Navigating the infodemic: A qualitative study of university students' information strategies during the COVID-19 pandemic.

Objectives: We aimed to study the strategies which university students developed for vetting information during the COVID-19 pandemic and associated infodemic. Methods: We conducted semi-structured interviews with 34 students, using a piloted topic guide which explored several areas of pandemic experiences, including students' use of media. Transcripts were analysed inductively following the thematic approach. Higher order themes were finalised following a coding exercise undertaken by two of the authors. Results: Participants were acutely aware of misinformation during the pandemic. They rated legacy news media (print and broadcast media with pre-Internet origins) higher than social media for reliable information about the pandemic. However, strikingly, not all legacy media were automatically trusted and not all social media were uniformly distrusted. Participants identified a set of mechanisms for establishing whether a piece of information was truthful and accurate. These mechanisms had four main focal points: (1) the source, (2) the message, (3) individual media literacy and (4) the trustworthiness of others. Despite possessing a critical awareness of misinformation, participants avoided posting anything in relation to the pandemic for fear of becoming the target of online abuse. Conclusions: In addition to underscoring the role of media literacy, our research foregrounds the need to attend to the importance of fostering media confidence. We define media confidence as the ability of digital media users to challenge and interrogate questionable or inaccurate information safe in the knowledge that there are adequate regulatory mechanisms in place to curb abuse, trolling and intimidation.

Novel method for prediction of combinatorial phase-variable gene expression states.

Short sequence repeat mediated phase variation results in diverse phenotype presentation in many bacteria including Campylobacter and Neisseria species. Current methods for identifying the expression states of phase-variable genes involve taking a high number of single colonies. This approach is subject to bias, sampling effects and high workloads that reduce the ability to perform intermediary sampling. The use of high concentration colony sweeps provides a work around but reduces the resolution of combinatorial expression profiles (termed phasotypes). A parsimonious approach combining both single colony and sweep data was developed to overcome these limitations. The critical methodological advance is the use of an algorithm that utilises the experimental data from the two sample types and a parsimonious, iterative mathematical analysis that outputs the phasotype distribution with the highest likelihood of underpinning the experimental data sets. The advantages of this unified method are increased resolution and accuracy of gene expression state combinations as compared to conventional single colony sampling, reduced requirement for sampling large numbers of colonies leading to reduced costs, and a higher capacity for collecting samples and replicates.•Inputting of sweep and single colony data into an algorithm for a rapid determination of the combinatorial phase variation states (phasotypes) for repeat-mediated phase-variable bacterial genes•This method reduces the number of single colony samples required to produce accurate estimates of phasotypes•This method will reduce the costs of phasotype analyses and increase potential to analyse more time points or sample sites leading to an improved understanding of how phase variation contributes to bacterial host persistence and the ability to cause disease.

Development of a robust and quantitative high-throughput screening method for assessing phenotypic variation in large Neisseria meningitidis isolate collections.

Genome-wide association studies are a powerful approach for identifying determinants of disease. For infectious diseases, high throughput assays are required for measuring the variance in multiple virulence-related phenotypes of large bacterial isolate collections and for association of this phenotypic variance with genotype. The primary limiting factors are cost, effectiveness and a standardized inoculum. A method was developed to create an inoculum array of multiple isolates that could be used for a series of high-throughput multi-isolate phenotypic investigations in a laboratory setting. A key starting point was the standardisation of the inoculum by production of identical batches of each isolate from cells grown to mid-log phase. Cultures with pre-determined optical densities were aliquoted in set patterns into multiple multi-well plates containing 50% glycerol and stored at -80 °C. Prior to a specific assay, an inoculum plate was defrosted and subjected to a brief period of incubation. Control strains can be placed on each plate in order to control for intra-assay variability. A high throughput screen is described in detail for quantification of biofilm formation. This example utilised the crystal violet staining method and multi-assay stock plates containing 16 meningococcal isolates.•Multi-assay stock plate of exponentially growing isolates is cost-effective and simple to implement in a laboratory setting.•This method would predict realistic standard deviations for multiple isolates in phenotypic assays and generate data for performance of power calculations for genotyping.•This method has the potential to identify both known and unknown genetic determinants of phenotypic variability for each tested isolate when paired with genetic analysis of whole genome sequencing data.

Variable disruption of epithelial monolayers by Neisseria meningitidis carriage isolates of the hypervirulent MenW cc11 and MenY cc23 lineages.

Colonization of mucosal tissues by Neisseria meningitidis requires adhesion mediated by the type IV pilus and multiple outer-membrane proteins. Penetration of the mucosa and invasion of epithelial cells are thought to contribute to host persistence and invasive disease. Using Calu-3 cell monolayers grown at an air-liquid interface, we examined adhesion, invasion and monolayer disruption by carriage isolates of two clonal complexes of N. meningitidis. Carriage isolates of both the serogroup Y cc23 and the hypervirulent serogroup W cc11 lineages exhibited high levels of cellular adhesion, and a variable disruption phenotype across independent isolates. Inactivation of the gene encoding the main pilus sub-unit in multiple cc11 isolates abrogated both adhesive capacity and ability to disrupt epithelial monolayers. Contrastingly, inactivation of the phase-variable opa or nadA genes reduced adhesion and invasion, but not disruption of monolayer integrity. Adherence of tissue-disruptive meningococci correlated with loss of staining for the tight junction protein, occludin. Intriguingly, in a pilus-negative strain background, we observed compensatory ON switching of opa genes, which facilitated continued adhesion. We conclude that disruption of epithelial monolayers occurs in multiple meningococcal lineages but can vary during carriage and is intimately linked to pilus-mediated adhesion.

University Students' Mental Health and Well-Being during the COVID-19 Pandemic: Findings from the UniCoVac Qualitative Study.

The worldwide spread of the coronavirus disease 2019 (COVID-19) pandemic in early 2020 affected all major sectors, including higher education. The measures to contain the spread of this deadly disease led to the closure of colleges and universities across the globe, disrupting the lives of millions of students and subjecting them to a new world of online learning. These sudden disturbances coupled with the demands of a new learning system and the experiences of living through a pandemic have placed additional strains on the mental health of university students. Research on university students' mental health, conducted during the pandemic, have found high levels of stress, anxiety and depression among students. In this qualitative study, we aimed to understand how pandemic experiences have affected student well-being by conducting in-depth interviews with 34 undergraduate students enrolled in a UK university. All interviews were conducted through Microsoft Teams and recorded with prior permission. Transcripts of recorded interviews were thematically analysed which identified two broad themes: (i) University students' mental health and well-being experiences during the pandemic; (ii) factors that influenced students' mental health and well-being. These factors were further distributed across six sub-themes: (a) isolation; (b) health and well-being; (c) bereavement; (d) academic concerns; (e) financial worries and; (f) support, coping, and resilience. Our study identifies the importance of mental health support to university students during pandemics and calls for measures to improve access to support services through these crisis points by universities. Findings can also inform students' mental health and risk assessments in the aftermath of the pandemic.

Cross-Sectional Study of University Students' Attitudes to 'On Campus' Delivery of COVID-19, MenACWY and MMR Vaccines and Future-Proofing Vaccine Roll-Out Strategies.

University students are a critical group for vaccination programmes against COVID-19, meningococcal disease (MenACWY) and measles, mumps and rubella (MMR). We aimed to evaluate risk factors for vaccine hesitancy and views about on-campus vaccine delivery among university students. Data were obtained through a cross-sectional anonymous online questionnaire study of undergraduate students in June 2021 and analysed by univariate and multivariate tests to detect associations. Complete data were obtained from 827 participants (7.6% response-rate). Self-reporting of COVID-19 vaccine status indicated uptake by two-thirds (64%; 527/827), willing for 23% (194/827), refusal by 5% (40/827) and uncertain results for 8% (66/827). Hesitancy for COVID-19 vaccines was 5% (40/761). COVID-19 vaccine hesitancy was associated with Black ethnicity (aOR, 7.01, 95% CI, 1.8-27.3) and concerns about vaccine side-effects (aOR, 1.72; 95% CI, 1.23-2.39). Uncertainty about vaccine status was frequently observed for MMR (11%) and MenACWY (26%) vaccines. Campus-associated COVID-19 vaccine campaigns were favoured by UK-based students (definitely, 45%; somewhat, 16%) and UK-based international students (definitely, 62%; somewhat, 12%). Limitations of this study were use of use of a cross-sectional approach, self-selection of the response cohort, slight biases in the demographics and a strict definition of vaccine hesitancy. Vaccine hesitancy and uncertainty about vaccine status are concerns for effective vaccine programmes. Extending capabilities of digital platforms for accessing vaccine information and sector-wide implementation of on-campus vaccine delivery are strategies for improving vaccine uptake among students. Future studies of vaccine hesitancy among students should aim to extend our observations to student populations in a wider range of university settings and with broader definitions of vaccine hesitancy.

Phase Variation During Host Colonization and Invasion by Campylobacter jejuni and Other Campylobacter Species.

Phase variation (PV) is a phenomenon common to a variety of bacterial species for niche adaption and survival in challenging environments. Among Campylobacter species, PV depends on the presence of intergenic and intragenic hypermutable G/C homopolymeric tracts. The presence of phase-variable genes is of especial interest for species that cause foodborne or zoonotic infections in humans. PV influences the formation and the structure of the lipooligosaccharide, flagella, and capsule in Campylobacter species. PV of components of these molecules is potentially important during invasion of host tissues, spread within hosts and transmission between hosts. Motility is a critical phenotype that is potentially modulated by PV. Variation in the status of the phase-variable genes has been observed to occur during colonization in chickens and mouse infection models. Interestingly, PV is also involved in bacterial survival of attack by bacteriophages even during chicken colonization. This review aims to explore and discuss observations of PV during model and natural infections by Campylobacter species and how PV may affect strategies for fighting infections by this foodborne pathogen.

How does feedback from phage infections influence the evolution of phase variation in Campylobacter?

Campylobacter jejuni (C. jejuni) causes gastroenteritis following the consumption of contaminated poultry meat, resulting in a large health and economic burden worldwide. Phage therapy is a promising technique for eradicating C. jejuni from poultry flocks and chicken carcasses. However, C. jejuni can resist infections by some phages through stochastic, phase-variable ON/OFF switching of the phage receptors mediated by simple sequence repeats (SSR). While selection strength and exposure time influence the evolution of SSR-mediated phase variation (PV), phages offer a more complex evolutionary environment as phage replication depends on having a permissive host organism. Here, we build and explore several continuous culture bacteria-phage computational models, each analysing different phase-variable scenarios calibrated to the experimental SSR rates of C. jejuni loci and replication parameters for the F336 phage. We simulate the evolution of PV rates via the adaptive dynamics framework for varying levels of selective pressures that act on the phage-resistant state. Our results indicate that growth reducing counter-selection on a single PV locus results in the stable maintenance of the phage, while compensatory selection between bacterial states affects the evolutionary stable mutation rates (i.e. very high and very low mutation rates are evolutionarily disadvantageous), whereas, in the absence of either selective pressure the evolution of PV rates results in mutation rates below the basal values. Contrastingly, a biologically-relevant model with two phase-variable loci resulted in phage extinction and locking of the bacteria into a phage-resistant state suggesting that another counter-selective pressure is required, instance, the use of a distinct phage whose receptor is an F336-phage-resistant state. We conclude that a delicate balance between counter-selection and phage-attack can result in both the evolution of phase-variable phage receptors and persistence of PV-receptor-specific phage.

Does Blood Transfusion Have an Effect on Outcomes After Aortic Valve Replacement Surgery?

OBJECTIVE: Long-term outcomes following surgical aortic valve replacement (AVR) are excellent. However, there is a significant early morbidity burden. Red cell transfusion is common in the perioperative period and deleterious effects of receiving a transfusion on early postoperative morbidity are well described in observational studies. This study aimed to assess the effect of transfusion on ischaemic or infective outcomes after aortic valve replacement. METHODS: Data from 270 patients enrolled in the Manubrium-limited ministernotomy versus conventional sternotomy for aortic valve replacement (MAVRIC) randomised controlled trial was used to create two cohorts, patients that received red cell transfusions following AVR and those that did not. Propensity score matching was performed to limit the effect of confounding variables. Strict transfusion thresholds were maintained, with patients receiving a transfusion if haemoglobin concentration fell below 80 g/L, or if significant bleeding or haemodynamic instability occurred. The primary outcome was a composite of ischaemic event (myocardial infarction, permanent stroke, gut ischaemia or acute kidney injury) or serious infection (sepsis, endocarditis, respiratory tract or wound infection). Patients were followed for 12 weeks following surgery. RESULTS: Sixty-three (63) of 270 patients received a red cell transfusion (23.3%). Transfused patients had significantly lower body mass index (BMI), a higher proportion of females, a lower preoperative haemoglobin and haematocrit, a higher EuroSCORE II score, worse renal function and were more likely to have undergone urgent surgery compared to the unadjusted control cohort. Once matched, there was no difference in the primary outcome between cohorts. There was a significantly increased length of hospital stay in the transfused group (median 7 days transfused, median 5 days not-transfused, p=0.001). CONCLUSIONS: Red cell transfusion, using a transfusion threshold of 80 g/L, does not appear to be associated with adverse ischaemic or infective outcomes after aortic valve replacement.

Rapid Transmission of a Hyper-Virulent Meningococcal Clone Due to High Effective Contact Numbers and Super Spreaders.

Rapid transmission, a critical contributory factor in outbreaks of invasive meningococcal disease, requires naïve populations of sufficient size and intermingling. We examined genomic variability and transmission dynamics in a student population subject to an 11-fold increase in carriage of a hypervirulent Neisseria meningitidis serogroup W ST-11 clone. Phylogenetic clusters, mutation and recombination rates were derived by bioinformatic analyses of whole-genome sequencing data. Transmission dynamics were determined by combining observed carriage rates, cluster sizes and distributions with simple SIS models. Between 9 and 15 genetically-distinct clusters were detected and associated with seven residential halls. Clusters had low mutation accumulation rates and infrequent recombination events. Modeling indicated that effective contacts decreased from 10 to 2 per day between the start and mid-point of the university term. Transmission rates fluctuated between 1 and 4% while the R(t) for carriage decreased from an initial rate of 47 to 1. Decreases in transmission values correlated with a rise in vaccine-induced immunity. Observed carriage dynamics could be mimicked by populations containing 20% of super spreaders with 2.3-fold higher effective contact rates. We conclude that spread of this hypervirulent ST-11 meningococcal clone depends on the levels of effective contacts and immunity rather than genomic variability. Additionally, we propose that super-spreaders enhance meningococcal transmission and that a 70% MenACWY immunization level is sufficient to retard, but not fully prevent, meningococcal spread in close-contact populations.

Broad vaccine protection against Neisseria meningitidis using factor H binding protein.

Neisseria meningitidis, the causative agent of invasive meningococcal disease (IMD), is classified into different serogroups defined by their polysaccharide capsules. Meningococcal serogroups A, B, C, W, and Y are responsible for most IMD cases, with serogroup B (MenB) causing a substantial percentage of IMD cases in many regions. Vaccines using capsular polysaccharides conjugated to carrier proteins have been successfully developed for serogroups A, C, W, and Y. However, because the MenB capsular polysaccharide is poorly immunogenic, MenB vaccine development has focused on alternative antigens. The 2 currently available MenB vaccines (MenB-4C and MenB-FHbp) both include factor H binding protein (FHbp), a surface-exposed protein harboured by nearly all meningococcal isolates that is important for survival of the bacteria in human blood. MenB-4C contains a nonlipidated FHbp from subfamily B in addition to other antigens, including Neisserial Heparin Binding Antigen, Neisserial adhesin A, and outer membrane vesicles, whereas MenB-FHbp contains a lipidated FHbp from each subfamily (A and B). FHbp is highly immunogenic and a main target of bactericidal activity of antibodies elicited by both licensed MenB vaccines. FHbp is also an important vaccine component, in contrast to some other meningococcal antigens that may have limited cross-protection across strains, as FHbp-specific antibodies can provide broad cross-protection within each subfamily. Limited cross-protection between subfamilies necessitates the inclusion of FHbp variants from both subfamilies to achieve broad FHbp-based vaccine coverage. Additionally, immune responses to the lipidated form of FHbp have a superior cross-reactive profile to those elicited by the nonlipidated form. Taken together, the inclusion of lipidated FHbp variants from both FHbp subfamilies is expected to provide broad protection against the diverse disease-causing meningococcal strains expressing a wide range of FHbp sequence variants. This review describes the development of vaccines for MenB disease prevention, with a focus on the FHbp antigen.

Meningococcal core and accessory phasomes vary by clonal complex.

Neisseria meningitidis is a Gram-negative human commensal pathogen, with extensive phenotypic plasticity afforded by phase-variable (PV) gene expression. Phase variation is a stochastic switch in gene expression from an ON to an OFF state, mediated by localized hypermutation of simple sequence repeats (SSRs). Circulating N. meningitidis clones vary in propensity to cause disease, with some clonal complexes (ccs) classified as hypervirulent and others as carriage-associated. We examined the PV gene repertoires, or phasome, of these lineages in order to determine whether phase variation contributes to disease propensity. We analysed 3328 genomes representative of nine circulating meningococcal ccs with PhasomeIt, a tool that identifies PV genes by the presence of SSRs and homologous gene clusters. The presence, absence and functions of all identified PV gene clusters were confirmed by annotation or blast searches within the Neisseria PubMLST database. While no significant differences were detected in the number of PV genes or the core, conserved phasome content between hypervirulent and carriage lineages, individual ccs exhibited major variations in PV gene numbers. Phylogenetic clusters produced by phasome or core genome analyses were similar, indicating co-evolution of PV genes with the core genome. While conservation of PV clusters is high, with 76 % present in all meningococcal isolates, maintenance of an SSR is variable, ranging from conserved in all isolates to present only in a single cc, indicating differing evolutionary trajectories for each lineage. Diverse functional groups of PV genes were present across the meningococcal lineages; however, the majority directly or indirectly influence bacterial surface antigens and could impact on future vaccine development. Finally, we observe that meningococci have open pan phasomes, indicating ongoing evolution of PV gene content and a significant potential for adaptive changes in this clinically relevant genus.

Localized Hypermutation is the Major Driver of Meningococcal Genetic Variability during Persistent Asymptomatic Carriage.

Host persistence of bacteria is facilitated by mutational and recombinatorial processes that counteract loss of genetic variation during transmission and selection from evolving host responses. Genetic variation was investigated during persistent asymptomatic carriage of Neisseria meningitidis Interrogation of whole-genome sequences for paired isolates from 25 carriers showed that de novo mutations were infrequent, while horizontal gene transfer occurred in 16% of carriers. Examination of multiple isolates per time point enabled separation of sporadic and transient allelic variation from directional variation. A comprehensive comparative analysis of directional allelic variation with hypermutation of simple sequence repeats and hyperrecombination of class 1 type IV pilus genes detected an average of seven events per carrier and 2:1 bias for changes due to localized hypermutation. Directional genetic variation was focused on the outer membrane with 69% of events occurring in genes encoding enzymatic modifiers of surface structures or outer membrane proteins. Multiple carriers exhibited directional and opposed switching of allelic variants of the surface-located Opa proteins that enables continuous expression of these adhesins alongside antigenic variation. A trend for switching from PilC1 to PilC2 expression was detected, indicating selection for specific alterations in the activities of the type IV pilus, whereas phase variation of restriction modification (RM) systems, as well as associated phasevarions, was infrequent. We conclude that asymptomatic meningococcal carriage on mucosal surfaces is facilitated by frequent localized hypermutation and horizontal gene transfer affecting genes encoding surface modifiers such that optimization of adhesive functions occurs alongside escape of immune responses by antigenic variation.IMPORTANCE Many bacterial pathogens coexist with host organisms, rarely causing disease while adapting to host responses. Neisseria meningitidis, a major cause of meningitis and septicemia, is a frequent persistent colonizer of asymptomatic teenagers/young adults. To assess how genetic variation contributes to host persistence, whole-genome sequencing and hypermutable sequence analyses were performed on multiple isolates obtained from students naturally colonized with meningococci. High frequencies of gene transfer were observed, occurring in 16% of carriers and affecting 51% of all nonhypermutable variable genes. Comparative analyses showed that hypermutable sequences were the major mechanism of variation, causing 2-fold more changes in gene function than other mechanisms. Genetic variation was focused on genes affecting the outer membrane, with directional changes in proteins responsible for bacterial adhesion to host surfaces. This comprehensive examination of genetic plasticity in individual hosts provides a significant new platform for rationale design of approaches to prevent the spread of this pathogen.

Selective and non-selective bottlenecks as drivers of the evolution of hypermutable bacterial loci.

Bottlenecks reduce the size of the gene pool within populations of all life forms with implications for their subsequent survival. Here, we examine the effects of bottlenecks on bacterial commensal-pathogens during transmission between, and dissemination within, hosts. By reducing genetic diversity, bottlenecks may alter individual or population-wide adaptive potential. A diverse range of hypermutable mechanisms have evolved in infectious agents that allow for rapid generation of genetic diversity in specific genomic loci as opposed to the variability arising from increased genome-wide mutation rates. These localised hypermutable mechanisms include multi-gene phase variation (PV) of outer membrane components, multi-allele PV of restriction systems and recombination-driven antigenic variation. We review selected experimental and theoretical (mathematical) models pertaining to the hypothesis that localised hypermutation (LH) compensates for fitness losses caused by bottlenecks and discuss whether bottlenecks have driven the evolution of hypermutable loci.

Corrigendum: Variant Signal Peptides of Vaccine Antigen, FHbp, Impair Processing Affecting Surface Localization and Antibody-Mediated Killing in Most Meningococcal Isolates.

[This corrects the article DOI: 10.3389/fmicb.2019.02847.].

Phage-Resistant Phase-Variant Sub-populations Mediate Herd Immunity Against Bacteriophage Invasion of Bacterial Meta-Populations.

Hypermutable loci are widespread in bacteria as mechanisms for rapid generation of phenotypic diversity within a population that enables survival of fluctuating, often antagonistic, selection pressures. Localized hypermutation can mediate phase variation and enable survival of bacteriophage predation due to high frequency, reversible alterations in the expression of phage receptors. As phase variation can also generate population-to-population heterogeneity, we hypothesized that this phenomenon may facilitate survival of spatially-separated bacterial populations from phage invasion in a manner analogous to herd immunity to infectious diseases in human populations. The lic2A gene of Haemophilus influenzae is subject to "ON" and "OFF" switches in expression mediated by mutations in a 5'CAAT repeat tract present within the reading frame. The enzyme encoded by lic2A mediates addition of a galactose moiety of the lipopolysaccharide. This moiety is required for attachment of the HP1C1 phage such that the ON state of the lic2A gene is associated with HP1c1 susceptibility while the OFF state is resistant to infection. We developed an "oscillating prey assay" to examine phage spread through a series of sub-populations of Haemophilus influenzae whose phage receptor is in an ON or OFF state. Phage extinction was frequently observed when the proportion of phage-resistant sub-populations exceeded 34%. In silico modeling indicated that phage extinction was interdependent on phage loss during transfer between sub-populations and the frequency of resistant sub-populations. In a fixed-area oscillating prey assay, heterogeneity in phage resistance was observed to generate vast differences in phage densities across a meta-population of multiple bacterial sub-populations resulting in protective quarantining of some sub-populations from phage attack. We conclude that phase-variable hypermutable loci produce bacterial "herd immunity" with resistant intermediary-populations acting as a barricade to reduce the viral load faced by phage-susceptible sub-populations. This paradigm of meta-population protection is applicable to evolution of hypermutable loci in multiple bacteria-phage and host-pathogen interactions.

Gastro-duodenal disease in Africa: Literature review and clinical data from Accra, Ghana.

Gastroduodenal disease (GDD) was initially thought to be uncommon in Africa. Amongst others, lack of access to optimal health infrastructure and suspicion of conventional medicine resulted in the reported prevalence of GDD being significantly lower than that in other areas of the world. Following the increasing availability of flexible upper gastro-intestinal endoscopy, it has now become apparent that GDD, especially peptic ulcer disease (PUD), is prevalent across the continent of Africa. Recognised risk factors for gastric cancer (GCA) include Helicobater pylori (H. pylori), diet, Epstein-Barr virus infection and industrial chemical exposure, while those for PUD are H. pylori, non-steroidal anti-inflammatory drug (NSAID)-use, smoking and alcohol consumption. Of these, H. pylori is generally accepted to be causally related to the development of atrophic gastritis (AG), intestinal metaplasia (IM), PUD and distal GCA. Here, we perform a systematic review of the patterns of GDD across Africa obtained with endoscopy, and complement the analysis with new data obtained on pre-malignant gastric his-topathological lesions in Accra, Ghana which was compared with previous data from Maputo, Mozambique. As there is a general lack of structured cohort studies in Africa, we also considered endoscopy-based hospital or tertiary centre studies of symptomatic individuals. In Africa, there is considerable heterogeneity in the prevalence of PUD with no clear geographical patterns. Furthermore, there are differences in PUD within-country despite universally endemic H. pylori infection. PUD is not uncommon in Africa. Most of the African tertiary-centre studies had higher prevalence of PUD when compared with similar studies in western countries. An additional intriguing observation is a recent, ongoing decline in PUD in some African countries where H. pylori infection is still high. One possible reason for the high, sustained prevalence of PUD may be the significant use of NSAIDs in local or over-the-counter preparations. The prevalence of AG and IM, were similar or modestly higher over rates in western countries but lower than those seen in Asia. . In our new data, sampling of 136 patients in Accra detected evidence of pre-malignant lesions (AG and/or IM) in 20 individuals (14.7%). Likewise, the prevalence of pre-malignant lesions, in a sample of 109 patients from Maputo, were 8.3% AG and 8.3% IM. While H. pylori is endemic in Africa, the observed prevalence for GCA is rather low. However, cancer data is drawn from country cancer registries that are not comprehensive due to considerable variation in the availability of efficient local cancer reporting systems, diagnostic health facilities and expertise. Validation of cases and their source as well as specificity of outcome definitions are not explicit in most studies further contributing to uncertainty about the precise incidence rates of GCA on the continent. We conclude that evidence is still lacking to support (or not) the African enigma theory due to inconsistencies in the data that indicate a particularly low incidence of GDD in African countries.

Potentiation of Phase Variation in Multiple Outer-Membrane Proteins During Spread of the Hyperinvasive Neisseria meningitidis Serogroup W ST-11 Lineage.

BACKGROUND: Since 2009, increases in the incidence of invasive meningococcal disease have occurred in the United Kingdom due to a sublineage of the Neisseria meningitidis serogroup W ST-11 clonal complex (hereafter, the "original UK strain"). In 2013, a descendent substrain (hereafter, the "2013 strain") became the dominant disease-causing variant. Multiple outer-membrane proteins of meningococci are subject to phase-variable switches in expression due to hypermutable simple-sequence repeats. We investigated whether alterations in phase-variable genes may have influenced the relative prevalence of the original UK and 2013 substrains, using multiple disease and carriage isolates. METHODS: Repeat numbers were determined by either bioinformatics analysis of whole-genome sequencing data or polymerase chain reaction amplification and sizing of fragments from genomic DNA extracts. Immunoblotting and sequence-translation analysis was performed to identify expression states. RESULTS: Significant increases in repeat numbers were detected between the original UK and 2013 strains in genes encoding PorA, NadA, and 2 Opa variants. Invasive and carriage isolates exhibited similar repeat numbers, but the absence of pilC gene expression was frequently associated with disease. CONCLUSIONS: Elevated repeat numbers in outer-membrane protein genes of the 2013 strain are indicative of higher phase-variation rates, suggesting that rapid expansion of this strain was due to a heightened ability to evade host immune responses during transmission and asymptomatic carriage.

Determination of Repeat Number and Expression States of Phase-Variable Loci Through Next Generation Sequencing and Bioinformatic Analysis.

Phase variation (PV) enables high frequency, reversible switches in expression of genetic loci across numerous species of bacteria. A major mechanism of PV in bacteria is the use of slipped strand mispairing across simple sequence repeats (SSRs). The generation and online availability of genomic datasets enables a comprehensive analysis of the distribution and composition of SSRs across multiple bacterial genomes of a species. PhasomeIt is a program that was developed to rapidly identify SSRs, to determine whether these SSRs mediate PV and to find homologous PV loci across multiple genomes. We describe use of this program for analysis of neisserial genomes. We further describe a method to reassemble specific PV loci to allow analysis of large repeat tracts which are often poorly assembled due to inherent drawbacks of the Illumina next generation sequencing (NGS) platform. These methodologies allow for rapid analysis of a major mechanism of PV across numerous species of Neisseria and other bacterial species.

Assessment of fHbp Expression Level by Reverse Transcriptase Quantitative PCR and Promoter Sequence Analysis.

Reverse transcriptase quantitative PCR (RT qPCR) is widely used for assessing the levels of expression of specific genes in various organisms. Here we describe a RT qPCR assay designed to determine the level of expression of fHbp in multiple isolates of Neisseria meningitidis. The level of expression is measured by a two-step qPCR and is associated with a promoter region analysis.