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Background: Meningococcal disease is caused by the bacterium Neisseria meningitidis, which is a commensal in the human upper airways. Teenagers and young adults are the main reservoir for the bacteria. The aim of this study was to assess the prevalence of meningococcal carriage and risk factors for carriage among high school and university students in Northwestern Ethiopia.

Methods: Oropharyngeal swabs and buccal samples were collected and stored on Whatman FTA-cards pending PCR analysis. DNA was extracted and a Multiplex TaqMan® custom assay was performed to detect N. meningitidis bacteria using the genes sodC and porA as species-specific targets. Genogrouping was performed for the samples positive for N. meningitidis using multiplex real-time PCR for the groups A, B, C, W, Y and X using a Rotor-Gene Q real-time PCR system.

Result: Out of the 1025 participants, 52% were female with a mean and median ages of 17 and 16 years, respectively. A total of 19 throat swab samples were positive for N. meningitidis giving an overall carriage rate of 1.9%. The carriage rate of N. meningitidis among high school students was 1.0% and 2.5% for the University students, respectively (p-value = 0.051). A significantly higher carriage rate was observed among participants with the age of 23 and above (p-value = 0.021). Among the N. meningitidis isolates, 16 out of 19 were non-groupable, while 3 were groupable. Of the groupable isolates, two belonged to group W and one to group B. Among the risk factors assessed, sharing a glass or bottle for drinking (86%), not having received a meningitis vaccine (66%), and having had a tonsillectomy (47%), were the most common risk factors. Among participants with positive meningococcal carriage, 89% of them have shared glass for drinking and 74% of them had not received previous meningococcal vaccine, while this figure is reduced to 66% and 65%, respectively among the non-carriers.

Conclusion: The overall meningococcal carriage rate was lower than those reported in previous Ethiopian studies, with age as the only identified risk factor.

BACKGROUND: Bacterial meningitis (BM) represents the most severe variant of meningitis, with a mortality rate that may reach up to 100% in the absence of appropriate treatment. The success of therapeutic interventions is depends upon prompt and precise diagnostic evaluations. However, there exists a significant deficiency in the literature regarding the diagnostic efficacy within the Ethiopian context. Consequently, this study aims to evaluate and compare the diagnostic precision of microbial culture and polymerase chain reaction (PCR) methodologies in individuals with suspected meningitis in Northwest Ethiopia.

METHODS: Cerebrospinal fluid (CSF) samples were procured from 400 patients who were clinically suspected of having meningitis and were admitted to the University of Gondar Specialized Hospital (UoGSH), located in Northwest Ethiopia. Real-time PCR, microbial culture, Gram staining, and cell enumeration were conducted at both the UoGSH laboratory and the Armauer Hansen Research Institute in Addis Ababa.

RESULTS: Of the total patients enrolled in the study, 58% were male. Clinical manifestations such as fever, headache, and neck stiffness were reported in 94%, 90%, and 81% of the patients, respectively, whereas altered consciousness was recorded in 37% of the cohort. The real-time PCR methodology identified 38 patients (10%) as positive for meningitis, in contrast to microbial culture, which detected only 10 (3%) of these positive cases. The two diagnostic modalities exhibited a correlation coefficient of 0.4 (p < 0.05).

CONCLUSION: The traditional microbiological culture technique, in conjunction with Gram staining, was found to have a limited sensitivity in identifying bacterial meningitis compared with the real-time PCR methodology. Consequently, the integration of molecular approaches with higher sensitivity, such as real-time PCR, facilitates prompt diagnosis and precise treatment, while simultaneously sustaining the overarching meningitis surveillance framework.

BACKGROUND: Neisseria meningitidis is a human pathogen, carried asymptomatically in the nasopharynx, that can also cause invasive meningococcal disease. Understanding the carriage/invasiveness balance is crucial, and bacterial genetic factors may impact this balance. A previous genome-wide association study reported that the gene porB class 3 was significantly associated with carriage isolates. This study aimed to examine the impact of porB variants on virulence in carriage and invasive meningococcal isolates.

RESULTS: For this, 24 isolates were used (13 invasive and 11 carriage) belonging to different genogroups (B, C, W, Y, and cnl) and selected based on the presence of the genetic variant porB class 2 or class 3. Transgenic BALB/c mice expressing human transferrin were infected intraperitoneally with these isolates. After 3 and 24 h of infection, clinical scores (fur quality, strength, and temperature) and bacterial load in blood were used to evaluate bacterial virulence. The concentrations of inflammatory cytokines were determined from blood. PorB- mutants were created from a carriage and an invasive isolate, and were tested in transgenic mice. The invasive isolates provoked significantly more severe infections compared to the carriage isolates, and the carriage isolates of porB class 3 were significantly less virulent than the invasive isolates of porB class 2 or 3. The invasive PorB- isolate caused milder infections than the parental isolate.

CONCLUSIONS: This study confirms the ability of invasive isolates of N. meningitidis to cause more severe infections than carriage isolates in transgenic mice. The porB expressed in invasive isolates seems to contribute to their higher virulence compared to carriage isolates, although this effect may depend on the genomic context. Notably, differences in virulence were mainly observed among serogroup C and W isolates.

BACKGROUND: The severity of infectious disease outcomes is dependent on the virulence factors of the pathogen and the host immune response. CARD8 is a major regulator of the innate immune proinflammatory response and has been suggested to modulate the host response to common inflammatory diseases. In the present study, the C10X genetic polymorphism in the CARD8 gene was investigated in relation to bacterial meningitis.

METHODS: A total of 400 clinically suspected meningitis patients hospitalized at the University of Gondar Hospital were enrolled in the study. Cerebrospinal fluid (CSF) and blood samples were collected for laboratory investigations. The collected CSF was cultured, and all the results obtained from the culture were confirmed using direct RT‒PCR. Genotyping of whole-blood samples was performed using a TaqMan assay. The results were compared with apparently healthy controls and with PCR-negative meningitis suspected patients.

RESULTS: Of the included patients, 57% were men and the most common clinical signs and symptoms were fever (81%), headache (80%), neck stiffness (76%), nausea (68%), and vomiting (67%). Microbiology culture identified 7 patients with bacterial meningitis caused by Neisseria meningitidis (n = 4) and Streptococcus pneumoniae (n = 3). The RT-PCR revealed 39 positive samples for N. meningitidis (n = 10) and S. pneumoniae (n = 29). A total of 332 whole-blood samples were genotyped with the following results: 151 (45.5%) C10X heterozygotes, 59 (17.7%) C10X homozygotes and 122 (36.7%) wild genotypes. The polymorphic gene carriers among laboratory confirmed, clinically diagnosed meningitis and healthy controls were 23(46%), 246(40%), and 1526(39%), respectively with OR = 1.27 (0.7-2.3) and OR = 1.34 (0.76-2.4). The presence of the C10X polymorphism in the CARD8 gene was more prevalent in suspected meningitis patients than in healthy controls (OR 1.2; 1.00-1.5). Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease. The presence of viable or active bacterial infection was found to be associated with the presence of heterozygous C10X carriers.

CONCLUSIONS: A greater proportion of C10X in the CARD8 gene in confirmed bacterial meningitis patients and clinically diagnosed meningitis patients than in healthy controls. Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease than heterozygote gene carriers and healthy controls.

Neisseria meningitidis can be a human commensal in the upper respiratory tract but is also capable of causing invasive diseases such as meningococcal meningitis and septicaemia. No specific genetic markers have been detected to distinguish carriage from disease isolates. The aim here was to find genetic traits that could be linked to phenotypic outcomes associated with carriage versus invasive N. meningitidis disease through a bacterial genome-wide association study (GWAS). In this study, invasive N. meningitidis isolates collected in Sweden (n=103) and carriage isolates collected at Örebro University, Sweden (n=213) 2018-2019 were analysed. The GWAS analysis, treeWAS, was applied to single-nucleotide polymorphisms (SNPs), genes and k-mers. One gene and one non-synonymous SNP were associated with invasive disease and seven genes and one non-synonymous SNP were associated with carriage isolates. The gene associated with invasive disease encodes a phage transposase (NEIS1048), and the associated invasive SNP glmU S373C encodes the enzyme N-acetylglucosamine 1-phosphate (GlcNAC 1-P) uridyltransferase. Of the genes associated with carriage isolates, a gene variant of porB encoding PorB class 3, the genes pilE/pilS and tspB have known functions. The SNP associated with carriage was fkbp D33N, encoding a FK506-binding protein (FKBP). K-mers from PilS, tbpB and tspB were found to be associated with carriage, while k-mers from mtrD and tbpA were associated with invasiveness. In the genes fkbp, glmU, PilC and pilE, k-mers were found that were associated with both carriage and invasive isolates, indicating that specific variations within these genes could play a role in invasiveness. The data presented here highlight genetic traits that are significantly associated with invasive or carriage N. meningitidis across the species population. These traits could prove essential to our understanding of the pathogenicity of N. meningitidis and could help to identify future vaccine targets.

The bacterium Neisseria meningitidis causes life-threatening disease worldwide, typically with a clinical presentation of sepsis or meningitis, but can be carried asymptomatically as part of the normal human oropharyngeal microbiota. The aim of this study was to examine N. meningitidis carriage with regard to prevalence, risk factors for carriage, distribution of meningococcal lineages and persistence of meningococcal carriage. Throat samples and data from a self-reported questionnaire were obtained from 2744 university students (median age: 23 years) at a university in Sweden on four occasions during a 12-month period. Meningococcal isolates were characterised using whole-genome sequencing. The carriage rate among the students was 9.1% (319/3488; 95% CI 8.2-10.1). Factors associated with higher carriage rate were age ≤22 years, previous tonsillectomy, cigarette smoking, drinking alcohol and attending parties, pubs and clubs. Female gender and sharing a household with children aged 0-9 years were associated with lower carriage. The most frequent genogroups were capsule null locus (cnl), group B and group Y and the most commonly identified clonal complexes (cc) were cc198 and cc23. Persistent carriage with the same meningococcal strain for 12 months was observed in two students. Follow-up times exceeding 12 months are recommended for future studies investigating long-term carriage of N. meningitidis.

Infections in the central nervous system (CNS) include meningitis and encephalitis and are associated with high mortality and morbidity. A large number of different pathogens can cause these infections, including Neisseria meningitidis. It’s crucial to find the causative pathogen in order to provide the best treatment to the patient and for disease surveillance. 

In Paper I, molecular methods were used to investigate the microbial etiology in patients presenting with CNS infections at United Mission Hospital in Tansen, Nepal. Although the cerebrospinal fluid samples were analyzed for a large number of microbes using two commercial multiplex PCR panels and additional in-house real-time PCR, the etiology of the infections was still unknown in a large number of patients. This calls for further development of diagnostic methods for CNS infections.

Neisseria meningitidis, the meningococcus, is a strictly human commensal but also capable to cause severe disease, typically in the form of sepsis and meningitis. The aim of Paper II and III was to study the clinical presentation of N. meningitidis serogroup Y and W infections, which increased unexpectedlyin Sweden from 2007 and 2014, respectively. By reviewing medical records of these infection episodes, the conclusion was drawn that atypical presentations with respiratory and gastrointestinal symptoms were common, rather than meningitis and petechiae.

In Paper IV, meningococcal carriage was studied among students at Örebro University. Age ≤22 years, smoking, previous tonsillectomy, frequent partying and male gender were associated with higher carrier rates. The so far longest observation of carriage of the same meningococcal strain was presented, with a duration of at least one year.

In conclusion, the results from these studies highlight the importance of early detection of meningococcal infections with atypical presentations and the need of improved diagnostics for CNS infections

Since 2015, the incidence of invasive meningococcal disease (IMD) caused by serogroup W (MenW) has increased in Sweden, due to the introduction of the 2013 strain belonging to clonal complex 11. The aim of this study was to describe the clinical presentation of MenW infections, in particular the 2013 strain, including genetic associations. Medical records of confirmed MenW IMD cases in Sweden during the years 1995-2019 (n = 113) were retrospectively reviewed and the clinical data analysed according to strain. Of all MenW patients, bacteraemia without the focus of infection was seen in 44%, bacteraemic pneumonia in 26%, meningitis in 13% and epiglottitis in 8%, gastrointestinal symptoms in 48% and 4% presented with petechiae. Phylogenetic analysis was used for possible links between genetic relationship and clinical picture. The 2013 strain infections, particularly in one cluster, were associated with more severe disease compared with other MenW infections. The patients with 2013 strain infections (n = 68) were older (52 years vs. 25 years for other strains), presented more often with diarrhoea as an atypical presentation (P = 0.045) and were more frequently admitted for intensive care (P = 0.032). There is a risk that the atypical clinical presentation of MenW infections, with predominantly gastrointestinal or respiratory symptoms rather than neck stiffness or petechiae, may lead to delay in life-saving treatment.

BACKGROUND: Neisseria meningitidis serogroups W and Y are the most common serogroups causing invasive meningococcal disease in Sweden. The majority of cases are caused by the serogroup W UK 2013 strain of clonal complex (cc) 11, and subtype 1 of the serogroup Y, YI strain of cc23. In this study, virulence factors of several lineages within cc11 and cc23 were investigated in transgenic BALB/c mice expressing human transferrin. Transgenic mice were infected intraperitoneally with serogroup W and Y isolates. Levels of bacteria and the proinflammatory cytokine CXCL1 were determined in blood collected 3 h and 24 h post-infection. Apoptosis was investigated in immune cells from peritoneal washes of infected mice. Adhesion and induction of apoptosis in human epithelial cells were also scored.

RESULTS: The levels of bacteraemia, CXCL1, and apoptosis were higher in serogroup W infected mice than in serogroup Y infected mice. Serogroup W isolates also induced higher levels of apoptosis and adhesion in human epithelial cells. No significant differences were observed between different lineages within cc11 and cc23.

CONCLUSIONS: N. meningitidis Serogroup W displayed a higher virulence in vivo in transgenic mice, compared to serogroup Y. This was reflected by higher bacteremia, proinflammatory activity, and ability to induce apoptosis in mouse immune cells and human epithelial cells.