- Email: [email protected]
Populations of pharyngeal meningococci in Niger
Vaccine 25S (2007) A53–A57
Populations of pharyngeal meningococci in Niger Pierre Nicolas a,∗ , Saacou Djibo b , Bernard Tenebray a , Philippe Castelli a , Richard Stor a , Amina Amadou Hamidou b , Pascal Boisier b , Suzanne Chanteau b a
Institut de M´edecine Tropicale du Service de Sant´e des Arm´ees (IMTSSA), WHO Collaborating Center for Reference and Research on Meningococci, BP 46, 13998 Marseille, France b Centre de Recherches M´ edicales et Sanitaires, Niamey, Niger Available online 7 May 2007
Abstract This study investigated the carriage of Neisseria meningitidis group W135 (NmW135) belonging to sequence type (ST)-2881, ST-11 and NmA ST-7, as these three lineages have been responsible for sporadic cases in 2003 in Niamey (Niger). ST-7 and ST-11 were also the two genotypes involved in recent outbreaks in the African meningitis belt. Among the 97 Nm isolates obtained from 287 schoolchildren swabbed three times, 1 was identified as NmA, 34 as NmW135, 8 as NmY and 54 were non-groupable (NG). Among the 86 isolates genotyped, 59.3% belonged to ST-192, 24.4% to ST-2881, 5.8% to ST-2880, 4.6% to ST-175, 3.5% to ST-4899, 1.2% to ST-11 and 1.2% to ST-7. Most of the isolates recovered were weakly pathogenic Nm NG ST-192 and NmW135 ST-2881. These results, although preliminary, are important to consider before introduction of a NmA conjugate meningococcal vaccine in Africa. © 2007 Elsevier Ltd. All rights reserved. Keywords: Neisseria meningitidis; Pharyngeal carriage; Multilocus sequence typing
1. Introduction Neisseria meningitidis (Nm) colonizes the nasopharynx without affecting the host. In non-epidemic settings, 10% of healthy individuals carry Nm [1]. Carriage does represent the major route of transmission of meningococci but few studies have been focussed on meningococcal carriage in healthy subjects in Africa. To characterize the Nm strains isolated from carriage, which are often non-capsulated (non-groupable) and/or non-typeable, it is necessary to use molecular biology tools. The multilocus sequence typing (MLST) assay, described by Maiden et al. [2], identifies directly, from the nucleotide sequence of internal fragments, the allelic forms of seven housekeeping genes, which allows one to characterize each strain by its sequence type (ST). Closely related STs are grouped into clonal complexes or lineages. Relatively few hyperinvasive lineages, defined on the basis of their frequency of isolation from patients relative
∗
Corresponding author. Tel.: +33 4 91 15 01 15; fax: +33 4 91 59 44 77. E-mail addresses: [email protected], [email protected] (P. Nicolas).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.04.041
to a low isolation rate from healthy carriers, are responsible for most cases of invasive disease worldwide [3]. Carried isolates look more diverse [4,5]. In sub-Saharan Africa, meningococcal meningitis occurs every year during the dry season between December and May and large epidemics are reported every 5–10 years. Introduction of a new strain into a susceptible population and environmental factors such as dryness are necessary for an epidemic to occur [6]. Most of the sporadic cases of meningococcal meningitis that were notified in 2003 in Niamey, the capital town of Niger, were due to NmA strains that belonged to ST-7 and NmW135 strains belonging to ST-11 or ST-2881 [7]. ST-7 and ST-11 have been responsible for most of the cases and outbreaks in the meningitis belt these last years and are considered as hyperinvasive lineages [8]. ST-2881 emerged in 2002 and was responsible for sporadic cases. To enhance our understanding of the population biology of meningococcal isolates and to look for strains belonging to ST-11, ST-7 and ST-2881, a study was carried out on pharyngeal carriage of Nm in schoolchildren from two schools in Niamey Niger. Characterization of pharyngeal meningococci was performed using grouping, typing/subtyping, pulsed field
A54
P. Nicolas et al. / Vaccine 25S (2007) A53–A57
gel electrophoresis (PFGE) and multilocus sequence typing (MLST).
2. Materials and methods 2.1. Study population The study was carried out on a population of 287 schoolchildren in two primary schools close to the CERMES (Centre d’Etudes et de Recherches M´edicales, Niamey, Niger). These studies were approved by the national ethics committee of Niger in February 2003. Written consent was obtained from the parents or legal guardians for all participants. Previous anti-meningococcal vaccinations using A + C polysaccharide vaccine were reported for 281 children, of whom 213 were vaccinated in 2001 and 101 in 2002. 2.2. Bacterial isolates Two hundred and eighty-seven schoolchildren were swabbed three times between February and June 2003, with an interval of 1.5 months apart between each swab. Nasopharyngeal swabs were immediately plated on chocolate agar medium supplemented with polyvitex, vancomycin, colistin and nystatin (VCN) (bioM´erieux, Lyon, France). The plates were incubated within 1 h from swab collection at 35–37 ◦ C in 5–10% CO2 atmosphere. N. meningitidis colonies were identified using biochemical tests as oxidase-positive, catalase-positive, glucose-positive, maltose-positive and ␥GT-positive Gram-negative cocci. The isolates were serogrouped using immune sera (Difco, Fischer Scientific, Paris, France). Non-groupable and polyagglutinable meningococci were also tested using polymerase chain reaction (PCR) to identify groups A, B, C, Y, W135 [9,10]. Meningococci were preserved in brain–heart infusion broth with 10% sterile glycerol at −80 ◦ C then shipped using egg transport media [11] to the WHO Collaborating Centre for Reference and Research on Meningococci in Marseille, France. 2.3. Assays Serotypes and subtypes were determined using monoclonal antibodies from the National Institute of Public Health and the Environment (Bilthoven, The Netherlands) by the whole-cell enzyme immunoassay technique, as described elsewhere [12]. For pulsed field gel electrophoresis (PFGE), the whole chromosomal DNA macrorestriction fragments generated by Spe I endonuclease were separated by PFGE as previously described [13]. The DNA fragments were separated in a Chef-DR II system (Bio-Rad Laboratories, France). Fingerprint patterns were analysed using Tenover criteria [14]. To reduce cost and time, rather than sequencing all the isolates, we assumed that all the fingerprints that were strictly indis-
tinguishable by PFGE belonged to the same sequence type (ST). All the others, even differing by only one band were sequenced and analysed using multilocus sequence typing (MLST). Fragments from seven housekeeping genes were used for typing: abcZ (putative ABC transporter), adk (adenylate kinase), aroE (shikimate dehydrogenase), fumC (fumarase), gdh (glucose-6 phosphate dehydrogenase), pdhC (pyruvate dehydrogenase subunit) and pgm (phosphoglucomutase), as indicated on the MLST website (http://pubmlst.org/neisseria/). After DNA preparation and amplification by PCR, each locus sequence was analyzed on an ABI Prism 373 DNA sequencer or an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). Sequence analysis was performed using the Vector NTI suite software (InforMax, Bethesda, MD) and the Sequence Navigator DNA and Protein Sequence comparison software (Applied Biosystems). The sequences were compared with the existing alleles on the MLST website for determination of allele numbers, sequence types (STs) and clonal complexes of the isolate [2,15].
3. Results 3.1. Groups Ninety-seven strains of Nm were isolated in Niamey: 38 from swab #1 (carriage rate: 13.2%), 28 from swab #2 (carriage rate: 10.1%) and 31 from swab #3 (carriage rate: 11.4%). One was identified as NmA (1%), 34 (35%) as NmW135, 8 as NmY (8%) and 54 (55%) were non-groupable (NG) by either serum agglutination and/or PCR techniques. Unfortunately, after transport to the WHO Collaborating Centre in Marseille, only 86 isolates could be cultivated and analysed (34 from swab# 1, 27 from swab# 2 and 25 from swab #3). 3.2. Types and subtypes All the 86 isolates were typed/subtyped: 51 were non-typeable (NT) and non-subtypeable (NST); 25 were NT:P1.5,2; five were 14:P1.5; three were 4:NST; one was 4:P1.9 and one 2a:P1.5,2 (Table 1). 3.3. STs and lineages All 86 isolates were analysed by PFGE and 24 were sequenced: 51 (59.3%) were found to belong to ST-192 and 21 (24.4%) to ST-2881, which differs from ST-11 at six loci. Five isolates belonged to ST-2880 (5.81%), 4 to ST-175 (4.65%), 3 to ST-4899 (3.48%). One isolate was ST-11 belonging to ST-11 complex (1.16%) and another one to ST-7 belonging to the ST-5 complex (1.16%) (Table 1).
P. Nicolas et al. / Vaccine 25S (2007) A53–A57 Table 1 Groups, types, subtypes and sequence types of 86 meningococci from pharyngeal carriage of 287 schoolchildren in two schools in Niamey (Niger) in 2003 Group:type:subtype
NG:NT:NST W135:NT:NST Y:NT:NST NG:NT:P1.5,2 Y:NT:P1.5,2 W135:NT:P1.5,2 Y:NT:P1.5,2 Y:14:P1.5 NG:14:P1.5 NG:2a:P1.5,2 A:4:P1.9 NG:4:NST
Sequence type
ST-192 ST-192 ST-192 ST-175 ST-175 ST-2881 ST-2881 ST-2880 ST-2880 ST-11 ST-7 ST-4899
Number of isolates Swab 1
Swab 2
Swab 3
17 7 0 1 0 5 1 1 1 1 0 0
12 2 0 2 1 7 0 0 0 0 1 2
10 2 1 0 0 7 1 2 1 0 0 1
Columns 3–5 correspond to swabs 1, 2 and 3 taken 1.5 months apart between February and June. NG = non-groupable; NT = non-typeable; NST = nonsubtypeable; ST = sequence type; P1. = subtype.
3.4. Serogroup diversity among the lineages Comparison of serogroups and lineages showed that 39 NG Nm isolates, 11 NmW135 and one NmY isolate shared the ST-192 characteristics, whereas one NmY and 20 NmW135 isolates were characterized as being ST-2881. ST2880 characterized three NmY and two NG isolates, ST-175, two NmY and two NG isolates, ST-4899, three NG isolates, ST-11 one NG isolate, and ST-7 one NmA isolate (Table 1). 4. Discussion The use of the MLST technique to characterize meningococcal isolates [2] has shown that between 1988 and 2004 most of the outbreaks in the African meningitis belt were caused by NmA strains that belonged to type 4, subtype P1.9 (A:4:P1.9), sequence type (ST)-5. Subsequently, ST-5 disappeared and was replaced by ST-7 [8]. ST-5 and ST-7 are closely related genotypes that belong to the ST-5 complex. In 2000, the global outbreak of meningitis which began in Saudi Arabia was caused by a NmW135:2a:P1.5,2 strain belonging to ST-11 [16]. Even though a similar clone had already been isolated in Africa, it is probable that the annual Hajj amplified its spread into the meningitis belt countries, resulting in an overall increase in the number of cases in the region and in the severe 2002 epidemic in Burkina Faso, with more than 12,000 cases [8,17]. As they display the capability to expand clonally, ST-5, ST-7 and ST-11 are “hypervirulent lineages”. ST-2881, which includes non-typeable NmW135 isolates of subtype P1.5,2 (W135:NT:P1.5,2) and differs from ST-11 by six out seven loci, also emerged in 2002, but until now has been responsible for only sporadic cases in Nigeria, Benin and Niger [7,8]. In 2003 in Niger, 8082 meningitis cases and 636 deaths were notified. In the regions of Zinder and Maradi, the epidemic was due to NmA:4:P1.9 strains that belonged to ST-7
A55
[8]. In Niamey, the capital town, where only sporadic cases were notified, 55% of the Nm strains were NmA ST-7, but 38% were NmW135 that could be ranged either in ST-11, identical to the 2002 Burkina Faso epidemic clone, or in ST2881. The latter represented more than 50% of the NmW135 cases [7]. This study shows that, at the same moment, swabbing performed on school children in Niamey resulted in the identification of one NmA, 34 NmW135 and 8 NmY strains together with 54 non-groupable isolates. The high percentage of non-groupable strains is not much of a surprise, as, in other studies, approximately 50% of the strains isolated from carriers were reported to lack capsule and to, therefore, be non-groupable [4,5]. These strains are assumed to be non-pathogenic, but it is known that capsule production can switch on and off. The loss of the capsule enhances the capacity of the bacteria to colonize the human nasopharynx and to escape the defence systems of the host. The 86 pharyngeal isolates that were characterized in Marseille belonged to seven lineages. Most of them (83.7%) belonged to either ST-192 (59.3%), which is rarely responsible for meningitis cases, or to ST-2881 (24.4%), as was the case for the W135:NT:P1.5,2 isolates responsible for sporadic cases of meningitis in Niamey. Three of the isolates were ST-4899: this new ST was described for the first time in this study and characterizes non-groupable isolates. Only one isolate belonged to ST-7 (NmA:4:P1.9), which has recently been involved in outbreaks in the meningitis belt [8] and which was responsible for half of the sporadic cases in Niamey. Only one strain isolated from healthy carriers was ST-11 (NG:2a:P1.5,2), the type which was involved in the 2002 Burkina Faso outbreak. So in this study, only 2.3% of the strains isolated from carriers represented the hyperinvasive clones. Caugant et al. found that such clones represented 8.8% of carriage isolates in Norway [4] and Jolley et al. 20.2% in the Czech Republic [5]. Hyperinvasive lineages are therefore underrepresented among healthy carriers, in sharp contrast with the situation in patients. Although Niger is a neighbouring country of Burkina Faso and belongs to the meningitis belt, there has been so far no NmW135 outbreak in the country. However, we showed in this study that in the year following the great outbreak in Burkina Faso, numerous NmW135 isolates were circulating among the schoolchildren in Niamey. Among the carried strains, 35% were NmW135. It is not known, however, if the immunity induced by carriage of NmW135:NT:P1.5,2 ST-2881 would be sufficient to prevent an epidemic caused by the virulent clonal complex ST-11. The serogroup specific capsular polysaccharides have long been recognized as the target of the serum bactericidal antibodies (SBA). However, subcapsular antigens also contribute to the development and maintenance of natural immunity against PorA (OMP1 = P1.) and lipooligosaccharides [18]. We speculate that the carriage of W135 isolates of the ST-2881 lineage might have favoured immunity against virulent NmW135 ST-11 strains, perhaps because outer membrane protein P1.5,2 epitopes are shared by both lineages.
A56
P. Nicolas et al. / Vaccine 25S (2007) A53–A57
Only one NmA isolate was found in this study even though more than half of the sporadic cases of meningitis in the capital town were due to this serogroup. It has already been observed that after epidemics only few people carry serogroup A strains [19]. Another explanation for the weak carriage of group A strains might be that most of the schoolchildren of this study were vaccinated 1 or 2 years before the study began, using A + C polysaccharide vaccines, but polysaccharide vaccines are known to have only a moderate or transient impact on carriage [20–22]. This study also shows the importance of carriage of weak pathogenic ST-192 Nm strains, most of which were non-groupable. This lineage has rarely been responsible for meningitis in the region. In a similar study in the Czech Republic most of the carried strains belonged to nonpathogenic or weakly pathogenic Neisseria [5]. Can the carriage of a less virulent strain hamper colonisation by a hypervirulent one? As the carriage of several clones is probably rare (1%) [23], we can speculate that the carriage of the less virulent genotypes might have hampered the colonisation of the pharynx by hyperinvasive lineages. Carried meningococci represent a highly diverse recombining population, and a given lineage may be found in several serogroups. In this study, the same STs (ST-192, ST2881) were shared by serogroup Y, serogroup W135 and non-groupable isolates (Table 1). Among ST-2881, NmY and NmW135 isolates often had indistinguishable fingerprint patterns as characterized by PFGE. These data show that capsule switches between W135 and Y strains did probably occur, as already reported [24]. This fact is of concern as the trivalent A/C/W135 vaccine that was introduced to fight serogroup A and W135 epidemics, does not contain the Y polysaccharide, so that the emergence of serogroup Y in response to vaccine pressure after large vaccination campaigns is possible. It will thus be important to continue the genotyping of meningococci using MLST to trace NmW135 and NmY strains, to monitor changes in the epidemiology of meningococci in Africa and to survey the eventuality of polysaccharide switches. Our results are important also in regard of the future introduction of a NmA conjugate vaccine in Africa. In Niamey, only one child carried a group A ST-7 isolate. In view of these data, to show an impact of a conjugate NmA vaccine on NmA carriage, it will be necessary to study about 20,000 persons (10,000 in the vaccinated group, 10,000 in the control group) [25]. These studies are certainly possible but they will have to be planned and carried out carefully as they will take place in countries where laboratories, technicians and biologists are very rare.
Acknowledgements The part of the work performed in Marseilles was supported by funds from Direction Centrale du Service de Sant´e des Arm´ees, Minist`ere de la D´efense, France and WHO funds
(C11/286/2). The part of the work performed in Niger was supported by the Institut Pasteur, Minist`ere de la Recherche and Niger Representation. Christophe Rogier is thanked for help with statistical analysis. This publication made use of the MLST Web site http://pubmlst.org, developed by Keith Jolley and Man-Suen Chan and seated at the Wellcome Trust centre for the Epidemiology of Infectious Diseases, University of Oxford. The development of this site has been funded by the Wellcome Trust and the European Union.
References [1] Yazdankhah SP, Caugant DA. Neisseria meningitidis: an overview of the carriage state. J Med Microbiol 2004;53:821–32. [2] Maiden MJC, Bygraves JA, Feil E, Morelli G, Russel J, Urwin R, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998;95:3140–5. [3] Caugant DA. Population genetics and molecular epidemiology of Neisseria meningitidis. APMIS 1998;106:505–25. [4] Caugant DA, Hoiby EA, Magnus P, Scheel O, Hoel T, Bjune G, et al. Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population. J Clin Microbiol 1994;32:323–30. [5] Jolley KA, Kalmusova J, Feil EJ, Gupta S, Musilek M, Kriz P, et al. Carried meningococci in the Czech Republic: a diverse recombining population. J Clin Microbiol 2000;8:4492–8. [6] Lapeyssonnie L. La m´eningite c´er´ebrospinale en Afrique. Bull WHO 1963;28(suppl´ement):1–100. [7] Nicolas P, Djibo S, Moussa A, Tenebray B, Boisier P, Chanteau S. Molecular epidemiology of meningococci isolated in Niger in 2003 shows serogroup A sequence type (ST)-7 and serogroup W135 ST-11 or ST-2881 strains. J Clin Microbiol 2005;43:1437–8. [8] Nicolas P, Norheim G, Garnotel E, Djibo S, Caugant DA. Molecular epidemiology of Neisseria meningitidis isolated in the African Meningitis Belt between 1988 and 2003 shows the dominance of the sequence type (ST-) 5 and ST-11 complexes. J Clin Microbiol 2005;43(10):5129–35. [9] Taha MK. Simultaneous approach for non-culture PCR-based identification and serogroup prediction of Neisseria meningitidis. J Clin Microbiol 2000;38:4492–8. [10] Sidkou F, Djibo S, Taha MK, Alonso JM, Djibo A, Kairo KK, et al. Polymerase chain reaction assay and bacterial meningitis surveillance in remote areas, Niger. Emerg Infect Dis 2003;9(11):1486–8. [11] Vandekerkove M, Bradstreet P, Faucon R, Benech S. Milieu de transport pour m´eningocoques. Ann Inst Pasteur 1967;113:260–3. [12] Poolman JT, Abdillahi H. Outer membrane protein serosubtyping of Neisseria meningitides. Eur J Clin Microbiol Infect Dis 1998;7:291– 2. [13] Nicolas P, Parzy D, Martet G. Pulsed-field gel electrophoresis analysis of clonal relationships among Neisseria meningitidis A strains from different outbreaks. Eur J Clin Microbiol Infect Dis 1997;16:541–4. [14] Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–9. [15] Jolley KA, Chan MS, Maiden MC. MlstdbNet-distributed multi-locus sequence typing (MLST) databases. BMC Bioinform 2004;5:86. [16] Taha MK, Achtman M, Alonso JM, Greenwood B, Ramsay M, Fox A, et al. Serogroup W135 meningococcal disease in Hajj pilgrims. Lancet 2000;356:2159. [17] World Health Organization. Meningococcal disease, serogroup W135, Burkina Faso: preliminary report. Wkly Epidemiol Rec 2002;18:152– 5.
P. Nicolas et al. / Vaccine 25S (2007) A53–A57 [18] Kremastinou J, Tzanakaki G, Pagalis A, Theodondou M, Weir DM, Blackwell CC. Detection of IgG and IgM to meningococcal outer membrane proteins in relation to carriage of Neisseria meningitidis or Neisseria lactamica. FEMS Med Microbiol 2003;24:73–8. [19] Djibo S, Nicolas P, Campagne G, Chippaux JP. Pharyngeal carriage of Neisseria meningitidis in a school of Niamey, Niger. Med Trop 2004;64:363–6. [20] Hassan-King M, Wall RA, Greenwood BM. Meningococcal carriage, meningococcal disease and vaccination. J Infect 1988;16(1):55–9. [21] Blakebrough IS, Greenwood BM, Whittle HC, Bradley AK, Gilles HM. Failure of meningococcal vaccination to stop the transmission of meningococci in Nigerian schoolboys. Ann Trop Med Parasitol 1983;12:175–8.
A57
[22] Jodar L, Feavers I, Salisbury D, Granoff DM. Development of vaccines against meningococcal disease. Lancet 2002;359:1499–508. [23] Andersen J, Berthelsen L, Bech Jensensen B, Lind I. Dynamics of the meningococcal carrier state and characteristics of the carrier strains: a longitudinal study within three cohorts of military recruits. Epidemiol Infect 1998;121:85–94. [24] Swartley JS, Marfin A, Edupuganti S, Liu LJ, Cieslak P, Perkins B, et al. Capsule switching of Neisseria meningitides. Proc Natl Acad Sci USA 1997;94:271–6. [25] Maiden MJC, Stuart JM, Bramley JC, MacLennan JM, Gray S, Andrew N. Carriage of serogroup C meningococci 1 year after meningococcal C conjugate polysaccharide vaccination. Lancet 2002;359:1829– 31.
Populations of pharyngeal meningococci in Niger Pierre Nicolas a,∗ , Saacou Djibo b , Bernard Tenebray a , Philippe Castelli a , Richard Stor a , Amina Amadou Hamidou b , Pascal Boisier b , Suzanne Chanteau b a
Institut de M´edecine Tropicale du Service de Sant´e des Arm´ees (IMTSSA), WHO Collaborating Center for Reference and Research on Meningococci, BP 46, 13998 Marseille, France b Centre de Recherches M´ edicales et Sanitaires, Niamey, Niger Available online 7 May 2007
Abstract This study investigated the carriage of Neisseria meningitidis group W135 (NmW135) belonging to sequence type (ST)-2881, ST-11 and NmA ST-7, as these three lineages have been responsible for sporadic cases in 2003 in Niamey (Niger). ST-7 and ST-11 were also the two genotypes involved in recent outbreaks in the African meningitis belt. Among the 97 Nm isolates obtained from 287 schoolchildren swabbed three times, 1 was identified as NmA, 34 as NmW135, 8 as NmY and 54 were non-groupable (NG). Among the 86 isolates genotyped, 59.3% belonged to ST-192, 24.4% to ST-2881, 5.8% to ST-2880, 4.6% to ST-175, 3.5% to ST-4899, 1.2% to ST-11 and 1.2% to ST-7. Most of the isolates recovered were weakly pathogenic Nm NG ST-192 and NmW135 ST-2881. These results, although preliminary, are important to consider before introduction of a NmA conjugate meningococcal vaccine in Africa. © 2007 Elsevier Ltd. All rights reserved. Keywords: Neisseria meningitidis; Pharyngeal carriage; Multilocus sequence typing
1. Introduction Neisseria meningitidis (Nm) colonizes the nasopharynx without affecting the host. In non-epidemic settings, 10% of healthy individuals carry Nm [1]. Carriage does represent the major route of transmission of meningococci but few studies have been focussed on meningococcal carriage in healthy subjects in Africa. To characterize the Nm strains isolated from carriage, which are often non-capsulated (non-groupable) and/or non-typeable, it is necessary to use molecular biology tools. The multilocus sequence typing (MLST) assay, described by Maiden et al. [2], identifies directly, from the nucleotide sequence of internal fragments, the allelic forms of seven housekeeping genes, which allows one to characterize each strain by its sequence type (ST). Closely related STs are grouped into clonal complexes or lineages. Relatively few hyperinvasive lineages, defined on the basis of their frequency of isolation from patients relative
∗
Corresponding author. Tel.: +33 4 91 15 01 15; fax: +33 4 91 59 44 77. E-mail addresses: [email protected], [email protected] (P. Nicolas).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.04.041
to a low isolation rate from healthy carriers, are responsible for most cases of invasive disease worldwide [3]. Carried isolates look more diverse [4,5]. In sub-Saharan Africa, meningococcal meningitis occurs every year during the dry season between December and May and large epidemics are reported every 5–10 years. Introduction of a new strain into a susceptible population and environmental factors such as dryness are necessary for an epidemic to occur [6]. Most of the sporadic cases of meningococcal meningitis that were notified in 2003 in Niamey, the capital town of Niger, were due to NmA strains that belonged to ST-7 and NmW135 strains belonging to ST-11 or ST-2881 [7]. ST-7 and ST-11 have been responsible for most of the cases and outbreaks in the meningitis belt these last years and are considered as hyperinvasive lineages [8]. ST-2881 emerged in 2002 and was responsible for sporadic cases. To enhance our understanding of the population biology of meningococcal isolates and to look for strains belonging to ST-11, ST-7 and ST-2881, a study was carried out on pharyngeal carriage of Nm in schoolchildren from two schools in Niamey Niger. Characterization of pharyngeal meningococci was performed using grouping, typing/subtyping, pulsed field
A54
P. Nicolas et al. / Vaccine 25S (2007) A53–A57
gel electrophoresis (PFGE) and multilocus sequence typing (MLST).
2. Materials and methods 2.1. Study population The study was carried out on a population of 287 schoolchildren in two primary schools close to the CERMES (Centre d’Etudes et de Recherches M´edicales, Niamey, Niger). These studies were approved by the national ethics committee of Niger in February 2003. Written consent was obtained from the parents or legal guardians for all participants. Previous anti-meningococcal vaccinations using A + C polysaccharide vaccine were reported for 281 children, of whom 213 were vaccinated in 2001 and 101 in 2002. 2.2. Bacterial isolates Two hundred and eighty-seven schoolchildren were swabbed three times between February and June 2003, with an interval of 1.5 months apart between each swab. Nasopharyngeal swabs were immediately plated on chocolate agar medium supplemented with polyvitex, vancomycin, colistin and nystatin (VCN) (bioM´erieux, Lyon, France). The plates were incubated within 1 h from swab collection at 35–37 ◦ C in 5–10% CO2 atmosphere. N. meningitidis colonies were identified using biochemical tests as oxidase-positive, catalase-positive, glucose-positive, maltose-positive and ␥GT-positive Gram-negative cocci. The isolates were serogrouped using immune sera (Difco, Fischer Scientific, Paris, France). Non-groupable and polyagglutinable meningococci were also tested using polymerase chain reaction (PCR) to identify groups A, B, C, Y, W135 [9,10]. Meningococci were preserved in brain–heart infusion broth with 10% sterile glycerol at −80 ◦ C then shipped using egg transport media [11] to the WHO Collaborating Centre for Reference and Research on Meningococci in Marseille, France. 2.3. Assays Serotypes and subtypes were determined using monoclonal antibodies from the National Institute of Public Health and the Environment (Bilthoven, The Netherlands) by the whole-cell enzyme immunoassay technique, as described elsewhere [12]. For pulsed field gel electrophoresis (PFGE), the whole chromosomal DNA macrorestriction fragments generated by Spe I endonuclease were separated by PFGE as previously described [13]. The DNA fragments were separated in a Chef-DR II system (Bio-Rad Laboratories, France). Fingerprint patterns were analysed using Tenover criteria [14]. To reduce cost and time, rather than sequencing all the isolates, we assumed that all the fingerprints that were strictly indis-
tinguishable by PFGE belonged to the same sequence type (ST). All the others, even differing by only one band were sequenced and analysed using multilocus sequence typing (MLST). Fragments from seven housekeeping genes were used for typing: abcZ (putative ABC transporter), adk (adenylate kinase), aroE (shikimate dehydrogenase), fumC (fumarase), gdh (glucose-6 phosphate dehydrogenase), pdhC (pyruvate dehydrogenase subunit) and pgm (phosphoglucomutase), as indicated on the MLST website (http://pubmlst.org/neisseria/). After DNA preparation and amplification by PCR, each locus sequence was analyzed on an ABI Prism 373 DNA sequencer or an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). Sequence analysis was performed using the Vector NTI suite software (InforMax, Bethesda, MD) and the Sequence Navigator DNA and Protein Sequence comparison software (Applied Biosystems). The sequences were compared with the existing alleles on the MLST website for determination of allele numbers, sequence types (STs) and clonal complexes of the isolate [2,15].
3. Results 3.1. Groups Ninety-seven strains of Nm were isolated in Niamey: 38 from swab #1 (carriage rate: 13.2%), 28 from swab #2 (carriage rate: 10.1%) and 31 from swab #3 (carriage rate: 11.4%). One was identified as NmA (1%), 34 (35%) as NmW135, 8 as NmY (8%) and 54 (55%) were non-groupable (NG) by either serum agglutination and/or PCR techniques. Unfortunately, after transport to the WHO Collaborating Centre in Marseille, only 86 isolates could be cultivated and analysed (34 from swab# 1, 27 from swab# 2 and 25 from swab #3). 3.2. Types and subtypes All the 86 isolates were typed/subtyped: 51 were non-typeable (NT) and non-subtypeable (NST); 25 were NT:P1.5,2; five were 14:P1.5; three were 4:NST; one was 4:P1.9 and one 2a:P1.5,2 (Table 1). 3.3. STs and lineages All 86 isolates were analysed by PFGE and 24 were sequenced: 51 (59.3%) were found to belong to ST-192 and 21 (24.4%) to ST-2881, which differs from ST-11 at six loci. Five isolates belonged to ST-2880 (5.81%), 4 to ST-175 (4.65%), 3 to ST-4899 (3.48%). One isolate was ST-11 belonging to ST-11 complex (1.16%) and another one to ST-7 belonging to the ST-5 complex (1.16%) (Table 1).
P. Nicolas et al. / Vaccine 25S (2007) A53–A57 Table 1 Groups, types, subtypes and sequence types of 86 meningococci from pharyngeal carriage of 287 schoolchildren in two schools in Niamey (Niger) in 2003 Group:type:subtype
NG:NT:NST W135:NT:NST Y:NT:NST NG:NT:P1.5,2 Y:NT:P1.5,2 W135:NT:P1.5,2 Y:NT:P1.5,2 Y:14:P1.5 NG:14:P1.5 NG:2a:P1.5,2 A:4:P1.9 NG:4:NST
Sequence type
ST-192 ST-192 ST-192 ST-175 ST-175 ST-2881 ST-2881 ST-2880 ST-2880 ST-11 ST-7 ST-4899
Number of isolates Swab 1
Swab 2
Swab 3
17 7 0 1 0 5 1 1 1 1 0 0
12 2 0 2 1 7 0 0 0 0 1 2
10 2 1 0 0 7 1 2 1 0 0 1
Columns 3–5 correspond to swabs 1, 2 and 3 taken 1.5 months apart between February and June. NG = non-groupable; NT = non-typeable; NST = nonsubtypeable; ST = sequence type; P1. = subtype.
3.4. Serogroup diversity among the lineages Comparison of serogroups and lineages showed that 39 NG Nm isolates, 11 NmW135 and one NmY isolate shared the ST-192 characteristics, whereas one NmY and 20 NmW135 isolates were characterized as being ST-2881. ST2880 characterized three NmY and two NG isolates, ST-175, two NmY and two NG isolates, ST-4899, three NG isolates, ST-11 one NG isolate, and ST-7 one NmA isolate (Table 1). 4. Discussion The use of the MLST technique to characterize meningococcal isolates [2] has shown that between 1988 and 2004 most of the outbreaks in the African meningitis belt were caused by NmA strains that belonged to type 4, subtype P1.9 (A:4:P1.9), sequence type (ST)-5. Subsequently, ST-5 disappeared and was replaced by ST-7 [8]. ST-5 and ST-7 are closely related genotypes that belong to the ST-5 complex. In 2000, the global outbreak of meningitis which began in Saudi Arabia was caused by a NmW135:2a:P1.5,2 strain belonging to ST-11 [16]. Even though a similar clone had already been isolated in Africa, it is probable that the annual Hajj amplified its spread into the meningitis belt countries, resulting in an overall increase in the number of cases in the region and in the severe 2002 epidemic in Burkina Faso, with more than 12,000 cases [8,17]. As they display the capability to expand clonally, ST-5, ST-7 and ST-11 are “hypervirulent lineages”. ST-2881, which includes non-typeable NmW135 isolates of subtype P1.5,2 (W135:NT:P1.5,2) and differs from ST-11 by six out seven loci, also emerged in 2002, but until now has been responsible for only sporadic cases in Nigeria, Benin and Niger [7,8]. In 2003 in Niger, 8082 meningitis cases and 636 deaths were notified. In the regions of Zinder and Maradi, the epidemic was due to NmA:4:P1.9 strains that belonged to ST-7
A55
[8]. In Niamey, the capital town, where only sporadic cases were notified, 55% of the Nm strains were NmA ST-7, but 38% were NmW135 that could be ranged either in ST-11, identical to the 2002 Burkina Faso epidemic clone, or in ST2881. The latter represented more than 50% of the NmW135 cases [7]. This study shows that, at the same moment, swabbing performed on school children in Niamey resulted in the identification of one NmA, 34 NmW135 and 8 NmY strains together with 54 non-groupable isolates. The high percentage of non-groupable strains is not much of a surprise, as, in other studies, approximately 50% of the strains isolated from carriers were reported to lack capsule and to, therefore, be non-groupable [4,5]. These strains are assumed to be non-pathogenic, but it is known that capsule production can switch on and off. The loss of the capsule enhances the capacity of the bacteria to colonize the human nasopharynx and to escape the defence systems of the host. The 86 pharyngeal isolates that were characterized in Marseille belonged to seven lineages. Most of them (83.7%) belonged to either ST-192 (59.3%), which is rarely responsible for meningitis cases, or to ST-2881 (24.4%), as was the case for the W135:NT:P1.5,2 isolates responsible for sporadic cases of meningitis in Niamey. Three of the isolates were ST-4899: this new ST was described for the first time in this study and characterizes non-groupable isolates. Only one isolate belonged to ST-7 (NmA:4:P1.9), which has recently been involved in outbreaks in the meningitis belt [8] and which was responsible for half of the sporadic cases in Niamey. Only one strain isolated from healthy carriers was ST-11 (NG:2a:P1.5,2), the type which was involved in the 2002 Burkina Faso outbreak. So in this study, only 2.3% of the strains isolated from carriers represented the hyperinvasive clones. Caugant et al. found that such clones represented 8.8% of carriage isolates in Norway [4] and Jolley et al. 20.2% in the Czech Republic [5]. Hyperinvasive lineages are therefore underrepresented among healthy carriers, in sharp contrast with the situation in patients. Although Niger is a neighbouring country of Burkina Faso and belongs to the meningitis belt, there has been so far no NmW135 outbreak in the country. However, we showed in this study that in the year following the great outbreak in Burkina Faso, numerous NmW135 isolates were circulating among the schoolchildren in Niamey. Among the carried strains, 35% were NmW135. It is not known, however, if the immunity induced by carriage of NmW135:NT:P1.5,2 ST-2881 would be sufficient to prevent an epidemic caused by the virulent clonal complex ST-11. The serogroup specific capsular polysaccharides have long been recognized as the target of the serum bactericidal antibodies (SBA). However, subcapsular antigens also contribute to the development and maintenance of natural immunity against PorA (OMP1 = P1.) and lipooligosaccharides [18]. We speculate that the carriage of W135 isolates of the ST-2881 lineage might have favoured immunity against virulent NmW135 ST-11 strains, perhaps because outer membrane protein P1.5,2 epitopes are shared by both lineages.
A56
P. Nicolas et al. / Vaccine 25S (2007) A53–A57
Only one NmA isolate was found in this study even though more than half of the sporadic cases of meningitis in the capital town were due to this serogroup. It has already been observed that after epidemics only few people carry serogroup A strains [19]. Another explanation for the weak carriage of group A strains might be that most of the schoolchildren of this study were vaccinated 1 or 2 years before the study began, using A + C polysaccharide vaccines, but polysaccharide vaccines are known to have only a moderate or transient impact on carriage [20–22]. This study also shows the importance of carriage of weak pathogenic ST-192 Nm strains, most of which were non-groupable. This lineage has rarely been responsible for meningitis in the region. In a similar study in the Czech Republic most of the carried strains belonged to nonpathogenic or weakly pathogenic Neisseria [5]. Can the carriage of a less virulent strain hamper colonisation by a hypervirulent one? As the carriage of several clones is probably rare (1%) [23], we can speculate that the carriage of the less virulent genotypes might have hampered the colonisation of the pharynx by hyperinvasive lineages. Carried meningococci represent a highly diverse recombining population, and a given lineage may be found in several serogroups. In this study, the same STs (ST-192, ST2881) were shared by serogroup Y, serogroup W135 and non-groupable isolates (Table 1). Among ST-2881, NmY and NmW135 isolates often had indistinguishable fingerprint patterns as characterized by PFGE. These data show that capsule switches between W135 and Y strains did probably occur, as already reported [24]. This fact is of concern as the trivalent A/C/W135 vaccine that was introduced to fight serogroup A and W135 epidemics, does not contain the Y polysaccharide, so that the emergence of serogroup Y in response to vaccine pressure after large vaccination campaigns is possible. It will thus be important to continue the genotyping of meningococci using MLST to trace NmW135 and NmY strains, to monitor changes in the epidemiology of meningococci in Africa and to survey the eventuality of polysaccharide switches. Our results are important also in regard of the future introduction of a NmA conjugate vaccine in Africa. In Niamey, only one child carried a group A ST-7 isolate. In view of these data, to show an impact of a conjugate NmA vaccine on NmA carriage, it will be necessary to study about 20,000 persons (10,000 in the vaccinated group, 10,000 in the control group) [25]. These studies are certainly possible but they will have to be planned and carried out carefully as they will take place in countries where laboratories, technicians and biologists are very rare.
Acknowledgements The part of the work performed in Marseilles was supported by funds from Direction Centrale du Service de Sant´e des Arm´ees, Minist`ere de la D´efense, France and WHO funds
(C11/286/2). The part of the work performed in Niger was supported by the Institut Pasteur, Minist`ere de la Recherche and Niger Representation. Christophe Rogier is thanked for help with statistical analysis. This publication made use of the MLST Web site http://pubmlst.org, developed by Keith Jolley and Man-Suen Chan and seated at the Wellcome Trust centre for the Epidemiology of Infectious Diseases, University of Oxford. The development of this site has been funded by the Wellcome Trust and the European Union.
References [1] Yazdankhah SP, Caugant DA. Neisseria meningitidis: an overview of the carriage state. J Med Microbiol 2004;53:821–32. [2] Maiden MJC, Bygraves JA, Feil E, Morelli G, Russel J, Urwin R, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998;95:3140–5. [3] Caugant DA. Population genetics and molecular epidemiology of Neisseria meningitidis. APMIS 1998;106:505–25. [4] Caugant DA, Hoiby EA, Magnus P, Scheel O, Hoel T, Bjune G, et al. Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population. J Clin Microbiol 1994;32:323–30. [5] Jolley KA, Kalmusova J, Feil EJ, Gupta S, Musilek M, Kriz P, et al. Carried meningococci in the Czech Republic: a diverse recombining population. J Clin Microbiol 2000;8:4492–8. [6] Lapeyssonnie L. La m´eningite c´er´ebrospinale en Afrique. Bull WHO 1963;28(suppl´ement):1–100. [7] Nicolas P, Djibo S, Moussa A, Tenebray B, Boisier P, Chanteau S. Molecular epidemiology of meningococci isolated in Niger in 2003 shows serogroup A sequence type (ST)-7 and serogroup W135 ST-11 or ST-2881 strains. J Clin Microbiol 2005;43:1437–8. [8] Nicolas P, Norheim G, Garnotel E, Djibo S, Caugant DA. Molecular epidemiology of Neisseria meningitidis isolated in the African Meningitis Belt between 1988 and 2003 shows the dominance of the sequence type (ST-) 5 and ST-11 complexes. J Clin Microbiol 2005;43(10):5129–35. [9] Taha MK. Simultaneous approach for non-culture PCR-based identification and serogroup prediction of Neisseria meningitidis. J Clin Microbiol 2000;38:4492–8. [10] Sidkou F, Djibo S, Taha MK, Alonso JM, Djibo A, Kairo KK, et al. Polymerase chain reaction assay and bacterial meningitis surveillance in remote areas, Niger. Emerg Infect Dis 2003;9(11):1486–8. [11] Vandekerkove M, Bradstreet P, Faucon R, Benech S. Milieu de transport pour m´eningocoques. Ann Inst Pasteur 1967;113:260–3. [12] Poolman JT, Abdillahi H. Outer membrane protein serosubtyping of Neisseria meningitides. Eur J Clin Microbiol Infect Dis 1998;7:291– 2. [13] Nicolas P, Parzy D, Martet G. Pulsed-field gel electrophoresis analysis of clonal relationships among Neisseria meningitidis A strains from different outbreaks. Eur J Clin Microbiol Infect Dis 1997;16:541–4. [14] Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–9. [15] Jolley KA, Chan MS, Maiden MC. MlstdbNet-distributed multi-locus sequence typing (MLST) databases. BMC Bioinform 2004;5:86. [16] Taha MK, Achtman M, Alonso JM, Greenwood B, Ramsay M, Fox A, et al. Serogroup W135 meningococcal disease in Hajj pilgrims. Lancet 2000;356:2159. [17] World Health Organization. Meningococcal disease, serogroup W135, Burkina Faso: preliminary report. Wkly Epidemiol Rec 2002;18:152– 5.
P. Nicolas et al. / Vaccine 25S (2007) A53–A57 [18] Kremastinou J, Tzanakaki G, Pagalis A, Theodondou M, Weir DM, Blackwell CC. Detection of IgG and IgM to meningococcal outer membrane proteins in relation to carriage of Neisseria meningitidis or Neisseria lactamica. FEMS Med Microbiol 2003;24:73–8. [19] Djibo S, Nicolas P, Campagne G, Chippaux JP. Pharyngeal carriage of Neisseria meningitidis in a school of Niamey, Niger. Med Trop 2004;64:363–6. [20] Hassan-King M, Wall RA, Greenwood BM. Meningococcal carriage, meningococcal disease and vaccination. J Infect 1988;16(1):55–9. [21] Blakebrough IS, Greenwood BM, Whittle HC, Bradley AK, Gilles HM. Failure of meningococcal vaccination to stop the transmission of meningococci in Nigerian schoolboys. Ann Trop Med Parasitol 1983;12:175–8.
A57
[22] Jodar L, Feavers I, Salisbury D, Granoff DM. Development of vaccines against meningococcal disease. Lancet 2002;359:1499–508. [23] Andersen J, Berthelsen L, Bech Jensensen B, Lind I. Dynamics of the meningococcal carrier state and characteristics of the carrier strains: a longitudinal study within three cohorts of military recruits. Epidemiol Infect 1998;121:85–94. [24] Swartley JS, Marfin A, Edupuganti S, Liu LJ, Cieslak P, Perkins B, et al. Capsule switching of Neisseria meningitides. Proc Natl Acad Sci USA 1997;94:271–6. [25] Maiden MJC, Stuart JM, Bramley JC, MacLennan JM, Gray S, Andrew N. Carriage of serogroup C meningococci 1 year after meningococcal C conjugate polysaccharide vaccination. Lancet 2002;359:1829– 31.