7752

39.

198.

7488.

7490.

2146.

7489.

7491.

7492.

254.

3808.

Clonal Complex

ST-60

Clonal

ST-198

60.61.

3793.7542.7543.

7545.7535.538. 380

222.

6389.

7323.7325.7509.7521.



7328.7515

.7516.2820.7495.7519.

7569.

472.

2670.

7480.

7481.

35.457.7344.

Clonal Complex

ST-35

ST 461

170.

2284.

303.

43.

691.

522.42.

2136.

547.

7549.

7546.

7550.

7548.

7558.

7559.13

6.1770.7329.183

8.7514.7328. .

7551.7552.7487.53.21 2 6 .

7863.

Clonal ComplexST-41/44 Clonal

ComplexST-53

267527577 7863.8625.7865.7908 .7 864 .

7 9 0 9 .103.791536


Clonal Complex5 .

3641 .

7 4 9 6 .

4 940 .

7 4 8 2 .

7 4 8 6 .

1 2 4 9 .

7 4 7 8 .

7 3 1 9 .

1 5 7 2 .

7503.

7497.

7555.

0.05 Clonal ComplexST-32



pST-103


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Figure 2. Seven Locus MLST Clonal Frame tree of Carriage Isolates. STs unassigned to a clonal complex are represented by an open circleSTs unassigned to a clonal complex are represented by an open circle and ST number, clonal complexes are shown by colour and ST number. When present, the central genotype is indicated by a coloured 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, Grey – clonal complex ST-269, Blue –clonal complex ST-32, Black – clonal complex ST-22, Mauve – clonal complex ST-461, Light Blue – clonal p , p , p , gcomplex ST-103.


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Table 1. Carriage of N. meningitidis by intake group, service time, and time of sampling

Intake group Service time Carriage rate %

Entry Sick visits End of service

July 2004 6 mo 1/235 (0.4%) 9/148 (6%) 27/235 (11.5%) 9 mo 0/22 1/18 (5.6%) 5/22(22.7%) 12 mo 1/125 (0.8%) 3/85 (3.2%) 24/125 (19.2%) Dropouts 0/38 2/21 (9.5%) NA*

January 2005 6 mo 9/283 (3.2%) 18/269 (6.7%) 68/283 (24%) 9 mo 1/33 (3%) 0/32 7/33 (21%) 12 mo 8/120 (6.7%) 9/125 (7.2%) 20/120 (17%) Dropouts 0/36 2/20 (10%) NA* Total carriage rate 20/892 (2.2%) 44/718 (6.1%) 151/818 (18.5%)

*NA, not applicable.


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Table 2. Distribution of carriage isolate (n=215) profiles within clonal complexes.

Clonal complex Number of isolates*

Number of STs

Serogroup(s) Most common strain profile(s)

ST-60 complex 60 20 NG (98%), B (2%) NG:NT:P1.5,2 (93%) ST-41/44 complex 46 25 B (85%), NG (15%) B:15:P1.17,16-3 (28%) ST-23 complex 25 11 Y (72%), NG (28%) Y:14:P1.5-2,10-1 (24%)

Y:NT:P1.5-1,2-2 (24%) ST-254 complex 18 6 NG (100%) NG:4:P1.5-1,10-6 (39%)

NG:4:P1,5-1,10-8 (39%) ST-35 complex 17 10 NG (76%), B (24%) NG:4:P1.22-1,14 (59%) Unassociated 14 13 B (57%), NG (43%) heterogenous ST-178 complex 11 8 NG (100%) NG:NT:P1.5,10-44 (18%) ST-198 complex 9 6 NG (100%) NG:15:P1.18,25-1 (44%) ST-53 complex 4 2 NG (100%) NG:21:P1.7,30-5 (75%) ST-213 complex 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 complex 2 2 B (50%), NG (50%) NG:15:P1.7-2,13-1 (50%) B:1:P1.21,26 (50%)

ST-32 complex 2 2 NG (100%) NG:15:P1.7,16 (100%) ST-22 complex 1 1 W135 (100%) W135:NT:P1.18-1,3 (100%) ST-461 complex 1 1 B (100%) B:1:P1.19-35,13-1 (100%) ST-103 complex 1 1 NG (100%) NG:NT:P1.5-2,10 (100%) *One isolate with incomplete MLST profile

** Serogroup:serotype:porA type (VR1, VR2)


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Table 3. Association of clonal complexes of carried meningococci with respiratory infection episodes.

Clonal complex Arrival n=20

Acute respiratory infection episode n=44

Departure n=151

p-value*

OR**

ST-60 complex 35.0% 16% 30.5% 0.045 0.4 (0.18-1.01) ST-41/44 complex 10.0% 27.3% 21.2% 0.061 1.5 (0.71-3.3) Unassociated 20.0% 4.5% 5.3% - - ST-23 complex 5.0% 31.8% 6.6% <0.001 7.1 (2.9-17.2) ST-254 complex 0% 9.1% 9.3% 0.9 1.1 (0.4-3.6) ST-35 complex 10.0% 4.5% 8.6% 0.5 0.5 (0.1-2.2) ST-178 complex 5.0% 0% 6.6% - -

*Pearson Chi-square test for the comparison of isolates collected from the military recruits on arrival or at the departure and during acute respiratory infection episode.

**Odds ratio (95% confidence interval) was adjusted for the intake group for the comparison of isolates collected from the military recruits on arrival or at the departure and during acute respiratory infection episode.


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Table 4. Strain characteristics of isolates collected from conscripts with multiple positive oropharyngeal cultures

Recruit no

Sample Isolate profile* Clonal complex ST Time** (days)

1 I (During) NG:4:P1.5-1,10-6 ST-254 complex 254 45 II (End) NG:4:P1.5-1,10-6 ST-254 complex 254 2 I (During) Y:NT:P1.5-1,2-2 ST-23 complex 23 15, 294 II (During) Y:NT:P1.5-1,2-2 ST-23 complex 23 III (End) NG:NT:P1.5,2 ST-60 complex 7543 3 I (Entering) NG:4:P1.22-1,14 ST-35 complex 35 27 II (During) NG:4:P1.22-1,14 ST-35 complex 35 4 I (During) NG:15:P1.7,16 ST-32 complex 1249 7 II (End) NG:15:P1.7,16 ST-32 complex 7478 5 I (Entering) Y:NT:P1.5-1,2-2 ST-23 complex 7518 61 II (During) Y:NT:P1.5-1,2-2 ST-23 complex 3228 6 I (Entering) NG:21:P1.7,30-5 ST-53 complex 2126 159 II (End) NG:21:P1.7,30-5 ST-53 complex 53 7 I (During) NG:NT:P1.5,2 unassociated 7561 170 II (End) NG:NT:P1.5,2 ST-60 complex 4146 8 I (During) B:NT:P1.5-1,2-2 ST-41/44 complex 42 26 II (End) B:NT:P1.5-1,2-2 ST-41/44 complex 2136 9 I (Entering) B:15:P1.17,16-3 unassociated 7559 80, 38 II (During) NG:NT:P1.5,2 ST-60 complex 7536 III (During) NG:NT:P1.5,2 ST-60 complex 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 complex 7535 11 I (During) B:15:P1.22-1,14 ST-41/44 complex 136 139 II (During) NG:14:P1.5-2,10-1 ST-23 complex 7568 12 I (During) Y:14:P1.5-2,10-28 ST-23 complex 23 25 II (End) B:4:P1.7-1,1 ST-35 complex 7487 13 I (During) NG:NT:P1.5,2 ST-60 complex 4146 52 II (End) NG:NT:P1.5,10-82 ST-178 complex 7497 14 I (Entering) NG:NT:P1.5-32,10-44 ST-178 complex 7864 165 II (End) NG:4:P1.5-32,10-44 ST-178 complex 7865 15 I (Entering) NG:NT:P1.ND***,ND*** unassociated 7915 339 II (End) NG:NT:P1.5,2 ST-60 complex 7541 16 I (During) NG:14:P1.5-2,10-28 ST-23 complex 23 105 II (End) NG:NT:P1.5,2 ST-60 complex 7538 17 I (During) B:15:P1.17,16-3 ST-41/44 complex 136 246 II (End) NG:NT:P1.5,2 ST-60 complex 7545 18 I (During) NG:4:P1.7-2,4 ST-41/44 complex 303 238 II (End) NG:14:P1.19-2,15 ST-35 complex 472 19 I (During) B:NT:P1.19,15-1 ST-41/44 complex 2691 240 II (End) NG:NT:P1.22,14 ST-213 complex 7496 20 I (Entering) B:15:P1.17,16-3 unassociated 7558 163 II (End) NG:15:P1.17,16-3 ST-41/44 complex 136 *Isolate profile was defined as serogroup:serotype:porA type (VR1, VR2). ** Time between samples I and II or II and III. *** ND, not done.


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Table 5. Distribution of invasive meningococcal disease isolate (n=84) profiles within clonal complexes.

*Three isolates with incomplete MLST profile

** Serogroup:serotype:porA type (VR1, VR2)

Clonal complex Number of isolates*

Number of STs*

Serogroup(s) Most common strain profile(s)**

Unassociated 27 23 B (89%), C (11%) heterogenous ST-41/44 complex 21 12 B (100%) B:4:P1.7-2,4 (42.9%) ST-32 complex 12 9 B (84%), C (8%), Y (8%) B:15:P1.7,16-6 (25%) ST-23 complex 5 4 Y (100%) Y:14:P1.5-2,10-1 (60%) ST-22 complex 2 2 B (50%), W135 (50%) B:NT:P1.18-1,3 (50%)

W13:NT:P1.18-1,3 (50%) ST-269 complex 2 2 B (50%), C (50%) B:4:P1.18,25/25-7 (50%)

C:21:P1.12-1,13-1 (50%) ST-60 complex 2 2 B (50%), W135 (50%) B:15:P1.7,16-6 (50%)

W135:NT:P1.22-1,14 (50%) ST-865 complex 2 1 B (100%) B:14:P1.7-2,13-2 (100%) ST-162 complex 1 1 B (100%) B:NT:P1.7-2,4 (100%) ST-167 complex 1 1 Y (100%) Y:1:P1.5-1,10-1 (100%) ST-174 complex 1 1 B (100%) B:14:P1.5-1,10-4 (100%) ST-334 complex 1 1 B (100%) B:2b:P1.7-2,13-1 (100%) ST-364 complex 1 1 B (100%) B:NT:P1.12-1,13-1 (100%) ST-53 complex 1 1 B (100%) B:4:P1.22-1,14 (100%) ST-549 complex 1 1 B (100%) B:4:P1.5-2,10-2 (100%) ST-18 complex 1 1 B (100%) B:NT:P1.5-1,10-4 (100%)


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