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Find video protocols related to scientific articles indexed in Pubmed.
An In Vitro Deletion in ribE Encoding Lumazine Synthase Contributes to Nitrofurantoin Resistance in Escherichia coli.
Antimicrob. Agents Chemother.
PUBLISHED: 09-22-2014
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Nitrofurantoin has been used for decades for the treatment of urinary tract infections (UTIs), but clinically significant resistance in Escherichia coli is uncommon. Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two nitrofurantoin-susceptible intestinal E. coli strains (ST540-p and ST2747-p) to increasing nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole-genome sequencing was performed for both susceptible isolates and selected mutants that exhibited the highest nitrofurantoin resistance levels aerobically (ST540-a and ST2747-a) and anaerobically (ST540-an and ST2747-an). ST540-a/ST540-an and ST2747-a (aerobic MICs of >64 ?g/ml) harbored mutations in the known nitrofurantoin resistance determinants nfsA and/or nfsB, which encode oxygen-insensitive nitroreductases. ST2747-an showed reduced nitrofurantoin susceptibility (aerobic MIC of 32 ?g/ml) and exhibited remarkable growth deficits but did not harbor nfsA/nfsB mutations. We identified a 12-nucleotide deletion in ribE, encoding lumazine synthase, an essential enzyme involved in the biosynthesis of flavin mononucleotide (FMN), which is an important cofactor for NfsA and NfsB. Complementing ST2747-an with a functional wild-type lumazine synthase restored nitrofurantoin susceptibility. Six NIT-R E. coli isolates (NRCI-1 to NRCI-6) from stools of UTI patients treated with nitrofurantoin, cefuroxime, or a fluoroquinolone harbored mutations in nfsA and/or nfsB but not ribE. Sequencing of the ribE gene in six intestinal and three urinary E. coli strains showing reduced nitrofurantoin susceptibility (MICs of 16 to 48 ?g/ml) also did not identify any relevant mutations. NRCI-1, NRCI-2, and NRCI-5 exhibited up to 4-fold higher anaerobic MICs, compared to the mutants generated in vitro, presumably because of additional mutations in oxygen-sensitive nitroreductases.
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Early inflammatory responses following cell grafting in the CNS trigger activation of the sub-ventricular zone: a proposed model of sequential cellular events.
Cell Transplant
PUBLISHED: 09-09-2014
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While multiple rodent pre-clinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP(+) mouse embryonic fibroblasts (mEFs) in the CNS of immune-competent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically-derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neo-angiogenesis, microglia/macrophage recruitment, astrogliosis and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the sub-ventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses, and will eventually allow for successful manipulation of this intervention.
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Histological characterization and quantification of cellular events following neural and fibroblast(-like) stem cell grafting in healthy and demyelinated CNS tissue.
Methods Mol. Biol.
PUBLISHED: 09-01-2014
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Preclinical animal studies involving intracerebral (stem) cell grafting are gaining popularity in many laboratories due to the reported beneficial effects of cell grafting on various diseases or traumata of the central nervous system (CNS). In this chapter, we describe a histological workflow to characterize and quantify cellular events following neural and fibroblast(-like) stem cell grafting in healthy and demyelinated CNS tissue. First, we provide standardized protocols to isolate and culture eGFP(+) neural and fibroblast(-like) stem cells from embryonic mouse tissue. Second, we describe flow cytometric procedures to determine cell viability, eGFP transgene expression, and the expression of different stem cell lineage markers. Third, we explain how to induce reproducible demyelination in the CNS of mice by means of cuprizone administration, a validated mouse model for human multiple sclerosis. Fourth, the technical procedures for cell grafting in the CNS are explained in detail. Finally, an optimized and validated workflow for the quantitative histological analysis of cell graft survival and endogenous astroglial and microglial responses is provided.
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Comparison of biofilm formation between major clonal lineages of methicillin resistant Staphylococcus aureus.
PLoS ONE
PUBLISHED: 08-08-2014
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Epidemic methicillin-resistant S. aureus (MRSA) clones cause infections in both hospital and community settings. As a biofilm phenotype further facilitates evasion of the host immune system and antibiotics, we compared the biofilm-forming capacities of various MRSA clones.
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Whole genome mapping as a fast-track tool to assess genomic stability of sequenced Staphylococcus aureus strains.
BMC Res Notes
PUBLISHED: 06-13-2014
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Whole genome (optical) mapping (WGM), a state-of-the-art mapping technology based on the generation of high resolution restriction maps, has so far been used for typing clinical outbreak strains and for mapping de novo sequence contigs in genome sequencing projects. We employed WGM to assess the genomic stability of previously sequenced Staphylococcus aureus strains that are commonly used in laboratories as reference standards.
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Prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness.
Eur. Respir. J.
PUBLISHED: 05-29-2014
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Rhinovirus infections occur frequently throughout life and have been reported in about one-third of asymptomatic cases. The clinical significance of sequential rhinovirus infections remains unclear. To determine the incidence and clinical relevance of sequential rhinovirus detections, nasopharyngeal samples from 2485 adults with acute cough/lower respiratory illness were analysed. Patients were enrolled prospectively by general practitioners from 12 European Union countries during three consecutive years (2007-2010). Nasopharyngeal samples were collected at the initial general practitioner consultation and 28 days thereafter and symptom scores were recorded by patients over that period. Rhinovirus RNA was detected in 444 (18%) out of 2485 visit one samples and in 110 (4.4%) out of 2485 visit two respiratory samples. 21 (5%) of the 444 patients had both samples positive for rhinovirus. Genotyping of both virus detections was successful for 17 (81%) out of 21 of these patients. Prolonged rhinovirus shedding occurred in six (35%) out of 21 and re-infection with a different rhinovirus in 11 (65%) out of 21. Rhinovirus re-infections were significantly associated with chronic obstructive pulmonary disease (p=0.04) and asthma (p=0.02) and appeared to be more severe than prolonged infections. Our findings indicate that in immunocompetent adults rhinovirus re-infections are more common than prolonged infections, and chronic airway comorbidities might predispose to more frequent rhinovirus re-infections.
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Clinical and microbiological impact of discontinuation of fluoroquinolone prophylaxis in patients with prolonged profound neutropenia.
Eur. J. Haematol.
PUBLISHED: 04-13-2014
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Infections remain a leading cause of morbidity and mortality in patients with reduced immunity caused by haematological disease and chemotherapy-induced neutropenia. We evaluated the clinical and microbiological impact of discontinuing fluoroquinolone prophylaxis in these patients.
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Complete Genome Sequences of Nitrofurantoin-Sensitive and -Resistant Escherichia coli ST540 and ST2747 Strains.
Genome Announc
PUBLISHED: 04-12-2014
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Widespread multidrug resistance in Escherichia coli has necessitated the reintroduction of older antibiotics, such as nitrofurantoin. However, mechanisms by which resistance to nitrofurantoin emerges in E. coli are not well elucidated. Toward this aim, we sequenced two nitrofurantoin-sensitive E. coli sequence types (ST540 and ST2747) and their four nitrofurantoin-resistant derivatives generated in vitro under aerobic and anaerobic growth conditions.
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Employing whole genome mapping for optimal de novo assembly of bacterial genomes.
BMC Res Notes
PUBLISHED: 04-08-2014
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De novo genome assembly can be challenging due to inherent properties of the reads, even when using current state-of-the-art assembly tools based on de Bruijn graphs. Often users are not bio-informaticians and, in a black box approach, utilise assembly parameters such as contig length and N50 to generate whole genome sequences, potentially resulting in mis-assemblies.
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Influence of lung function on course of disease and response to antibiotic therapy in adult primary care patients with acute cough: a post hoc analysis of patients enrolled in a prospective multicentre study.
NPJ Prim Care Respir Med
PUBLISHED: 03-21-2014
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In acute cough patients, impaired lung function as present in chronic lung conditions like asthma and chronic obstructive pulmonary disease (COPD) are often thought to negatively influence course of disease, but clear evidence is lacking.
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Antibiotic use in eastern Europe: a cross-national database study in coordination with the WHO Regional Office for Europe.
Lancet Infect Dis
PUBLISHED: 03-20-2014
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There are no reliable data on antibiotic use in non-European Union (EU) southern and eastern European countries and newly independent states. We aimed to collect valid, representative, comparable data on systemic antimicrobial use in these non-EU countries of the WHO European region.
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Measuring trends of outpatient antibiotic use in Europe: jointly modelling longitudinal data in defined daily doses and packages.
J. Antimicrob. Chemother.
PUBLISHED: 03-12-2014
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To complement analyses of the linear trend and seasonal fluctuation of European outpatient antibiotic use expressed in defined daily doses (DDD) by analyses of data in packages, to assess the agreement between both measures and to study changes in the number of DDD per package over time.
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Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.
Lab Chip
PUBLISHED: 03-10-2014
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In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI.
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Distinct spatial distribution of microglia and macrophages following mesenchymal stem cell implantation in mouse brain.
Immunol. Cell Biol.
PUBLISHED: 03-07-2014
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Although implantation of cellular material in the central nervous system (CNS) is a key direction in CNS regenerative medicine, this approach is currently limited by the occurrence of strong endogenous immune cell responses. In a model of mesenchymal stem cell (MSC) grafting in the CNS of immune-competent mice, we previously described that MSC grafts become highly surrounded and invaded by Iba1(+) myeloid cells (microglia and/or macrophages). Here, following grafting of blue fluorescent protein (BFP)-expressing MSC in the CNS of CX3CR1(+/-) and CX3CR1(-/-) mice, our results indicate: (1) that the observed inflammatory response is independent of the fractalkine signalling axis, and (2) that a significant spatial distribution of Iba1(+) inflammatory cells occurs, in which Iba1(+) CX3CR1(+) myeloid cells mainly surround the MSC graft and Iba1(+) CX3CR1(-) myeloid cells mainly invade the graft at 10 days post transplantation. Although Iba1(+) CX3CR1(+) myeloid cells are considered to be of resident microglial origin, Iba1(+) CX3CR1(-) myeloid cells are most likely of peripheral monocyte/macrophage origin. In order to confirm the latter, we performed MSC-BFP grafting experiments in the CNS of eGFP(+) bone marrow chimeric C57BL/6 mice. Analysis of MSC-BFP grafts in the CNS of these mice confirmed our observation that peripheral monocytes/macrophages invade the MSC graft and that resident microglia surround the MSC graft site. Furthermore, analysis of major histocompatibility complex class II (MHCII) expression revealed that mainly macrophages, but not microglia, express this M1 pro-inflammatory marker in the context of MSC grafting in the CNS. These results again highlight the complexity of cell implantation immunology in the CNS.
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Medication use in European primary care patients with lower respiratory tract infection: an observational study.
Br J Gen Pract
PUBLISHED: 02-26-2014
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BACKGROUND It is largely unknown what medication is used by patients with lower respiratory tract infection (LRTI). AIM To describe the use of self-medication and prescribed medication in adults presenting with LRTI in different European countries, and to relate self-medication to patient characteristics. DESIGN AND SETTING An observational study in 16 primary care networks in 12 European countries. METHOD A total of 2530 adult patients presenting with LRTI in 12 European countries filled in a diary on any medication used before and after a primary care consultation. Patient characteristics related to self-medication were determined by univariable and multivariable logistic regression analysis. RESULTS The frequency and types of medication used differed greatly between European countries. Overall, 55.4% self-medicated before consultation, and 21.5% after consultation, most frequently with paracetamol, antitussives, and mucolytics. Females, non-smokers, and patients with more severe symptoms used more self-medication. Patients who were not prescribed medication during the consultation self-medicated more often afterwards. Self-medication with antibiotics was relatively rare. CONCLUSION A considerable amount of medication, often with no proven efficacy, was used by adults presenting with LRTI in primary care. There were large differences between European countries. These findings should help develop patient information resources, international guidelines, and international legislation concerning the availability of over-the-counter medication, and can also support interventions against unwarranted variations in care. In addition, further research on the effects of symptomatic medication is needed.
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Amoxicillin for acute lower respiratory tract infection in primary care: subgroup analysis of potential high-risk groups.
Br J Gen Pract
PUBLISHED: 02-26-2014
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Antibiotics are of limited overall clinical benefit for uncomplicated lower respiratory tract infection (LRTI) but there is uncertainty about their effectiveness for patients with features associated with higher levels of antibiotic prescribing.
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Clinicians' Views and Experiences of Interventions to Enhance the Quality of Antibiotic Prescribing for Acute Respiratory Tract Infections.
J Gen Intern Med
PUBLISHED: 02-17-2014
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Evidence shows a high rate of unnecessary antibiotic prescriptions in primary care in Europe and the United States. Given the costs of widespread use and associated antibiotic resistance, reducing inappropriate use is a public health priority.
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Exploring patients' views of primary care consultations with contrasting interventions for acute cough: a six-country European qualitative study.
NPJ Prim Care Respir Med
PUBLISHED: 02-17-2014
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In a pan-European randomised controlled trial (GRACE INTRO) of two interventions, (i) a point-of-care C-reactive protein test and/or (ii) training in communication skills and use of an interactive patient booklet, both interventions resulted in large reductions in antibiotic prescribing for acute cough.
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HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV-positive tumour cells.
J. Cell. Mol. Med.
PUBLISHED: 02-17-2014
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Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.
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A systematic review and meta-analysis of the effects of antibiotic consumption on antibiotic resistance.
BMC Infect. Dis.
PUBLISHED: 01-09-2014
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Greater use of antibiotics during the past 50 years has exerted selective pressure on susceptible bacteria and may have favoured the survival of resistant strains. Existing information on antibiotic resistance patterns from pathogens circulating among community-based patients is substantially less than from hospitalized patients on whom guidelines are often based. We therefore chose to assess the relationship between the antibiotic resistance pattern of bacteria circulating in the community and the consumption of antibiotics in the community.
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Public beliefs on antibiotics and symptoms of respiratory tract infections among rural and urban population in Poland: a questionnaire study.
PLoS ONE
PUBLISHED: 01-01-2014
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General public views and expectations around the use of antibiotics can influence general practitioners' antibiotic prescribing decisions. We set out to describe the knowledge, attitudes and beliefs about the use of antibiotics for respiratory tract infections in adults in Poland, and explore differences according to where people live in an urban-rural continuum.
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Metagenomic analysis of a sample from a patient with respiratory tract infection reveals the presence of a ?-papillomavirus.
Front Microbiol
PUBLISHED: 01-01-2014
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Previously unknown or unexpected pathogens may be responsible for that proportion of respiratory diseases in which a causative agent cannot be identified. The application of broad-spectrum, sequence independent virus discovery techniques may be useful to reduce this proportion and widen our knowledge about respiratory pathogens. Thanks to the availability of high-throughput sequencing (HTS) technology, it became today possible to detect viruses which are present at a very low load, but the clinical relevance of those viruses must be investigated. In this study we used VIDISCA-454, a restriction enzyme based virus discovery method that utilizes Roche 454 HTS system, on a nasal swab collected from a subject with respiratory complaints. A ?-papillomavirus was detected (complete genome: 7142 bp) and its role in disease was investigated. Respiratory samples collected both during the acute phase of the illness and 2 weeks after full recovery contained the virus. The patient presented antibodies directed against the virus but there was no difference between IgG levels in blood samples collected during the acute phase and 2 weeks after full recovery. We therefore concluded that the detected ?-papillomavirus is unlikely to be the causative agent of the respiratory complaints and its presence in the nose of the patient is not related to the disease. Although HTS based virus discovery techniques proved their great potential as a tool to clarify the etiology of some infectious diseases, the obtained information must be subjected to cautious interpretations. This study underlines the crucial importance of performing careful investigations on viruses identified when applying sensitive virus discovery techniques, since the mere identification of a virus and its presence in a clinical sample are not satisfactory proofs to establish a causative link with a disease.
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Distinct in vitro properties of embryonic and extra?embryonic fibroblast?like cells are reflected in their in vivo behaviour following grafting in the adult mouse brain.
Cell Transplant
PUBLISHED: 12-18-2013
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Although intracerebral transplantation of various fibroblast (-like) cell populations has been shown feasible, little is known about the actual in vivo remodelling of these cellular grafts and their environment. In this study, we aimed to compare the in vitro and in vivo behaviour of two phenotypically similar ? but developmentally distinct ? fibroblast?like cell populations, namely mouse embryonic fibroblasts (mEF) and mouse foetal membrane?derived stromal cells (mFMSC). While both mEF and mFMSC are readily able to reduce TNF? secretion by LPS/IFN??activated BV2 microglia, mFMSC and mEF display a strikingly opposite behaviour with regard to VEGF production under normal and inflammatory conditions. Whereas mFMSC downregulate VEGF production upon co?culture with LPS/IFN??activated BV2 microglia, mEF upregulate VEGF production in the presence of LPS/IFN??activated BV2 microglia. Subsequently, in vivo grafting of mFMSC and mEF revealed no difference in microglial and astroglial responses towards the cellular grafts. However, mFMSC grafts displayed a lower degree of neo?angiogenesis as compared to mEF grafts, thereby potentially explaining the lower cell number able to survive in mFMSC grafts. In summary, our results suggest that physiological differences between fibroblast?like cell populations might lie at the basis of variations in histopathological and/or clinical outcome following cell grafting in mouse brain.
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Antibiotic resistance-the need for global solutions.
Lancet Infect Dis
PUBLISHED: 11-17-2013
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The causes of antibiotic resistance are complex and include human behaviour at many levels of society; the consequences affect everybody in the world. Similarities with climate change are evident. Many efforts have been made to describe the many different facets of antibiotic resistance and the interventions needed to meet the challenge. However, coordinated action is largely absent, especially at the political level, both nationally and internationally. Antibiotics paved the way for unprecedented medical and societal developments, and are today indispensible in all health systems. Achievements in modern medicine, such as major surgery, organ transplantation, treatment of preterm babies, and cancer chemotherapy, which we today take for granted, would not be possible without access to effective treatment for bacterial infections. Within just a few years, we might be faced with dire setbacks, medically, socially, and economically, unless real and unprecedented global coordinated actions are immediately taken. Here, we describe the global situation of antibiotic resistance, its major causes and consequences, and identify key areas in which action is urgently needed.
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Interventions to reduce colonisation and transmission of antimicrobial-resistant bacteria in intensive care units: an interrupted time series study and cluster randomised trial.
Lancet Infect Dis
PUBLISHED: 10-23-2013
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Intensive care units (ICUs) are high-risk areas for transmission of antimicrobial-resistant bacteria, but no controlled study has tested the effect of rapid screening and isolation of carriers on transmission in settings with best-standard precautions. We assessed interventions to reduce colonisation and transmission of antimicrobial-resistant bacteria in European ICUs.
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Appropriate international measures for outpatient antibiotic prescribing and consumption: recommendations from a national data comparison of different measures.
J. Antimicrob. Chemother.
PUBLISHED: 10-01-2013
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Many European countries have engaged in awareness campaigns to decrease outpatient antibiotic use and several measures have been proposed, e.g. the number of defined daily doses (DDDs) or packages per 1000 inhabitants per day, producing conflicting findings. Therefore, we set out to explore what measure is most appropriate.
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Active liquid degassing in microfluidic systems.
Lab Chip
PUBLISHED: 09-24-2013
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We present a method for efficient air bubble removal in microfluidic applications. Air bubbles are extracted from a liquid chamber into a vacuum chamber through a semipermeable membrane, consisting of PDMS coated with amorphous Teflon(®) AF 1600. Whereas air is efficiently extracted through the membrane, water loss is greatly reduced by the Teflon even at elevated temperatures. We present the water loss and permeability change with the amount of added Teflon AF to the membrane. Also, we demonstrate bubble-free, multiplex DNA amplification using PCR in a PDMS microfluidic device.
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Comparison of strategies to reduce meticillin-resistant Staphylococcus aureus rates in surgical patients: a controlled multicentre intervention trial.
BMJ Open
PUBLISHED: 09-24-2013
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To compare the effect of two strategies (enhanced hand hygiene vs meticillin-resistant Staphylococcus aureus (MRSA) screening and decolonisation) alone and in combination on MRSA rates in surgical wards.
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Circulating dendritic cells of multiple sclerosis patients are proinflammatory and their frequency is correlated with MS-associated genetic risk factors.
Mult. Scler.
PUBLISHED: 09-20-2013
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The role of the adaptive immune system and more specifically T cells in the pathogenesis of multiple sclerosis (MS) has been studied extensively. Emerging evidence suggests that dendritic cells (DCs), which are innate immune cells, also contribute to MS.
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IFITM3 and susceptibility to respiratory viral infections in the community.
J. Infect. Dis.
PUBLISHED: 08-30-2013
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Interferon-inducible transmembrane proteins 1, 2 and 3 (IFITM1,2 and 3) are viral restriction factors that mediate cellular resistance to several viruses. We have genotyped a possible splice-site altering SNP (rs12252) in the IFITM3 gene in 34 H1N1 influenza cases with severe pneumonia and over 5000 individuals comprising cases of community-acquired mild lower respiratory tract infection and matched controls of Caucasian ancestry. We found evidence of an association between rs12252 rare allele homozygotes and susceptibility to mild influenza (in patients attending primary care), but could not confirm a previously reported association between this SNP and susceptibility to severe H1N1 infection.
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Point prevalence survey on antibiotic use in a Croatian Infectious Disease Hospital.
J Chemother
PUBLISHED: 08-03-2013
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Antibiotic use is the driving force for increasing antibiotic resistance. A large proportion of antibiotics in hospitals are used inadequately. The objective of this study was to evaluate antibiotic use at the Hospital for Infectious Diseases through point-prevalence surveys conducted in 2006, 2008, and 2009. Point prevalence surveys were part of the European Surveillance on Antimicrobial Consumption (ESAC) Hospital Care Subproject and patients data were collected following ESAC protocol. Additionally, the adequacy of antimicrobial therapy and administration of the first line antibiotic according to the local guidelines were assessed by an infectious disease doctor and a clinical microbiologist. In the study period among the 599 patients admitted to hospital, 352 (58·8%) received antibiotics. Out of 448 antimicrobial treatments, 313 (69·9%) were administered parenterally and 135 (30·1%) orally. Altogether in years 2006, 2008, and 2009 the most commonly prescribed antibiotics were ceftriaxone (19·9%), co-amoxiclav (15·4%), ciprofloxacin (12·3%), narrow spectrum penicillins (6·5%), and penicillinase resistant penicillins (5·6%). Most (82·6%) of the treated infections were community acquired infections. The predominating diagnoses were urinary tract infections and infections with no primary site defined, followed by skin, soft tissue and bone and joint infections. The overall adequacy of antimicrobial therapy was 82% and the first line antibiotic according to the local guidelines was administered with high frequency for central nervous system and cardiovascular infections (100%), and low for ear, nose, and throat infections, urinary tract infections, lower respiratory tract and bone and joint infections (23·0%, 51·6%, 52·5%, 65·0%, respectively) which indicates a significant overuse of antibiotics for diagnoses listed. The results of an individual point prevalence survey provided reliable and representative data for the hospital. Point-prevalence surveys proved to be a valuable method for detecting targets for antibiotic prescribing improvement and they clearly showed that our local hospital guidelines offered too many choices of antibiotic treatment for each clinical indication and needed revision.
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Effects of internet-based training on antibiotic prescribing rates for acute respiratory-tract infections: a multinational, cluster, randomised, factorial, controlled trial.
Lancet
PUBLISHED: 07-31-2013
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High-volume prescribing of antibiotics in primary care is a major driver of antibiotic resistance. Education of physicians and patients can lower prescribing levels, but it frequently relies on highly trained staff. We assessed whether internet-based training methods could alter prescribing practices in multiple health-care systems.
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The antibiotic resistance and prescribing in European Children project: a neonatal and pediatric antimicrobial web-based point prevalence survey in 73 hospitals worldwide.
Pediatr. Infect. Dis. J.
PUBLISHED: 07-11-2013
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The neonatal and pediatric antimicrobial point prevalence survey (PPS) of the Antibiotic Resistance and Prescribing in European Children project (http://www.arpecproject.eu/) aims to standardize a method for surveillance of antimicrobial use in children and neonates admitted to the hospital within Europe. This article describes the audit criteria used and reports overall country-specific proportions of antimicrobial use. An analytical review presents methodologies on antimicrobial use.
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Patient and prescriber determinants for the choice between amoxicillin and broader-spectrum antibiotics: a nationwide prescription-level analysis.
J. Antimicrob. Chemother.
PUBLISHED: 05-16-2013
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Bacterial resistance to antibiotics, driven by antibiotic consumption, imposes a major threat to the effective treatment of bacterial infections. In addition to reducing the amount of antibiotics prescribed, avoiding broad-spectrum antibiotics could extend the lifetime of the current arsenal of antibiotic substances. Therefore, we documented prescriber and patient characteristics associated with the choice between amoxicillin and broader-spectrum alternatives (co-amoxiclav or moxifloxacin) in recent years in Belgium.
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Outpatient systemic antimycotic and antifungal use in Europe: new outcome measure provides new insight.
Int. J. Antimicrob. Agents
PUBLISHED: 05-03-2013
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To broaden our understanding of outpatient systemic antimycotic and antifungal use in Europe, use data in defined daily doses (DDD) were complemented with data in packages and the results were compared. Within the European Surveillance of Antimicrobial Consumption project and using the Anatomical Therapeutic Chemical (ATC) classification, data on outpatient use of all 14 antimycotics (12) and antifungals (2) for systemic use (ATC J02 and D01B), aggregated at the level of the active substance, were collected for 2009. Their use was expressed in DDD per 1000 inhabitants per day (DID) and in packages per 1000 inhabitants per day (PID) (WHO Collaborating Centre for Drug Statistics Methodology ATC/DDD version 2011). In total, 24 countries delivered data in DID; 13 countries also delivered data in PID. In DID, Belgium had the highest (3.24 DID) and Romania the lowest (0.38 DID) total outpatient antimycotic and antifungal use. In PID, Greece had the highest (0.44 PID) and Sweden the lowest (0.08 PID) use. In DID, terbinafine was the most used substance in 19/24 countries (10/13 countries providing DID and PID data). In PID, fluconazole was the most used substance in all 13 countries. Combining DID and PID data substantially improved the interpretation of total outpatient antimycotic and antifungal use in Europe, and both outcome measures should be used for surveillance of these compounds. High use of fluconazole in PID might be more relevant for surveillance of antimicrobial consumption in relation to resistance than high use of terbinafine in DID.
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Differences in gut microbiota composition between obese and lean children: a cross-sectional study.
Gut Pathog
PUBLISHED: 03-18-2013
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An altered gut microbiota composition has recently been linked to obesity. The principal aim of this study is to investigate and compare the gut microbiota composition in obese and lean children. Secondly, associations between analysed gut bacterial species, dietary compounds, energy intake and biochemical blood parameters are evaluated.
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Prevalence and resistance of commensal Staphylococcus aureus, including meticillin-resistant S aureus, in nine European countries: a cross-sectional study.
Lancet Infect Dis
PUBLISHED: 03-06-2013
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Information about the prevalence of Staphylococcus aureus resistance to antimicrobial drugs has mainly been obtained from invasive strains, although the commensal microbiota is thought to be an important reservoir of resistance. We aimed to compare the prevalence of nasal S aureus carriage and antibiotic resistance, including meticillin-resistant S aureus (MRSA), in healthy patients across nine European countries.
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Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism.
J. Virol.
PUBLISHED: 02-20-2013
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The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains.
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Diagnosing pneumonia in patients with acute cough: clinical judgment compared to chest radiography.
Eur. Respir. J.
PUBLISHED: 01-24-2013
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Pneumonia is often diagnosed and treated empirically. We set out to determine the diagnostic accuracy of clinical judgment based on signs and symptoms to detect radiographic pneumonia in patients presenting with acute cough in primary care. In 2810 European patients with acute cough, general practitioners (GPs) recorded whether they considered pneumonia to be present ("yes" or "no") immediately after history and physical examination. Chest radiography was performed within 1 week by local radiologists blind to other patient characteristics. 140 patients had radiographic pneumonia (5%), of whom 41 (29%) had been diagnosed as such. 31 (1%) patients had a clinical diagnosis that was not confirmed by radiography (n=2670). In clinically suspected pneumonia, 57% of subjects were subsequently diagnosed with radiographic pneumonia. Negative predictive value (NPV), sensitivity and specificity of GPs clinical judgment were 96%, 29% and 99%, respectively. Compared to patients with a clinical diagnosis of pneumonia, less severe symptoms were found in radiographic pneumonia cases not suspected clinically (p<0.05). The predictive values of GPs clinical judgment, particularly the high NPVs, are helpful in routine care. Nonetheless, the majority of diagnoses of radiographic pneumonias was not suspected on clinical grounds. There is a need to further support the detection of clinically relevant pneumonia in primary care.
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Antimicrobial resistance: a global view from the 2013 World Healthcare-Associated Infections Forum.
Antimicrob Resist Infect Control
PUBLISHED: 01-01-2013
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Antimicrobial resistance (AMR) is now a global threat. Its emergence rests on antimicrobial overuse in humans and food-producing animals; globalization and suboptimal infection control facilitate its spread. While aggressive measures in some countries have led to the containment of some resistant gram-positive organisms, extensively resistant gram-negative organisms such as carbapenem-resistant enterobacteriaceae and pan-resistant Acinetobacter spp. continue their rapid spread. Please start the sentence as follows: Antimicrobial conservation/stewardship programs have seen some measure of success in reducing antimicrobial overuse in humans, but their reach is limited to acute-care settings in high-income countries. Outside the European Union, there is scant or no oversight of antimicrobial administration to food-producing animals, while evidence mounts that this administration leads directly to resistant human infections. Both horizontal and vertical infection control measures can interrupt transmission among humans, but many of these are costly and essentially limited to high-income countries as well. Novel antimicrobials are urgently needed; in recent decades pharmaceutical companies have largely abandoned antimicrobial discovery and development given their high costs and low yield. Against this backdrop, international and cross-disciplinary collaboration appears to be taking root in earnest, although specific strategies still need defining. Educational programs targeting both antimicrobial prescribers and consumers must be further developed and supported. The general public must continue to be made aware of the current scale of AMRs threat, and must perceive antimicrobials as they are: a non-renewable and endangered resource.
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Autologous antibody capture to enrich immunogenic viruses for viral discovery.
PLoS ONE
PUBLISHED: 01-01-2013
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Discovery of new viruses has been boosted by novel deep sequencing technologies. Currently, many viruses can be identified by sequencing without knowledge of the pathogenicity of the virus. However, attributing the presence of a virus in patient material to a disease in the patient can be a challenge. One approach to meet this challenge is identification of viral sequences based on enrichment by autologous patient antibody capture. This method facilitates identification of viruses that have provoked an immune response within the patient and may increase the sensitivity of the current virus discovery techniques. To demonstrate the utility of this method, virus discovery deep sequencing (VIDISCA-454) was performed on clinical samples from 19 patients: 13 with a known respiratory viral infection and 6 with a known gastrointestinal viral infection. Patient sera was collected from one to several months after the acute infection phase. Input and antibody capture material was sequenced and enrichment was assessed. In 18 of the 19 patients, viral reads from immunogenic viruses were enriched by antibody capture (ranging between 1.5x to 343x in respiratory material, and 1.4x to 53x in stool). Enriched reads were also determined in an identity independent manner by using a novel algorithm Xcompare. In 16 of the 19 patients, 21% to 100% of the enriched reads were derived from infecting viruses. In conclusion, the technique provides a novel approach to specifically identify immunogenic viral sequences among the bulk of sequences which are usually encountered during virus discovery metagenomics.
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Are patient views about antibiotics related to clinician perceptions, management and outcome? A multi-country study in outpatients with acute cough.
PLoS ONE
PUBLISHED: 01-01-2013
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Outpatients with acute cough who expect, hope for or ask for antibiotics may be more unwell, benefit more from antibiotic treatment, and be more satisfied with care when they are prescribed antibiotics. Clinicians may not accurately identify those patients.
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European Surveillance of Antimicrobial Consumption (ESAC): quality appraisal of antibiotic use in Europe.
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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To assess quality of outpatient antibiotic use in Europe in 2009 based on the 12 European Surveillance of Antimicrobial Consumption (ESAC) drug-specific quality indicators and to evaluate changes in quality between 2004 and 2009.
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European Surveillance of Antimicrobial Consumption (ESAC): outpatient use of tetracyclines, sulphonamides and trimethoprim, and other antibacterials in Europe (1997-2009).
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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Data on more than a decade of outpatient use of tetracyclines, sulphonamides and trimethoprim, and other antibacterials in Europe were collected from 33 countries as part of the European Surveillance of Antimicrobial Consumption (ESAC) project, funded by the European Centre for Disease Prevention and Control (ECDC).
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European Surveillance of Antimicrobial Consumption (ESAC): outpatient quinolone use in Europe (1997-2009).
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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Data on more than a decade of outpatient quinolone use were collected from 33 European countries within the European Surveillance of Antimicrobial Consumption (ESAC) project, funded by the European Centre for Disease Prevention and Control (ECDC).
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European Surveillance of Antimicrobial Consumption (ESAC): outpatient macrolide, lincosamide and streptogramin (MLS) use in Europe (1997-2009).
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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Data on more than a decade of outpatient macrolide, lincosamide and streptogramin (MLS) use in Europe were collected from 33 countries within the European Surveillance of Antimicrobial Consumption (ESAC) project, funded by the European Centre for Disease Prevention and Control (ECDC), using the WHO Anatomical Therapeutic Chemical (ATC)/defined daily dose (DDD) methodology.
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European Surveillance of Antimicrobial Consumption (ESAC): outpatient antibiotic use in Europe (1997-2009).
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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To describe total outpatient systemic antibiotic use in Europe from 1997 to 2009 and to analyse statistically trends of total use and composition of use over time.
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European Surveillance of Antimicrobial Consumption (ESAC): outpatient cephalosporin use in Europe (1997-2009).
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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Data on 13 years of outpatient cephalosporin use were collected from 33 European countries within the European Surveillance of Antimicrobial Consumption (ESAC) project, funded by the European Centre for Disease Prevention and Control (ECDC), and analysed in detail.
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European Surveillance of Antimicrobial Consumption (ESAC): outpatient penicillin use in Europe (1997-2009).
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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Data on 13 years (1997-2009) of outpatient penicillin use were collected from 33 European countries within the European Surveillance of Antimicrobial Consumption (ESAC) project and analysed in detail.
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Analysing the composition of outpatient antibiotic use: a tutorial on compositional data analysis.
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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In this tutorial, statistical methods for studying outpatient use of antibiotics in Europe are described, using data provided by IMS Health. The methods are applied to two related research questions, namely the assessment of changes in the relative volume of use of different antibiotic subclasses over time and changes in the absolute volume of antibiotic use.
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Application of mixed-effects models to study the country-specific outpatient antibiotic use in Europe: a tutorial on longitudinal data analysis.
J. Antimicrob. Chemother.
PUBLISHED: 11-19-2011
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Resistance to antibiotics is a major public health problem and antibiotic use is being increasingly recognized as the main selective pressure driving this resistance. Yearly and quarterly data on outpatient antibiotic use were collected by the European Surveillance of Antimicrobial Consumption (ESAC) project for the period 1997-2009 from 33 and 27 European countries, respectively, and expressed in defined daily doses per 1000 inhabitants per day. Since repeated measures were taken for the countries, correlation has to be taken into account when analysing the data. This paper illustrates the application of mixed-effects models to the study of country-specific outpatient antibiotic use in Europe. Mixed models are useful in a wide variety of disciplines in the biomedical, physical and social sciences. In this application for outpatient antibiotic use, the linear mixed model is extended to a non-linear mixed model, allowing analysis of seasonal variation on top of a global trend, with country-specific effects for global mean use and amplitude, and trends over time in use and in amplitude.
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Parenteral versus oral administration of systemic antimicrobials in European nursing homes: a point-prevalence survey.
Drugs Aging
PUBLISHED: 10-06-2011
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Residents in long-term care facilities are predisposed to healthcare-associated infections that are likely caused by antimicrobial-resistant micro-organisms. Long-term care facilities are increasingly able to offer parenteral antimicrobial treatment but there are few data on the use and appropriateness of such treatment in this setting. Information on the use of parenteral antimicrobials and associated factors in long-term care facilities is necessary to assess the risks and benefits of this treatment and to support the development of antimicrobial policies aimed at minimizing the emergence and spread of antimicrobial resistance.
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The body gets used to them: patients interpretations of antibiotic resistance and the implications for containment strategies.
J Gen Intern Med
PUBLISHED: 09-26-2011
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Interventions promoting evidence based antibiotic prescribing and use frequently build on the concept of antibiotic resistance but patients and clinicians may not share the same assumptions about its meaning.
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Building an international network for a primary care research program: reflections on challenges and solutions in the set-up and delivery of a prospective observational study of acute cough in 13 European countries.
BMC Fam Pract
PUBLISHED: 07-27-2011
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Implementing a primary care clinical research study in several countries can make it possible to recruit sufficient patients in a short period of time that allows important clinical questions to be answered. Large multi-country studies in primary care are unusual and are typically associated with challenges requiring innovative solutions. We conducted a multi-country study and through this paper, we share reflections on the challenges we faced and some of the solutions we developed with a special focus on the study set up, structure and development of Primary Care Networks (PCNs).
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Implementation of e-Bug in Belgium.
J. Antimicrob. Chemother.
PUBLISHED: 06-18-2011
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Antimicrobial resistance is an important issue in Belgium. For more than a decade, national projects aiming to promote the prudent use of antimicrobials and to decrease antimicrobial resistance in ambulatory, nursing-home and hospital settings have been launched by the Belgian Antibiotic Policy Coordination Committee (BAPCOC), coordinated by the Federal Public Service of Public Health, Food Chain Safety and Environment. In this paper, we describe the implementation of the e-Bug Project in Belgium by the University of Antwerp in collaboration with BAPCOC and supported by the Ministries of Education, and how e-Bug complemented a new national campaign focusing on antibiotic use in children first started on the European Antibiotic Awareness Day in 2008. We also describe the impact of the organization of education in Belgium on the implementation process, as well as plans for the future of e-Bug in our country.
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A multi-country qualitative study of clinicians and patients views on point of care tests for lower respiratory tract infection.
Fam Pract
PUBLISHED: 06-08-2011
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Point of care tests (POCTs) are being promoted to better target antibiotic prescribing with the aim of improving outcomes and containing antibiotic resistance.
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The appropriateness of prescribing antibiotics in the community in Europe: study design.
BMC Infect. Dis.
PUBLISHED: 05-31-2011
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Over 90% of all antibiotics in Europe are prescribed in primary care. It is important that antibiotics are prescribed that are likely to be effective; however, information about antibiotic resistance in the community is incomplete. The aim of our study is to investigate the appropriateness of antibiotic prescribing in primary care in Europe by collecting and combining patterns of antibiotic resistance patterns and antibiotic prescription patterns in primary care. We will also evaluate the appropriateness of national antibiotic prescription guidelines in relation to resistance patterns.
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European Surveillance of Antimicrobial Consumption (ESAC): systemic antiviral use in Europe.
J. Antimicrob. Chemother.
PUBLISHED: 05-27-2011
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To assess the total systemic antiviral use in Europe and to identify the antiviral substances most commonly used.
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Antimicrobial prescribing in European nursing homes.
J. Antimicrob. Chemother.
PUBLISHED: 05-19-2011
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The aim of this study was to investigate antimicrobial prescribing in nursing homes in countries across Europe.
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Presentation, antibiotic management and associated outcome in Polish adults presenting with acute cough/LRTI.
Fam Pract
PUBLISHED: 05-09-2011
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In-depth knowledge of existing practice is required to inform interventions aimed at antibiotic prescribing quality improvement. We set out to describe the presentation, antimicrobial management and associated outcome of adults presenting in general practice with acute cough/lower respiratory tract infection (LRTI) in Poland.
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Towards clinical definitions of lower respiratory tract infection (LRTI) for research and primary care practice in Europe: an international consensus study.
Prim Care Respir J
PUBLISHED: 04-22-2011
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Antibiotic prescriptions for lower respiratory tract infection (LRTI) account for a large proportion of antibiotic consumption. Many of these prescriptions do not benefit patients and contribute to antibiotic resistance. Research to improve evidence-based management requires clear definitions of clinical entities. We aimed to generate definitions for common LRTIs that are applicable to clinical practice and low-intensity investigation research settings in European primary care.
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European Surveillance of Antimicrobial Consumption (ESAC): value of a point-prevalence survey of antimicrobial use across Europe.
Drugs
PUBLISHED: 04-21-2011
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All 27 EU member states and another seven countries participate in the European Surveillance of Antimicrobial Consumption (ESAC) project. ESAC carried out three hospital point-prevalence surveys on antimicrobial use. Point-prevalence surveys linked antimicrobial use to indication and also assessed dosing using a standardized methodology for data collection and online data submission with feedback capability using a dedicated web-based tool. The objectives of the ESAC hospital point-prevalence surveys were to first determine the feasibility of a pan-European survey and identify targets for quality improvement. Hospitals were voluntarily selected by the lead national or hospital representatives for each country. The WHO Anatomical Therapeutic Chemical Classification of drugs was used for classification of antimicrobials. The three surveys were carried out during a maximum of 2 weeks in the second quarter of 2006, 2008 and 2009. Each department had to be surveyed in 1 day. All systemic antibacterials (J01), rifampicin (J04AB), oral vancomycin (A07AA) and oral/rectal metronidazole (P01AB) were the antimicrobials surveyed, including the prescribed regimen. The number of participating hospitals increased from 20 to 172 from 2006 to 2009. The patient demographics and indications for treatment were similar throughout the three point-prevalence surveys. Reason in notes and surgical prophylaxis >24 hours were also similar. Guideline compliance (51%) was only introduced in the 2009 point-prevalence survey, replacing sample for culture and sensitivity (<50% in 2006 and 2008) since samples were either not taken or no information was available for the majority (>50%) of patients. The use of combination therapy, although exhibiting a wide range within each category, was related to hospital type, with teaching and tertiary hospitals having a significantly higher use of combination therapy (teaching?:?non-teaching hospitals [p?
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Intestinal microflora and body mass index during the first three years of life: an observational study.
Gut Pathog
PUBLISHED: 04-11-2011
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Recent research on obesity has demonstrated that the intestinal microflora can have an important influence on host energy balance. The aim of the study was to investigate the relationship between the intestinal microflora and the body mass index in the first 3 years of life.
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Denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma.
BMC Microbiol.
PUBLISHED: 04-10-2011
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The extended hygiene hypothesis suggests that the initial composition of the infant gut microbiota is a key determinant in the development of atopic disease. Several studies have demonstrated that the microbiota of allergic and non-allergic infants are different even before the development of symptoms, with a critical time window during the first 6 months of life. The aim of the study was to investigate the association between early intestinal colonisation and the development of asthma in the first 3 years of life using DGGE (denaturing gradient gel electrophoresis).
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European Surveillance of Antimicrobial Consumption (ESAC): disease-specific quality indicators for outpatient antibiotic prescribing.
BMJ Qual Saf
PUBLISHED: 03-29-2011
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Background In 2007, ESAC (http://www.esac.ua.ac.be) published a set of 12 valid drug-specific quality indicators for outpatient antibiotic use in Europe. In this study, the authors aimed to develop evidence-based disease-specific quality indicators for outpatient antibiotic prescribing in Europe. Methods Two meetings were convened to produce a list of disease-specific quality indicators for outpatient antibiotic prescribing which conform to internationally agreed recommendations, building on a similar development of drug-specific quality indicators, and in collaboration with CHAMP and HAPPY AUDIT. 62 experts were asked to complete two scoring rounds of the proposed indicators on seven dimensions: their relevance to (1) reducing antimicrobial resistance, (2) patient health benefit, (3) cost-effectiveness, (4) policy makers, (5) individual prescribers, (6) their evidence base and (7) their range of acceptable use, using a scale ranging from 1 (=completely disagree) to 9 (=completely agree). Scores were judged according to the UCLA-RAND appropriateness method. Results For the six main indications for antibiotic prescribing (acute otitis media, acute upper-respiratory infection, acute/chronic sinusitis, acute tonsillitis, acute bronchitis/bronchiolitis, cystitis/other urinary infection) and for pneumonia, three quality indicators were proposed, the percentage prescribed (a) antibiotics; (b) recommended antibiotics; (c) quinolones. This set was scored by 40 experts from 25 countries. After one scoring round, all indicators were already rated as relevant on all dimensions, except one. Conclusion All proposed disease-specific quality indicators for outpatient antibiotic prescribing have face validity and are potentially applicable. They could be used to better describe antibiotic use and assess the quality of antibiotic prescribing patterns in ambulatory care.
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A sensitive assay for virus discovery in respiratory clinical samples.
PLoS ONE
PUBLISHED: 01-24-2011
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In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.