Invasion of a murine in vitro blood-brain barrier co-culture model by dengue virus serotypes 1 to 4.

The blood-brain barrier (BBB) is a physical barrier that restricts the passage of cells and molecules as well as pathogens into the central nervous system (CNS). Some viruses enter the CNS by disrupting the BBB, while others can reach the CNS without altering the integrity of the BBB. Even though dengue virus (DENV) is not a distinctive neurotropic virus, the virus is considered to be one of the leading causes of neurological manifestations. In this study, we found that DENV is able to compromise the integrity of a murine in vitro blood-brain barrier (BBB) model, resulting in hyperpermeability, as shown by a significant increase in sucrose and albumin permeability. Infection of brain endothelial cells (ECs) was facilitated by the presence of glycans, in particular, mannose and N-acetyl glucosamine residues, on cell surfaces and viral envelope proteins, and the requirement for glycan moieties for cell infection was serotype-specific. Direct viral disruption of brain ECs was observed, leading to a significant decrease in tight-junction protein expression and peripheral localization, which contributed to the changes in BBB permeability. In conclusion, the hyperpermeability and breaching mechanism of BBB by DENV are primarily due to direct consequences of viral infection of ECs, as shown in this in vitro study.

Serotyping of Brunei pneumococcal clinical strains and the investigation of their capability to adhere and invade a brain endothelium model.

INTRODUCTION: Pneumococcal infections have caused morbidity and mortality globally. Streptococcus pneumoniae (pneumococci) are commensal bacteria that colonize the nasopharynx, asymptomatically. From there, pneumococci can spread in the lungs causing pneumonia and disseminate in the bloodstream causing bacteremia (sepsis) and reach the brain leading to meningitis. Endothelial cells are one of the most important components of the blood-brain barrier that separates the blood from the brain and plays the first protective role against pneumococcal entry. Thus this study aimed to investigate on the ability of non-meningitis pneumococcal clinical strains to adhere and invade a brain endothelium model. METHODS: Two pneumococcal Brunei clinical strains were serotyped by multiplex PCR method using oligonucleotide sequences derived from Centers for Disease Control and Prevention. A validated immortalised mouse brain endothelial cell line (bEnd.3) was used as a brain endothelium model for the study of the pneumococcal breach of the blood-brain barrier using an adherence and invasion assay. RESULTS: Both of the pneumococcal clinical strains were found to be serotype 19F, a common circulating serotype in Southeast Asia and globally and possess the ability to adhere and invade the brain endothelial cells. CONCLUSION: In addition, this is the first report on the serotype identification of pneumococci in Brunei Darussalam and their application on a brain endothelium model. Further studies are required to understand the virulence capabilities of the clinical strains.

Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection.

A preliminary assessment of asymptomatic bacteriuria of pregnancy in brunei darussalam.

BACKGROUND: Asymptomatic bacteriuria describes a condition in which urine culture reveals a significant growth of pathogenic bacteria, specifically greater than 10(5) of colony-forming units per millilitre of urine. It has a direct bearing on the health of a pregnant woman, her pregnancy and consequently the foetus. Thus, this study investigated the prevalence of asymptomatic bacteriuria of pregnancy in Raja Isteri Pengiran Anak Saleha (RIPAS) Hospital, the premiere tertiary hospital in Brunei Darussalam. METHODS: A total of 170 pregnant women who were visiting the Department of Obstetrics and Gynaecology at RIPAS Hospital for routine antenatal care between February and March 2011 volunteered for this cross-sectional study. They did not present with any clinical symptoms of bacteriuria or indeed any other illness. They were investigated for bacteriuria by urine microscopy, culture, and sensitivity. RESULTS: Urine samples from seven of the women produced significant bacterial growth, showing a prevalence of 4.1%. The organisms isolated were Klebsiella species (2.94%) and Escherichia coli (1.18%); these bacteria were both sensitive to amoxicillin, vancomycin, tetracycline, and erythromycin. CONCLUSION: Brunei has a similar prevalence of asymptomatic bacteriuria to other South-East Asian countries.

The prevalence of dengue virus in Brunei Darussalam during January-November 2010.

The aim of this study was to retrospectively determine the prevalence of dengue virus from April 2010 to November 2010 in Brunei Darussalam. A total of 250 serum samples from dengue diagnosed patients were examined. All serum samples were tested for dengue IgM and IgG antibodies and dengue NS1 antigen using the PanBio dengue ELISA commercial kit. To determine the prevalence of dengue virus serotype in the country, serotyping was performed for the 14 samples that were positive for NS1 antigen. Dengue virus serotyping was carried out using the conventional reverse transcriptase-polymerase chain reaction (RT-PCR). Of the 250 serum samples included in the study, 196 were laboratory dengue confirmed cases. Dengue virus serotype 1 (DENV-1) was the predominant circulating serotype, followed by DENV-3 and DENV-2. This is the first report of DENV-3 isolation in Brunei Darussalam.

Protocol for a three-phase prospective study to develop educational resource of antimicrobial resistance and stewardship for medical programme.

INTRODUCTION: Antimicrobial resistance (AMR) is one of the critical medical issues of the 21st century. Medical professionals are the primary prescribers of antimicrobials; their undergraduate education of antimicrobial stewardship (AMS) is considered one of the fundamental approaches in combating the issue of AMR. This education level provides a platform to bridge any gaps in their knowledge and competency in AMS. This study aims to develop an educational resource on microbes, hygiene and prudent antimicrobial use for the undergraduate medical programme. The guideline produced will then be assimilated into the existing curriculum which will help to improve the quality of education which in turn will improve rationale as the use of antimicrobials in the future. METHODS AND ANALYSIS: A three-step approach consensus approach will be adopted for this study for the development of a validated medical curriculum guideline on AMR. A preliminary curriculum for the programme will be drafted from reviews of published literature including syllabi as well as national and international guidelines. A total of 26 potential sources were found to be relevant, and selected for this study. Subsequently, the drafted curriculum will be subjected for validation via online surveys by various infectious disease experts. Finally, a Delphi technique will be employed to obtain consensus on heterogeneous findings to the revised curriculum. The quantitative and qualitative responses will be analysed and discussed among the panel of researchers. ETHICS AND DISSEMINATION: This study protocol has been approved by the Institute of Health Sciences Research Ethics Committee of Universiti Brunei Darussalam (Reference: UBD/PAPRSBIHSREC/2020/124). Informed consent declaration will be collected prior to data collections as indication of agreement of participation in the study. Results will be made available to medical educators and also researchers on AMR and stewardship. The results also will be disseminated at feedback sessions to officers at Ministry of Health and Ministry of Education, Brunei Darussalam.

Educational resource for antimicrobial resistance and stewardship for dentistry programmes: a research protocol.

INTRODUCTION: Antimicrobial resistance (AMR) is a global public and patient safety issue. With the high AMR risk, ensuring that the next generation of dentists that have optimal knowledge and confidence in the area of AMR is crucial. A systematic approach is vital to design an AMR content that is comprehensive and clinically relevant. The primary objective of this research study will be to implement a consensus-based approach to elucidate AMR content and curriculum priorities for professional dentistry programmes. This research aims to establish consensus along with eliciting opinion on appropriate AMR topics to be covered in the Bachelor of Dental Surgery syllabus. METHODS AND ANALYSIS: A three-phase approach to validate content for curriculum guidelines on AMR will be adopted. First, literature review and content analysis were conducted to find out the available pertinent literature in dentistry programmes. A total of 23 potential literature have been chosen for inclusion within this study following literature review and analysis in phase 1. The materials found will be used to draft curriculum on antimicrobials for dentistry programmes. The next phase involves the validation of the drafted curriculum content by recruiting local and foreign experts via a survey questionnaire. Finally, Delphi technique will be conducted to obtain consensus on the important or controversial modifications to the revised curriculum. ETHICS AND DISSEMINATION: An ethics application is currently under review with the Institute of Health Science Research Ethics Committee, Universiti Brunei Darussalam. All participants are required to provide a written consent form. Findings will be used to identify significant knowledge gaps on AMR aspect in a way that results in lasting change in clinical practice. Moreover, AMR content priorities related to dentistry clinical practice will be determined in order to develop need-based educational resource on microbes, hygiene and prudent antimicrobial use for dentistry programmes.

University Students' Antibiotic Use and Knowledge of Antimicrobial Resistance: What Are the Common Myths?

We aimed to assess antibiotic usage and knowledge regarding antibiotics and antimicrobial resistance (AMR) among undergraduate students of the Universiti Brunei Darussalam (UBD), public university located in Brunei Darussalam. A cross-sectional study was performed using a self-administered questionnaire. The questionnaire was adapted from the World Health Organization's (WHO) "Antibiotic resistance: Multi-country public awareness" survey distributed online. Students at the UBD were invited to participate in the online survey through internal email. The questionnaire consisted of five sections: demographic information, antibiotic usage, knowledge on antibiotics, antibiotic resistance (AMR), and use of antibiotics in agriculture. The data were analyzed descriptively and appropriate inferential statistics were used accordingly. A total of 130 students returned a completed questionnaire. The result of the study found that 51% (n = 66) of the students had good level of knowledge of antibiotic and antimicrobial resistance with a mean total knowledge score of nine out of 14. Of note, 76% (n = 99) of the respondents mistakenly believed that antibiotic resistance is the result of the body becoming resistant to antibiotics. Only 14% (n = 18) of the respondents were found to have poor knowledge on antibiotics and antimicrobial resistance in the study. Misconceptions in regards to the use of antibiotics for conditions related to viral illnesses like cold and flu (41%, n = 53) were noticed among the respondents in our study. Thus, improving knowledge on antibiotics is crucial to address these beliefs.

Immortalized endothelial cell lines for in vitro blood-brain barrier models: A systematic review.

Endothelial cells play the most important role in construction of the blood-brain barrier. Many studies have opted to use commercially available, easily transfected or immortalized endothelial cell lines as in vitro blood-brain barrier models. Numerous endothelial cell lines are available, but we do not currently have strong evidence for which cell lines are optimal for establishment of such models. This review aimed to investigate the application of immortalized endothelial cell lines as in vitro blood-brain barrier models. The databases used for this review were PubMed, OVID MEDLINE, ProQuest, ScienceDirect, and SpringerLink. A narrative systematic review was conducted and identified 155 studies. As a result, 36 immortalized endothelial cell lines of human, mouse, rat, porcine and bovine origins were found for the establishment of in vitro blood-brain barrier and brain endothelium models. This review provides a summary of immortalized endothelial cell lines as a guideline for future studies and improvements in the establishment of in vitro blood-brain barrier models. It is important to establish a good and reproducible model that has the potential for multiple applications, in particular a model of such a complex compartment such as the blood-brain barrier.

N-acylhomoserine lactones antagonize virulence gene expression and quorum sensing in Staphylococcus aureus.

Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 muM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.