Genotypic and Phenotypic Characterization of the O-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates.

Species within the genus Neisseria display significant glycan diversity associated with the O-linked protein glycosylation (pgl) systems due to phase variation and polymorphic genes and gene content. The aim of this study was to examine in detail the pgl genotype and glycosylation phenotype in meningococcal isolates and the changes occurring during short-term asymptomatic carriage. Paired meningococcal isolates derived from 50 asymptomatic meningococcal carriers, taken about 2 months apart, were analyzed with whole-genome sequencing. The O-linked protein glycosylation genes were characterized in detail using the Genome Comparator tool at the https://pubmlst.org/ database. Immunoblotting with glycan-specific antibodies (Abs) was used to investigate the protein glycosylation phenotype. All major pgl locus polymorphisms identified in Neisseria meningitidis to date were present in our isolate collection, with the variable presence of pglG and pglH, both in combination with either pglB or pglB2 We identified significant changes and diversity in the pgl genotype and/or glycan phenotype in 96% of the paired isolates. There was also a high degree of glycan microheterogeneity, in which different variants of glycan structures were found at a given glycoprotein. The main mechanism responsible for the observed differences was phase-variable expression of the involved glycosyltransferases and the O-acetyltransferase. To our knowledge, this is the first characterization of the pgl genotype and glycosylation phenotype in a larger strain collection. This report thus provides important insight into glycan diversity in N. meningitidis and into the phase variability changes that influence the expressed glycoform repertoire during meningococcal carriage.IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis, which is also a common commensal in the upper respiratory tract of healthy humans. In bacteria, numerous loci involved in biosynthesis of surface-exposed antigenic structures that are involved in the interaction between bacteria and host are frequently subjected to homologous recombination and phase variation. These mechanisms are well described in Neisseria, and phase variation provides the ability to change these structures reversibly in response to the environment. Protein glycosylation systems are becoming widely identified in bacteria, and yet little is known about the mechanisms and evolutionary forces influencing glycan composition during carriage and disease.

New molecular tools for meningitis diagnostics in Ethiopia - a necessary step towards improving antimicrobial prescription.

BACKGROUND: Meningitis remains a top cause of premature death and loss of disability-adjusted life years in low-income countries. In resource-limited settings, proper laboratory diagnostics are often scarce and knowledge about national and local epidemiology is limited. Misdiagnosis, incorrect treatment and overuse of antibiotics are potential consequences, especially for viral meningitis. METHODS: A prospective study was conducted over three months in a teaching hospital in Ethiopia with limited laboratory resources. Cerebrospinal fluid (CSF) samples from patients with suspected meningitis were analysed using a multiplex PCR-based system (FilmArray, BioFire), in addition to basic routine testing with microscopy and culture. Clinical data, as well as information on treatment and outcome were collected. RESULTS: Two hundred and eighteen patients were included; 117 (54%) neonates (0-29 days), 63 (29%) paediatrics (1 month-15 years) and 38 (17%) adults (≥16 years). Of 218 CSF samples, 21 (10%) were PCR positive; 4% in neonates, 14% in paediatrics and 18% in adults. Virus was detected in 57% of the PCR positive samples, bacteria in 33% and fungi in 10%. All CSF samples that were PCR positive for a bacterial agent had a white cell count ≥75 cells/mm3 and/or turbid appearance. The majority (90%) of patients received more than one antibiotic for treatment of the meningitis episode. There was no difference in the mean number of different antibiotics received or in the cumulative number of days with antibiotic treatment between patients with a microorganism detected in CSF and those without. CONCLUSIONS: A rapid molecular diagnostic system was successfully implemented in an Ethiopian setting without previous experience of molecular diagnostics. Viral meningitis was diagnosed for the first time in routine clinical practice in Ethiopia, and viral agents were the most commonly detected microorganisms in CSF. This study illustrates the potential of rapid diagnostic tests for reducing antibiotic usage in suspected meningitis cases. However, the cost of consumables for the molecular diagnostic system used in this study limits its use in low-income countries.

Whole genome sequencing reveals within-host genetic changes in paired meningococcal carriage isolates from Ethiopia.

BACKGROUND: Meningococcal colonization is a prerequisite for transmission and disease, but the bacterium only very infrequently causes disease while asymptomatic carriage is common. Carriage is highly dynamic, showing a great variety across time and space within and across populations, but also within individuals. The understanding of genetic changes in the meningococcus during carriage, when the bacteria resides in its natural niche, is important for understanding not only the carriage state, but the dynamics of the entire meningococcal population. RESULTS: Paired meningococcal isolates, obtained from 50 asymptomatic carriers about 2 months apart were analyzed with whole genome sequencing (WGS). Phylogenetic analysis revealed that most paired isolates from the same individual were closely related, and the average and median number of allelic differences between paired isolates defined as the same strain was 35. About twice as many differences were seen between isolates from different individuals within the same sequence type (ST). In 8%, different strains were detected at different time points. A difference in ST was observed in 6%, including an individual who was found to carry three different STs over the course of 9 weeks. One individual carried different strains from the same ST. In total, 566 of 1605 cgMLST genes had undergone within-host genetic changes in one or more pairs. The most frequently changed cgMLST gene was relA that was changed in 47% of pairs. Across the whole genome, pilE, differed mostly, in 85% of the pairs. The most frequent mechanisms of genetic difference between paired isolates were phase variation and recombination, including gene conversion. Different STs showed variation with regard to which genes that were most frequently changed, mostly due to absence/presence of phase variation. CONCLUSIONS: This study revealed within-host genetic differences in meningococcal isolates during short-term asymptomatic carriage. The most frequently changed genes were genes belonging to the pilin family, the restriction/modification system, opacity proteins and genes involved in glycosylation. Higher resolution genome-wide sequence typing is necessary to resolve the diversity of isolates and reveals genetic differences not discovered by traditional typing schemes, and would be the preferred choice of technology.

Prevalence and epidemiology of meningococcal carriage in Southern Ethiopia prior to implementation of MenAfriVac, a conjugate vaccine.

BACKGROUND: Neisseria meningitidis colonizes humans and transmits mainly by asymptomatic carriage. We sought to determine the prevalence and epidemiology of meningococcal carriage in Ethiopia prior to the introduction of MenAfriVac, a serogroup A meningococcal conjugate vaccine. METHODS: A cross-sectional meningococcal carriage study was conducted in Arba Minch, southern Ethiopia. A total of 7479 oropharyngeal samples were collected from 1 to 29 year old volunteers, between March and October, 2014. The swabs were cultured for N. meningitidis and Neisseria lactamica in Ethiopia. N. meningitidis isolates were confirmed and characterized by their serogroup, sequence type (ST) and PorA:FetA profile in Norway. RESULTS: Overall carriage prevalence was 6.6 %. There was no significant difference in overall carriage between male (6.7 %) and female (6.4 %) participants. Highest carriage prevalence (10.9 %) for females was found in the 15-19 years of age, while prevalence among males was highest (11.3 %) in the 20-24 age group. Non-groupable isolates dominated (76.4 %), followed by serogroups X (14.0 %) and W (5.9 %) isolates. No serogroup A was found. Most non-groupable isolates were ST-192. Serogroup W isolates were assigned to the ST-11 clonal complex, and serogroup X isolates to the ST-181 and ST-41/44 clonal complexes. Overall carriage prevalence of N. lactamica was 28.1 %. Carriage of N. meningitidis and N. lactamica varied depending on age and geographic area, but there was no association between carriage of the two species. CONCLUSIONS: Epidemic strains of serogroups W and X were circulating in this area of Ethiopia. As no serogroup A was found among the carriage isolates the immediate impact of mass-vaccination with MenAfriVac on transmission of N. meningitidis in this population is expected to be marginal.

Clinical Profile of Neonates Admitted with Sepsis to Neonatal Intensive Care Unit of Jimma Medical Center, A Tertiary Hospital in Ethiopia.

BACKGROUND: Globally, over 3 million newborn die each year, one million of these attributed to infections. The objective of this study was to determine the etiologies and clinical characteristics of sepsis in neonates admitted to intensive care unit of a tertiary hospital in Ethiopia. METHODS: A longitudinal hospital based cohort study was conducted from April 1 to October 31, 2018 at the neonatal intensive care unit of Jimma Medical Center, southwest Ethiopia. Diagnosis of sepsis was established using the World Health Organization's case definition. Structured questionnaires and case specific recording formats were used to capture the relevant data. Venous blood and cerebrospinal fluid from neonates suspected to have sepsis were collected. RESULTS: Out of 304 neonates enrolled in the study, 195 (64.1%) had clinical evidence for sepsis, majority (84.1%; 164/195) of them having early onset neonatal sepsis. The three most frequent presenting signs and symptoms were fast breathing (64.6%; 122/195), fever (48.1%; 91/195) and altered feeding (39.0%; 76/195). Etiologic agents were detected from the blood culture of 61.2% (115/195) neonates. Bacterial pathogens contributed for 94.8% (109/115); the rest being fungal etiologies. Coagulase negative staphylococci (25.7%; 28/109), Staphylococcus aureus (22.1%; 24/109) and Klebsiella species (16.5%; 18/109) were the most commonly isolated bacteria. CONCLUSION: Majority of the neonates had early onset neonatal sepsis. The major etiologies isolated in our study markedly deviate from the usual organisms causing neonatal sepsis. Multicentre study and continuous surveillance are essential to tackle the current challenge to reduce neonatal mortality due to sepsis in Ethiopia.

Development and Evaluation of a Multiplex Microsphere Assay for Quantitation of IgG and IgA Antibodies against Neisseria meningitidis Serogroup A, C, W, and Y Polysaccharides.

We developed and evaluated a rapid and simple multiplex microsphere assay for the quantification of specific IgG and IgA antibodies against meningococcal serogroup A, C, W, and Y capsular polysaccharides in serum and saliva. Meningococcal polysaccharides were conjugated to distinct magnetic carboxylated microspheres, and the performance of the assay was assessed using the CDC1992 standard meningococcal reference serum and a panel of serum and saliva samples. The standard curve was linear over an eight 3-fold dilution range in the IgG assay and a seven 3-fold dilution range in the IgA assay. No cross-reactivity was discovered, and the assay showed high specificity with ≥91% homologous inhibition and ≤11% heterologous inhibition for all serogroups and immunoglobulin classes. Lower limits of detections were ≤280 pg/ml for IgG and ≤920 pg/ml for IgA antibodies. The assay was reproducible, with a mean coefficient of variation of ≤5% for intra-assay duplicates, a mean coefficient of variation of ≤20% for interassay repeated analysis with different conjugations of microspheres, and a mean coefficient of variation within 25.8% for interoperator variation. The assay showed good correlation to the standard meningococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies. This multiplex assay is robust and reliable and requires less sample volume, and less time and workload are needed than for ELISA, making this method highly relevant for serological and salivary investigations on the effect of meningococcal vaccines and for immunosurveillance studies.