A protocol for a systematic review of the diagnostic accuracy of Loop-mediated-isothermal AMPlification (LAMP) in diagnosis of invasive meningococcal disease in children.

BACKGROUND: Meningococcal disease (MD) is notoriously difficult to diagnose in the early stages of the illness and presents similarly to many self-limiting viral infections. This mandates a cautious approach to diagnosis and initial management of suspected MD with many children receiving precautionary broad-spectrum intravenous antibiotics. Despite this approach, some children are still diagnosed late. In the last 10 years, there have been advances in nucleic acid amplification techniques, and there is now a rapid test that can detect meningococcal DNA in under 30 min. This Loop-mediated-isothermal AMPlification (LAMP) technology may make it possible to diagnose MD at initial presentation thereby greatly improving outcomes and minimising harms through unnecessary treatment. The aim of this systematic review is to determine the diagnostic accuracy of LAMP technology in cases of suspected MD. The review has been registered with PROSPERO [CRD42017078026]. METHODS: To identify relevant studies, we will search MEDLINE, Embase, Web of Science, Scopus and The Cochrane Library. In additional, we will hand-search reference lists and grey literature including contacting the manufacturers of commercially available LAMP tests for MD for any unpublished data. Two reviewers will independently screen study eligibility and extract data. Methodological quality will be assessed, by two authors, according to the revised tool for the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2); any discrepancies will be resolved by a third author. The following test characteristics will be extracted into 2 × 2 tables for all included studies: true positives, false positives, true negatives, and false negatives. Study-specific estimates of sensitivity and specificity with 95% confidence intervals will be displayed in forest plots. To investigate heterogeneity, we will include covariates such as age, sample type, and study type into a bivariate random-effects model. DISCUSSION: This review will help determine the diagnostic accuracy of LAMP technology in diagnosing MD from blood, CSF and throat swabs in children. The data will help to define where in the diagnostic pathway LAMP could be useful including potential as a point-of-care test for children at first presentation.

Epitope imprinting of iron binding protein of Neisseria meningitidis bacteria through multiple monomers imprinting approach.

Epitope imprinting is a promising technique for fabrication of novel diagnostic tools. In this study, an epitope imprinted methodology for recognition of target epitope sequence as well as targeted protein infused by bacterial infection in blood samples of patients suffering from brain fever is developed. Template sequence chosen is a ferric iron binding fbp A protein present in Neisseria meningitidis bacteria. To orient the imprinting template peptide sequence on gold surface of electrochemical quartz crystal microbalance (EQCM), thiol chemistry was utilized to form the self-assembled monolayer on EQCM electrode. Here, synergistic effects induced by various noncovalent interactions extended by multiple monomers (3-sulfopropyl methacrylate potassium-salt and benzyl methacrylate) were used in fabricating the imprinting polymeric matrix with additional firmness provided by N,N-methylene-bis-acrylamide as cross-linker and azo-isobutyronitrile as initiator. Extraction of template molecule was carried out with phosphate buffer solution. After extraction of epitope molecules from the polymeric film, epitope molecularly imprinted polymeric films were fabricated on EQCM electrode surface. Nonimprinted polymers were also synthesized in the similar manner without epitope molecule. Detection limit of epitope molecularly imprinted polymers and imprinting factor (epitope molecularly imprinted polymers/nonimprinted polymers) was calculated 1.39 ng mL-1 and 12.27 respectively showing high binding capacity and specific recognition behavior toward template molecule. Simplicity of present method would put forward a fast, facile, cost-effective diagnostic tool for mass health care.

Meningococcal meningitis with neurological complications and meningococcemia due to serogroup W sequence type 11 complex.

Invasive meningococcal disease (IMD) caused by the serogroup W (MenW) sequence type-11 complex strain has recently emerged worldwide. Meningococcal infections due to this strain are associated with high case fatality and often atypical clinical manifestations. However, the annual IMD incidence was low, and MenW is rare in Japan. We described the first Japanese case of meningococcal meningitis and meningococcemia caused by this strain in a previously healthy 27-year-old woman. This case showed various neurological complications such as abducens palsy, cerebellitis, and cerebellar infarction, and reactive arthritis. This case provides useful information on the possibility of spreading IMD strains and the cause of various complications.

Evidence for Rise in Meningococcal Serogroup C Bactericidal Antibody Titers in the Absence of Booster Vaccination in Previously Vaccinated Children.

BACKGROUND: The introduction of meningococcal serogroup C (MenC) conjugate vaccines in the United Kingdom and Australia led to an impressive decline in the incidence of invasive disease. This study examined bactericidal antibody titers over time in the UK and Australian children who received a MenC conjugate vaccine in early childhood to test the hypothesis that ongoing boosting of immunity in the absence of further doses of vaccine in some children may contribute to ongoing protection from disease. METHODS: Serum bactericidal assay using rabbit complement (rSBA) titers at each follow-up visit were compared with all preceding visits to identify any ≥4-fold rise in titers. The proportion of children with a ≥4-fold rise in rSBA titers in paired sera at any visit-to-visit comparison was calculated. RESULTS: Of 392 children with at least one set of paired sera in the Australian cohort, 72 (18.4%) had a ≥4-fold increase in rSBA titers at least one year after vaccination, including six children (1.5%) who showed evidence of boosting twice. Of 234 children with at least one set of paired sera in the UK cohort, 39 (16.7%) had a ≥4-fold rise in rSBA titers at least one year after vaccination including 2 children (0.9%) with evidence of boosting twice. CONCLUSIONS: A substantial minority of children immunized with MenC conjugate vaccine in early childhood had a rise in bactericidal antibody titers in the years after immunization in the absence of booster vaccination. This occurs most commonly at around 6-7 years of age corresponding to school entry and greater social mixing and might indicate exposure to MenC carriage.

Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence.

BACKGROUND: Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence. RESULTS: Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region. CONCLUSIONS: Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.

Large scale genomic analysis shows no evidence for pathogen adaptation between the blood and cerebrospinal fluid niches during bacterial meningitis.

Recent studies have provided evidence for rapid pathogen genome diversification, some of which could potentially affect the course of disease. We have previously described such variation seen between isolates infecting the blood and cerebrospinal fluid (CSF) of a single patient during a case of bacterial meningitis. Here, we performed whole-genome sequencing of paired isolates from the blood and CSF of 869 meningitis patients to determine whether such variation frequently occurs between these two niches in cases of bacterial meningitis. Using a combination of reference-free variant calling approaches, we show that no genetic adaptation occurs in either invaded niche during bacterial meningitis for two major pathogen species, Streptococcus pneumoniae and Neisseria meningitidis. This study therefore shows that the bacteria capable of causing meningitis are already able to do this upon entering the blood, and no further sequence change is necessary to cross the blood-brain barrier. Our findings place the focus back on bacterial evolution between nasopharyngeal carriage and invasion, or diversity of the host, as likely mechanisms for determining invasiveness.

Circadian Variation of Plasminogen-Activator-Inhibitor-1 Levels in Children with Meningococcal Sepsis.

OBJECTIVE: To study whether the circadian variation of plasminogen-activator-inhibitor-1 (PAI-1) levels, with high morning levels, is associated with poor outcome of children with meningococcal sepsis presenting in the morning hours. DESIGN: Retrospective analysis of prospectively collected clinical and laboratory data. SETTING: Single center study at Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands. SUBJECTS: 184 patients aged 3 weeks to 18 years with meningococcal sepsis. In 36 of these children, PAI-1 levels at admission to the PICU were measured in plasma by ELISA. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Circadian variation was studied by dividing one day in blocks of 6 hours. Patients admitted between 6:00 am and 12:00 am had increased illness severity scores and higher PAI-1 levels (n = 9, median 6912 ng/mL, IQR 5808-15600) compared to patients admitted at night (P = 0.019, n = 9, median 3546 ng/mL, IQR 1668-6118) or in the afternoon (P = 0.007, n = 7, median 4224 ng/mL, IQR 1804-5790). In 184 patients, analysis of circadian variation in relation to outcome showed more deaths, amputations and need for skin grafts in patients admitted to the PICU between 6:00 am and 12:00 am than patients admitted during the rest of the day (P = 0.009). CONCLUSIONS: Circadian variation of PAI-1 levels is present in children with meningococcal sepsis and is associated with illness severity, with a peak level in the morning. Whether circadian variation is an independent risk factor for morbidity and mortality in meningococcal sepsis needs to be explored in future studies.

A Seroepidemiological Study of Serogroup A Meningococcal Infection in the African Meningitis Belt.

The pattern of epidemic meningococcal disease in the African meningitis belt may be influenced by the background level of population immunity but this has been measured infrequently. A standardised enzyme-linked immunosorbent assay (ELISA) for measuring meningococcal serogroup A IgG antibodies was established at five centres within the meningitis belt. Antibody concentrations were then measured in 3930 individuals stratified by age and residence from six countries. Seroprevalence by age was used in a catalytic model to determine the force of infection. Meningococcal serogroup A IgG antibody concentrations were high in each country but showed heterogeneity across the meningitis belt. The geometric mean concentration (GMC) was highest in Ghana (9.09 µg/mL [95% CI 8.29, 9.97]) and lowest in Ethiopia (1.43 µg/mL [95% CI 1.31, 1.57]) on the margins of the belt. The force of infection was lowest in Ethiopia (λ = 0.028). Variables associated with a concentration above the putative protective level of 2 µg/mL were age, urban residence and a history of recent vaccination with a meningococcal vaccine. Prior to vaccination with the serogroup A meningococcal conjugate vaccine, meningococcal serogroup A IgG antibody concentrations were high across the African meningitis belt and yet the region remained susceptible to epidemics.

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage.

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.

Thrombin-activatable fibrinolysis inhibitor influences disease severity in humans and mice with pneumococcal meningitis.

BACKGROUND: Mortality and morbidity in patients with bacterial meningitis result from the proinflammatory response and dysregulation of coagulation and fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is activated by free thrombin or thrombin in complex with thrombomodulin, and plays an antifibrinolytic role during fibrin clot degradation, but also has an anti-inflammatory role by inactivating proinflammatory mediators, such as complement activation products. OBJECTIVE: To assess the role of TAFI in pneumococcal meningitis. METHODS: We performed a prospective nationwide genetic association study in patients with bacterial meningitis, determined TAFI and complement levels in cerebrospinal fluid (CSF), and assessed the function of TAFI in a pneumococcal meningitis mouse model by using Cpb2 (TAFI) knockout mice. RESULTS: Polymorphisms (reference sequences: rs1926447 and rs3742264) in the CPB2 gene, coding for TAFI, were related to the development of systemic complications in patients with pneumococcal meningitis. Higher protein levels of TAFI in CSF were significantly associated with CSF complement levels (C3a, iC3b, and C5b-9) and with more systemic complications in patients with bacterial meningitis. The risk allele of rs1926447 (TT) was associated with higher levels of TAFI in CSF. In the murine model, consistent with the human data, Cpb2-deficient mice had decreased disease severity, as reflected by lower mortality, and attenuated cytokine levels and bacterial outgrowth in the systemic compartment during disease, without differences in the brain compartment, as compared with wild-type mice. CONCLUSIONS: These findings suggest that TAFI plays an important role during pneumococcal meningitis, which is likely to be mediated through inhibition of the complement system, and influences the occurrence of systemic complications and inflammation.

The impact of aggregating serogroups in dynamic models of Neisseria meningitidis transmission.

BACKGROUND: Neisseria meningitidis (Nm) is a pathogen of multiple serogroups that is highly prevalent in many populations. Serogroups associated with invasive meningococcal disease (IMD) in Canada, for example, include A, B, C, W-135, X and Y. IMD is a rare but serious outcome of Nm infection, and can be prevented with vaccines that target certain serogroups. This has stimulated the development of dynamic models to evaluate vaccine impact. However, these models typically aggregate the various Nm serogroups into a small number of combined groups, instead of modelling each serogroup individually. The impact of aggregation on dynamic Nm model predictions is poorly understood. Our objective was to explore the impact of aggregation on dynamic model predictions. METHODS: We developed two age-structured agent-based models--a 2-strain model and a 4-strain model--to simulate vaccination programs in the Canadian setting. The 2-strain model was used to explore two different groupings: C, versus all other serogroups combined; and B, versus all other serogroups combined. The 4-strain model used the four groupings: C, B, Neisseria lactamica, versus all other serogroups combined. We compared the predicted impact of monovalent C vaccine, quadrivalent ACWY vaccine (MCV-4), and monovalent B vaccine (4CMenB) on the prevalence of serogroup carriage under these different models. RESULTS: The 2-strain and 4-strain models predicted similar overall impacts of vaccines on carriage prevalence, especially with respect to the vaccine-targeted serogroups. However, there were some significant quantitative and qualitative differences. Declines in vaccine-targeted serogroups were more rapid in the 2-strain model than the 4-strain model, for both the C and the 4CMenB vaccines. Sustained oscillations, and evidence for multiple attractors (i.e., different types of dynamics for the same model parameters but different initial conditions), occurred in the 4-strain model but not the 2-strain model. Strain replacement was also more pronounced in the 4-strain model, on account of the 4-strain model spreading prevalence more thinly across groups and thus enhancing competitive interactions. CONCLUSIONS: Simplifying assumptions like aggregation of serogroups can have significant impacts on dynamic model predictions. Modellers should carefully weigh the advantages and disadvantages of aggregation when formulating models for multi-strain pathogens.

Antibody persistence in Australian adolescents following meningococcal C conjugate vaccination.

BACKGROUND: In Australia, following the introduction of serogroup C meningococcal (MenC) conjugate vaccine for toddlers and catch-up immunization through adolescence, MenC disease incidence plummeted and remains low. However, individual protection following MenC conjugate vaccination, particularly in young children, may be short-lived. We investigated the persistence of MenC serum bactericidal antibody (SBA) titers in adolescents, more than 7 years after a single "catch-up" dose of MenC conjugate vaccine. We also investigated their exposure and susceptibility to meningococcal serogroups A, W and Y. METHODS: MenC SBA titers and Men A, C, W and Y IgG geometric mean concentration were measured in 240 healthy 11- to 16-year-old adolescents. The correlate of protection was an rSBA titer of ≥8. RESULTS: An rSBA≥8 was observed in 105 [44% (95% confidence interval {CI}, 37-50%)] of 240 adolescents (mean age, 13.2 years, mean interval since MenC immunization, 8.2 years). The proportion with an rSBA≥8, geometric mean rSBA titer and geometric mean IgG concentration increased with age, from 22% to 75%, 3.7 to 33.4 and 0.13 to 0.52 µg/mL, in participants who received MenC vaccine at mean age 2.8 to 7.5 years, respectively. Natural acquired antibody to Men A, W and Y was low with IgG geometric mean concentrations of 1.26, 0.38 and 0.47 µg/mL, respectively. CONCLUSIONS: More than half of Australian adolescents have inadequate serological protection against MenC disease and low natural immunity to MenA, W and Y.

A complement C5 gene mutation, c.754G>A:p.A252T, is common in the Western Cape, South Africa and found to be homozygous in seven percent of Black African meningococcal disease cases.

Patients with genetically determined deficiency of complement component 5 are usually diagnosed because of recurrent invasive Neisseria meningitidis infections. Approximately 40 individual cases have been diagnosed worldwide. Nevertheless, reports of the responsible genetic defects have been sporadic, and we know of no previous reports of C5 deficiency being associated with a number of independent meningococcal disease cases in particular communities. Here we describe C5 deficiency in seven unrelated Western Cape, South African families. Three different C5 mutations c.55C>T:p.Q19X, c.754G>A:p.A252T and c.4426C>T:p.R1476X were diagnosed in index cases from two families who had both presented with recurrent meningococcal disease. p.Q19X and p.R1476X have already been described in North American Black families and more recently p.Q19X in a Saudi family. However, p.A252T was only reported in SNP databases and was not associated with disease until the present study was undertaken in the Western Cape, South Africa. We tested for p.A252T in 140 patients presenting with meningococcal disease in the Cape Town area, and found seven individuals in five families who were homozygous for the mutation p.A252T. Very low serum C5 protein levels (0.1-4%) and correspondingly low in vitro functional activity were found in all homozygous individuals. Allele frequencies of p.A252T in the Black African and Cape Coloured communities were 3% and 0.66% and estimated homozygosities are 1/1100 and 1/22,500 respectively. In 2012 we reported association between p.A252T and meningococcal disease. Molecular modelling of p.A252T has indicated an area of molecular stress in the C5 molecule which may provide a mechanism for the very low level in the circulation. This report includes seven affected families indicating that C5D is not rare in South Africa.

[POSSIBILITY OF OBTAINING ALLOGENEIC ANTIMENINGOCOCCUS FRESH FROZEN PLASMA AND CONTROL OF ITS QUALITY INDICATORS].

Results of the study identifying the titer of natural antimeningococcus antibodies serogroup B of non-immunized donor population of Zhytomyr Region aged 18-54 years, all AB0 blood groups are presented for further use of these results in the organization of the immune donation. Indicators of quality of antimeningococcus fresh frozen plasma obtained by different methods are characterized.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Investigating the immunodominance of carbohydrate antigens in a bivalent unimolecular glycoconjugate vaccine against serogroup A and C meningococcal disease.

Multicomponent constructs, obtained by coupling different glycans to the carrier protein, have been proposed as a way to co-deliver multiple surface carbohydrates targeting different strains of one pathogen and reduce the number of biomolecules in the formulation of multivalent vaccines. To assess the feasibility of this approach for anti-microbial vaccines and investigate the potential immunodominance of one carbohydrate antigen over the others in these constructs, we designed a bivalent unimolecular vaccine against serogroup A (MenA) and C (MenC) meningococci, with the two different oligomers conjugated to same molecule of carrier protein (CRM197). The immune response elicited in mice by the bivalent MenAC construct was compared with the ones induced by the monovalent MenA and MenC vaccines and their combinations. After the second dose, the bivalent construct induced good levels of anti-MenA and anti-MenC antibodies with respect to the controls. However, the murine sera from the MenAC construct exhibited good anti-MenC bactericidal activity, and very low anti-MenA functionality when compared to the monovalent controls. This result was explained with the diverse relative avidities against MenA and MenC polysaccharides, which were measured in the generated sera. The immunodominant effect of the MenC antigen was fully overcome following the third immunization, when sera endowed with higher avidity and excellent bactericidal activity against both MenA and MenC expressing strains were elicited. Construction of multicomponent glycoconjugate vaccines against microbial pathogens is a feasible approach, but particular attention should be devoted to study and overcome possible occurrence of immune interference among the carbohydrates.

Non-activated plasma-derived PC improves amputation rate of children undergoing sepsis.

Low circulating protein C (PC) levels have been observed in sepsis, especially in patients with Neisseriae Meningitides infections. Poor clinical outcome and high limb amputation rates have been associated in infected patients with low circulating PC levels. Published studies using activated PC replacement therapy patients with sepsis have shown reduced mortality rates, however, its use has been associated with severe bleeding events. Paediatric sepsis studies using non-activated plasma-derived PC (Ceprotin®) are lacking. We present a retrospective study in children with sepsis who were treated with Ceprotin® focusing on amputation rate post treatment. Thirty subjects were identified. Median age at diagnosis was 2 years. Twenty-one (70%) were treated for Nesseria Meningitides and one (3%) for Streptococcus-A ß-haemolyticus, another 8 (26%) patients with malignancies were treated for neutropenic sepsis. Following Ceprotin® administration, a significant increase in leukocyte count (p=0.004), neutrophil count (p=0.001) and PC (pretreatment=13%, posttreatment=88.5%; p=0.0001) was seen. Prothrombin time (pretreatment =30.3 seconds, posttreatment =16.5; p=0.000) and activated partial thromboplastin time (pretreatment =61.8 sec, postreatment =42.6 sec; p=0.000) were significantly reduced, while fibrinogen levels were significantly elevated (pretreatment =1.9 g/dL, posttreatment =4.4 g/dL; p=0.000). The median time between admission to intensive care and Ceprotin® administration was 10 hrs. Limb amputation rate was reduced (16-23% versus 30-50% from previous studies) and there were no haemorrhagic events observed. This study demonstrates the safe administration of non-activated plasma-derived PC concentrate in patients with sepsis who are coagulopathic and it associated with a reduction in amputation rates.

Avidity of IgG antibodies against meningococcal serogroup a polysaccharide and correlations with bactericidal activity in sera from meningitis patients and controls from Ethiopia.

Meningococcal meningitis is a significant global health challenge, especially for sub-Saharan area: the African meningitis belt. Neisseria meningitidis of serogroup A (MenA) is responsible for the large number of epidemics that have been recorded in these countries. To determine the level of antibodies against meningococcal A polysaccharide (APS) that correlates with protection against MenA disease in the African meningitis belt, it may be important to consider antibody avidity along with quantity. In this study, two ELISA methods using the chaotropic agent ammonium thiocyanate were compared and employed to measure avidity indexes (AI) of IgG antibodies against APS in controls and in acute and convalescent sera from Ethiopian meningococcal patients. High statistical correlations between the AIs determined by the two methods were observed. The geometric mean AI (GMAI) increased with time from acute to convalescent sera indicating affinity maturation. GMAI was significantly higher in convalescent sera from the MenA patients and in sera from the controls than in acute sera from patients with meningococcal disease. A significant correlation between serum bactericidal activity titres (SBA) and concentration of IgG antibodies against APS was observed; however, our results did not indicate that determination of antibody avidities by the thiocyanate elution method gave a better correlation with SBA than anti-APS IgG concentrations determined by the standard ELISA method.

Microparticle-associated tissue factor activity correlates with plasma levels of bacterial lipopolysaccharides in meningococcal septic shock.

INTRODUCTION: The plasma level of bacterial lipopolysaccharides (LPS) is associated with activation of the coagulation system, inhibition of fibrinolysis and the nature of the clinical presentation and outcome in patients with meningococcal disease. Tissue factor (TF)-bearing microparticles (MPs) appear to contribute to the pathogenesis of disseminated intravascular coagulation (DIC). The aim of this study was to investigate the relationship between MP-associated TF activity and the level of bacterial LPS in plasma from patients with meningococcal septic shock and meningitis. MATERIALS AND METHODS: MPs isolated from citrated plasmas were assessed for TF-dependent activity with both a plasma-based thrombin generation assay (CAT) and whole blood-based thromboelastometry (ROTEM). The LPS level was measured using a chromogenic Limulus amebocyte lysate assay. RESULTS: MPs obtained from patients with meningococcal septic shock initiated significantly more efficient and TF-dependent thrombin generation in the CAT assay compared to MPs from patients with meningococcal meningitis. Differences in MP-associated TF activity between the septic shock patients and the meningitis patients were also evident when MPs were added to whole blood using ROTEM. The level of plasma LPS in patients with septic shock (range 2-2,100 EU/mL) was correlated with thrombogram parameters in the CAT assay; lagtime (r(s)=-0.84), time to peak (rs=-0.83), peak (r(s)=0.85) and ETP (r(s)=0.83). CONCLUSIONS: MPs obtained from patients with meningococcal septic shock displayed more efficient TF-dependent thrombin generation and clot formation compared to MPs from meningitis patients. MP-associated TF activity was closely associated with plasma LPS levels in the septic shock group.