Polyclonal antibodies specific for serogroup X of N. meningitidis and uses thereof in diagnosis

Abstract

The present invention is directed to polyclonal antibodies, specific for the capsular polysaccharides of Neisseria meningitidis serogroup X (NmX), wherein said antibodies are suitable for in vitro detection of Neisseria meningitidis serogroup X in biological fluids without culture. The invention also concerns said polyclonal antibodies in different diagnostic tests and methods, in order to detect NmX. The invention discloses also a rapid diagnostic test for detecting NmX in a biological fluid, as well as a method for obtaining polyclonal antibodies useful for detecting NmX in biological fluids such as cerebrospinal fluid, serum and urine.

Claims

1. A method of detecting Neisseria meningitidis serogroup X bacteria (NmX) in a biological fluid, the method comprising: (a) contacting a biological fluid from a subject having or suspected of having an infection by NmX with polyclonal antibodies specific to the capsular polysaccharides of the NmX and (b) determining the presence or the absence of the NmX in said fluid, wherein the polyclonal antibodies are obtained by immunization of a mammal with whole inactivated Neisseria meningitidis serogroup X bacteria followed by purification of the antibodies from serum of the immunized mammal by first affinity chromatography with protein G followed by second affinity purification with purified Neisseria meningitidis serogroup X capsular polysaccharides (cpsX), wherein said purified antibodies detect the NmX in the biological fluid sample in an immunoassay performed without culturing said biological fluid and wherein said antibodies do not cross-react with Neisseria meningitidis bacteria of serogroups A, B, C, Y and W.

2. The method of claim 1, wherein the polyclonal antibodies are linked directly or indirectly, covalently or noncovalently to a detection label.

3. The method of claim 1, wherein step (b) comprises an immunoassay.

4. The method of claim 1, wherein the immunoassay has a sensitivity of at least 90%.

5. The method of claim 1, wherein the biological fluid sample is chosen from cerebrospinal fluid, blood, serum, and urine.

6. The method of claim 1, wherein the presence of the capsular polysaccharides of the NmX in said fluid is indicative of the NmX infection.

7. The method of claim 1, wherein the polyclonal antibodies detect the NmX but do not cross-react with Neisseria meningitidis bacteria of serogroups A, B, C, Y and W.

8. The method of claim 1, wherein step (b) comprises use of a membrane, wherein said membrane comprises: (c) a first zone comprising the polyclonal antibodies specific for the capsular polysaccharides of the NmX conjugated to a detection label; (d) a control zone comprising an immobilized control antibody that recognizes the polyclonal antibodies of the first zone; and (e) a capture zone comprising the polyclonal antibodies specific for the capsular polysaccharides of the NmX immobilized thereon as capture antibodies.

9. The method of claim 8, wherein the polyclonal antibodies are rabbit antibodies and the immobilized control antibodies are anti-rabbit antibodies.

Description

LEGENDS OF FIGURES

(1) FIG. 1. Dot blotting analysis of rabbit sera. Sera from two rabbits prior to immunization (day 0) and 7 days after injection of the third dose of NmX strain 19504 (day 28) were used at 1:1000 dilutions in immunoblotting. Four meningococcal isolates were spotted at 2.10.sup.5 colony forming units, CFU/mL (1: strain 19404, 2: strain 23557, 3: strain 24196, 4: strain 24287).

(2) FIGS. 2A to 2C. Specific recognition of the purified rabbit anti-cpsX IgG antibodies. (A) Dot blotting analysis against whole bacteria. Serogroups are indicated above the dots and amounts of loaded bacteria in each spot are indicated on the right (in colony forming units, 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). Data are expressed as OD 492 nm absorption for each concentration of antibodies (in pg/mL). Data correspond to the means of two independent experiments. The corresponding serogroups are indicated on the right. (C) Detection cut-off 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).

(3) FIG. 3. Predictive values for N. meningitidis diagnosis. Positive Predictive Values and Negative Predictive Values (PPV and NPV, respectively) for the diagnosis of NmX were calculated according to a disease prevalence ranging between 0 and 100%.

(4) FIGS. 4A to 4B. (A) structure of the capsular polysaccharide of meningococci serogroup X: homopolymer of 1ζ4-linked N-acetyl-D-glucosamine 1-phosphate. (B) 1H NMR spectrum recorded on a Bruck Avance 400 spectrometer type, with a frequency of 400 MHz. The sample was dissolved in deuterium oxide (D.sub.2O). The chemical shifts (δ) are expressed in parts per million (ppm) and the reference used is the 4,4-dimethyl-4-silapentane 1-sulfonic acid (DSS). The coupling constants are given in Herz (Hz). The peaks are indicated according to the position (1 to 6) on the repeated unts of the cpsX (see FIG. 4A).

(5) FIG. 5. Detection of meningococci serogroup X in urine and serum using the serogroup X dipstick. The dipsticks disclosed in example 2 were used to detect NmX on urine samples (above) and serum sample (below) at different concentrations of meningococci, namely at 0 CFU/mL (labeled “negative” on the left side), at 10.sup.5 CFU/mL (middle) and at 10.sup.6 CFU/mL (right side). The detection line is indicated by an arrow.

EXAMPLES

(6) The inventors 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 a NmX-specific immunochromatography dipstick RDT. The test was validated against purified cpsX and meningococcal strains of different serogroups. Its performance was evaluated against PCR on a collection of 369 cerebrospinal fluid (CSF) samples obtained from patients living in countries within the meningitis belt (Cameroon, Côte d'lvoire and Niger) or in France. The RDT was highly specific for NmX strains. A cut-off of 10.sup.5 CFU/mL and 1 ng/mL was observed for the reference NmX strain and purified cpsX, respectively. Sensitivity and specificity were 94% and 100%, respectively. A high agreement between PCR and RDT (Kappa coefficient of 0.98) was observed. The RDT test gave a high positive likelihood ratio and a low negative likelihood (0.07) indicating almost 100% probability to declare disease or not when the test is positive or negative, respectively. This unique NmX-specific test could be added to the available set RDT tests for the detection of meningococcal meningitis in Africa as a major tool to reinforce epidemiological surveillance after the introduction of the NmA conjugate vaccine.

Example 1: Materials and Methods

(7) Bacterial Strains and Samples

(8) N. meningitidis isolates used in this study were isolates from cases of meningococcal disease (see Table 1 for details). Bacteria were cultured on GCB medium (GC Agar Base, Difco, Detroit Mich., 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'lvoire 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 the frame of these centres' mission 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 that are all anonymized.

(9) 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. PCR conditions and primers were as previously described (8). Culture was not used as it has been constantly shown to be less sensitive than PCR (26). Culture data were available only for 26 of the 369 tested CSF samples.

(10) TABLE-US-00001 TABLE 1 Strains used in the study and their characteristics Strain reference Serogroup:serotype/serosub-type  21525* A:4:P1.9 21526 A:4:P1.9 19256 B:NT:P1.5, 2 19257 B:2a:P1.5, 2 19324 B:2b:P1.5, 2  21721* B:NT:P1.4 22733 B:15:P1.4 22590 B:14:P1.7, 16 22644 C:15:P1.7, 16 22639 C:2a:P.5 20137 C:2b:P1.5, 2 19008 C:2a:P1.5, 2 20134 C:NT:P1.10 19456 Y:14:NST  19336* Y:NT:P1.5  19995* W:2a:P1.5, 2 19481 W:NT:P1.5 19836 W:NT:P1.6 19383 E:NT:P1.5, 2  19504* X:NT:P1.5, 2 24196 X:4:P1.12 24287 X:4:P1.16 23557 X:NT:P1.5 NT: Nontypeable, NST: Nonsubtypeable *Strains that were used for capsular polysaccharide purification

(11) Purification of the Capsular Polysaccharide from NmX

(12) The capsular polysaccharide of serogroup X (cpsX, see FIG. 4A for structural definition) 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 (18). Briefly, bacteria (1 L) at late-logarithmic phase of growth were formaldehyde-inactivated (1% v/v) and then treated with CETAVLON (0.1% w/v) (Sigma Aldrich, France). After centrifugation, the pellet was dissolved in cold aqueous CaCl.sub.2 (0.9M). The solubilised 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 (Na.sub.2HPO.sub.4, NaH.sub.2PO.sub.4, 0.2 M) 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 mg of the preparation were dissolved in 2 mL of phosphate buffer K.sub.2HPO.sub.4, KH.sub.2PO.sub.4 (0.05 M), pH 7, and purified by gel filtration on a Biosep-SEC-S3000 column (300×21.2 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 cut-off size of 10K-15K, and the residue was lyophilized. The yield was about 20 mg/L of culture. The profile of the purified cpsX was checked by proton nuclear magnetic resonance (.sup.1H NMR) (FIG. 4B) as previously described (19). CpsA, cpsB, cpsC, cpsY and cpsW were similarly purified from five strains of serogroups A, B, C, Y and W (strains 21524, 21721, 22639, 16366 and 19995 respectively, Table 1).

(13) Rabbit Immunization and Purification of Specific Anti-cpsX IgG Antibodies

(14) Two New Zealand White female rabbits (3 kg) were immunised intravenously three times with doses of 1 mL of a suspension of 10.sup.9 colony forming units (CFU) of freshly heat-inactivated Nmx strain 19504 (30 min at 56° C.), at day 0, 7 and 21. Sera were taken before immunization and at day 28 after the first injection to evaluate the immune response by ELISA (see below). Dot blotting with rabbit sera (1:1000 serum dilution) was performed using Amersham ECL kits (GE Healthcare Life Sciences Velizy-Villacoublay, France) as previously described (20). Rabbit immunisation 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. The inventors' laboratory has the administrative authorization for animal experimentation (Permit Number 75-1554) and the protocol was approved by the Institut Pasteur Review Board that is part of in 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's sera were passed through a HiTrap Protein G HP column (GE Healthcare, France) and eluted with glycine-HCl 0.1 M pH 2.7. Fractions of 1 mL were recovered in 50 μL of Tris-HCl buffer (1 M, pH 9). 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 manufacturer recommendations (Thermo Scientific, Rockford, Ill. 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 of purified cpsX or 100 μL of a bacterial suspension of 3.10.sup.8 bacteria/mL (NmX strain 19504). The purified antibodies (at a 500 μg/ml concentration) were tested against serial dilutions of bacteria from serogroup 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 2 μg/mL concentration.

(15) Production and Validation of a RDT Against NmX

(16) A one-step vertical flow immune-chromatography dipstick was set up using purified cpsX-pAbs that were conjugated to gold particles (British Biocell International, Cardiff, UK) as previously described (21). Unconjugated cpsX-pAbs were used as capture antibodies and goat anti-rabbit IgG (ICN Biomedicals, Aurora, Ohio, United States) were 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 centimeter respectively. For the test evaluation, dipsticks were dipped, for a 10-15 min period at room temperature, in 100 μL of PBS containing bacterial suspensions or CSF samples.

(17) Data Analysis

(18) Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) were calculated using a 2×2 contingency table. The positive likelihood ratios LR (LR.sup.+=Se/[1−Sp]) and the negative LR (LR.sup.−=[1−Se]/Sp), were also calculated (22). These values give an indication of the likelihood that the sample is positive or negative prior to testing. The diagnostic odds ratio (DOR), defined as the ratio of the odds of positive test results in specimens with NmX on the odds of positive test results in specimens negative for NmX, was calculated as follows DOR=(Se/[1−Se])/([1−Sp/Sp] (23). Finally, the Cohen's kappa (j) statistic was calculated to measure concordance between PCR and RDT (24). K may range from 0 to 1, and a j value higher than 0.8 is thought to reflecting almost perfect.

Example 2: Results and Discussion

(19) 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 bacteria detection was obtained with control pre-immune sera, a 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 were purified by affinity chromatography on a NmX cps activated column. Dot plot analysis of the purified IgG response against decreasing numbers of bacteria (from 5×10.sup.5 to 5×10.sup.3 cells per spot) from serogroups A, B, C, Y, W and X showed that antibodies only recognized serogroup X strain (FIG. 2A). The absence of recognition of the other serogroups (A, B, C, Y and VV) 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).

(20) A dipstick rapid diagnostic test for NmX was produced (see Material and Methods), and its detection limits were established. For the purified cpsX, this limit was 1 ng/mL (FIG. 2C) and was 10.sup.5 CFU/mL for NmX bacteria (strain 19504). The cut-off analysis was repeated 3 times with identical findings that were not affected by dipstick storage for 3 weeks at 25° C. The inventors also tested the RDT on a collection of bacterial suspension (Table 1) at 10.sup.6 CFU/mL. Only the serogroup X isolates were detectable.

(21) The detection limit of 1 ng CpsX/ml is similar to that of ELISA assays and lower than that of latex agglutination assays (10-100 ng CpsX/ml), explaining the higher specificities and sensitivities of RDTs compared with the agglutination kit.

(22) Use of the NmX Dipsticks on Clinical Samples

(23) The NmX dipstick was tested on a panel of 369 CSF selected from historical collections kept in National Reference Centre/Laboratory from four different countries, differing in terms of meningitis incidence (Cameroon, Côte d'lvoire, 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 only available for 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 S. agalactiae and 12 CSF samples were sterile by culture).

(24) Among these isolates, 52% (n=191) were positive for Nm, 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 Nm positive CSF, 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 Nm 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 positive for NmX by PCR, 86 were also positive by RDT. All the 26 CSF samples with culture data were tested negative by NmX-specific RDT.

(25) This validation under laboratory conditions took place during the epidemic season in the three laboratories located in countries of the meningitis belt. Therefore, the inventors took advantage of the epidemic season and tested the new NmX-specific RDT on all 153 CSF samples that were received in the three laboratories in Cameroon, Côte d'lvoire and Niger. No NmX was detected by PCR or by RDT in any of the samples. In contrast, several samples were positive by PCR for S. pneumoniae (14%), NmW (7%) and H. influenzae (3%).

(26) TABLE-US-00002 TABLE 2 Results of CSF samples obtained by PCR and by RDT Geographical origins CP IP Côte RDT PCR IP Paris CERMES Garoua d'Ivoire Total NmX.sup.+ NmX.sup.− NmA 6 15 6 0 27 0 27 NmB 6 0 0 2 8 0 8 NmC 7 0 0 0 7 0 7 NmY 2 0 0 0 2 0 2 NmW 6 10 4 18 38 0 38 NmX 7 80 5 0 92 86 6 Nm NG 0 0 16 1 17 0 17 S. 0 0 10 13 23 0 23 pneumoniae H. 0 0 1 3 4 0 4 influenzae S. 1 0 0 0 1 0 1 agalactiae Negative* 10 0 77 63 150 0 150 Total 45 105 119 100 369 86 283 *PCR Negative for N. meningitidis, S. pneumoniae and H. influenzae CSF: cerebrospinal fluid; RDT: Rapid Diagnostic test; IP, Institut Pasteur: CP: centre Pasteur; Nm: Neisseria meningitidis; NG: non groupable

(27) Performance of the NmX-Specific RDT: Sensitivity, Specificity, Likelihood Ratios, and Predictive Values

(28) RDT data showed a good correlation with PCR data, indicating a Kappa correlation coefficient of 98%. The sensitivity, specificity and 95% CI (confident interval) data 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%. Calculating the positive likelihood LR.sup.+ and DOR was not feasible due to a Sp value of 100%. LR.sup.+ and DOR values were therefore calculated using a value for the specificity that corresponded to the lower 95% confidence interval for specificity (0.99) (Table 3).

(29) TABLE-US-00003 TABLE 3 Performance of the RDT for NmX Test parameter Value 95% confidence interval Sensitivity (Se) 0.94 0.86 to 0.98 Specificity (Sp) 1 0.99 to 1   Positive Likelihood ratio (LH.sup.+)* 94  32 to 8252 Negative Likelihood ratio (LH.sup.−) 0.07 0.03 to 0.15 Positive predictive value (PPV) 1 0.96 to 1   Negative predictive value (NPV) 0.98 0.95 to 0.99 Diagnostic odd ratio (DOR)* 1567   379 to 118420 Dividing by zero; the values of LH.sup.+ and DOR were calculated using a value for specificity that corresponded to the lower 95% confidence interval (0.99).

(30) The prevalence of NmX among the 369 tested CSF 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 also vary across time and countries within the meningitis belt and elsewhere. We therefore calculated the negative and positive predictive values (NPV and PPV) according to a prevalence varying from 0 to 100%, using the Se and Sp obtained from the CSF samples in this study (FIG. 3).

DISCUSSION

(31) Reliable tests for the identification of cases of meningococcal meningitis and serogroup-determination are crucial to ensure proper individual (case-by-case) as well as collective management of cases and epidemiological surveillance. Culturing N. meningitidis may frequently fail due to early antibiotic treatment and fragility of this bacterial species (25). During the last two decades, PCR-based nonculture methods have been developed, enabling a significant improvement of the management and surveillance of bacterial meningitis (26). PCR-based methods require specific laboratory equipment and trained staff and can not be used as a bedside method (i.e. for physicians to make a decision on individual treatment). Nevertheless, the PCR technology was implemented in several reference laboratories located in countries within the African meningitis belt (26). However, PCR may not be sufficiently set to ensure country-wide surveillance, especially in populations leaving 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 temperature up to 45° C. at least. They are easy to use and to 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 a RDT test 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 positive test (Table 3). The inventors also evaluated its usefulness that 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, the 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 in 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 of 0.95 or higher) if this prevalence increased to 50%. These considerations seem realistic and reflect the current epidemiological situation in the meningitis belt after the introduction of MenAfriVac™ that was associated with significant decrease of NmA (9). Indeed, the small scale prospective use of the new RDT in the three centres located in this area (Abidjan, Garoua and Niamey), which is disclosed herein, suggests, on the basis of the sensitivities of RDT and PCR (that are less than 100%) that NmX may be present albeit not as a dominating pathogen. In contrast, NmW was the most frequently isolated Nm species, while most cases were associated to S. pneumoniae. However, a large-scale multi-site prospective study comparing PCR and all the available RDT (A, C, Y, W, Y and X) is warranted in the future. The new RDT described here will be crucial in vaccination decision making to implement large scale vaccination with the available broad serogroup coverage vaccine that can target NmX (5) or with NmX-specific vaccines under development (14).

Example 3: Detection of Meningococci Serogroup X in Urine and Serum Using the Serogroup X Dipstick

(32) Non-infected urine samples and serum samples were spiked with Neisseria meningitidis strain of serogroup X (LNP19504) at a final concentration of 10.sup.5 and 10.sup.6 CFU/ml; this technique is indeed frequently used for validation of kits for bacterial detection, including kits for N. meningitidis detection as the sensitivity data obtained with spiked samples are known to correlate well with sensitivity data obtained on corresponding bodily fluids of infected patients. These samples were then tested by the dipstick that has been previously described in example 2 to detect meningococcal serogroup X (see also 28).

(33) As shown in FIG. 5, serogroup X was detectable for both concentrations in the urine and no band was detectable in non infected urine. These data are similar to those obtained in CSF samples in example 2.

(34) In the serum, serogroup X was detected at the concentration of 10.sup.6 CFU/ml but not at 10.sup.5 CFU/ml suggesting lower sensitivity in serum.

(35) These results demonstrate the feasibility of diagnosing N. meningitidis infection in serum and urine samples of individuals to be tested.

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