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Vector-borne protozoal diseases (VBPD) represent an enormous health and economic burden, particularly in low- and middle-income countries. Their control requires integrated approaches that consider not only therapeutic interventions for affected human and animal populations but also preventive tools. Environmental contamination can lead to therapeutic ineffectiveness. Effective intervention must consider in-depth knowledge of the environmental factors that regulate the exposure, transmission and pathogenicity of VBPD within a One Health approach. In recent decades, the incidence and prevalence of VBPD have been substantially reduced in many regions of the world, although there are still hot spots and emerging epidemiological cycles. Except for a partially protective vaccine against malaria, vaccination is not available for any other human VBPD, and therefore epidemiological control and chemotherapy are the main control tools. Current therapeutics have several drawbacks, including reduced efficacy, toxicity and high price of safer formulations. In addition, the industrial pipeline is limited, and no therapeutic breakthroughs are expected. Integrated control of VBPD requires multitarget control systems adapted to the disease and region. In this scenario, harmonized surveillance systems, accurate reporting and increased public and private investment will ensure more rational use of the few available and new drugs.

Plasma 25-dihydroxyvitamin D levels appear reduced in patients with obesity or type 2 diabetes, as reported in several observational studies. However, the association between these reduced hormone levels and metabolic parameters is unclear. In any case, vitamin D supplementation in patients with Metabolic Syndrome is standard. Still, the impacts of this supplementation on conditions such as glycemia, blood pressure, and lipidemia are debatable. Based on this question, we carried out a systematic review and meta-analysis of randomized clinical trials in Brazil, Europe, and the United States that analyzed the effects of vitamin D supplementation on Metabolic Syndrome parameters in patients with obesity or type 2 diabetes. Our search yielded 519 articles and included 12 randomized controlled trials in the meta-analysis. Vitamin D supplementation had no effect on any of the outcomes analyzed (fasting blood glucose and insulinemia, glycated hemoglobin, HOMA index, systolic and diastolic blood pressure, weight, waist circumference, total cholesterol, LDL and HDL, and triglycerides). However, subgroup analyses indicated that using vitamin D up to 2000 IU daily reduced participants' fasting blood glucose and glycated hemoglobin. Furthermore, the intervention reduced diastolic blood pressure only in participants with vitamin D deficiency. At least two studies showed a high risk of bias using the Rob2 protocol. According to the GRADE protocol, the evidence quality varied from moderate to very low. These results indicate that vitamin D supplementation does not improve patients' metabolic parameters and that the association between plasma 25-dihydroxyvitamin D levels and Metabolic Syndrome may not be causal but caused by other confounding characteristics. However, in any case, the quality of evidence is still low, and more randomized clinical trials are essential to clarify these relationships., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The European Medicines Agency adopted their Geriatric Medicines Strategy more than a decade ago. The strategy aims at elucidating the evidence basis for marketing authorization of new medicines which will be used in the older population, and at ensuring the appropriate communication of findings to the patient and healthcare provider. During the past decade new tools and data sources have emerged to support the strategy goals, and their use should be considered. Possible concrete actions are presented to improve the design of clinical trials, the data collection both pre- and post-approval, the assessment of the findings, and the communication to assist informed prescription and safe medicine taking. Implementation and prioritization of these actions should be done from the perspective of addressing the needs of patients while maximizing efficient use of resources, with the aim of integrating geriatric aspects into routine medicines development and assessment., (© 2024 The Authors. Journal of the American Geriatrics Society published by Wiley Periodicals LLC on behalf of The American Geriatrics Society.)

Background: Over the last twenty years of orphan drug regulation in Europe, the regulatory framework has increased its complexity, with different regulatory paths and tools engineered to facilitate the innovation and accelerate approvals. Recently, the proposal of the new Pharmaceutical Legislation for the European Union, which will replace at least three Regulations and one Directive, was released and its new framework is raising many questions. The aim of this study was to present a characterisation of the Orphan Medicinal Products (OMPs) authorised by the European Commission (EC), between 2010 and 2022, looking into eighteen variables, contributing to the ongoing discussion on the proposal and implementation of the new Pharmaceutical Legislation proposed., Methods: Data of the OMPs identified and approved between 2010 and 2022 were extracted from the European Public Assessment Reports (EPARs) produced by the European Medicines Agency. Information regarding legal basis of the application, applicant, protocol assistance received, type of authorization, registration status, type of molecule, ATC code, therapeutic area, target age, disease prevalence, number of pivotal clinical trials supporting the application, clinical trial designs, respective efficacy endpoints and number of patients enrolled in the pivotal clinical trials were extracted. A descriptive statistical analysis was applied., Results: We identified 192 OMPs approved in the period between 2010 and 2022. 89% of the OMPs have legal basis of "full application". 86% of the sponsors received protocol assistance whereas 64% of the MAA benefited from the accelerated assessment. 53% of the active substances are small molecules; about 1 in 5 molecules are repurposed. 40% of the OMPs have oncological therapeutic indications and 56% of the OMPs are intended to treat only adults. 71% of the products were approved based on a single pivotal trial., Conclusions: This analysis of OMPs approved between 2010 and 2022 shows that a shift has occurred in the rare disease medicine development space. Through the period studied we observe an increase of non-small molecules approved, accelerated assessment received and non-standard MA's granted., (© 2024. The Author(s).)

One of the most frequent causes of acute viral hepatitis is hepatitis E virus (HEV) causing 20 million infections worldwide each year and 44,000 deaths. Studies on HEV in the Iberian Peninsula have been increasing through time with HEV infection being identified in humans and animals. The aim of the present systematic review was to compile and evaluate all the published data on HEV from studies performed in humans, animals and environmental samples in the Iberian Peninsula. The electronic databases Mendeley, PubMed, Scopus, and Web of Science were thoroughly searched, and research published up until February 01, 2023 were included. Resulting in a total of 151 eligible papers by full reading and application of PRISMA exclusion/inclusion criteria. Overall, the present review shows that several HEV genotypes, namely HEV-1, 3, 4, and 6 as well as Rocahepevirus, are circulating in humans, animals, and in the environment in the Iberian Peninsula. HEV-3 was the most common genotype circulating in humans in Portugal and Spain, as expected for developed countries, with HEV-1 only being detected in travelers and emigrants from HEV endemic regions. Spain is the biggest pork producer in Europe and given the high circulation of HEV in pigs, with HEV-3 being primarily associated to zoonotic transmission through consumption of swine meat and meat products, in our opinion, the introduction of an HEV surveillance system in swine and inclusion of HEV in diagnostic routines for acute and chronic human hepatitis would be important. Additionally, we propose that establishing a monitoring mechanism for HEV is crucial in order to gain a comprehensive understanding of the prevalence of this illness and the various strains present in the Iberian Peninsula, as well as their potential impact on public health., (© 2023. The Author(s).)

Background: It is important to understand the effects of population ageing on disease burden and explore conditions that drive poor health in later life to prevent or manage these. We examined the development of disease burden and its components for major disease groups among older adults in Europe over the last 30 years., Methods: Using data from the Global Burden of Disease 2019 Study, we analyzed burden of disease trends between 1990 and 2019 measured by years of life lost (YLL), years lived with disability (YLD) and disability-adjusted life years (DALYs) among older adults (65+ years) in Western, Central and Eastern Europe using cause groups for diseases and injuries., Results: Between 1990 and 2019, the crude numbers of DALYs for all causes increased substantially among older Western Europeans. In Eastern Europe, the absolute DALYs also increased from 1990 to 2005 but then decreased between 2006 and 2013. However, DALY rates declined for all European regions over time, with large differences in the magnitude by region and gender. Changes in the YLL rate were mainly driven by the contribution of cardiovascular diseases., Conclusions: This study found an increased overall absolute disease burden among older Europeans between 1990 and 2019. The demographic change that has taken place in Eastern European countries implies a potential problem of directed resource allocation to the health care sector. Furthermore, the findings highlight the potential health gains through directing resources to health promotion and treatment to reduce YLDs and to prevent YLLs, primarily from cardiovascular diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Public Health Association.)

Background: Understanding variability in prescribing patterns through comparative drug utilization studies can contribute to improve an efficient, effective and safe use of medicines., Objectives: To perform a cross-country comparison of consumption patterns of ambulatory high expenditure therapeutic groups between Portugal and six European countries and simulate potential cost-saving scenarios through the adoption of the different prescribing patterns of studied countries., Methods: Cross-country comparison of 2019 drug consumption patterns between Portugal, Denmark, England, Finland, the Netherlands, Norway, and Spain. Analysis comprised antihypertensive drugs, glucose lowering drugs (GLD), insulins, lipid lowering drugs (LLD) and oral anticoagulants. Cost-saving analysis were performed using the Portugal average annual cost/daily defined dose and the potential reduction in expenditure simulating other European countries consumption pattern scenarios., Results: Portugal had the lowest consumption uptake of metformin and the highest consumption of GLD (30.1%) and LLD (8.5% vs <3%) fixed-dose combinations. Annual cost-savings scenarios showed that Portugal would have saved between 53 M€ and 305 M€ if it had the same prescribing patterns than Norway or the Netherlands, respectively., Conclusions: Different utilization patterns across countries were found. Although Portugal has the lowest gross domestic product per capita among the countries studied, it had the highest uptake of newly and costly drugs.

Introduction: unCoVer-Unravelling data for rapid evidence-based response to COVID-19-is a Horizon 2020-funded network of 29 partners from 18 countries capable of collecting and using real-world data (RWD) derived from the response and provision of care to patients with COVID-19 by health systems across Europe and elsewhere. unCoVer aims to exploit the full potential of this information to rapidly address clinical and epidemiological research questions arising from the evolving pandemic., Methods and Analysis: From the onset of the COVID-19 pandemic, partners are gathering RWD from electronic health records currently including information from over 22 000 hospitalised patients with COVID-19, and national surveillance and screening data, and registries with over 1 900 000 COVID-19 cases across Europe, with continuous updates. These heterogeneous datasets will be described, harmonised and integrated into a multi-user data repository operated through Opal-DataSHIELD, an interoperable open-source server application. Federated data analyses, without sharing or disclosing any individual-level data, will be performed with the objective to reveal patients' baseline characteristics, biomarkers, determinants of COVID-19 prognosis, safety and effectiveness of treatments, and potential strategies against COVID-19, as well as epidemiological patterns. These analyses will complement evidence from efficacy/safety clinical trials, where vulnerable, more complex/heterogeneous populations and those most at risk of severe COVID-19 are often excluded., Ethics and Dissemination: After strict ethical considerations, databases will be available through a federated data analysis platform that allows processing of available COVID-19 RWD without disclosing identification information to analysts and limiting output to data aggregates. Dissemination of unCoVer's activities will be related to the access and use of dissimilar RWD, as well as the results generated by the pooled analyses. Dissemination will include training and educational activities, scientific publications and conference communications., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Food allergy affects up to 6% of Europeans. Allergen identification is important for the risk assessment and management of the inadvertent presence of allergens in foods. The VITAL® initiative for voluntary incidental trace allergen labeling suggests protein reference doses, based on clinical reactivity in food challenge studies, at or below which voluntary labelling is unnecessary. Here, we investigated if current analytical methodology could verify the published VITAL® 2.0 doses, that were available during this analysis, in serving sizes between 5 and 500 g. Available data on published and commercial ELISA, PCR and mass spectrometry methods, especially for the detection of peanuts, soy, hazelnut, wheat, cow's milk and hen's egg were reviewed in detail. Limit of detection, quantitative capability, matrix compatibility, and specificity were assessed. Implications by the recently published VITAL® 3.0 doses were also considered. We conclude that available analytical methods are capable of reasonably robust detection of peanut, soy, hazelnut and wheat allergens for levels at or below the VITAL® 2.0 and also 3.0 doses, with some methods even capable of achieving this in a large 500 g serving size. Cow's milk and hen's egg are more problematic, largely due to matrix/processing incompatibility. An unmet need remains for harmonized reporting units, available reference materials, and method ring-trials to enable validation and the provision of comparable measurement results., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This study aims to evaluate the instrumental activities of daily living (iADLs) limitations in Europe and its association with socio-demographic characteristics, economic parameters and physical and mental health status. We used data from the wave 6 of SHARE database. Individuals were classified as having either none or one or more limitations on iADLs. Participants aged 65 or more years who answered all questions for the variables included in this work were selected. A total of 54.8% of participants were female and had a mean age of 74.37 (SD = 7.08) years. A global prevalence of 1 or more iADLs in Europe was shown to be 23.8% and more prevalent in women than in men (27.1% vs. 17.6%) and in people aged 85 years or more (51.5%). Older age, female gender, lower education, physical inactivity, frailty, having two or more chronic diseases, presence of depression, polypharmacy, poor self-perception of health and lower network satisfaction were found to be factors associated with the presence of 1 or more iADLs limitation. This study highlights the burden of iADLs limitations at the European level. These are based on a multidimensional biopsychosocial model and are associated with both health conditions and environmental factors. This intersection between the physical and social world underscores its potential as a health indicator and can, to some extent, explain some of the pronounced differences seen among European countries. Different inter-tasks can also stress different dimensions of health indicators in distinct and specific groups of individuals. Minimizing the impact of iADL limitations can improve the quality and sustainability of public health systems.

Linezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally from multiple sources, but we lack a comprehensive analysis of human and animal optrA -LREfs strains. To assess if optrA is dispersed in isolates with varied genetic backgrounds or with common genetic features, we investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), virulence, prophages, plasmidome] and optrA -containing platforms of 27 publicly available optrA -positive E. faecalis genomes from different hosts in seven countries. At the genome-level analysis, an in-house database with 64 virulence genes was tested for the first time. Our analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera from Firmicutes . Phylogenies of core and accessory genomes were not congruent, and at least PAI-associated and prophage genes contribute to such differences. Epidemiologically unrelated clones (ST21, ST476-like and ST489) obtained from human clinical and animal hosts in different continents over eight years (2010-2017) could be phylogenetically related (3-126 SNPs difference). optrA was located on the chromosome within a Tn 6674 -like element ( n =10) or on medium-size plasmids (30-60 kb; n =14) belonging to main plasmid families (RepA&#95;N/Inc18/Rep&#95;3). In most cases, the immediate gene vicinity of optrA was generally identical in chromosomal (Tn 6674 ) or plasmid ( impB-fexA-optrA ) backbones. Tn 6674 was always inserted into the same ∆ radC integration site and embedded in a 32 kb chromosomal platform common to strains from different origins (patients, healthy humans, and animals) in Europe, Africa, and Asia during 2012-2017. This platform is conserved among hundreds of E. faecalis genomes and proposed as a chromosomal hotspot for optrA integration. The finding of optrA in strains sharing common adaptive features and genetic backgrounds across different hosts and countries suggests the occurrence of common and independent genetic events occurring in distant regions and might explain the easy de novo generation of optrA -positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious impact on the efficacy of linezolid for the treatment of Gram-positive infections.

Objectives: As the world population ages, psychiatrists will increasingly need instruments for measuring constructs of psychopathology that are generalizable to diverse elders. The study tested whether syndromes of co-occurring problems derived from self-ratings of psychopathology by US elders would fit self-ratings by elders in 19 other societies., Methods/design: The Older Adult Self-Report (OASR) was completed by 12 826 adults who were 60 to 102 years old in 19 societies from North and South America, Asia, and Eastern, Northern, Southern, and Western Europe, plus the United States. Individual and multigroup confirmatory factor analyses (CFAs) tested the fit of the seven-syndrome OASR model, consisting of the Anxious/Depressed, Worries, Somatic Complaints, Functional Impairment, Memory/Cognition Problems, Thought Problems, and Irritable/Disinhibited syndromes., Results: In individual CFAs, the primary model fit index showed good fit for all societies, while the secondary model fit indices showed acceptable to good fit. The items loaded strongly on their respective factors, with a median item loading of .63 across 20 societies, and 98.7% of the loadings were statistically significant. In multigroup CFAs, 98% of items demonstrated approximate or full metric invariance. Fifteen percent of items demonstrated approximate or full scalar invariance, and another 59% demonstrated scalar invariance across more than half of societies., Conclusions: The findings supported the generalizability of OASR syndromes across societies. The seven syndromes offer empirically based clinical constructs that are relevant for elders of different backgrounds. They can be used to assess diverse elders and as a taxonomic framework to facilitate communication, services, research, and training in geriatric psychiatry., (© 2020 John Wiley & Sons Ltd.)

Obesity is a severe worldwide epidemic. Obesity comorbidities, such as type 2 diabetes mellitus, hypertension, and atherosclerosis, are costly for patients and governments. The treatment of obesity involves several facets, including lifestyle changes, bariatric surgery, and pharmacotherapy. As changes in lifestyle require considerable patient commitment that is sometimes unachievable, and surgery is expensive and invasive, pharmacotherapy is the primary option for most patients. This review describes the pharmacotherapy currently available in the USA, Europe, and Brazil, focusing on its limitations. We then analyze the results from clinical trials of new drug candidates. Most drugs cause weight loss of < 4 kg compared with controls, and severe adverse effects have caused a number of drugs to be withdrawn from the market in several countries. Drugs under development have not shown more significant weight loss or reduced adverse effects. We conclude that a significant portion of obese patients have few treatment options because of the adverse effects and minimal weight loss associated with current pharmacotherapy. However, drugs currently under development appear unable to change this scenario in the near future. Thus, it is essential that new compounds are developed and new molecular targets studied so obesity can be efficiently treated in all patients in the future.

Honey is the natural sweet substance produced by Apis mellifera honeybees in Europe. Depending on the country/region, the A. mellifera subspecies native to Europe belong to three different lineages: A (A. m. iberiensis), M (A. m. iberiensis and A. m. mellifera) and C (A. m. ligustica and A. m. carnica). In this work, two DNA-based approaches were developed with the aim of entomological authentication of European honeys. A cytb specific PCR assay was proposed to identify A-lineage honeybees, while a second method based on real-time PCR coupled to high resolution melting analysis targeting the COI gene was developed to differentiate C- and M-lineages honeybees. The proposed methodologies were validated successfully with honeys of known origin and applied to the entomological authentication of 20 commercial samples from different European countries. The results highlight the predominance of honeys from C-lineage honeybees in Europe, except in Iberian Peninsula countries (honey from A-lineage honeybees)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Gene therapy medicinal products (GTMPs) are innovative and promising treatment strategies. In Europe, the Committee for Advanced Therapies (CAT) is responsible for making marketing authorization recommendations to the Committee of Human Medicinal Products (CHMP). In the current study, we identified major objections, issues, or concerns raised during the Marketing Authorization Application (MAA) process for products resulting from the interaction of both committees between 2008 and 2017. During the first few years following CAT establishment, quality issues were often identified as major deficiencies, whereas issues at the nonclinical level appeared to be less frequent. Clinical efficacy and safety issues appeared to have a major role in unsuccessful MAA outcome for GTMPs. Most deficiencies were addressed through clarification during the MAA review or in postmarketing settings. The MAA procedure for GTMPs is complex and it is anticipated that continuous MAA submissions will further enhance the experience of both regulators and applicants, reducing the attrition rate for approval., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microorganisms are intentionally added at different stages of the food and feed chain (food or feed additive, novel food or plant protection product) and are subjected to regulation and safety assessment by the European Food Safety Authority. Safety evaluation is based on application dossiers for market authorisation to the European Commission. The qualified presumption of safety (QPS) concept was developed in 20031 to provide a harmonised generic safety pre-appraisal of the above microorganisms. Unambiguously defined biological taxonomic units (TUs) are assessed for their body of knowledge, their safety and their end use. Identified safety concerns for a certain TU can be, where reasonable in number and not universally present, reflected as 'qualifications.' Strains belonging to TUs having QPS status may benefit of a fast track evaluation. The lowest TU for which the QPS status is granted is the species level for bacteria and yeasts and the family for viruses. The QPS concept is also applicable to genetically modified microorganisms used for production purposes. Based on the current body of knowledge and/or the ambiguous taxonomic position, some TUs, such as filamentous fungi, bacteriophages, Enterococcus faecium, Escherichia coli, Streptomyces spp. and Oomycetes, are not considered liable for QPS status.

The review uses the Helicobacter pylori , the gastric bacterium that colonizes the human stomach, to address how to obtain information from bacterial genomes about prophage biology. In a time of continuous growing number of genomes available, this review provides tools to explore genomes for prophage presence, or other mobile genetic elements and virulence factors. The review starts by covering the genetic diversity of H. pylori and then moves to the biologic basis and the bioinformatics approaches used for studding the H. pylori phage biology from their genomes and how this is related with the bacterial population structure. Aspects concerning H. pylori prophage biology, evolution and phylogeography are discussed.

Background: Market forces may not be sufficient to stimulate research and development of medicines for small patient populations, such as children and patients with rare diseases. Both the European Union Orphan and Paediatric Regulations were introduced to address the unmet public health needs of these smaller patient populations through the use of incentives, rewards and obligations. Developers for new medicines for rare diseases must agree a paediatric investigation plan (PIP) or waiver with the European Medicines Agency's (EMA) Paediatric Committee (PDCO), and can also apply for an orphan designation (OD) from the EMA's Committee of Orphan Medicinal Products (COMP). The scope of both the OD and the PIP (or waiver) is defined by the agreed condition., Objectives: The aim of this study was to analyse the approach of PDCO and COMP in defining the appropriate condition for a PIP or OD, respectively, in order to investigate potential challenges in the paediatric development of orphan medicines which have to meet the requirements of both legislations., Methods: A comparative analysis of PIP conditions and OD conditions was performed for medicines that have been reviewed by both Committees., Results: We found that in the substantial majority of cases there is no divergence between the conclusions of COMP and PDCO with regard to the condition for which a medicine is to be developed., Conclusion: These findings demonstrate that a collaborative approach allows both Regulations to work synergistically to foster pharmaceutical development for rare diseases in childhood., Competing Interests: Competing interests: None declered., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Honey can be produced by different species of honeybees, with two being of economic importance due to their use in apiculture, namely Apis mellifera (known as European honeybee) and Apis cerana (known as Asian honeybee). Due to the decline of the wild populations of the Asian honeybee, this honey generally attains much higher market value, being prone to adulteration. This work aims at proposing new tools, based on the use of molecular markers, for the entomological authentication of honey. To this end, new species-specific primers were designed targeting the tRNA leu -cox2 intergenic region and allowing the detection of A. cerana DNA by qualitative polymerase chain reaction (PCR). Additionally, a novel real-time PCR method with high resolution melting analysis was developed to target the 16S rRNA gene of both bee species, allowing their discrimination in different clusters. The proposed methodologies were further applied with success in the authentication of Asian and European honey samples by the identification of honeybee DNA, demonstrating the usefulness of these simple and cost-effective new approaches., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Biofilm formation has been associated with the persistence of Acinetobacter baumannii in hospital settings and its propensity to cause infection. We investigated the adhesion ability and clonality of 128 A. baumannii isolates recovered from urine and urinary catheters of patients admitted to 5 European hospitals during 1991-2013., Methods: Isolates identification was confirmed by rpoB sequencing and by the presence of blaOXA-51. The presence of carbapenemases was detected by PCR. Clonality was determined by Sequence Group (SG) identification, Pulsed field gel electrophoresis (PFGE) and Multilocus sequence typing. Adhesion ability was defined by quantitative biofilm production assay and biofilms were characterized by Confocal Laser Microscopy and Scanning Electron Microscopy., Results: The 128 isolates, either resistant (85.9%) or susceptible (14.1%) to carbapenems, and belonging to 50 different PFGE types and 24 different STs, were distributed among SG1 (67.2%), SG2 (10.2%) and other allelic profiles (22.7%). ST218 was the most frequent ST, corresponding to 54,5% of the isolates collected between 2011 and 2013. Among the 109 isolates showing resistance to at least 1 carbapenem, 55% revealed the presence of an acquired carbapenem-hydrolyzing class D - lactamases (CHDL): blaOXA-23 were the most frequent gene detected from 2008 onwards (75%). Among all the clinical isolates, 42.2% were strong biofilm producers, with the older isolates having the highest adhesion ability. Most isolates recovered later, belonging to ST218 and harbouring blaOXA-23, were homogeneously less adhesive., Conclusions: An evolution towards a decrease in adhesion ability and a CHDL content change was observed along the years in several European countries.

Prophages of Helicobacter pylori, a bacterium known to co-evolve in the stomach of its human host, were recently identified. However, their role in the diversity of H. pylori strains is unknown. We demonstrate here and for the first time that the diversity of the prophage genes offers the ability to distinguish between European populations, and that H. pylori prophages and their host bacteria share a complex evolutionary history. By comparing the phylogenetic trees of two prophage genes (integrase and holin) and the multilocus sequence typing (MLST)-based data obtained for seven housekeeping genes, we observed that the majority of the strains belong to the same phylogeographic group in both trees. Furthermore, we found that the Bayesian analysis of the population structure of the prophage genes identified two H. pylori European populations, hpNEurope and hpSWEurope, while the MLST sequences identified one European population, hpEurope. The population structure analysis of H. pylori prophages was even more discriminative than the traditional MLST-based method for the European population. Prophages are new players to be considered not only to show the diversity of H. pylori strains but also to more sharply define human populations.

The occurrence of acquired metal tolerance genes in emerging MDR Salmonella enterica serotype 4,[5],12:i:- clones was assessed and their associated platforms and tolerance phenotype were characterised. Salmonella 4,[5],12:i:- from different sources belonging to European, Spanish and Southern European clones were studied. Screening for copper (pcoA-pcoD/tcrB), silver/copper (silA-silE), mercury (merA), arsenic (arsB) and tellurite (terF) tolerance genes was performed by PCR/sequencing. CuSO(4)/AgNO(3) MICs were determined in aerobic/anaerobic atmospheres by agar dilution. Conjugation assays, genomic location and plasmid analysis were performed by standard procedures. Most isolates from European (98%) and Spanish (74%) clones carried silA-silE, contrasting with the Southern European clone (26%). merA/62% (European and Spanish clones) and pcoA-pcoD/50% (European clone) were also detected. merA±pco+sil were chromosomally located in the European clone, whereas in Spanish and Southern European clones sil±merA were within plasmids, both with antibiotic resistance genes. The pcoA-pcoD/silA-silE(+) isolates showed higher MICCuSO(4) in anaerobiosis than those without these genes (MIC(50)=24-28 vs. 2 mM). Different MICAgNO(3) of silA-silE(+) (MIC(50)=0.25 mM) and silA-silE(-)(MIC(50)=0.16 mM) isolates were observed in both atmospheres, with an MIC increment after prior exposure to silver (>3 vs. 0.08-0.125 mM) in aerobiosis. A high frequency of copper and silver tolerance, particularly among the two major Salmonella 4,[5],12:i:- MDR clones (European/Spanish) circulating in Europe and causing human infections, might facilitate adaptation/expansion of these strains in metal-contaminated environments, particularly copper in anaerobiosis. Furthermore, metal toxic concentrations in food-animal environments can contribute to persistence of genetic platforms carrying metal/antibiotic resistance genes in this foodborne zoonotic pathogen., (Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 10,641 viruses collected by WHO-recognized National Influenza Centres between May 2013 and May 2014 to determine 50% inhibitory concentration (IC50) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. In addition, neuraminidase (NA) sequence data, available from the WHO CCs and from sequence databases (n=3206), were screened for amino acid substitutions associated with reduced NAI susceptibility. Ninety-five per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 2% (n=172) showed highly reduced inhibition (HRI) against at least one of the four NAIs, commonly oseltamivir, while 0.3% (n=32) showed reduced inhibition (RI). Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=169), A(H3N2) with NA E119V (n=1), B/Victoria-lineage with NA E117G (n=1) and B/Yamagata-lineage with NA H273Y (n=1); amino acid position numbering is A subtype and B type specific. Although approximately 98% of circulating viruses tested during the 2013-2014 period were sensitive to all four NAIs, a large community cluster of A(H1N1)pdm09 viruses with the NA H275Y substitution from patients with no previous exposure to antivirals was detected in Hokkaido, Japan. Significant numbers of A(H1N1)pdm09 NA H275Y viruses were also detected in China and the United States: phylogenetic analyses showed that the Chinese viruses were similar to those from Japan, while the United States viruses clustered separately from those of the Hokkaido outbreak, indicative of multiple resistance-emergence events. Consequently, global surveillance of influenza antiviral susceptibility should be continued from a public health perspective., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Molecular characterization of patients with Duchenne or Becker muscular dystrophies is essential for establishing a differential diagnosis, allowing appropriate clinical follow-up, patient management and genetic counseling. In light of the recent mutation-based therapeutic approaches, DMD gene analysis has gained further relevance. Owing to the size and complexity of the DMD gene and the diversity of mutation types, molecular analysis is not always a straightforward task requiring the combination of several methodologies. Our national genetic diagnostic service genetically characterized 308 dystrophinopathy patients (284 unrelated families), leading to the identification of 175 distinct mutations, including 39 unpublished variants. These studies revealed several potential diagnostic pitfalls (because of technical limitations or related with DMD's genetic heterogeneity) that may be overlooked even considering the international disease-specific diagnostic guidelines. Comprehensive analysis involved expression studies at the mRNA level, the identification of splicing changes and ultimately providing evidence for apparent exceptions to the reading-frame rule. Besides increasing the mutation detection rate, this detailed molecular characterization is indispensable for the identification of suitable candidates for the new mutation-centered therapies. As patient registries are internationally recognized as essential for clinical trial recruitment, this led us to develop the Portuguese Duchenne and Becker Muscular Dystrophy registry in collaboration with the Translational Research in Europe-Assessment and Treatment of Neuromuscular Diseases network.

Use of single and multiple-dose studies is required to establish the bioequivalence between two extended-release oral dosage forms under the current European Guidelines. However, FDA is less strict in this regard and only requires a single-dose study. The objective of this work is to use a computer simulation in order to test the two approaches. Three pharmacokinetic models, representing different release mechanisms, were considered, and Monte Carlo simulations with intra- and inter-individual variabilities were performed. Five different bioequivalence protocols were used and a new pharmacokinetic metric -C(τ), the concentration at the end of the intended dosing interval obtained in the single-dose study - is proposed in order to avoid the need for steady-state studies while keeping the ability to detect differences between formulations. Results have shown that the European requirements are more capable to discriminate between two potentially different formulations but at the cost of the multiple-dose study and with an increased number of subjects when compared to the FDA requirements. However, the use of C(max) and AUC(0-)(t) obtained on a single-dose study with the added C(τ) metric equals the discriminatory ability of the current EMA requirements, without the need of a multiple-dose study. This proposed approach results in the reduction in the number of studies and volunteers enrolled in clinical bioequivalence trials, without compromising the quality assurance of a new extended-release oral formulation., (Copyright © 2011 Elsevier B.V. All rights reserved.)