Preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

Combining T2-preparation with spectral and spatial selectivity for improved coronary MRA

Cardiovascular disease is the leading cause of death worldwide. Within this subset, coronary artery disease (CAD) is the most prevalent. Magnetic resonance angiography (MRA) is an emerging technique that provides a safe, non-invasive way of assessing CAD progression. To generate contrast between tissues, MR images are weighted according to the magnetic properties of those tissues. In cardiac MRI, T2 contrast, which is governed by the rate of transverse signal loss, is often created through the use of a T2-Preparation module. T2-Preparation, or T2-Prep, is a magnetization preparation scheme used to improve blood/myocardium contrast in cardiac MRI. T2-Prep methods generally use a non-selective +90°, 180°, 180°, -90° train of radiofrequency (RF) pulses (or variant thereof), to tip magnetization into the transverse plane, allow it to evolve, and then to restore it to the longitudinal plane. A key feature in this process is the combination of a +90° and -90° RF pulse. By changing either one of these, a mismatch occurs between signal excitation and restoration. This feature can be exploited to provide additional spectral or spatial selectivity. In this work, both of these possibilities are explored. The first - spectral selectivity - has been examined as a method of improving fat saturation in coronary MRA. The second - spatial selectivity - has been examined as a means of reducing imaging time by decreasing the field of view, and as a method of reducing artefacts originating from the tissues surrounding the heart. Two additional applications, parallel imaging and self-navigation, are also presented. This thesis is thus composed of four sections. The first, "A Fat Signal Suppression for Coronary MRA at 3T using a Water-Selective Adiabatic T2-Preparation Technique", was originally published in the journal Magnetic Resonance in Medicine (MRM) with co-authors Ruud B. van Heeswijk and Matthias Stuber. The second, "Combined T2-Preparation and 2D Pencil Beam Inner Volume Selection", again with co-authors Ruud van Heeswijk and Matthias Stuber, was also published in the journal MRM. The third, "A cylindrical, inner volume selecting 2D-T2-Prep improves GRAPPA-accelerated image quality in MRA of the right coronary artery", written with co-authors Jerome Yerly and Matthias Stuber, has been submitted to the "Journal of Cardiovascular Magnetic Resonance", and the fourth, "Combined respiratory self-navigation and 'pencil-beam' 2D-T2 -Prep for free-breathing, whole-heart coronary MRA", with co¬authors Jerome Chaptinel, Giulia Ginami, Gabriele Bonanno, Simone Coppo, Ruud van Heeswijk, Davide Piccini, and Matthias Stuber, is undergoing internal review prior to submission to the journal MRM. -- Les maladies cardiovasculaires sont la cause principale de décès dans le monde : parmi celles-ci, les maladies coronariennes sont les plus répandues. L'angiographie par résonance magnétique (ARM) est une technique émergente qui fournit une manière sûre, non invasive d'évaluer la progression de la coronaropathie. Pour obtenir un contraste entre les tissus, les images d'IRM sont pondérées en fonction des propriétés magnétiques de ces tissus. En IRM cardiaque, le contraste en T2, qui est lié à la décroissance du signal transversal, est souvent créé grâce à l'utilisàtion d'un module de préparation T2. La préparation T2, ou T2-Prep, est un système de préparation de l'aimantation qui est utilisé pour améliorer le contraste entre le sang et le myocarde lors d'une IRM cardiaque. Les méthodes de T2-Prep utilisent généralement une série non-sélective d'impulsions de radiofréquence (RF), typiquement [+ 90°, 180°, 180°, -90°] ou une variante, qui bascule l'aimantation dans le plan transversal, lui permet d'évoluer, puis la restaure dans le plan longitudinal. Un élément clé de ce processus est la combinaison des impulsions RF de +90° et -90°. En changeant l'une ou l'autre des impulsions, un décalage se produit entre l'excitation du signal et de la restauration. Cette fonction peut être exploitée pour fournir une sélectivité spectrale ou spatiale. Dans cette thèse, les deux possibilités sont explorées. La première - la sélectivité spectrale - a été examinée comme une méthode d'améliorer la saturation de la graisse dans l'IRM coronarienne. La deuxième - la sélectivité spatiale - a été étudiée comme un moyen de réduire le temps d'imagerie en diminuant le champ de vue, et comme une méthode de réduction des artefacts provenant des tissus entourant le coeur. Deux applications supplémentaires, l'imagerie parallèle et la self-navigation, sont également présentées. Cette thèse est ainsi composée de quatre sections. La première, "A Fat Signal Suppression for Coronary MRA at 3T using a Water-Selective Adiabatic T2-Preparation Technique", a été publiée dans la revue médicale Magnetic Resonance .in Medicine (MRM) avec les co-auteurs Ruud B. van Heeswijk et Matthias Stuber. La deuxième, Combined T2-Preparation and 2D Pencil Beam Inner Volume Selection", encore une fois avec les co-auteurs Ruud van Heeswijk et Matthias Stuber, a également été publiée dans le journal MRM. La troisième, "A cylindrical, inner volume selecting 2D-T2-Prep improves GRAPPA- accelerated image quality in MRA of the right coronary artery", écrite avec les co-auteurs Jérôme Yerly et Matthias Stuber, a été présentée au "Journal of Cardiovascular Magnetic Resonance", et la quatrième, "Combined respiratory self-navigation and 'pencil-beam' 2D-T2 -Prep for free-breathing, whole-heart coronary MRA", avec les co-auteurs Jérôme Chaptinel, Giulia Ginami, Gabriele Bonanno , Simone Coppo, Ruud van Heeswijk, Davide Piccini, et Matthias Stuber, subit un examen interne avant la soumission à la revue MRM.

Discrepancies over the onset of surfactant monomer aggregation interpreted by fluorescence, conductivity and surface tension methods

Molecular probe techniques have made important contributions to the determination of microstructure of surfactant assemblies such as size, stability, micropolarity and conformation. Conductivity and surface tension were used to determine the critical aggregation concentration (cac) of polymer-surfactant complexes and the critical micellar concentration (cmc) of aqueous micellar aggregates. The results are compared with those of fluorescent techniques. Several surfactant systems were examined, 1-butanol-sodium dodecylsulfate (SDS) mixtures, solutions containing poly(ethylene oxide)-SDS, poly(vinylpyrrolidone)-SDS and poly(acrylic acid)-alkyltrimethylammonium bromide complexes. We found differences between the cac and cmc values obtained by conductivity or surface tension and those obtained by techniques which use hydrophobic probe.

Oxochloroalkoxide of the Cerium (IV) and Titanium (IV) as oxides precursor

The Cerium (IV) and Titanium (IV) oxides mixture (CeO2-3TiO2) was prepared by thermal treatment of the oxochloroisopropoxide of Cerium (IV) and Titanium (IV). The chemical route utilizing the Cerium (III) chloride alcoholic complex and Titanium (IV) isopropoxide is presented. The compound Ce5Ti15Cl16O30 (iOPr)4(OH-Et)15 was characterized by elemental analysis, FTIR and TG/DTG. The X-ray diffraction patterns of the oxides resulting from the thermal decomposition of the precursor at 1000 °C for 36 h indicated the formation of cubic cerianite (a = 5.417Å) and tetragonal rutile (a = 4.592Å) and (c = 2.962 Å), with apparent crystallite sizes around 38 and 55nm, respectively.

Food Safety Testing: Rapid Molecular Methods for Chemical and Biological Hazards

The central goal of food safety policy in the European Union (EU) is to protect consumer health by guaranteeing a high level of food safety throughout the food chain. This goal can in part be achieved by testing foodstuffs for the presence of various chemical and biological hazards. The aim of this study was to facilitate food safety testing by providing rapid and user-friendly methods for the detection of particular food-related hazards. Heterogeneous competitive time-resolved fluoroimmunoassays were developed for the detection of selected veterinary residues, that is coccidiostat residues, in eggs and chicken liver. After a simplified sample preparation procedure, the immunoassays were performed either in manual format with dissociation-enhanced measurement or in automated format with pre-dried assay reagents and surface measurement. Although the assays were primarily designed for screening purposes providing only qualitative results, they could also be used in a quantitative mode. All the developed assays had good performance characteristics enabling reliable screening of samples at concentration levels required by the authorities. A novel polymerase chain reaction (PCR)-based assay system was developed for the detection of Salmonella spp. in food. The sample preparation included a short non-selective pre-enrichment step, after which the target cells were collected with immunomagnetic beads and applied to PCR reaction vessels containing all the reagents required for the assay in dry form. The homogeneous PCR assay was performed with a novel instrument platform, GenomEra^™, and the qualitative assay results were automatically interpreted based on end-point time-resolved fluorescence measurements and cut-off values. The assay was validated using various food matrices spiked with sub-lethally injured Salmonella cells at levels of 1-10 colony forming units (CFU)/25 g of food. The main advantage of the system was the exceptionally short time to result; the entire process starting from the pre-enrichment and ending with the PCR result could be completed in eight hours. In conclusion, molecular methods using state-of-the-art assay techniques were developed for food safety testing. The combination of time-resolved fluorescence detection and ready-to-use reagents enabled sensitive assays easily amenable to automation. Consequently, together with the simplified sample preparation, these methods could prove to be applicable in routine testing.

A comparative study of difficulties in accounting preparation and judgement in agriculture using fair value and historical cost accounting for biological assets valuation

This paper presents empirical research comparing the accounting difficulties that arise from the use of two valuation methods for biological assets, fair value (FV) and historical cost (HC) accounting, in the agricultural sector. It also compares how reliable each valuation method is in the decision-making process of agents within the sector. By conducting an experiment with students, farmers, and accountants operating in the agricultural sector, we find that they have more difficulties, make larger miscalculations and make poorer judgements with HC accounting than with FV accounting. In-depth interviews uncover flawed accounting practices in the agricultural sector in Spain in order to meet HC accounting requirements. Given the complexities of cost calculation for biological assets and the predominance of small family business units in advanced Western countries, the study concludes that accounting can be more easily applied in the agricultural sector under FV than HC accounting, and that HC conveys a less accurate grasp of the real situation of a farm.

Stability-indicating methods for quantitative determination of zidovudine and stavudine in capsules

Zidovudine (AZT) and stavudine (D4T) are nucleoside reverse transcriptase inhibitors extensively used in human immunodeficiency virus (HIV) infected patients. In order to evaluate the quality of these drugs, two stability indicating HPLC methods were developed. The validated methods were applied in quantitative determination of AZT, D4T and their induced degradation products in capsule preparations. The stability studies were conducted at controlled temperature and relative humidity conditions based on the International Conference on Harmonization stability studies protocol for Zone IV areas. Easy sample preparation and low-cost make these methods especially useful for quality control and stability studies of AZT and D4T in drug products.

Adsorção de íons dicromato nos compósitos celulose/ZrO2.nH2O preparados pelos métodos da precipitação convencional e em solução homogênea

This article describes the preparation and characterization of the cellulose/ hydrated zirconium oxide composites prepared by conventional precipitation (PC) and homogeneous solution precipitation (PSH) methods. The composite obtained by the PC method was prepared by using an ammonia solution as the precipitating agent, while the composite obtained by the PSH method was prepared by using urea as the precipitating agent. The adsorption of dichromate ions on the composites was studied using factorial design 2³. The variables were: initial concentration, agitation time and mass of the composite. The data obtained agree better for the composite obtained by the PC method.

Preparation of glasses and glass ceramics of heavy metal oxides containing silver: optical, structural and electrochemical properties

Silver containing heavy metal oxide glasses and glass ceramics of the system WO3-SbPO4-PbO-AgCl with different AgCl contents have been prepared and their thermal, structural and optical properties characterized. Glass ceramics containing metallic silver nanoparticles have been prepared by annealing glass samples at temperatures above the glass transition and analyzed by transmission electron microscopy and energy dispersive X-ray microanalysis. The presence of the metallic clusters has been also confirmed by the observation of a surface plasmon resonance band in the visible range. Cyclic voltammetric measurements indicated the presence of metallic silver into the glasses, even before to perform the thermal treatment.

Development and validation of a dissolution test for primaquine/polyethylene oxide matrix tablets

A simple, precise, specific, repeatable and discriminating dissolution test for primaquine (PQ) matrix tablets was developed and validated according to ICH and FDA guidelines. Two UV assaying methods were validated for determination of PQ released in 0.1 M hydrochloric acid and water media. Both methods were linear (R²>0.999), precise (R.S.D.<1.87%) and accurate (97.65-99.97%). Dissolution efficiency (69-88%) and equivalence of formulations (f2) was assessed in different media and apparatuses (basket/100 rpm and paddle/50 rpm) tested. Discriminating condition was 900 mL aqueous medium, basket at 100 rpm and sampling times at 1, 4 and 8 h. Repeatability (R.S.D.<2.71%) and intermediate precision (R.S.D.<2.06%) of dissolution method were satisfactory.

Modalities for soil preparation and gypsum application in ultisol: stem productivity of sugarcane

The study was conducted in an area of expansion of sugarcane at Vale do Paraná factory in Suzanápolis city - São Paulo (SP), in Brazil, in the northwestern region of the State of São Paulo. It was used the sugarcane variety RB92-5345, 1.5m of spacing between rows, in an Ultisol. The study aimed to evaluate the productivity of sugarcane and first ratoon and some soil chemical attributes in function of soil tillage and application or not of gypsum. The experimental design was randomized blocks with six treatments, in a factorial 3x2 and six replicates, the main treatments were soil tillage with three equipments, moldboard plow, chisel plow, and heavy harrow, and two secondary treatments with application of 1 t ha-1of gypsum and no gypsum. After each harvest of cane, the soil was characterized as to its fertility indicators in layers of 0.0-0.15; 0.15-0.30 and 0.30-0.45m. Differences in values of soil chemical attributes due to the methods of preparation occurred in the sugarcane did not last until the harvest of the 1st ratoon cane, and also did not influence the crop productivity. The gypsum application resulted in higher values ​​of total recoverable sugar (TRS) and the productivity of tons of stems per hectare (TSH) to sugarcane and 1st ratoon cane, respectively, confirming the initial hypothesis.

Application of capillary electrophoretic methods for determining carbohydrates and aliphatic carboxylic acids formed during wood processing

Knowledge of the behaviour of cellulose, hemicelluloses, and lignin during wood and pulp processing is essential for understanding and controlling the processes. Determination of monosaccharide composition gives information about the structural polysaccharide composition of wood material and helps when determining the quality of fibrous products. In addition, monitoring of the acidic degradation products gives information of the extent of degradation of lignin and polysaccharides. This work describes two capillary electrophoretic methods developed for the analysis of monosaccharides and for the determination of aliphatic carboxylic acids from alkaline oxidation solutions of lignin and wood. Capillary electrophoresis (CE), in its many variants is an alternative separation technique to chromatographic methods. In capillary zone electrophoresis (CZE) the fused silica capillary is filled with an electrolyte solution. An applied voltage generates a field across the capillary. The movement of the ions under electric field is based on the charge and hydrodynamic radius of ions. Carbohydrates contain hydroxyl groups that are ionised only in strongly alkaline conditions. After ionisation, the structures are suitable for electrophoretic analysis and identification through either indirect UV detection or electrochemical detection. The current work presents a new capillary zone electrophoretic method, relying on in-capillary reaction and direct UV detection at the wavelength of 270 nm. The method has been used for the simultaneous separation of neutral carbohydrates, including mono- and disaccharides and sugar alcohols. The in-capillary reaction produces negatively charged and UV-absorbing compounds. The optimised method was applied to real samples. The methodology is fast since no other sample preparation, except dilution, is required. A new method for aliphatic carboxylic acids in highly alkaline process liquids was developed. The goal was to develop a method for the simultaneous analysis of the dicarboxylic acids, hydroxy acids and volatile acids that are oxidation and degradation products of lignin and wood polysaccharides. The CZE method was applied to three process cases. First, the fate of lignin under alkaline oxidation conditions was monitored by determining the level of carboxylic acids from process solutions. In the second application, the degradation of spruce wood using alkaline and catalysed alkaline oxidation were compared by determining carboxylic acids from the process solutions. In addition, the effectiveness of membrane filtration and preparative liquid chromatography in the enrichment of hydroxy acids from black liquor was evaluated, by analysing the effluents with capillary electrophoresis.

Modeling and Control of the Power Conversion Unit in a Solid Oxide Fuel Cell Environment

In this doctoral thesis, a power conversion unit for a 10 kWsolid oxide fuel cell is modeled, and a suitable control system is designed. The need for research was identified based on an observation that there was no information available about the characteristics of the solid oxide fuel cell from the perspective of power electronics and the control system, and suitable control methods had not previously been studied in the literature. In addition, because of the digital implementation of the control system, the inherent characteristics of the digital system had to be taken into account in the characteristics of the solid oxide fuel cell (SOFC). The characteristics of the solid oxide fuel cell as well the methods for the modeling and control of the DC/DC converter and the grid converter are studied by a literature survey. Based on the survey, the characteristics of the SOFC as an electrical power source are identified, and a solution to the interfacing of the SOFC in distributed generation is proposed. A mathematical model of the power conversion unit is provided, and the control design for the DC/DC converter and the grid converter is made based on the proposed interfacing solution. The limit cycling phenomenon is identified as a source of low-frequency current ripple, which is found to be insignificant when connected to a grid-tied converter. A method to mitigate a second harmonic originating from the grid interface is proposed, and practical considerations of the operation with the solid oxide fuel cell plant are presented. At the theoretical level, the thesis discusses and summarizes the methods to successfully derive a model for a DC/DC converter, a grid converter, and a power conversion unit. The results of this doctoral thesis can also be used in other applications, and the models and methods can be adopted to similar applications such as photovoltaic systems. When comparing the results with the objectives of the doctoral thesis, we may conclude that the objectives set for the work are met. In this doctoral thesis, theoretical and practical guidelines are presented for the successful control design to connect a SOFC-based distributed generation plant to the utility grid.

Novel technology for preparation of optically active chemicals

Asymmetric synthesis using modified heterogeneous catalysts has gained lots of interest in the production of optically pure chemicals, such as pharmaceuticals, nutraceuticals, fragrances and agrochemicals. Heterogeneous modified catalysts capable of inducing high enantioselectivities are preferred in industrial scale due to their superior separation and handling properties. The topic has been intensively investigated both in industry and academia. The enantioselective hydrogenation of ethyl benzoylformate (EBF) to (R)-ethyl mandelate over (-)-cinchonidine (CD)-modified Pt/Al2O3 catalyst in a laboratory-scale semi-batch reactor was studied as a function of modifier concentration, reaction temperature, stirring rate and catalyst particle size. The main product was always (R)-ethyl mandelate while small amounts of (S)-ethyl mandelate were obtained as by product. The kinetic results showed higher enantioselectivity and lower initial rates approaching asymptotically to a constant value as the amount of modifier was increased. Additionally, catalyst deactivation due to presence of impurities in the feed was prominent in some cases; therefore activated carbon was used as a cleaning agent of the raw material to remove impurities prior to catalyst addition. Detailed characterizations methods (SEM, EDX, TPR, BET, chemisorption, particle size distribution) of the catalysts were carried out. Solvent effects were also studied in the semi-batch reactor. Solvents with dielectric constant (e) between 2 and 25 were applied. The enantiomeric excess (ee) increased with an increase of the dielectric coefficient up to a maximum followed by a nonlinear decrease. A kinetic model was proposed for the enantioselectivity dependence on the dielectric constant based on the Kirkwood treatment. The non-linear dependence of ee on (e) successfully described the variation of ee in different solvents. Systematic kinetic experiments were carried out in the semi-batch reactor. Toluene was used as a solvent. Based on these results, a kinetic model based on the assumption of different number of sites was developed. Density functional theory calculations were applied to study the energetics of the EBF adsorption on pure Pt(1 1 1). The hydrogenation rate constants were determined along with the adsorption parameters by non-linear regression analysis. A comparison between the model and the experimental data revealed a very good correspondence. Transient experiments in a fixed-bed reactor were also carried out in this work. The results demonstrated that continuous enantioselective hydrogenation of EBF in hexane/2-propanol 90/10 (v/v) is possible and that continuous feeding of (-)-cinchonidine is needed to maintain a high steady-state enantioselectivity. The catalyst showed a good stability and high enantioselectivity was achieved in the fixed-bed reactor. Chromatographic separation of (R)- and (S)-ethyl mandelate originating from the continuous reactor was investigated. A commercial column filled with a chiral resin was chosen as a perspective preparative-scale adsorbent. Since the adsorption equilibrium isotherms were linear within the entire investigated range of concentrations, they were determined by pulse experiments for the isomers present in a post-reaction mixture. Breakthrough curves were measured and described successfully by the dispersive plug flow model with a linear driving force approximation. The focus of this research project was the development of a new integrated production concept of optically active chemicals by combining heterogeneous catalysis and chromatographic separation technology. The proposed work is fundamental research in advanced process technology aiming to improve efficiency and enable clean and environmentally benign production of enantiomeric pure chemicals.

Lipase and omega-transaminase catalysis in preparation of alcohol and amine enantiomers

Enantiopure intermediates are of high value in drug synthesis. Biocatalysis alone or combined with chemical synthesis provides powerful tools to access enantiopure compounds. In biocatalysis, chemo-, regio- and enantioselectivity of enzymes are combined with their inherent environmentally benign nature. Enzymes can be applied in versatile chemical reactions with non-natural substrates under synthesis conditions. Immobilization of an enzyme is a crucial part of an efficient biocatalytic synthesis method. Successful immobilization enhances the catalytic performance of an enzyme and enables its reuse in successive reactions. This thesis demonstrates the feasibility of biocatalysis in the preparation of enantiopure secondary alcohols and primary amines. Viability and synthetic usability of the studied biocatalytic methods have been addressed throughout this thesis. Candida antarctica lipase B (CAL-B) catalyzed enantioselective O-acylation of racemic secondary alcohols was successfully incorporated with in situ racemization in the dynamic kinetic resolution, affording the (R)-esters in high yields and enantiopurities. Side reactions causing decrease in yield and enantiopurity were suppressed. CAL-B was also utilized in the solvent-free kinetic resolution of racemic primary amines. This method produced the enantiomers as (R)-amides and (S)-amines under ambient conditions. An in-house sol-gel entrapment increased the reusability of CAL-B. Arthrobacter sp. omega-transaminase was entrapped in sol-gel matrices to obtain a reusable catalyst for the preparation enantiopure primary amines in an aqueous medium. The obtained heterogeneous omega-transaminase catalyst enabled the enantiomeric enrichment of the racemic amines to their (S)-enantiomers. The synthetic usability of the sol-gel catalyst was demonstrated in five successive preparative kinetic resolutions.