downloaded from on october 7, 2019 by ...7 martin c.j. maiden 5, maija leinonen 4, helena käyhty 8,...
Post on 28-Jan-2020
4 Views
Preview:
TRANSCRIPT
1
Genotypic and phenotypic characterization of the carriage and invasive 1
disease isolates of Neisseria meningitidis in Finland 2
3
4
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: ujounio@paju.oulu.fi 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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
2
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
3
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
74
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
4
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
5
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
6
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
149
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
7
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
8
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
9
(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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
10
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
11
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
273
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
12
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
13
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
14
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
15
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
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
16
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
REFERENCES 400
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
17
401
1. Block, C., M. Gdalevich, R. Buber, I. Ashkenazi, S. Ashkenazi, and N. Keller. 1999. Factors 402 associated with pharyngeal carriage of Neisseria meningitidis among Israel Defense Force 403 personnel at the end of their compulsory service. Epidemiol. Infect. 122:51-57. 404
2. Brehony, C., K. A. Jolley, and M. C. Maiden. 2007. Multilocus sequence typing for global 405 surveillance of meningococcal disease. FEMS Microbiol. Rev. 31:15-26. 406
3. Cartwright, K. A., J. M. Stuart, D. M. Jones, and N. D. Noah. 1987. The Stonehouse survey: 407 nasopharyngeal carriage of meningococci and Neisseria lactamica. Epidemiol. Infect. 99:591-408 601. 409
4. Caugant, D. A. 2008. Genetics and evolution of Neisseria meningitidis: importance for the 410 epidemiology of meningococcal disease. Infect. Genet. Evol. 8:558-565. 411
5. Caugant, D. A., C. Fogg, F. Bajunirwe, P. Piola, R. Twesigye, F. Mutebi, L. O. Froholm, E. 412 Rosenqvist, V. Batwala, I. S. Aaberge, J. A. Rottingen, and P. J. Guerin. 2006. Pharyngeal 413 carriage of Neisseria meningitidis in 2-19-year-old individuals in Uganda. Trans. R. Soc. Trop. 414 Med. Hyg. 100:1159-1163. 415
6. Caugant, D. A., E. A. Hoiby, P. Magnus, O. Scheel, T. Hoel, G. Bjune, E. Wedege, J. Eng, 416 and L. O. Froholm. 1994. Asymptomatic carriage of Neisseria meningitidis in a randomly 417 sampled population. J. Clin. Microbiol. 32:323-330. 418
7. Caugant, D. A., E. A. Hoiby, E. Rosenqvist, L. O. Froholm, and R. K. Selander. 1992. 419 Transmission of Neisseria meningitidis among asymptomatic military recruits and antibody 420 analysis. Epidemiol. Infect. 109:241-253. 421
8. Caugant, D. A., B. E. Kristiansen, L. O. Froholm, K. Bovre, and R. K. Selander. 1988. 422 Clonal diversity of Neisseria meningitidis from a population of asymptomatic carriers. Infect. 423 Immun. 56:2060-2068. 424
9. Caugant, D. A., L. F. Mocca, C. E. Frasch, L. O. Froholm, W. D. Zollinger, and R. K. 425 Selander. 1987. Genetic structure of Neisseria meningitidis populations in relation to 426 serogroup, serotype, and outer membrane protein pattern. J. Bacteriol. 169:2781-2792. 427
10. Claus, H., M. C. Maiden, D. J. Wilson, N. D. McCarthy, K. A. Jolley, R. Urwin, F. Hessler, 428 M. Frosch, and U. Vogel. 2005. Genetic analysis of meningococci carried by children and 429 young adults. J. Infect. Dis. 191:1263-1271. 430
11. Dellicour, S., and B. Greenwood. 2007. Systematic review: Impact of meningococcal 431 vaccination on pharyngeal carriage of meningococci. Trop. Med. Int. Health. 12:1409-1421. 432
12. Didelot, X., and D. Falush. 2007. Inference of bacterial microevolution using multilocus 433 sequence data. Genetics. 175:1251-1266. 434
13. Didelot, X., R. Urwin, M. C. Maiden, and D. Falush. 2009. Genealogical typing of Neisseria 435 meningitidis. Microbiology. 155:3176-3186. 436
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
18
14. EU-IBIS Network. Invasive Neisseria meningitidis and invasive Haemophlus influenzae in 437 Europe 2005. Health Protection Agency, London 2007.:. 438
15. Fraser, P. K., G. K. Bailey, J. D. Abbott, J. B. Gill, and D. J. Walker. 1973. The 439 meningococcal carrier-rate. Lancet. 1:1235-1237. 440
16. Frosch, M., I. Gorgen, G. J. Boulnois, K. N. Timmis, and D. Bitter-Suermann. 1985. NZB 441 mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of 442 an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B 443 meningococci. Proc. Natl. Acad. Sci. U. S. A. 82:1194-1198. 444
17. Gelman, A., and Rubin D. 1992. Inference from iterative simulation using multiple 445 sequences. Statistical Science. 7: 457–472. 446
18. Glitza, I. C., I. Ehrhard, B. Muller-Pebody, R. Reintjes, T. Breuer, A. Ammon, and H. G. 447 Sonntag. 2008. Longitudinal study of meningococcal carrier rates in teenagers. Int. J. Hyg. 448 Environ. Health. 211:263-272. 449
19. Harrison, L. H., K. A. Jolley, K. A. Shutt, J. W. Marsh, M. O'Leary, L. T. Sanza, M. C. 450 Maiden, and Maryland Emerging Infections Program. 2006. Antigenic shift and increased 451 incidence of meningococcal disease. J. Infect. Dis. 193:1266-1274. 452
20. Holmes, E. C., R. Urwin, and M. C. Maiden. 1999. The influence of recombination on the 453 population structure and evolution of the human pathogen Neisseria meningitidis. Mol. Biol. 454 Evol. 16:741-749. 455
21. Jansen, A. G., E. A. Sanders, A. VAN DER Ende, A. M. VAN Loon, A. W. Hoes, and E. 456 Hak. 2008. Invasive pneumococcal and meningococcal disease: association with influenza 457 virus and respiratory syncytial virus activity? Epidemiol. Infect. 136:1448-1454. 458
22. Juvonen, R., A. Bloigu, A. Peitso, S. Silvennoinen-Kassinen, P. Saikku, M. Leinonen, and 459 T. Harju. 2008. Risk factors for acute respiratory tract illness in military conscripts. 460 Respirology. 13:575-580. 461
23. Kaijalainen, T., E. Ruokokoski, P. Ukkonen, and E. Herva. 2004. Survival of Streptococcus 462 pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis frozen in skim milk- 463 tryptone-glucose-glycerol medium. J. Clin. Microbiol. 42:412-414. 464
24. Mackinnon, F. G., R. Borrow, A. R. Gorringe, A. J. Fox, D. M. Jones, and A. Robinson. 465 1993. Demonstration of lipooligosaccharide immunotype and capsule as virulence factors for 466 Neisseria meningitidis using an infant mouse intranasal infection model. Microb. Pathog. 467 15:359-366. 468
25. Maiden, M. C. 2004. Dynamics of bacterial carriage and disease: lessons from the 469 meningococcus. Adv. Exp. Med. Biol. 549:23-29. 470
26. Maiden, M. C., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. 471 Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt. 1998. 472 Multilocus sequence typing: a portable approach to the identification of clones within 473 populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. U. S. A. 95:3140-3145. 474
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
19
27. Peltola, H., P. H. Makela, O. ELO, O. Pettay, O. V. Renkonen, and A. Sivonen. 1976. 475 Vaccination against meningococcal group A disease in Finland 1974-75. Scand. J. Infect. Dis. 476 8:169-174. 477
28. Pether, J. V., N. F. Lightfoot, R. J. Scott, J. Morgan, A. P. Steele-Perkins, and S. C. 478 Sheard. 1988. Carriage of Neisseria meningitidis: investigations in a military establishment. 479 Epidemiol. Infect. 101:21-42. 480
29. Raza, M. W., O. R. El Ahmer, M. M. Ogilvie, C. C. Blackwell, A. T. Saadi, R. A. Elton, 481 and D. M. Weir. 1999. Infection with respiratory syncytial virus enhances expression of native 482 receptors for non-pilate Neisseria meningitidis on HEp-2 cells. FEMS Immunol. Med. 483 Microbiol. 23:115-124. 484
30. Renkonen, O. V., A. Sivonen, and R. Visakorpi. 1987. Effect of ciprofloxacin on carrier 485 rate of Neisseria meningitidis in army recruits in Finland. Antimicrob. Agents Chemother. 486 31:962-963. 487
31. Ristola, M., M. Jahkola, H. Käyhty, M. Sarvas, P. H. Mäkelä, D. Caugant, and Visakorpi 488 R. 1995. Meningooccal outbreak in southern Finland. Europ Commun Diseases Bull. June:2-489 3:. 490
32. Roberts, J., B. Greenwood, and J. Stuart. 2009. Sampling methods to detect carriage of 491 Neisseria meningitidis; literature review. J. Infect. 58:103-107. 492
33. Rosenstein, N. E., B. A. Perkins, D. S. Stephens, L. Lefkowitz, M. L. Cartter, R. Danila, P. 493 Cieslak, K. A. Shutt, T. Popovic, A. Schuchat, L. H. Harrison, and A. L. Reingold. 1999. The 494 changing epidemiology of meningococcal disease in the United States, 1992-1996. J. Infect. 495 Dis. 180:1894-1901. 496
34. Stephens, D. S. 1999. Uncloaking the meningococcus: dynamics of carriage and disease. 497 Lancet. 353:941-942. 498
35. Thulin Hedberg, S., B. Toros, H. Fredlund, P. Olcen, and P. Molling. 2011. Genetic 499 characterisation of the emerging invasive Neisseria meningitidis serogroup Y in Sweden, 2000 500 to 2010. Euro Surveill. 16:19885. 501
36. Toropainen M, Vainio A, Saarinen L, Kayhty H, Kuusi M, Virolainen A. Increase of 502 invasive meningococcal disease caused by serogroup Y in Finland, 2010. Poster. 21st European 503 Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Milan, Italy, May 7-10, 504 2011. 505
37. Toropainen, M., L. Saarinen, P. van der Ley, B. Kuipers, and H. Kayhty. 2001. Murine 506 monoclonal antibodies to PorA of Neisseria meningitidis show reduced protective activity in 507 vivo against B:15:P1.7,16 subtype variants in an infant rat infection model. Microb. Pathog. 508 30:139-148. 509
38. Tuite, A. R., L. M. Kinlin, S. P. Kuster, F. Jamieson, J. C. Kwong, A. McGeer, and D. N. 510 Fisman. 2010. Respiratory virus infection and risk of invasive meningococcal disease in 511 central Ontario, Canada. PLoS One. 5:e15493. 512
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
20
39. Vogel, U., G. Morelli, K. Zurth, H. Claus, E. Kriener, M. Achtman, and M. Frosch. 1998. 513 Necessity of molecular techniques to distinguish between Neisseria meningitidis strains 514 isolated from patients with meningococcal disease and from their healthy contacts. J. Clin. 515 Microbiol. 36:2465-2470. 516
40. Yazdankhah, S. P., and D. A. Caugant. 2004. Neisseria meningitidis: an overview of the 517 carriage state. J. Med. Microbiol. 53:821-832. 518
41. Yazdankhah, S. P., P. Kriz, G. Tzanakaki, J. Kremastinou, J. Kalmusova, M. Musilek, T. 519 Alvestad, K. A. Jolley, D. J. Wilson, N. D. McCarthy, D. A. Caugant, and M. C. Maiden. 2004. 520 Distribution of serogroups and genotypes among disease-associated and carried isolates of 521 Neisseria meningitidis from the Czech Republic, Greece, and Norway. J. Clin. Microbiol. 522 42:5146-5153. 523
524
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
7
75
200
751
2793. 29
97.
7528.
7527.
7534.
7525.
7524.
7529.
507.
Clonal ComplexST-549
Clonal ComplexST-334
Clonal C l
756
755.
7544.
60.
7553.
3735.
7530.
532.
03.
2. 750
7505.
7504.
7500.
9.
Clonal ComplexST-162
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
Clonal ComplexST-364
ComplexST-369
ST-35
Clonal ComplexST-174
Clonal ComplexST-167
Clonal Complex
ST-18
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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.
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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
125 swabs taken in July 2004
85 swabs taken in July 2004-July 125 swabs taken in
July 2005
9 mo. n=22
12 mo. n=125
22 swabs taken in July 2004
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
38 swabs taken in July 2004
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
283 swabs taken in July 2005
33 swabs taken in J 2005
32 swabs taken in January 2005- 33 swabs taken in
O t b 2005
283 swabs taken in January 2005
472 men enrolled
108 asthmatic 364 non-asthmatic
n=33
12 mo. n=120
120 swabs taken in January 2005
January 2005 January 2005October 2005 October 2005
20 swabs taken in
125 swabs taken in January 2005-January 2006
120 swabs taken in January 2006
Total number of swabs taken 472 446 436
no swabsDropouts n=36
36 swabs taken in July 2004
20 swabs taken in January 2005-
March 2005
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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.
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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.
Clonal ComplexST-254
Clonal ComplexST-461
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 ComplexST-178
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
Clonal ComplexST-269
Clonal ComplexST-213
pST-103
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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.
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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.
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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)
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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.
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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.
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
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%)
on February 12, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
top related