Research Article
23 January 2015

Development and Evaluation of a Dipstick Diagnostic Test for Neisseria meningitidis Serogroup X


The emergence of Neisseria meningitidis serogroup X (NmX) in the African meningitis belt has urged the development of diagnostic tools and vaccines for this serogroup, especially following the introduction of a conjugate vaccine against N. meningitidis serogroup A (NmA). We have developed and evaluated a new rapid diagnostic test (RDT) for detecting the capsular polysaccharide (cps) antigen of this emerging serogroup. Whole inactivated NmX bacteria were used to immunize rabbits. Following purification by affinity chromatography, the cpsX-specific IgG antibodies were utilized to develop an NmX-specific immunochromatography dipstick RDT. The test was validated against purified cpsX and meningococcal strains of different serogroups. Its performance was evaluated against that of PCR on a collection of 369 cerebrospinal fluid (CSF) samples obtained from patients living in countries within the meningitis belt (Cameroon, Côte d'Ivoire, and Niger) or in France. The RDT was highly specific for NmX strains. Cutoffs of 105 CFU/ml and 1 ng/ml were observed for the reference NmX strain and purified cpsX, respectively. Sensitivity and specificity were 100% and 94%, respectively. A high agreement between PCR and RDT (Kappa coefficient, 0.98) was observed. The RDT gave a high positive likelihood ratio and a low negative likelihood (0.07), indicating almost 100% probability of declaring disease or not when the test is positive or negative, respectively. This unique NmX-specific test could be added to the available set of RDT for the detection of meningococcal meningitis in Africa as a major tool to reinforce epidemiological surveillance after the introduction of the NmA conjugate vaccine.


Neisseria meningitidis is an exclusively human capsulated bacterium that can provoke severe invasive infections, such as meningitis and septicemia (1). Meningococcal disease is still a major public health concern due to potential epidemic spread. While the disease occurs sporadically in Europe and North America, it is responsible for major recurrent epidemics within the African meningitis belt (2). The bacterial capsular polysaccharide determines the 12 N. meningitidis serogroups currently described. Six serogroups (A, B, C, Y, W, and X) are responsible for the vast majority of cases of meningococcal disease worldwide. However, they differ in their global frequencies and geographical distribution (3). This distribution impacts vaccination strategies, which for the most part involve established polysaccharide-based vaccines against serogroups A, C, Y, and W. Besides, an innovative recombinant protein-based vaccine was recently licensed in Europe and Australia against meningococci of serogroup B (4). This multicomponent vaccine targets conserved proteins among meningococci, regardless of their serogroup. Therefore, it has the potential to cover non-serogroup-B isolates, such as those of serogroup X (5). In the meningitis belt, N. meningitidis serogroup A (NmA) predominated prior to the introduction of the NmA polysaccharide-protein conjugate vaccine (MenAfriVac) (6), while other serogroups (mainly serogroups W [NmW] and X [NmX]), were also detected and still are. Of particular concern, outbreaks due to isolates of NmW and NmX were recently reported in Africa (79). Surveillance of the distribution of meningococcal serogroups is therefore important, and its comprehensiveness will benefit from diagnostic tools that can be widely used at the bedside. In recent years, we have contributed to the development and validation of immunochromatography dipstick rapid diagnostic tests (RDT) for the identification of N. meningitidis serogroups A, C, Y, and W (10, 11). This major achievement was a first step in the improvement of bedside diagnosis of meningococcal infection in Niger, a country within the meningitis belt (10, 12). While NmX is still rare in Europe (13), its increasing importance in the meningitis belt supports the licensing of an efficient device to diagnose NmX infection as well as ongoing studies toward an NmX polysaccharide-based vaccine (14). Here, we report the design, development, and validation in the field of a new RDT for the detection of NmX isolates. Hence, this work contributes to the completion of the available tools for the diagnosis and surveillance of meningococcal meningitis in the meningitis belt.


Bacterial strains and samples.

The N. meningitidis isolates used in this study were isolates from cases of meningococcal disease (see Table 1 for details). The bacteria were cultured on GC medium base (GCB) (Difco, Detroit, MI, USA) supplemented with Kellogg supplements (15). The serogroup was determined by agglutination with serogroup-specific antisera, according to the standard procedure (16). Further phenotyping (serotyping and serosubtyping) was performed using monoclonal antibodies against the meningococcal proteins PorA and PorB, as previously described (17). The cerebrospinal fluid (CSF) samples tested in this study corresponded to suspected bacterial meningitis cases. They were obtained from the National Reference Laboratories for Meningococci located at the Institut Pasteur of Côte d'Ivoire and at the Institut Pasteur, Paris, France, as well as from the Centre de Recherche Médicale et Sanitaire (CERMES) in Niamey, Niger, and from the Centre Pasteur of Garoua, Cameroon. These samples were received in line with the mission of these centers for the surveillance of meningococcal diseases in the corresponding countries under approvals from the internal board of the Institut Pasteur to collect, characterize, and use these samples, which were all anonymized.
TABLE 1 Strains used in the study and their characteristics
Strain referenceSerogroup:serotype/serosubtypea
20137C:2b: P1.5,2
19008C:2a: P1.5,2
19383E:NT: P1.5,2
19504bX:NT: P1.5,2
NT, nontypeable; NST, nonsubtypeable.
Strains that were used for capsular polysaccharide purification.
The PCR analysis of these samples was used as a reference method to detect N. meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae, as well as to genogroup meningococcus-positive specimens. The PCR conditions and primers used were previously described (8). Culture was not used, as it has repeatedly been shown to be less sensitive than PCR (18). Culture data were available for only 26 of the 369 tested CSF samples.

Purification of the capsular polysaccharide from NmX.

The capsular polysaccharide of serogroup X (cpsX) was purified from the NmX strain 19504 (that gave the highest yield when cultured on GCB medium with Kellogg supplements) by the Cetavlon extraction method, as previously described (19). Briefly, 1 liter of bacteria at late-logarithmic phase of growth was formaldehyde inactivated (1% [vol/vol]) and then treated with Cetavlon (0.1% [wt/vol]) (Sigma-Aldrich, France). After centrifugation, the pellet was dissolved in cold aqueous CaCl2 (0.9 M). The solubilized materials were cleared by precipitation in 25% aqueous ethanol, and the remaining supernatant was precipitated by 80% aqueous ethanol. The pellet was dissolved in phosphate buffer (0.2 M Na2HPO4, NaH2PO4) and treated with DNase and RNase, followed by proteinase K treatment (Sigma-Aldrich, France) and cold phenol extraction. The extract was extensively dialyzed against distilled water and lyophilized to obtain the crude capsular polysaccharide. Ten milligrams of the preparation was dissolved in 2 ml of phosphate buffer 0.05 M K2HPO4 and KH2PO4 (pH 7) and purified by gel filtration on a BioSep-SEC-s3000 column (300 by 21.2 cm; Phenomenex, France) that was equilibrated with the same buffer. Elution was carried out with the same phosphate buffer at 5 ml/min and monitored at 214 nm and 280 nm. The void volume fractions containing cpsX in the high-molecular-weight range were pooled and dialyzed against distilled water at 4°C, using a dialysis membrane with a cutoff size of 10K to 15K, and the residue was lyophilized. The yield was about 20 mg/liter of culture. The profile of the purified cpsX was checked by proton nuclear magnetic resonance (1H NMR) (data not shown), as previously described (20). cpsA, cpsB, cpsC, cpsY, and cpsW were similarly purified from five strains of serogroups A, B, C, Y, and W (N. meningitidis strains 21524, 21721, 22639, 16366, and 19995, respectively; Table 1).

Rabbit immunization and purification of specific anti-cpsX IgG antibodies.

Two New Zealand White female rabbits (3 kg) were immunized intravenously three times with doses of 1 ml of a suspension of 109 CFU of freshly heat-inactivated NmX strain 19504 (30 min at 56°C) on days 0, 7 and 21. Serum samples were taken before immunization and at day 28 after the first injection to evaluate the immune response by enzyme-linked immunosorbent assay (ELISA) (see below). Dot blotting with rabbit serum (1:1,000 serum dilution) was performed using Amersham ECL kits (GE Healthcare Life Sciences, Vélizy-Villacoublay, France), as previously described (21). Rabbit immunization was performed according to the European Union Directive 2010/63/EU (and its revision, 86/609/EEC) on the protection of animals used for scientific purposes. Our laboratory has the administrative authorization for animal experimentation (permit no. 75-1554), and the protocol was approved by the Institut Pasteur review board that is part of the Regional Committee of Ethics of Animal Experiments of the Paris region (CETEA 2013-0190).
IgG antibody purification was performed by affinity chromatography in two steps. First, the rabbit serum samples was passed through a HiTrap Protein G HP column (GE Healthcare, France) and eluted with 0.1 M glycine-HCl (pH 2.7). Fractions of 1 ml were recovered in 50 μl of 1 M Tris-HCl buffer (pH 9). The fractions were tested for protein content by measuring their absorbance at 280 nm. Pooled fractions were passed through a cpsX affinity column obtained by chemical coupling of the amine functions of the CarboxyLink resin and the phosphate functions from cpsX, according to the manufacturer's recommendations (Thermo Scientific, Rockford, IL, USA). The eluted fractions were tested by ELISA against purified cpsX and whole inactivated NmX bacteria. To do so, ELISA wells were coated overnight with 100 μl of a solution containing 2 μg/ml purified cpsX or 100 μl of a bacterial suspension of 3 × 108 CFU/ml (NmX strain 19504). The purified antibodies (at a 500-pg/ml concentration) were tested against serial dilutions of bacteria from serogroups A, B, C, Y, W, and X in a dot blot experiment, and serial dilutions of the antibodies were then tested in ELISA on counterpart-coated cps at a concentration of 2 μg/ml.

Production and validation of a RDT against NmX.

A one-step vertical flow immunochromatography dipstick was set up using purified cpsX-PAbs that were conjugated to gold particles (British Biocell International, Cardiff, United Kingdom), as previously described (22). Unconjugated cpsX-PAbs were used as capture antibodies, and goat anti-rabbit IgG (ICN Biomedicals, Aurora, OH, USA) was used as control antibodies. Both types of antibodies were sprayed onto nitrocellulose (Schleicher & Schuell Bioscience, Ecquevilly, France) at 2 μg and 1 μg per line cm, respectively. For the test, dipsticks were dipped for a 10- to 15-min period at room temperature in 100 μl of phosphate-buffered saline (PBS) containing bacterial suspensions or CSF samples.

Data analysis.

Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) were calculated using a 2 by 2 contingency table. The positive likelihood ratios LR (LR+) (calculated as Se/[1 − Sp]) and the negative LR (LR) (calculated as [1 − Se]/Sp) were also calculated (23). These values give an indication of the likelihood that the sample is positive or negative, respectively, prior to testing. The diagnostic odds ratio (DOR), defined as the ratio of the odds of positive test results in specimens with NmX to the odds of positive test results in specimens negative for NmX, was calculated as follows: DOR = (Se/[1 − Se])/([1 − Sp/Sp] (24). Finally, the Cohen's kappa (κ) statistic was calculated to measure the concordance between PCR and RDT (25).


Characterization of rabbit anti-meningococcal serogroup X rabbit serum.

Following the three dose-immunization regimen with whole NmX bacteria, the rabbit sera were tested in dot blot analysis against spotted bacteria. While no bacterial detection was obtained with the control preimmune sera, strong detection was obtained with the sera from immunized rabbits (Fig. 1). Sera from the two responding rabbits were pooled, and anti-cpsX-specific IgG was purified by affinity chromatography on an NmX cps activated column. Dot plot analysis of the purified IgG response against decreasing numbers of bacteria (from 5 × 105 to 5 × 103 cells per spot) from serogroups A, B, C, Y, W, and X showed that antibodies recognized serogroup X strains only (Fig. 2A). The absence of recognition of the other serogroups (A, B, C, Y, and W) was further confirmed independently by ELISA analysis of the antibody response against coated (1 μg/ml) purified cps corresponding to the six serogroups (Fig. 2B).
FIG 1 Dot blotting analysis of rabbit sera. Serum samples from two rabbits collected prior to immunization (day 0) and 7 days after injection of the third dose of NmX strain 19504 (day 28) were used at 1:1,000 dilutions in immunoblotting. Four meningococcal isolates were spotted at 2 × 105 CFU/ml. Lane 1, strain 19504; lane 2, strain 23557; lane 3, strain 24196; lane 4, strain 24287).
FIG 2 Specific recognition of the purified rabbit anti-cpsX IgG antibodies. (A) Dot blot analysis against whole bacteria. The serogroups are indicated above the dots, and the amounts of loaded bacteria in each spot are indicated on the right (in CFU). Antibodies were used at a final concentration of 500 pg/ml. (B) ELISA analysis using coated purified capsular polysaccharide for serogroups A, B, C, Y, W, and X (Table 1). The data are expressed as the optical density at 492 nm (OD492) absorption for each concentration of antibodies (in pg/ml). The data correspond to the means from two independent experiments. The corresponding serogroups are indicated on the right. (C) Detection cutoff value for purified cpsX. The amounts are indicated in ng above each dipstick. A dipstick, before use, is shown on the left. The upper two arrows indicate the capture control line corresponding to the goat anti-rabbit IgG. The lower two arrows indicate the capture line corresponding to the anti-cpsX-specific IgG (cpsX line).
A dipstick rapid diagnostic test for NmX was produced (see Materials and Methods), and its detection limits were established. For the purified cpsX, this limit was 1 ng/ml (Fig. 2C), and it was 105 CFU/ml (data not shown) for NmX bacteria (strain 19504). The cutoff analysis was repeated 3 times with identical findings that were not affected by dipstick storage for 3 weeks at 25°C. We also tested the RDT on a collection of bacterial suspensions (Table 1) at 106 CFU/ml. Only the serogroup X isolates were detectable (data not shown). As different concentrations of antibodies were used in these assays, the data suggest that the concentrations of antibodies do not preclude detectable reactivity with other serogroups.

Use of the NmX dipsticks on clinical samples.

The NmX dipstick was tested on a panel of 369 CSF samples selected from historical collections kept in the National Reference Centre/Laboratory from four different countries, which differ in terms of meningitis incidence (Cameroon, Côte d'Ivoire, France, and Niger). Noticeably, three out of the four laboratories are located in countries within the meningitis belt. The CSF samples corresponded to suspected cases of acute bacterial meningitis. They were characterized by PCR for etiological diagnosis (Table 2). Culture results were available for only 26 samples (8 samples positive for S. pneumoniae, 4 positive for N. meningitidis [2 serogroup B and 2 serogroup W], 1 positive for H. influenzae, 1 positive for Streptococcus agalactiae, and 12 CSF samples were sterile by culture).
TABLE 2 Results of CSF samples obtained by PCR and by RDTa
PCR groupGeographical origin (no.)bNo. in RDT group:
IP ParisCERMESCP GarouaIP Côte d'IvoireTotalNmX+NmX
NG N. meningitidisc0016117017
S. pneumoniae00101323023
H. influenzae0013404
S. agalactiae1000101
CSF, cerebrospinal fluid; RDT, rapid diagnostic test.
IP, Institut Pasteur; CP, Centre Pasteur.
NG, nongroupable.
PCR negative for N. meningitidis, S. pneumoniae, and H. influenzae.
Among these isolates, 52% (n = 191) were positive for N. meningitidis, 8% (n = 28) were positive for other bacterial species, namely, S. pneumoniae, H. influenzae, and S. agalactiae, and 40% (n = 150) were negative by PCR for these species. Among the N. meningitidis-positive CSF samples, the six meningococcal capsular groups involved in invasive meningococcal infections were represented: group A (n = 27), group B (n = 8), group C (n = 7), group Y (n = 2), group W (n = 38), and group X (n = 92). In addition, 17 CSF samples were positive for N. meningitidis by PCR, although they were negative for groups A, B, C, Y, W, and X. All samples that were negative for NmX by PCR were also negative for this group by the new NmX-specific RDT. Among the 92 CSF samples positive for NmX by PCR, 86 were also positive by RDT. All 26 CSF samples with culture data tested negative by NmX-specific RDT (data not shown).
We also conducted a limited prospective analysis during the 2014 epidemic season in the three laboratories located in countries of the meningitis belt. We tested all 153 CSF samples that were received in the three laboratories in Cameroon, Côte d'Ivoire, and Niger between 1 January 2014 and 15 March 2014. No NmX was detected by PCR or RDT in any of the samples. In contrast, several samples were positive by PCR for S. pneumoniae (14%), NmW (7%), and H. influenzae (3%).

Performance of the NmX-specific RDT: sensitivity, specificity, likelihood ratios, and predictive values.

The RDT data showed a good correlation with the PCR data, indicating a Kappa correlation coefficient of 0.98. The sensitivity, specificity, and 95% confidence interval (95% CI) of the RDT obtained for the documented 369 CSF samples are summarized in Table 3. The specificity of RDT for CSF infected by NmX was 100%, while the sensitivity reached 94%. A calculation of the positive likelihood LR+ and DOR was not feasible due to an Sp value of 100%. LR+ and DOR values were therefore calculated using a value for the specificity that corresponded to the lower 95% confidence interval for specificity (99%) (Table 3).
TABLE 3 Performance of the RDT for NmX
Test parameteraValue95% confidence interval
Se, sensitivity; Sp, specificity; LH+, positive likelihood ratio; LH, negative likelihood ratio; PPV, positive predictive value; NPV, negative predictive value; DOR, diagnostic odds ratio.
Dividing by zero; the values of LH+ and DOR were calculated using a value for specificity that corresponded to the lower 95% confidence interval (99%).
The prevalence of NmX among the 369 tested CSF samples was 25%. Therefore, the NPV and PPV are given in Table 3 under this prevalence value. However, the tested samples were selected from the collections of the participating laboratories and may not reflect the real prevalence of the disease. Moreover, the frequency of NmX meningitis may vary across time and countries within the meningitis belt and elsewhere. We therefore calculated the negative and positive predictive values (NPV and PPV, respectively) according to a prevalence varying from 0 to 100%, using the Se and Sp obtained from the CSF samples in this study (Fig. 3).
FIG 3 Predictive values for N. meningitidis diagnosis. Positive predictive values (PPV) and negative predictive values (NPV) for the diagnosis of NmX were calculated according to a disease prevalence ranging between 0 and 100%.


Reliable tests for the identification of cases of meningococcal meningitis and serogroup determination are crucial to ensuring proper individual (case-by-case) and collective management of cases and epidemiological surveillance. Culturing N. meningitidis may frequently fail due to early antibiotic treatment and the fragility of this bacterial species (26). During the last 2 decades, PCR-based nonculture methods have been developed, enabling a significant improvement in the management and surveillance of bacterial meningitis (18). PCR-based methods require specific laboratory equipment and trained staff and cannot be used as a bedside method. Nevertheless, the PCR technology was implemented in several reference laboratories located in countries within the African meningitis belt (18). However, PCR may not be sufficiently set to ensure countrywide surveillance, especially in populations living in remote areas. Other tests, such as the currently available latex agglutination kits, require trained staff and an unbroken cold chain for storage and distribution of the kits.
The recent implementation of RDT for meningococci of serogroups A, C, Y, and W was a major breakthrough for individual diagnosis and for surveillance of meningococcal diseases in the African meningitis belt (12). These tests are stable at temperatures up to at least 45°C. They are easy to use and interpret in the absence of extensive training and therefore are adapted for bedside use. The emergence of meningococcal isolates of serogroup X urged the development of an RDT for this serogroup to complete the current RDT tools. We first analyzed the inherent quality of such a serogroup X-specific test. The specificity and sensitivity parameters were evaluated under laboratory conditions using a selected panel of relevant CSF samples. The good quality of the new RDT was reflected by its high sensitivity and specificity for NmX, with a very high likelihood ratio for a positive test (Table 3).
We also evaluated its usefulness, which depends not only on the quality of the test but also on the prevalence of the NmX meningitis in the tested population. The prevalence of NmX within the panel of CSF samples that was used to evaluate the RDT specificity and sensitivity was 25.7%. It may not properly reflect the real prevalence of NmX in areas at risk. Usefulness is usually evaluated using two parameters, PPV and NPV. When NmX prevalence was forced to vary between 0 and 100%, the PPV remained stable at 1, indicating that the test remained highly proficient at ruling in a case. Moreover, the NPV retained high values when the prevalence of NmX was very low. In addition, the test remained proficient (NPV, ≥0.95) if the prevalence increased to 50%. These considerations seem realistic and reflect the current epidemiological situation in the meningitis belt after the introduction of MenAfriVac, which was associated with a significant decrease in NmA (9). Indeed, this small-scale prospective use of the new RDT in the three centers located in this area (Abidjan, Garoua, and Niamey), suggests, on the basis of the sensitivities of RDT and PCR (which are <100%), that NmX may be present, albeit not as a dominating pathogen. In contrast, NmW was the most frequently isolated N. meningitidis serogroup, while most cases were associated with S. pneumoniae. However, a large-scale multisite prospective study comparing PCR and all the available RDT (for serogroups A, C, Y, W, Y, and X) is warranted in the future. Moreover, additional work is required to miniaturize the RDT to be applicable to small-volume samples (<100 μl).
In summary, this work reports a new reliable and rapid diagnostic test to detect serogroup X that should enhance the diagnosis of meningitis due to this serogroup and is expected to improve epidemiological surveillance. Epidemiological changes upon the implementation of MenAfriVac can be better monitored. This test would be helpful for the development and implementation of vaccines with broad serogroup coverage that can target NmX (5) or with NmX-specific vaccines (14).


We thank all the staff at the Institut Pasteur at Abidjan, Côte d'Ivoire, and at the Centre Pasteur at Garoua, Cameroon, for their warm hospitality. We also thank Maud Seguy for her excellent contribution to the management of this project.
The research work was supported by funding from the Fondation Total, including a fellowship to A.A.


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Information & Contributors


Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 53Number 2February 2015
Pages: 449 - 454
Editor: P. H. Gilligan
PubMed: 25411183


Received: 25 August 2014
Returned for modification: 22 October 2014
Accepted: 13 November 2014
Published online: 23 January 2015


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Alain Agnememel
Institut Pasteur, Invasive Bacterial Infection Unit, National Reference Centre for Meningococci, Paris, France
WHO Collaborating Centre for Meningococci, Institut Pasteur, Paris, France
Université Denis Diderot, Sorbonne Paris Cité, Paris, France
François Traincard
Institut Pasteur, Plateforme d'Ingénierie des Anticorps (PFIA), Paris, France
Sylvie Dartevelle
Institut Pasteur, Plateforme d'Ingénierie des Anticorps (PFIA), Paris, France
Laurence Mulard
Institut Pasteur, Unité de Chimie des Biomolécules, Paris, France
CNRS UMR 3523, Institut Pasteur, Paris, France
Ali Elhaji Mahamane
Centre de Recherche Médicale et Sanitaire (CERMES), Niamey, Niger
Odile Ouwe Missi Oukem-Boyer
Centre de Recherche Médicale et Sanitaire (CERMES), Niamey, Niger
Mélanie Denizon
Institut Pasteur, Invasive Bacterial Infection Unit, National Reference Centre for Meningococci, Paris, France
WHO Collaborating Centre for Meningococci, Institut Pasteur, Paris, France
Adèle Kacou-N′douba
Institut Pasteur de Côte d'Ivoire, Abidjan, Ivory Coast
Mireille Dosso
Institut Pasteur de Côte d'Ivoire, Abidjan, Ivory Coast
Bouba Gake
Centre Pasteur Annexe de Garoua, Garoua, Cameroon
Jean-Pierre Lombart
Centre Pasteur Annexe de Garoua, Garoua, Cameroon
Muhamed-Kheir Taha
Institut Pasteur, Invasive Bacterial Infection Unit, National Reference Centre for Meningococci, Paris, France
WHO Collaborating Centre for Meningococci, Institut Pasteur, Paris, France


P. H. Gilligan


Address correspondence to Muhamed-Kheir Taha, [email protected].

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Journal of Clinical Microbiology Vol.53 • Issue 2 • ASM Journals Pay Per View, PPV 25
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