Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Engineering microbes for consolidated bioprocessing: new approaches in the light of synthetic biology

Mitigating climate change calls for a reduction in emissions of greenhouse gases, mainly CO2. Twenty-seven per cent of all CO2 emissions come from sources hard to eliminate, including aviation, shipping, and long-distance land transportation. To scale-up production, a transition from first-generation biofuels (edible plant biomass feedstock) to second-generation biofuels (non-edible lignocellulosic plant biomass) is required. However, lignocellulosic bioprocesses struggle to reach economic viability due to the high cost of added enzymes required to digest cellulose. A microbe that was able to generate the cellulase to digest cellulose and able to generate desired products (in what is called a consolidated bioprocess) is not known to exist in nature. Decades of research in synthetic biology have not been successful in creating it due to multiple challenges, such as generating candidate microbial strains and testing them. In this work, novel methodological approaches were developed to overcome these challenges, thus increasing the likelihood of future research finding the ideal microbe. Firstly, the generation of candidate strains was improved by a re-design of the paradigm of modular cloning, based on a vector design called JUMP (Joint Universal Modular Plasmids). Complex multi-gene plasmids can be built from standard DNA parts in a reliable and automation-friendly way using modular cloning systems, based on Golden Gate cloning (which uses type IIS restriction enzymes). However, current standards lack the flexibility to change the microbial host and to perform assemblies to optimise genes of a group. JUMP vector backbones are based on the Standard European Vector Architecture (SEVA), and also have additional modular cloning sites to modify vectors for specific purposes. The experimental results presented here showed that these features allowed use of the vector system in different organisms and reduced the number of assemblies required to optimise and test multi-gene constructs. Secondly, among different explored approaches to test strains, using fluorescent growth reporters was found to have the properties required to screen strains in a faster and more relevant way. Cellulose is insoluble in water, and consequently, previous analytical methodologies to assess the cellulolytic capacity of microorganisms used soluble analogues of cellulase substrates or depended on separation steps which are difficult to do in a fast and high-throughput way. The data presented here showed that expression of fluorescence genes, providing a direct measure of growth, could be measured without separation of cellulose. An Escherichia coli strain expressing cellulases CenA and Chu2268, previously shown to bestow cellulolytic ability on E. coli, was confirmed to be able to grow using cellulose as sole carbon source, which demonstrated the use of fluorescent growth reporters to detect cellulolytic activity. Finally, a strain benchmark was built and characterised to allow screening of expression of cellulolytic genes. A collection of modular parts encoding cellulolytic proteins and secretion signal peptides was built to allow future screening, and the effect of genetic and environmental factors in the measurement of fluorescence as a reporter of growth was investigated. It was found that volumetric conditions and medium additives critically affected the capacity of E. coli to grow at the expense of cellulose. A methodology was developed to allow growth measurement in 96-well plates. In conclusion, this study lays the foundations to establish a faster research cycle to generate and screen microbes that can utilise cellulosic biomass to produce valuable bioproducts and biofuels

A Metabolically Integrated Lossen Rearrangement in Escherichia coli

Biocompatible chemistry enables abiotic reactions to be interfaced with metabolic pathways in living microorganisms. This includes both native and de novo biosynthetic processes to access abiotic feedstocks, intermediates and products in vivo. Herein we report a biocompatible Lossen rearrangement that is catalysed by phosphate in the bacterium Escherichia coli for the chemical transformation of activated acyl hydroxamates to primary amines in living cells. Using a para-carboxylated substrate, the biocompatible reaction can be used to generate the metabolite para-aminobenzoic acid (PABA) to rescue ∆pabA/B and ∆aroC auxotrophy and enable cell growth. The Lossen substrate can also be synthesised from polyethylene terephthalate (PET) and applied to whole-cell biocatalytic reactions and fermentations generating industrial small molecule products – including the analgesic and antipyretic drug paracetamol – paving the way for a general strategy to addict E. coli and other industrial chassis strains to PET plastic waste as a bioremediation strategy and for the upcycling of plastic waste using engineered biology. Together, this work showcases how non-enzymatic biocompatible reactions can be interfaced and cooperate with microbial metabolism to expand the available toolbox of metabolic chemistry for small molecule synthesis in native and engineered cellular systems

Contemporary use of cefazolin for MSSA infective endocarditis: analysis of a national prospective cohort

Objectives: This study aimed to assess the real use of cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infective endocarditis (IE) in the Spanish National Endocarditis Database (GAMES) and to compare it with antistaphylococcal penicillin (ASP). Methods: Prospective cohort study with retrospective analysis of a cohort of MSSA IE treated with cloxacillin and/or cefazolin. Outcomes assessed were relapse; intra-hospital, overall, and endocarditis-related mortality; and adverse events. Risk of renal toxicity with each treatment was evaluated separately. Results: We included 631 IE episodes caused by MSSA treated with cloxacillin and/or cefazolin. Antibiotic treatment was cloxacillin, cefazolin, or both in 537 (85%), 57 (9%), and 37 (6%) episodes, respectively. Patients treated with cefazolin had significantly higher rates of comorbidities (median Charlson Index 7, P <0.01) and previous renal failure (57.9%, P <0.01). Patients treated with cloxacillin presented higher rates of septic shock (25%, P = 0.033) and new-onset or worsening renal failure (47.3%, P = 0.024) with significantly higher rates of in-hospital mortality (38.5%, P = 0.017). One-year IE-related mortality and rate of relapses were similar between treatment groups. None of the treatments were identified as risk or protective factors. Conclusion: Our results suggest that cefazolin is a valuable option for the treatment of MSSA IE, without differences in 1-year mortality or relapses compared with cloxacillin, and might be considered equally effective