The major histocompatibility complex class I immunopeptidome of extracellular vesicles.

Extracellular vesicles (EVs) are released by most cell types and have been associated with multiple immunomodulatory functions. MHC class I molecules have crucial roles in antigen presentation and in eliciting immune responses and are known to be incorporated into EVs. However, the MHC class I immunopeptidome of EVs has not been established. Here, using a small-scale immunoisolation of the antigen serotypes HLA-A*02:01 and HLA-B*27:05 expressed on the Epstein-Barr virus-transformed B cell line Jesthom and MS of the eluted peptides from both cells and EVs, we identified 516 peptides that bind either HLA-A*02:01 or HLA-B*27:05. Of importance, the predicted serotype-binding affinities and peptide-anchor motifs did not significantly differ between the peptide pools isolated from cells or EVs, indicating that during EV biogenesis, no obvious editing of the MHC class I immunopeptidome occurs. These results, for the first time, establish EVs as a source of MHC class I peptides that can be used for the study of the immunopeptidome and in the discovery of potential neoantigens for immunotherapies.

Patient and public understanding and knowledge of antimicrobial resistance and stewardship in a UK hospital: should public campaigns change focus?

BACKGROUND: The rising global tide of antimicrobial resistance is a well-described phenomenon. Employing effective and innovative antimicrobial stewardship strategies is an essential approach to combat this public health threat. Education of the public and patients is paramount to enable the success of such strategies. METHODS: A panel of hospital multidisciplinary healthcare professionals was set up and a short quiz containing true/false statements around antimicrobial stewardship and resistance was designed and piloted. An educational leaflet with the correct replies and supporting information was also produced and disseminated. Participants were recruited on a single day (18 November 2015) from the hospital outpatient clinics and the hospital outpatient pharmacy waiting room. RESULTS: One hundred and forty-five completed quizzes were returned, providing a total of 1450 answers. Overall, 934 of 1450 (64%) statements were scored correctly whilst 481 (33%) were scored incorrectly; 35 (3%) statements were left unscored. We speculate that these results may demonstrate that respondents understood the statements, as only a small proportion of statements were left unanswered. The question dealing with the definition of antimicrobial resistance and the question dealing with the definition of antimicrobial stewardship obtained the most incorrect replies (85% and 72%, respectively). However, a specific factual recall question regarding only one microorganism (MRSA) received the most correct responses (99%). CONCLUSIONS: We describe a simple, innovative method of engagement with patients and the general public to help educate and disseminate important public health messages around antimicrobial resistance and stewardship. We also identified the need for public health campaigns to address the knowledge gaps found around this topic.

Exploring the coverage of antimicrobial stewardship across UK clinical postgraduate training curricula.

OBJECTIVES: Antimicrobial resistance (AMR) is a global political and patient safety issue. With ongoing strategic interventions to improve the shape of UK postgraduate clinical training, ensuring that all clinicians have appropriate knowledge and practical skills in the area of AMR is essential. To assess this, a cross-sectional analysis was undertaken of the coverage and quality of antimicrobial stewardship (AMS)/AMR within UK postgraduate clinical training curricula. METHODS: UK clinical specialty training curricula were identified. Topics and individual learning points relating to AMS or AMR were extracted for each specialty. Learning points were quality assessed against the expected level of clinical competence. Inter-specialty analysis was performed. RESULTS: Overall 37 specialties were assessed, equating to 2318 topics and 42 527 learning points. Of these, 8/2318 (0.3%) topics and 184/42 527 (0.4%) learning points were related to AMS/AMR. Infectious diseases represented all eight topics and 43/184 (23%) of the learning points. In contrast, primary care, which is responsible for the highest proportion of antimicrobial usage, had no topics and only 2/1368 (0.15%) of the AMS/AMR learning points. This paucity of representation was reflected across most of the remaining specialties. On quality assessment, the majority of learning points (111/184; 60%) required knowledge only, with no demonstration of behaviour in clinical practice required. CONCLUSIONS: Coverage of AMS/AMR is poor across the majority of UK postgraduate clinical training curricula, with little depth of learning required. Given the threat of AMR, and evolving changes in clinical training pathways, we call for cross-specialty action to address this current lack of engagement.

In Vitro Generation of Human Tolerogenic Monocyte-Derived Dendritic Cells.

Human monocyte-derived dendritic cells (moDC) are commonly used as a research tool to investigate interactions between antigen-presenting cells and T cells. Generation of these cells involves the isolation of CD14 positive monocytes from peripheral blood and their in vitro differentiation into immature moDC by the cytokines GM-CSF and IL-4. Their functional characteristics can then be manipulated by maturing these cells with a cocktail of agents, which can be tailored to induce either immune activating or tolerogenic properties. Here, we describe a protocol for the generation of moDC with stable tolerogenic function, referred to as tolerogenic dendritic cells. These cells have been developed as an immunotherapeutic tool for the treatment of autoimmune disease but have also proven useful to dissect mechanisms of T cell tolerance induction in vitro.

Means of Livelihood, Clean Environment to Women Empowerment: The Multi-Faceted Role of Donkeys.

Despite the substantial contribution donkeys make to the livelihood of the world's poorest populations, the existence of donkeys has received little notice worldwide. This article reviews the value of donkeys in a variety of sectors, including agriculture, construction industry, and mining, as well as their role in empowering women and achieving sustainable development goals. However, donkeys and mules are not given enough credit or attention in terms of developing strategies regarding their role in reducing poverty. There is a dearth of information and statistics on their impact across industries, the factors contributing to the donkey population dropping, the socioeconomic status of the dependent communities, and related animal and human welfare issues.

Problem based review: alcohol-use disorders on the Acute Medical Unit.

Alcohol-use disorders including acute intoxication and withdrawal are common in the acute medical setting. Acute physicians should be aware of the indications for inpatient detoxification, and be able to liase with specialist alcohol services in the hospital and in the community to determine those patients for whom community-based detoxification may be beneficial. Additionally, it is important to recognise the benefit of Brief Interventions for higher-risk drinkers who are not yet dependent. For patients with confusion and a possible history of high alcohol intake and malnutrition, acute physicians should maintain a high index of suspicion for Wernicke's Encephalopathy and treat appropriately with parenteral thiamine.

Fluorine labelling of therapeutic human tolerogenic dendritic cells for 19F-magnetic resonance imaging.

Tolerogenic dendritic cell (tolDC) therapies aim to restore self-tolerance in patients suffering from autoimmune diseases. Phase 1 clinical trials with tolDC have shown the feasibility and safety of this approach, but have also highlighted a lack of understanding of their distribution in vivo. Fluorine-19 magnetic resonance imaging (19F-MRI) promises an attractive cell tracking method because it allows for detection of 19F-labelled cells in a non-invasive and longitudinal manner. Here, we tested the suitability of nanoparticles containing 19F (19F-NP) for labelling of therapeutic human tolDC for detection by 19F-MRI. We found that tolDC readily endocytosed 19F-NP with acceptable effects on cell viability and yield. The MRI signal-to-noise ratios obtained are more than sufficient for detection of the administered tolDC dose (10 million cells) at the injection site in vivo, depending on the tissue depth and the rate of cell dispersal. Importantly, 19F-NP labelling did not revert tolDC into immunogenic DC, as confirmed by their low expression of typical mature DC surface markers (CD83, CD86), low secretion of pro-inflammatory IL-12p70, and low capacity to induce IFN-γ in allogeneic CD4+ T cells. In addition, the capacity of tolDC to secrete anti-inflammatory IL-10 was not diminished by 19F-NP labelling. We conclude that 19F-NP is a suitable imaging agent for tolDC. With currently available technologies, this imaging approach does not yet approach the sensitivity required to detect small numbers of migrating cells, but could have important utility for determining the accuracy of injecting tolDC into the desired target tissue and their efflux rate.

Collar pressure mapping: An evaluation of seven collar types used on working donkeys in Europe.

BACKGROUND: Working donkeys represent an important source of energy worldwide. Poor working conditions and equipment affect their ability to work. Poorly designed, ill-fitted harness causes inefficient transfer of power and leads to health and welfare issues. Using technology to assess different types of collars provides a better understanding of those that are most efficient for working donkeys. MATERIALS AND METHODS: Seven different collars were tested using pressure pads. Contact area, median, maximum and peak pressures were obtained for the whole collar and critical points. Eight donkeys pulled 20% and 40% of their body weight, using each collar, under controlled conditions. RESULTS: Contact area and pressures vary between collars and effort, with design and expansion capacity of the collars playing a major role. Simple collars designed specifically for donkeys performed well, with full collars designed for horses also having good results. Due to reduced expansion capacity and contact in the critical points, the breast collars were the least effective. CONCLUSION: Design, appropriate padding and manufacturing materials and adjustment capacity are key features for good collars, and such parameters are of paramount importance in terms of health and welfare for working donkeys.

First report of Salmonella enterica serotype paratyphi A azithromycin resistance leading to treatment failure.

The prevalence of Salmonella enterica serotype Paratyphi A infection is increasing, and multidrug resistance is a well-recognized problem. Resistance to fluoroquinolones is common and leads to more frequent use of newer agents like azithromycin. We report the first case of azithromycin resistance and treatment failure in a patient with S. Paratyphi A infection.

Community-associated methicillin-resistant Staphylococcus aureus infections.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been recognized for over a decade, and usually refers to MRSA identified in previously healthy individuals with no recognized MRSA risk factors. Infections range from minor skin and soft tissue infections, through to severe pneumonia and necrotizing fasciitis. This review summarizes the current data on the epidemiology and molecular features of CA-MRSA, in addition to diagnosis and therapeutic measures. We also refer to current national guidelines for the management of these infections. Areas of agreement include the important genotypic and phenotypic differences of community MRSA strains compared with hospital strains. Areas of controversy include the precise epidemiological definition of community-acquired/associated MRSA. Fortunately, true CA-MRSA can be differentiated from hospital MRSA by molecular techniques, as discussed herein. Recent interest has focused on the changing epidemiology of CA-MRSA. Worldwide, CA-MRSA is now seen outside of the initial specific population groups, and in the USA, the successful USA300 community strain is beginning to spread back into hospitals. Reasons why USA300 remains relatively uncommon in Europe are unclear. Topics timely for research include the investigation of the epidemiology of infections and evolutionary genomics.

Use of purified Clostridium difficile spores to facilitate evaluation of health care disinfection regimens.

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.

Healthcare-associated bacterial infections in the paediatric ICU.

BACKGROUND: An estimated 3.2 million patients annually develop healthcare-associated infections (HCAIs) in Europe alone amid the major challenge of increasing antimicrobial resistance. Critically ill children warrant specific evaluation because of differences in epidemiology, causative organisms and infection sites. OBJECTIVES: To examine the prevalence and antimicrobial susceptibility patterns of three types of HCAI in critically ill children and determine the effect on their disease course. MATERIALS AND METHODS: Retrospective cohort review of critically ill children admitted to a general paediatric ICU (PICU) at a regional academic tertiary referral centre over a 3 year period. RESULTS: There were 1930 admissions with a median age of 38 months. Children with HCAIs had a higher incidence of comorbidities (74% versus 24%) and a longer median length of stay (8 days versus 3 days). We identified 26 positive isolates (blood, lower respiratory and urine) taken 48 h or more after admission. The combined incidence was 1.34%. Hospital-acquired pneumonia accounted for 58% of HCAIs, urinary tract infections for 31% and bloodstream infections for 11%. The majority (61.5%) of HCAIs were caused by Gram-negative organisms. Seven isolates were resistant to antimicrobials used to treat HCAI. All of these were Gram-negative organisms (Pseudomonas aeruginosa, Klebsiella oxytoca and Escherichia coli). CONCLUSIONS: These data revealed a low incidence of HCAIs, 27% of which were resistant Gram-negative organisms. Critically ill children with HCAIs were more likely to have comorbidities and an increased length of stay. These factors may increasingly impact on PICU bed availability, an already limited resource.

Variation in Salmonella enterica serovar typhi IncHI1 plasmids during the global spread of resistant typhoid fever.

A global collection of plasmids of the IncHI1 incompatibility group from Salmonella enterica serovar Typhi were analyzed by using a combination of DNA sequencing, DNA sequence analysis, PCR, and microarrays. The IncHI1 resistance plasmids of serovar Typhi display a backbone of conserved gene content and arrangement, within which are embedded preferred acquisition sites for horizontal DNA transfer events. The variable regions appear to be preferred acquisition sites for DNA, most likely through composite transposition, which is presumably driven by the acquisition of resistance genes. Plasmid multilocus sequence typing, a molecular typing method for IncHI1 plasmids, was developed using variation in six conserved loci to trace the spread of these plasmids and to elucidate their evolutionary relationships. The application of this method to a collection of 36 IncHI1 plasmids revealed a chronological clustering of plasmids despite their difference in geographical origins. Our findings suggest that the predominant plasmid types present after 1993 have not evolved directly from the earlier predominant plasmid type but have displaced them. We propose that antibiotic selection acts to maintain resistance genes on the plasmid, but there is also competition between plasmids encoding the same resistance phenotype.

Report of neonatal meningitis due to Salmonella enterica serotype Agona and review of breast milk-associated neonatal Salmonella infections.

We present the first documented case of Salmonella enterica serotype Agona meningitis in a 6-day-old baby. S. enterica serotype Agona was isolated concurrently from infant cerebrospinal fluid and parental fecal samples, and Salmonella was isolated from breast milk. The role of breast milk in transmission of Salmonella enterica is discussed.

First report of human infection with Salmonella enterica serovar Apapa resulting from exposure to a pet lizard.

We present the first documented human case of Salmonella enterica serovar Apapa infection, isolated concurrently from a hospital inpatient and a pet lizard. The isolates were identical by biochemical profiling and pulsed-field gel electrophoresis. This rare serotype is known to be associated with reptiles. The current practice for avoiding reptile-associated infections is reviewed.

Adherence to antibiotic guidelines and reported penicillin allergy: pooled cohort data on prescribing and allergy documentation from two English National Health Service (NHS) trusts.

OBJECTIVE: To investigate documentation of antimicrobial allergy and to determine prescribing adherence to local antibiotic guidelines for inpatients with and without reported penicillin allergy treated for infection in a National Health Service (NHS) context. SETTING: Data were collected at two English hospital NHS trusts over two time-periods: June 2016 and February 2017. DESIGN: Cohort study. Trust 1 data were sourced from prospective point prevalence surveys. Trust 2 data were extracted retrospectively from an electronic report. PARTICIPANTS: Inpatients treated for urinary tract infection (UTI), community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP) and skin and soft tissue infection (SSTI). Data on allergy were collected, and antibiotic selection assessed for adherence to trust guidelines with differences between groups presented as adjusted ORs. RESULTS: A total of 1497 patients were included, with 2645 antibiotics orders. Patients were treated for CAP (n=495; 33.1%), UTI (407; 27.2%), HAP (330; 22%) and SSTI (265; 17.7%). There were 240 (16%) patients with penicillin allergy. Penicillin allergy was recorded as allergy (n=52; 21.7%), side effect (27; 11.3%) and no documentation (161; 67.1%). Overall, 2184 (82.6%) antibiotic orders were guideline-adherent. Adherence was greatest for those labelled penicillin allergy (453 of 517; 87.6%) versus no allergy (1731 of 2128; 81.3%) (OR 0.52 (95% CI 0.37 to 0.73) p<0.001). Guideline-adherence for CAP was higher if penicillin allergy (151 of 163; 92.6%) versus no allergy (582 of 810; 71.9%) (OR 0.20 (95% CI 0.10 to 0.37) p<0.001). There was no difference in adherence between those with and without penicillin allergy for UTI, HAP or SSTI treatment. CONCLUSIONS: A relatively high proportion of patients had a penicillin allergy and two thirds of these had no description of their allergy, which has important implications for patient safety. Patients with penicillin allergy treated for CAP, received more guideline adherent antibiotics than those without allergy. Future studies investigating the clinical impact of penicillin allergy should include data on adherence to antibiotic guidelines.

Characterization of the genomes of a diverse collection of Salmonella enterica serovar Typhimurium definitive phage type 104.

Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) has caused significant morbidity and mortality in humans and animals for almost three decades. We completed the full DNA sequence of one DT104 strain, NCTC13348, and showed that significant differences between the genome of this isolate and the genome of the previously sequenced strain Salmonella serovar Typhimurium LT2 are due to integrated prophage elements and Salmonella genomic island 1 encoding antibiotic resistance genes. Thirteen isolates of Salmonella serovar Typhimurium DT104 with different pulsed-field gel electrophoresis (PFGE) profiles were analyzed by using multilocus sequence typing (MLST), plasmid profiling, hybridization to a pan-Salmonella DNA microarray, and prophage-based multiplex PCR. All the isolates belonged to a single MLST type, sequence type ST19. Microarray data demonstrated that the gene contents of the 13 DT104 isolates were remarkably conserved. The PFGE DNA fragment size differences in these isolates could be explained to a great extent by differences in the prophage and plasmid contents. Thus, here the nature of variation in different Salmonella serovar Typhimurium DT104 isolates is further defined at the gene and whole-genome levels, illustrating how this phage type evolves over time.

A rare presentation of ventriculitis and brain abscess caused by Fusobacterium nucleatum.

Anaerobic ventriculitis is rare, and usually seen in patients with predisposing factors such as otitis media, mastoiditis, sinusitis or recent neurosurgery. We report what we believe to be the first case of ventriculitis and brain abscess due to Fusobacterium nucleatum infection in a man with no significant predisposing factors. He was successfully treated with antibiotic therapy.

Technical challenges of working with extracellular vesicles.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.