Dissertations / Theses on the topic 'CARE DIAGNOSTICS'

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2015.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 127-136).
Point of care testing is expanding the healthcare field towards personalized and early-detection medicine. Microfluidic platforms present an opportunity for low cost, portable diagnostic sensors through manipulation of small volumes of fluids on isolated, compact devices. One of the challenges of microfluidic sensors is the biological sample pretreatment steps that are manually performed prior to on-chip loading and sensing. This issue is especially prominent for human blood, which contains about a billion cells in one milliliter total volume. These blood cells can rupture, clog devices, block optical readouts, and foul electrodes. At the same time, the liquid portion of human blood, plasma, is rich in a variety of disease indicators, many of which have not yet been identified, and thus is an essential part in the diagnostic field. This thesis focuses on the design of a small, around 1 cm long, microfluidic device that separates out blood plasma from undiluted human blood. This design does not require any external field or equipment, beyond a loading syringe and collection tubing. The separation results show 10-100 times improvement in plasma purity over the literature values for passive separation designs. This separation system was then combined with a colorimetric malaria sensor that produced a visually detectable colored result with a 7.5 nM limit of detection in whole blood. This thesis details the design of a low power point of care diagnostic process that is capable of blood processing and detection, and which eliminates the need for any external laboratory-scale equipment. Advantages and challenges of other low power, microfluidic sensor constructs are also discussed.
by Tatyana A. Shatova.
Ph. D.

This thesis focuses on inductive base sensor design at MHz range frequency. The background theory, design, experiments and results for a new magnetic particles sensor is presented. A new magnetic sensor based on a planar coil was investigated for DNA pathogen detection. Change in inductance of the planar coil due to the presence of magnetic particles with varying mass was measured. The experimental set-up consisted of different sized planar coil with associated electronics for inductance measurements. The best sensor performance was accomplished using two different inductors while oscillating at frequencies 2.4MHz using 9.5μH inductor and 7.2MHz with 85μH inductor. The sensor has very large signal to noise ratio (580×103), while the average amount of frequency drift was 0.58. This sensor was tested with various types of magnetic particles. In addition, iron-oxide nanoparticles were synthesized through water in oil microemulsion method and with an average size of 25nm. The best sensitivity achieved for detection of 50μg iron-oxide particles was with the bead size of 10nm. 81Hz frequency shift was attained in regard to that amount of particles. This research shows that increasing the resonance frequency to 7.2MHz can cause the larger output signal difference (frequency shift) in the presence of magnetic particles; however, the sensor stability is the most important factor for determining the detection resolution and sensitivity. The sensitivity is better if the sensor can detect smaller amount of magnetic sample. The results of this research demonstrate that while the sample consists of smaller size particles, the sensor can detect the lower amount of sample. This is due to the heating effect of nanoparticles. On the other hand the sample distance from the sensor has a major impact on the sensitivity too; the shorter the distance, the higher the sensitivity. This technique can potentially be extended to detect several different types of bacterial pathogens and can be modified for multiplex quantitative detection. This sensing technique will be incorporated into a handheld, disposable microfluidic chip for point-of-care diagnostics for sexually transmitted diseases.

A simple, but innovative microfluidic Lab-on-a-chip (LOC) device which is broadly applicable in point of care diagnostics of biological pathogens was designed, fabricated and assembled utilising explicit microfluidic techniques. The purpose of this design was to develop a cartridge with the capability to perform multiplex DNA amplification reactions on a single device. To achieve this outcome, conventional laboratory protocols for sample preparation; involving DNA extraction, purification and elution were miniaturized to suit this lab-on-a-chip device of 75mm X 50mm cross-sectional area. The extraction process was carried out in a uniquely designed microchamber embedded with chitosan membrane that binds DNA at pH 5.0 and elutes when a different solution at pH 9.0 flows through. Likewise, purification protocol that occurs in the designed waste reservoir is very significant in biomedical field because it is concerned with waste treatment and cartridge disposability, was performed with a super absorbent powder that converts liquid to a gel like substance. This powder is known as sodium polyacrylate, which is also they treated with anti-bacterial chemicals to prevent environmental contamination. Furthermore, this process also employed the use of a passive valve for a precise fluid handling operation involving flow regulation from extraction to waste reservoir. In order to achieve the intended multiplexing function a multiplexer was created to distribute flow simultaneously through a bifurcated network of channels connected to six similar amplification microchambers. Prior to fabrication, computational fluid dynamics (CFD) simulation was utilized at flowrates less than 10μL/s as the means to test the effectiveness of each design components and also to specifically deduct empirical values that can be analyzed to improve or understand the relationship between the fluid and geometrical constraints of the microfluidic modular elements. The device produced was a hybrid cartridge composed of PDMS and glass which is the most widely used materials microfluidics research due to their low cost and simplicity of fabrication by soft lithography technique. The choice of material also took into account the various physical and chemical properties advantages and disadvantages in their bio-medical applications. Such properties include but not limited to surface energy that determines the wetting fluid characteristics, biocompatibility, optical transparency. Subsequently, after a prototype cartridge was developed fluid flow experimentation using liquid coloured dye was used on the fully fabricated cartridge to test the efficacy of its microfluidic functionalities before expensive DNA amplification reagents were utilised at similar flowrates to the CFD simulations. This gave rise to comparison between similar and dissimilar flow Peculiarities in the microfluidic circuit of both experiments. The final experiment was performed with the aid of a recent molecular technique in DNA amplification known as of RPA kit (recombinase polymerase amplification reaction). It involved performing two main reaction experiments; first, was the positive experiment that bears the sample DNA and the latter, negative that served as the control without DNA. In the end, quantitative analysis of results was done using an agarose gel that showed 143 base pairs, for the positive samples, thus validating the amplification experiment.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 129-134).
Diagnostic tests in resource-limited settings require technologies that are affordable and easy-to-use with minimal infrastructure. Colorimetric detection methods that provide results that are readable by eye, without reliance on specialized and expensive equipment, have great utility in these settings. Existing colorimetric methods based on enzymatic reactions and gold nanoparticles often produce results that must be read within a specified time interval to ensure their validity. In many instances, a user has to wait several minutes for the color to develop. Moreover, the result can be interpreted incorrectly because of low visual contrast. Therefore, a colorimetric detection technology that produces bright and unambiguous readout within a time interval of a few seconds to less than two minutes, and removes the burden of accurate time keeping from the user can be very beneficial in low-resource settings. Photo-initiated polymerization-based amplification (PBA) is a technology that allows detection of a surface-bound analyte through co-localization of a visible-light photoinitiator with the analyte present on the surface. In the presence of an appropriate dose of light and monomers, a subsequent free radical polymerization reaction results in formation of an interfacial hydrogel in areas where the initiator has been localized. In this thesis, we modified the eosin/tertiary amine-based PBA technology, which had previously been developed on transparent glass surfaces, for use with cellulose-based (paper) surfaces. Using Plasmodium falciparum histidine-rich protein as an example, we showed that paper-based PBA allowed high-contrast visual detection of proteins with a limit-of-detection of single digit nM concentration (~7 nM) in complex matrices such as human serum and plasma purified from blood samples through the use of a hand-operated microfluidic device. The paper-based immunoassay required only 10 [mu]L sample per test and the total time for signal amplification, from illumination to colorimetric detection, was 2-2.5 minutes per test. The method provided quantitative information regarding analyte levels when combined with cellphone-based imaging. It also allowed decoupling of the capture of analyte on the surface from the signal amplification and visualization steps. We showed that in comparison with enzymatic amplification methods and silver deposition on gold nanoparticles, PBA-based readout on paper was cheaper, easier to perceive at its limit-of-detection, and had the lowest incidence of false readouts due to timing errors. In addition to developing PBA for use in paper devices, we combined PBA with a dilution array approach for quantifying analyte levels by counting number of visible polymer spots on a biochip. We used an empirical design approach that did not depend on measurement of equilibrium and kinetic binding parameters of the antibodies used in the assay and provided a dynamic range of three orders of magnitude, 70 pM to 70 nM, for visual quantification of the analyte. We also built a portable, light-weight, and customizable LED-based device with automated timer functionality for use with PBA assays in point-of-care settings.
by Shefali Lathwal.
Ph. D.

Developing non-invasive and accurate diagnostics that are easily manufactured, robust and reusable will provide monitoring of high-risk individuals in any clinical or point-of-care environment, particularly in the developing world. There is currently no rapid, low-cost and generic sensor fabrication technique capable of producing narrow-band, uniform, reversible colorimetric readouts with a high-tuneability range. This thesis aims to present a theoretical and experimental basis for the rapid fabrication, optimisation and testing of holographic sensors for the quantification of pH, organic solvents, metal cations, and glucose in solutions. The sensing mechanism was computationally modelled to optimise its optical characteristics and predict the readouts. A single pulse of a laser (6 ns, 532 nm, 350 mJ) in holographic “Denisyuk” reflection mode allowed rapid production of sensors through silver-halide chemistry, in situ particle size reduction and photopolymerisation. The fabricated sensors consisted of off-axis Bragg diffraction gratings of ordered silver nanoparticles and localised refractive index changes in poly(2-hydroxyethyl methacrylate) and polyacrylamide films. The sensors exhibited reversible Bragg peak shifts, and diffracted the spectrum of narrow-band light over the wavelength range λpeak ≈ 500-1100 nm. The application of the holographic sensors was demonstrated by sensing pH in artificial urine over the physiological range (4.5-9.0), with a sensitivity of 48 nm/pH unit between pH 5.0 and 6.0. For sensing metal cations, a porphyrin derivative was synthesised to act as the crosslinker, the light absorbing material, the component of a diffraction grating, as well as the cation chelating agent. The sensor allowed reversible quantification of Cu2+ and Fe2+ ions (50 mM - 1 M) with a response time within 50 s. Clinical trials of a glucose sensor in the urine samples of diabetic patients demonstrated that the glucose sensor has an improved performance compared to a commercial high-throughput urinalysis device. The experimental sensitivity of the glucose sensor exhibited a limit of detection of 90 µM, and permitted diagnosis of glucosuria up to 350 mM. The sensor response was achieved within 5 min and the sensor could be reused about 400 times without compromising its accuracy. Holographic sensors were also tested in flake form, and integrated with paper-iron oxide composites, dyed filter and chromatography papers, and nitrocellulose-based test strips. Finally, a generic smartphone application was developed and tested to quantify colorimetric tests for both Android and iOS operating systems. The developed sensing platform and the smartphone application have implications for the development of low-cost, reusable and equipment-free point-of-care diagnostic devices.

Reduced sulphydryl thiols (RSH): cysteine, homocysteine and glutathione are fundamental cellular components having important biological functions, including roles within the pathogenesis of a variety of clinical conditions. Independent analysis of these species is problematic and analytical difficulties relating to instrumental selectivity and sensitivity need to be overcome. This thesis describes the work carried out on the development and characterisation of a range of systems that could be used to facilitate thiol detection, ideally at the point-of-care, focussing largely on electrochemical techniques. Silver-thiol interactions were studied as a route to assist the sample processing. Here a novel controlled silver release mechanism was assessed. Silver release was found to be dependent upon the thiol structure. This has possible future applications to the development of methods to prevent biofilm formation, although the full mechanism of silver-thiol release requires further understanding. The development of unique molecular imprinted polymers was attempted. These would facilitate the detection of amino acids and the relevant thiol species via the amine functionality. The polymers proved unstable in the presence of hydroxylamine. However, this property makes the polymers suitable for use as protective or sacrificial polymers which can potentially be exploited in the manufacture of patterned electrodes. The nucleophilic substitution reaction between thiols and quinones, or quinone type materials, was explored as a possible route to assist selective thiol detection via electrochemical or colorimetric methods. Development of such reagentless sensing platforms would be beneficial in clinical analysis. Selectivity of thiol determination was achieved, although sensitivity issues will restrict real-world applications. A pH sensor utilising uric acid redox sensitivity was developed and was integrated within a disposable electrode assembly to enable wound pH monitoring. This platform was adapted as a prototype generic sensor for thiol analysis.

De nos jours, efficacité et précision des diagnostics médicaux sont des éléments essentiels pour la prévention en termes de santé et permettre une prise en charge rapide des maladies des patients. Les récentes innovations technologiques, particulièrement dans les domaines de la microélectronique et des sciences des matériaux ont permis le développement de nouvelles plateformes personnalisées de diagnostics portatifs. Les matériaux électroniques organiques qui ont déjà par le passé démontré leur potentiel en étant intégrés dans des produits de grande consommation tels que les écrans de smartphones ou encore les cellules solaires montrent un fort potentiel pour une intégration dans des dispositifs biomédicaux. En effet, de par leurs natures et leurs propriétés physiques et chimiques, ils peuvent être à la fois en contact avec les milieux biologiques et constituer l’interface entre les éléments biologiques à l’étude, et les dispositifs électroniques. L’objectif de mes travaux de thèse et d’étudier et évaluer les performances des matériaux organiques électroniques intégrés dans des dispositifs biomédicaux en étudiant leurs interactions avec des milieux biologiques et par l’utilisation et l’optimisation de ces dispositifs permettre la détection de métabolites tel que le glucose ou lactate par exemple. Pendant ma thèse, j’ai notamment créé une plateforme de diagnostics combinant à la fois microfluidique et électronique organique permettant la multi détection de métabolites présents dans des fluides corporels humains, j’ai également conçu des capteurs intégrant des transistors organiques au sein des circuits électroniques classiques afin de détecter la présence des cellules tumorales. D’autres applications biologiques ont également été envisagées telles que la détection d’acides nucléiques par l’utilisation d’une approche simple de biofonctionnalisation. Bien que l’objectif ma thèse était de de créer des capteurs biomédicaux en utilisant une approche in vitro, il pourrait être également possible d’intégrer ces dispositifs « in vivo » ou encore dans des e-textiles
Rapid and early diagnosis of disease plays a major role in preventative healthcare. Undoubtedly, technological evolutions, particularly in microelectronics and materials science, have made the hitherto utopian scenario of portable, point-of-care personalized diagnostics a reality. Organic electronic materials, having already demonstrated a significant technological maturity with the development of high tech products such as displays for smartphones or portable solar cells, have emerged as especially promising candidates for biomedical applications. Their soft and fuzzy nature allows for an almost seamless interface with the biological milieu rendering these materials ideally capable of bridging the gap between electronics and biology. The aim of this thesis is to explore and validate the capabilities of organic electronic materials and devices in real-world biological sensing applications focusing on metabolite sensing, by combining both the right materials and device engineering. We show proof-of-concept studies including microfluidic integrated organic electronic platforms for multiple metabolite detection in bodily fluids, as well as more complex organic transistor circuits for detection in tumor cell cultures. We finally show the versatility of organic electronic materials and devices by demonstrating other sensing strategies such as nucleic acid detection using a simple biofunctionalization approach. Although the focus is on in vitro metabolite monitoring, the findings generated throughout this work can be extended to a variety of other sensing strategies as well as to applications including on body (wearable) or even in vivo sensing

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2018
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 115-121).
The pressure of increasing service demands and improving turnaround times of results in healthcare industry require the development of more rapid, point-of-care and personalized diagnostic tools. Light is ubiquitous in biology and offers various elegant solutions for diagnostic, therapies and theranostic applications. At present, there is not a single biomedical application where optical components are not applied. If we can come up with a framework that can miniaturize these optical components on a chip, it can offer various advantages in terms of scalability, portability, cost and improved performance for real-time monitoring and bedside treatment. On these lines, we propose bio-photonic integrated circuits (Bio-PICs) for point-of-care diagnostics. These circuits rely on moving photons in photonic waveguides (similar to electrons in your electronic chips) to provide on-chip sensing solution. Specifically, we present Bio-PICs for aerosol spectroscopy, blood coagulometry, and TB breath analyzer test.
by Robin Singh.
S.M.
S.M. Massachusetts Institute of Technology, Department of Mechanical Engineering

Correct diagnosis of disease is essential in the effort to save and improve lives. Point of care (POC) diagnostics are in-vitro tests that assist in patient diagnosis and can be used at the location of patient care. POC diagnostics are easy to use and provide near-instant readouts allowing medical providers and patients to make rapid decisions about treatment. Increased access to POC testing is especially beneficial to low-income and low resource areas that cannot afford expensive lab testing. The World Health Organization (WHO) has outlined at least 113 diseases for which POC diagnostics are needed. Because of this, developing effective, efficient, and economical methods for creating new POC tests is essential. Work in section one of this thesis describes strategies by which new POC bio-diagnostics can be created. The use of oxidized cellulose as a vector for antibody immobilization was explored in several cellulose-based materials to provide quick, economical tests while still obtaining effective limits of detection when used to detect the pregnancy hormone Human Chorionic Gonadotropin (HCG) in a proof of concept study. The majority of these tests could detect as low as 100 ng/mL of HCG well below the clinical level necessary for detection at 2400 ng/mL. The use of a hand-powered syringe-based POC named the fast flow immunoassay (FFI) was tested for its ability to increase observable signal in a sandwich immunoassay by passing the sample through the test filter multiple times. 10 passes through the filter resulted in a signal approximately 17x more intense than a 1-hour dot-blot sandwich immunoassay. Both oxidized cotton and FFI systems can be used to develop new POC assays quickly and economically. Future use of these POC systems could help expand the availability of diagnostic testing to disadvantaged areas. Gold-based drugs have been used and investigated as medications multiple times throughout history to treat various diseases such as Rheumatoid arthritis, parasitic infections, and cancer. In the last few decades, gold nanoparticles have been used as drug delivery agents and catalysts for various reactions. Recently catalytic gold nanocrystals have been characterized for their ability to treat neurodegenerative diseases. Although these results were promising, much is still unknown about their mechanism of action. Section two of this thesis investigates potential molecular pathways that gold nanocrystals could be affecting, specifically the IL-6/Jak/STAT3 inflammation pathway and the Nrf2 antioxidant pathway. The gold nanocrystals we tested did not affect these pathways at physiologically obtainable concentrations. Additional work was done to characterize protein interactome or protein corona of gold nanocrystals. Preliminary proteomic characterization of this protein corona in fetal bovine serum (FBS) identified 118 potential interactors and classified those based on function and structure. Future work will need to be done to follow up on these identifications and to determine what mechanistic implications they may have.

Infectious diseases pose a serious threat to global health. Molecular diagnostics provide solutions for effective control and prevention of infections, however suffers from expensive laboratory equipment, and infrastructure to be fully implemented at point of care (POC), especially at low-resource settings. Lab-on-a-chip that aims to integrate complex biochemical analyses into automated systems is promising for POC analysis. A major challenge is the integration of a complete molecular diagnostic assay, generally translating into complex microfluidics, with the requirement of low fabrication cost. This thesis explores the use of flexible electronics, plastic foils and roll-to-roll manufacturing to enable low-cost microfluidic systems, for molecular diagnostic assays especially targeted towards infectious diseases. Many biochemical assays rely on heat; hence a first aspect in this thesis is the integration of a microheater into microfluidics. In a first project a system for SNP-genotyping is presented using solid phase melting curve analysis to discriminate mutations at a single base resolution. Starting with a glass based concept (paper I) which is further developed to a foil based system (paper II), detection of the polymorphism in the neuropeptide Y associated with increased risk of type II diabetes is demonstrated as a proof of principle. Further development and optimization of the microheater concept has enabled roll-to-roll manufacturing compatibility and multiplexing of targets (paper III). A bacterial sub-typing and multiresistance detection in clinical Staphylococcus Aureus samples is demonstrated for applications in infectious diseases diagnostics. Finally, the microheater concept is further developed to enable μPCR (paper IV). Detection of genomic HIV-1 is demonstrated and a portable detection setup based on an LED light source and low cost CMOS camera for detection was developed. A second aspect of this thesis is integration of light sources and optical detection (paper V-VI). A multilayer system integrating an electroluminescent light source, reactive sensor dyes and organic semiconductor transistor for detection is demonstrated. The system could be used for amine detection in gases (paper V). System was made further roll-to-roll compatible. The system uses an external LED light source and a photodetector processed in only one screen printing- and one dispensing step (paper VI). As a proof of principle, absorbance based DNA hybridization was detected. Collectively, roll-to-roll manufacturing compatible “lab on foil” systems have the potential to improve our ability to diagnose at POC especially at resource-limited settings.

QC 20170426

The present thesis aims to develop a Lab-on-Chip (LOC) platform technology for mixing, separation, and trapping of biological samples, in particular for handling biological and non-biological particles with application in three-dimensional tissue printing and point of care diagnostics. Numerical models for the different manipulation tasks were first established. Physical parameters such as magnetic flux density, pressure, velocity of the fluids, particle trajectory, and species concentration were numerically simulated. The numerical model was then used to optimise mixing, separation, and trapping of biological particles such as cells and exosomes. Hydrodynamic trapping of three-dimensional (3D) spheroids was demonstrated as a passive separation method. The developed LOC device facilitates trapping, culturing and fusion of cell spheroids in a single system. The successful test of the device indicates its potential therapeutic application in the repair of nerve injuries. Furthermore, active manipulation was employed to initiate mixing and separation of non-magnetic particles using negative magnetophoresis. The proposed mixing platform offers an optimised and low-cost solution by separating the permanent magnets from the LOC device. Separation of nonmagnetic fluorescent particles with a subtle size difference was demonstrated. The problem of clogging the channel by magnetic nanoparticles was solved by introducing multiple ferrofluid streams with different concentrations. Finally, positive magnetophoresis was utilised to implement on-chip mixing and subsequent separation of a biological sample for cancer detection. The device with integrated sample preparation promises applications in Point-of-Care (POC) diagnostics of early stage cancer.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

Master of Science
Department of Biological & Agricultural Engineering
Mei He
Anemia affects about 25% of the world’s population and causes roughly 8% of all disability cases. The development of an affordable point-of-care (POC) device for detecting anemia could be a significant for individuals in underdeveloped countries trying to manage their anemia. The objective of this study was to design and fabricate a 3D printed, low cost microfluidic mixing chip that could be used for the diagnosis of anemia. Microfluidic mixing chips use capillary flow to move fluids without the aid of external power. With new developments in 3D printing technology, microfluidic devices can be fabricated quickly and inexpensively. This study designed and demonstrated a passive microfluidic mixing chip that used capillary force to mix blood and a hemoglobin detecting assay. A 3D computational fluid dynamic simulation model of the chip design showed 96% efficiency when mixing two fluids. The mixing chip was fabricated using a desktop 3D printer in one hour for less than $0.50. Blood samples used for the clinical validation were provided by The University of Kansas Medical Center Biospecimen Repository. During clinical validation, RGB (red, green, blue) values of the hemoglobin detection assay color change within the chip showed consistent and repeatable results, indicating the chip design works efficiently as a passive mixing device. The anemia detection assay tended to overestimate hemoglobin levels at lower values while underestimating them in higher values, showing the assay needs to go through more troubleshooting.

Thesis: S.M. in Engineering and Management, Massachusetts Institute of Technology, School of Engineering, System Design and Management Program, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 76-80).
Rapid disease diagnosis is critical during infectious disease outbreaks to enable early intervention measures and minimize risk of disease transmission. Recent outbreaks in low-resource settings have highlighted challenges with traditional laboratory-based diagnostic approaches including a dependence on supporting infrastructure and highly trained users. Limitations of laboratory-based devices often result in geographical separation of labs from cases creating delays and barriers for diagnosis. There is increasing interest in the use of point-of-care diagnostics during outbreaks to enable more dispersed field diagnostic approaches and improve accessibility of testing. Point-of-care diagnostics, however, are often less accurate than laboratory-based tests, which can make them a less trusted option. This thesis explores the possibility that accessible, less accurate point-of-care devices could enable more efficient containment of disease outbreaks compared to current practices that employ expensive, and often distant laboratory-based tests. Although the benefit of point-of-care devices has been discussed anecdotally, little work has been done to quantify the relative impact of point-of-care diagnostics on transmission characteristics during an outbreak. This thesis aims to establish a basic cross-domain simulation model that considers medical, engineering, and societal/cultural factors that contribute to disease outbreak outcomes. The simulation approach is used to assess the trade-off between diagnostic access and accuracy during the 2014 West Africa Ebola outbreak to determine if point-of-care devices could have offered a benefit. A sensitivity analysis is also conducted to assess the potential impact of diagnostics on future outbreaks. Simulation results support the hypothesis that deployment of point-of-care devices to increase accessibility of testing could significantly reduce the number of secondary infections during an outbreak. This finding is shown to be true across outbreaks of varying sizes and transmission characteristics and for devices with varying accuracy performance.
by Ashley L. Whitney.
S.M. in Engineering and Management

Protein biomarkers are important diagnostic tools for detection of non-communicable diseases, such as cancer and cardiovascular conditions. In order to be used as diagnostic tools they need to be detected at very low concentrations in biological samples (e.g. whole blood, serum or urine). This has been currently performed in central laboratories using expensive, bulky equipment and time consuming assays.

Cette thèse de doctorat porte dans l’ensemble une étude approfondie sur une technologie émergente pour l’amplification isotherme des acides nucléiques appelée recombinase polymerase amplification (RPA). L’introduction porte une description détaillée sur la RPA. Cette revue de littérature documente et discute les diverses applications de la RPA en soulignant les connaissances actuelles concernant les applications diagnostiques. Malgré la composition complexe de la RPA (6 à 7 protéines dans le même mélange réactionnel), cette dernière s’avère une technologie rapide (générant des résultats < 20 min), spécifique et sensible (détection de l’ordre de quelques copies de génome), et largement appliquée dans différentes disciplines. Ces avantages nous permettent de croire que la RPA possède la flexibilité nécessaire pour être utilisée comme outil de diagnostic rapide des maladies infectieuses en réduisant le temps d’obtention des résultats à moins d’une heure au lieu de 2 à 3 jours avec les tests de cultures standards. En conséquence, il sera possible d’intégrer la RPA dans des plateformes microfluidiques ou laboratoire sur puce qui permettent la préparation d’échantillons, l’amplification et la détection des acides nucléiques des microbes causant des infections. En premier lieu, les travaux de cette thèse ont généré des lignes directrices additionnelles pour la conception des amorces/sondes RPA. En second lieu, nos travaux ont permis de développer un essai diagnostic RPA pour la détection des streptocoques du groupe B, responsables de la septicémie et la méningite chez les nouveau-nés. Cet essai fut le premier à évaluer la performance de la RPA avec des échantillons cliniques humains. Ce test diagnostic RPA a été comparé à une méthode de référence, la réaction en chaîne par polymérase (PCR). Cette démonstration sur des échantillons cliniques nous à inciter à pousser notre étude pour réaliser le dernier objectif de ce projet qui consistait à automatiser la RPA par intégration dans un système microfluidique miniaturisé centripète. Une collaboration avec des experts en génies et en matériaux a permis de générer un dispositif microfluidique appelé blade ainsi de l’instrument impliqué dans l’opération des différentes tâches mécanistiques. Ces résultats préliminaires suggèrent qu’il sera important d’offrir un système automatisé complet applicable au chevet du patient. Par conséquence, il sera possible d’exécuter une analyse complète des agents infectieux en moins d’une heure sans le besoin des procédures complexes de préparation et de transport des échantillons cliniques ni le recours à du personnel qualifié.
This dissertation consists of an exhaustive study on an emerging technology for isothermal amplification of nucleic acids called recombinase polymerase amplification (RPA). The introduction of this thesis is a detailed description of the RPA. This review documents and discusses the various applications of this technology by pointing to the current knowledge about RPA for diagnostic applications. Despite the complex composition of RPA (6 to 7 proteins in the same reaction mixture), the latter was shown to be rapid (generating results in < 20 min), specific and sensitive (detecting few target genome copies), and applied widely in different fields. Based on these advantages, we assume that RPA has a flexibility allowing it to be used for the rapid diagnosis of infectious diseases thus reducing time-to-result to less than an hour. Consequently, it will be possible to integrate RPA in microfluidic platforms providing a lab-on chip system. The first part of this doctoral project generated additional guidelines for RPA primers/probes design to develop specific RPA diagnostic assays. Second, we developed an RPA diagnostic test for the detection of group B streptococci, responsible for sepsis and meningitis in newborns. This assay was the first to evaluate RPA with human clinical samples. This diagnostic test was compared to a reference method, the polymerase chain reaction (PCR). This demonstration with clinical samples served to carry out the final objective of this project that was to automate RPA in a miniaturized microfluidic centripetal system. Collaboration with engineers and experts in materials has generated the microfluidic device called "blade" and the instrument involved in the operation of various mechanistic tasks. These preliminary results suggested that it will be important to provide an automated system applicable at bedside. Consequently, it will be possible to perform a complete analysis of infectious agents in less than an hour without the need for complex procedures for the preparation and transport of clinical specimens or the assistance of qualified personnel.

Single Nucleotide Polymorphisms (SNPs) are common genetic variations associated to specific phenotypes. SNPs have high relevance in different biomedical areas including both preventive/personalized medicine and non-clinical contexts, such food traceability. However, current techniques for SNP genotyping are expensive, require specialized laboratories and costly instrumentations, and are time consuming. In this framework, the development of rapid, point-of-care (POC) SNP diagnostic technologies would be of great interest, opening new possibilities for early, on-site screenings in multiple fields. In this PhD thesis, starting from state-of-art approaches, a rapid and portable diagnostic strategy based on Loop Mediated Isothermal Amplification (LAMP) has been developed for SNPs genotyping. In particular, the developed strategy was applied and validated, in a naked-eye colorimetric approach, for food varietal discrimination (i.e. durum wheat variety) in a model case of industrial relevance. Moreover, the POC technology was targeted to different salivary biomarkers, to address nutrigenetics and pharmacogenetics issues. Notably, the possibility to rapidly diagnose the genetic predisposition to lactose intolerance or the impairment of folic acid metabolism was proven. Furthermore, the developed POC-compatible protocols can work with portable, battery-powered devices, allowing for POC operation of such diagnostic tests.

Dengue is a major public health problem that affects tens of millions of people annually in tropical and sub-tropical countries. This acute viral infection happens to be severe and even life threatening but there is still no available drug or vaccine. Previous studies have noted early higher viral burden in patients who develop more severe symptoms suggesting that administration of a potent and safe antiviral may prevent progression to severe dengue. To verify this hypothesis, we have conducted the first RCT directed towards reducing the viral burden in vivo by administrating chloroquine (CQ), a cheap and well-tolerated drug that inhibits DENV in vitro with concentrations achievable in vivo, to 307 Vietnamese adults with suspected dengue (257 of them were laboratory-confirmed cases). Unfortunately, we did not see an effect of CQ on the duration of infection. However in patients treated with CQ, we observed a trend towards a lower incidence of severe forms. We did not find any differences in the immune response that can explain this trend. We also found more adverse events, primarily vomiting, with CQ. In addition, we have explored the relationships between clinical features, antibody responses and virological markers in these patients. We found that the early magnitude of viremia is positively associated with disease severity and there are serotype dependent differences in infection kinetics. We found as well that DENV was cleared faster and earlier in patients with secondary infections. To complete this study, we have also evaluated 2 rapid lateral flow tests for the diagnosis of dengue in a panel of plasma samples from 245 RT-PCR confirmed dengue patients and 47 with other febrile illnesses. Our data suggest that the NS1 test component of these tests are highly specific and have similar levels of sensitivity (~60%). Both NS1 assays were significantly more sensitive for primary than secondary dengue. The IgM parameter in the SD Duo test improved overall test sensitivity without compromising specificity. All these findings are of major importance for further anti-viral drug testing.

Abstract Technical improvements in computed tomography (CT) scanners have provided new possibilities to exploit the resources of this imaging modality in the evaluation of patients with multiple injuries or patients being treated in an intensive care unit (ICU). The purpose of this study was to assess the significance of multi-detector computed tomography (MDCT) in diagnostics and treatment decisions concerning multiple trauma and critically ill patients. Findings of MDCT using a dedicated trauma protocol in 133 patients exposed to high-energy blunt trauma were retrospectively evaluated. Diagnostic information about the injuries that would enable planning of treatment was sought. The imaging protocol consisted of axial scanning of the head and helical scanning of the facial bones, cervical spine, thorax, abdomen, and pelvis. Ninety-nine of the patients (74%) had at least one finding consistent with trauma. Nineteen false negative findings and two false positive findings were made. The overall sensitivity of MDCT was 94%, specificity 100%, and accuracy 97%. The reliability of a structured 5-min evaluation of MDCT images from the scanner’s console was prospectively evaluated in 40 high-energy trauma patients. The dedicated trauma protocol covering the thorax, abdomen, and pelvis was used in MDCT scanning. The findings were compared with the final radiological diagnosis of the MDCT data made on a picture archiving and communicating system (PACS) workstation, the operative findings, and the clinical follow-up. The evaluation from the scanner’s console enabled diagnosis of all potentially life-threatening injuries, the sensitivity for all injuries being 60% and specificity 98%. The effects of MDCT on the treatment of patients in a 12-bed medical-surgical ICU were observed prospectively. Sixty-four patients with an ICU stay longer than 48 h had had inconclusive findings with other modalities of radiological imaging. They underwent altogether 82 MDCT examinations. Fifty examinations (61%) resulted in a change in treatment, and 20 (24%) of them otherwise contributed to or supported clinical decision-making. Twelve examinations (15%) failed to provide any additional information relevant to the patient’s treatment. MDCT examination was helpful in general ICU patients, with inconclusive findings with other imaging modalities. CT images of 127 mixed medical-surgical ICU patients were retrospectively reviewed for the previously determined findings. Forty-three of these patients underwent open cholecystectomy, revealing eight cases with a normal gallbladder (GB), 26 with an edematous GB, and nine with necrotic acute acalculous cholecystitis (AAC). Abnormal CT findings were present in 96% of all the ICU patients. Higher bile density in the GB body and subserosal edema were associated with an edematous GB. The most specific findings predicting necrotic AAC were gas in the GB wall or lumen, lack of GB wall enhancement, and edema around the GB. The frequent prevalence of nonspecific abnormal imaging findings in the GB of ICU patients limits the diagnostic value of CT scanning.

This thesis contributes to the development of Lab-on-a-Chip systems that enables reliable, rapid medical diagnostics at the point-of-care. These contributions are focused on microfluidic Lab-on-a-Chip systems for sepsis diagnosis, autonomous sample-to-answer tests, and dried blood spot sampling. Sepsis is a serious condition with high mortality and high costs for society and healthcare. To facilitate rapid and effective antibiotic treatment, improved sepsis diagnostics is needed. Diagnosis of sepsis requires the processing of relatively large blood volumes, creating a need for novel and effective techniques for the handling of large volume flows and pressures on chip. Components, materials, and manufacturing methods for pneumatically driven Lab-on-a-Chip systems have therefore been developed in this thesis. Microvalves, an essential component in many Lab-on-a-Chip systems have been the focus on several of the advances: a novel elastomeric material (Rubbery Off-Stoichiometric-Thiol-Ene-Epoxy) with low gas and liquid permeability; the first leak-tight vertical membrane microvalves, allowing large channel cross-sections for high volumetric flow throughput; and novel PDMS manufacturing methods enabling their realization. Additionally, two of the new components developed in this thesis focus on separation of bacteria from blood cells based on differences in particle size, and cell wall composition: inertial microfluidic removal of large particles in multiple parallel microchannels with low aspect ratio; and selective lysis of blood cells while keeping bacteria intact. How these components, materials and methods could be used together to achieve faster sepsis diagnostics is also discussed. Lab-on-a-Chip tests can not only be used for sepsis, but have implications in many point-of-care tests. Disposable and completely autonomous sampleto- answer tests, like pregnancy tests, are capillary driven. Applying such tests in more demanding applications has traditionally been limited by poor material properties of the paper-based products used. A new porous material, called “Synthetic Microfluidic Paper”, has been developed in this thesis. The Synthetic Microfluidic Paper features well-defined geometries consisting of slanted interlocked micropillars. The material is transparent, has a large surface area, large porous fraction, and results in low variability in capillary flowrates. The fact that Synthetic Microfluidic Paper can be produced with multiple pore sizes in the same sheet enables novel concepts for self-aligned spotting of liquids and well-controlled positioning of functional microbeads. Diagnostic testing can also be achieved by collecting the sample at the point-of-care while performing the analysis elsewhere. Easy collection of finger-prick blood in paper can be performed by a method called dried blood spots. This thesis investigates how the process of drying affects the homogeneity of dried blood spots, which can explain part of the variability that has been measured in the subsequent analysis. To reduce this variability, a microfluidic sampling chip has been developed in this thesis. The chip, which is capillary driven, autonomously collects a specific volume of plasma from a drop of blood, and dry-stores it in paper. After sampling, the chip can be mailed back to a central lab for analysis.

QC 20160122

Introduction: Deaths due to chronic liver disease have increased significantly in recent decades. This is due to increases in alcohol consumption and obesity during this time period, and insensitive screening tests (liver function blood tests) utilised in primary care. This thesis describes a new liver disease community diagnostic pathway which focussed upon defined risk factors for chronic liver disease and uses Transient Elastography (TE) as the primary investigation modality. The aims of the thesis are to assess the feasibility of this pathway for detecting liver disease due to alcohol or non-alcoholic fatty liver disease within the United Kingdom healthcare system, to quantify the number of new cases detected with this approach and to evaluate patient experience of these investigations. Methods: Following a systematic review of the literature, an investigation pathway was derived and piloted in 4 general practice sites in Nottinghamshire in two phases between February 2012 and September 2014. Patients with hazardous alcohol use, type 2 diabetes or persistently raised alanine aminotransferase (ALT) level and negative liver serology were eligible for study. TE was performed in the community; a liver stiffness reading of ≥8 kilopascals defined clinically significant liver disease and subsequent review in a consultant led community clinic. Risk factors for new diagnoses of liver disease and cirrhosis were identified and the association with obesity investigated. A qualitative interview substudy was conducted to explore the experiences of 20 patients undergoing investigation. Results: In a total adult population of 20,868 patients, 2,022 patients were eligible for study of whom 909 (45%) underwent TE. Valid liver stiffness measurements were possible in 98% of patients. Overall, 230 cases of elevated liver stiffness and 27 new cases of cirrhosis were identified. Minimum cirrhosis prevalence in patients with type 2 diabetes was 2%. Obesity was significantly associated with diagnosis of cirrhosis in type 2 diabetics (odds ratio 9.4 (95% CI 2.2-40.9)) and hazardous alcohol users (OR 5.6 (95% CI 1.6-19.7)). The majority of new cases of liver disease had normal ALT levels. From the initial pilot phase in two general practices in Rushcliffe (Nottingham), in which liver function test data from 378 patients undergoing TE was analysed, 72.4% with elevated liver stiffness measurement, 60% with biopsy proven cirrhosis and 90% with cirrhosis diagnosis had normal ALT. Patients felt that TE was a useful adjunct to lifestyle change and described a positive experience of liver disease investigation. Conclusion: A new non-invasive diagnostic pathway for liver disease was feasible to implement in Nottinghamshire primary care and resulted in significantly increased diagnosis of chronic liver disease and cirrhosis. These findings warrant exploration of the pathway in a larger primary care population.

Introduction: Augmented care is a potentially significant reservoir of antimicrobial resistance (AMR). In the context of augmented care, this thesis investigates AMR variation and its drivers, barriers to optimising antimicrobial use, and the role of rapid infection-related diagnostics in addressing this. Methods: Mixed methods were used. AMR and antimicrobial consumption were quantitatively analysed, and driving factors explored using (i) statistical algorithms and (ii) thematic analysis of semi-structured interviews. As a case study, the role of rapid microbiology diagnostics was investigated with interrupted time series analysis of introduction of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF). The added value of computerised decision support was then assessed. Results: Analysis of 174,434 isolates demonstrated significant AMR variation between and within hospitals, with an excess in augmented care. Multiple ‘mini-outbreaks’ partly account for this, as does variation in antimicrobial consumption. Communication, knowledge, and personalisation of prescribing significantly influence the latter. Microbiology results are suboptimally utilised in these three areas. MALDI-ToF introduction, as a case study of a rapid microbiology diagnostic, significantly improved bacterial identification, but did not alter antimicrobial consumption. A role in surveillance is apparent, through identifying and investigating outbreaks, and in infection control, through carbapenemase detection. A computerised decision support system, derived from analysis of augmented care antimicrobial prescribing, facilitates wider impact from such rapid microbiology diagnostics. Conclusion: Augmented care areas have high, yet heterogeneous, levels of AMR, in part due to previously undetected outbreaks, and in part from variation in prescribing. Lost opportunities in communication, personalisation, and knowledge-holding influence the latter. Increasing the rapidity and specificity of microbial identification plays only a limited role in antimicrobial stewardship, but wider roles in surveillance and infection control will further impact AMR in augmented care. Decision support systems add further value, facilitating integration of clinical data, and shaping empiric antimicrobial choice where results are not available.

Diseases are often diagnosed by detection of disease-specific biomarkers in fluid samples. However, many state-of-the-art detection methods require a lab with complex machinery, trained operators, and/or lengthy analysis time. In contrast, point-of-care (POC) devices are brought to the patient's location, they are easy to use, and results are obtained almost immediately. Many current POC devices are too difficult to be used without a skilled assistant, and although many are able to detect analytes above a threshold value, they give little or no quantitative information. This work presents the development of polymer-based microfluidic devices capable of sensing and quantifying biomarkers in fluid samples in a straightforward manner using a novel biomarker assay termed "flow valve diagnostics". In this assay, an antibody-modified polydimethylsiloxane (PDMS) microchannel constricts due to the binding force between antibodies and antigens, stopping fluid flow. The flow distance is measured and correlated to antigen concentration. This detection method is an improvement over other methods because it is an innovative, non-instrumented, label-free, easy-to-use approach. These devices are small, portable, disposable, inexpensive, and thus ideal for use in POC testing. I have successfully fabricated flow valve devices with standard micromachining techniques, including photolithography, replica molding with PDMS, and plasma oxidation. Following fabrication, I compared two methods for attaching receptor biomolecules (e.g., antibodies) to the microchannel surfaces: non-specific adsorption and silanization with 3-glycidoxytrimethoxypropylsilane (GOPS). I used laser-induced fluorescence to determine that silanization with GOPS was the better method for biomolecule attachment. Finally, I tested antibody-modified flow valve devices with target antigens to determine if the antibody/antigen binding force was strong enough to cause channel pinching and flow stoppage. By modifying the device design and using higher antigen concentrations, I was able to show that flow valve devices can detect antigens in a concentration-dependent manner. Future work to improve the device design and to modify and test these devices with different receptor/target pairs will bring flow valve diagnostics closer to becoming a valuable asset in biomarker detection and POC testing.

Els test de tipus 'point-of-care' (POC) presenten un gran potencial per al maneig i el diagnòstic de malalties. Els dispositius POC permeten la realització de proves clíniques prop del pacient, permetent així un diagnòstic ràpid, una ràpida iniciació de tractaments, i en cas necessari, una derivació ràpida a altres centres mèdics. Aquests dispositius tenen a més el potencial de ser més econòmics, més robustos, i més fàcils d'usar que els dispositius mèdics tradicionals. Per aquests motius, els dispositius mèdics de tipus POC es consideren prometedors per als països en vies de desenvolupament, els quals són també els que necessiten de forma més urgent noves tecnologies mèdiques. En aquest context, aquesta tesi se centra en el desenvolupament de dispositius mèdics de diagnòstic in-vitro de tipus POC per salut global. Tenint en compte que els recursos per al desenvolupament i explotació de dispositius POC per a països amb baixos recursos són limitats, el Capítol 2 s'enfoca en el desenvolupament de prioritats d'investigació en salut. Mitjançant l'establiment d'aquestes prioritats es pretén facilitar la selecció d'objectius a fabricants d'instruments mèdics, així com incrementar l'impacte de les noves tecnologies desenvolupades. Els criteris de priorització considerats són molt amplis i inclouen l'impacte d'un nou test en la incidència d'una malaltia, la disponibilitat i preu dels tractaments de les malalties, la inversió tecnològica per al desenvolupament d'un nou dispositiu, i els principis bioètics. El tercer capítol descriu el desenvolupament d'un dispositiu mèdic senzill que pot ser fabricat fàcilment en laboratoris amb escassos recursos: tires reactives de diagnòstic de paper per a la detecció de biomarcadors presents en fluids biològics fabricats amb impressores de raig de tinta domèstiques i amb receptes senzilles per la preparació de les tintes. Aquesta tècnica de fabricació de tires reactives de diagnòstic va ser provada per a la detecció de deficiència de iode, un problema sever de salut global al món. En aquest capítol es presenten experiments de preparació de tintes químiques, impressió en paper, detecció de iode en les concentracions presents en l'orina, i consells per al desenvolupament de noves tintes per a la detecció d'altres biomarcadors de malalties. Aquest simple i versàtil procés de fabricació de tests de diagnòstic permetria a hospitals i laboratoris amb pocs recursos dissenyar els seus propis diagnòstics per a malalties rellevants, i en la forma i quantitat adaptada a les necessitats de cada comunitat. Desafortunadament, no totes les malalties es poden diagnosticar usant senzilles tires reactives de diagnòstic, i freqüentment es necessiten dispositius més complexos. El capítol 4 està enfocat en el desenvolupament de dispositius de PCR i RT-PCR de baix cost, a temps real, i de tipus POC que permeten detectar quantitativament patògens basats en DNA i RNA respectivament. El nostre sistema es basa en PCR de flux continu, el qual manté zones de temperatura fixes i empeny la solució de PCR entre les àrees calefactades, permetent així una transferència de calor més ràpida i conseqüentment, una PCR més veloç. Tots dos sistemes de PCR i RT-PCR van ser fabricats a partir d'un xip microfluídic sol ús dissenyat per a ser produït a baix cost industrialment mitjançant mètodes de 'roll-to-roll'. El sistema òptic permet la detecció de patògens en temps real mitjançant mesures de fluorescència. Per demostrar la funció del xip, dos bacteris infeccioses i un virus van ser seleccionats: Chlamydia trachomatis, Escherichia coli O157: H7, i Ebola virus. Per als tres patògens, es van provar diferents velocitats de flux, es va determinar el límit de detecció del sistema, i es van calcular les eficiències de les PCRs. L'èxit dels resultats obtinguts i la versatilitat del sistema, fa que aquests dispositius es considerin prometedors per al diagnòstic d'altres patògens com Zika o chikungunya, que constitueixen amenaces mundials a la salut pública. Tots dos dispositius de diagnòstic in vitro presentats en aquesta tesi són bons exemples de dispositius de diagnòstic apropiats per a salut global.
Los test de tipo ‘point-of-care’ (POC) presentan un gran potencial para el manejo y el diagnóstico de enfermedades. Los dispositivos POC permiten la realización de pruebas clínicas cerca del paciente, permitiendo así un diagnóstico rápido, una pronta iniciación de tratamientos, y en caso necesario, una derivación rápida a otros centros médicos. Estos dispositivos tienen además el potencial de ser más económicos, más robustos, y más fáciles de usar que los dispositivos médicos tradicionales. Por estos motivos, los dispositivos médicos de tipo POC se consideran prometedores para los países en vías de desarrollo, los cuales son también los que necesitan de forma más urgente nuevas tecnologías médicas. En este contexto, esta tesis se centra en el desarrollo de dispositivos médicos de diagnóstico in vitro de tipo POC para salud global. Teniendo en cuenta que los recursos para el desarrollo de dispositivos POC para países con bajos recursos son limitados, el Capítulo 2 se enfoca en el desarrollo de prioridades de investigación en salud. Mediante el establecimiento de estas prioridades se pretende facilitar la selección de objetivos a fabricantes de dispositivos médicos, así como incrementar el impacto de las nuevas tecnologías desarrolladas. Los criterios de priorización considerados son muy amplios e incluyen el impacto de un nuevo test en la incidencia de una enfermedad, la disponibilidad y precio de los tratamientos de las enfermedades, la inversión tecnológica para el desarrollo de un nuevo dispositivo, y los principios bioéticos. El segundo Capítulo 3 describe el desarrollo de un dispositivo médico sencillo que puede ser fabricado fácilmente en laboratorios con escasos recursos: tiras reactivas de diagnóstico de papel para la detección de biomarcadores presentes en fluidos biológicos fabricados con impresoras de chorro de tinta domésticas y con recetas sencillas para la preparación de las tintas. Esta técnica de fabricación de tiras reactivas de diagnóstico fue probada para la detección de deficiencia de yodo, un problema severo de salud global en el mundo. En este capítulo se presentan experimentos de preparación de tintas químicas, impresión en papel, detección de yodo en las concentraciones presentes en la orina, y directrices para el desarrollo de nuevas tintas para la detección de otros biomarcadores de enfermedades. Este simple y versátil proceso de fabricación de tests de diagnóstico permitiría a hospitales y laboratorios con pocos recursos diseñar sus propios diagnósticos para enfermedades relevantes, en una forma y cantidad adaptada a las necesidades de cada comunidad. Desafortunadamente, no todas las enfermedades pueden diagnosticarse usando sencillas tiras reactivas de diagnóstico, y frecuentemente se necesitan dispositivos más complejos. El Capítulo 4 está enfocado en el desarrollo de dispositivos de PCR y RT-PCR de bajo coste, de tiempo-real, y de tipo POC que permiten detectar cuantitativamente patógenos basados en DNA y RNA respectivamente. Nuestro sistema se basa en PCR de flujo continuo, el cual mantiene zonas de temperatura fijas y empuja la solución de PCR entre las áreas calefactadas, permitiendo así una transferencia de calor más rápida y consecuentemente, PCR más veloces. Ambos sistemas de PCR y RT-PCR fueron fabricados en base a un chip microfluídico desechable diseñado para ser producido a bajo coste industrialmente mediante métodos de ‘roll-to-roll’. El sistema óptico permite la detección de patógenos en tiempo real mediante medidas de fluorescencia. Para demostrar la función del chip, dos bacterias infecciosas y un virus fueron seleccionados: Chlamydia trachomatis, Escherichia coli O157:H7, y Ebola virus. Para los tres patógenos, se probaron diferentes velocidades de flujo, se determinó el límite de detección del sistema, y se calcularon las eficiencias de las PCRs. El éxito de los resultados obtenidos y la versatilidad del sistema, hace que estos dispositivos se consideren prometedores para el diagnóstico de otros patógenos como Zika o chikungunya, que constituyen amenazas mundiales a la salud pública. Ambos dispositivos de diagnóstico in vitro presentados en esta tesis son buenos ejemplos de dispositivos de diagnóstico apropiados para salud global.
Point-of-care (POC) testing has great potential for the management and diagnosis of disease. POC devices allow for testing close to the patient permitting rapid diagnosis, prompt treatment initiation, and when needed, quick referral to other health-care units. They have the potential to be lower-cost, more robust, and more user-friendly than traditional medical devices. For these reasons, POC diagnostic tests are a promising approach for the developing world, where there is also the most urgent need for new health technologies. In this context, this thesis is focused in the development of POC in vitro diagnostic tests for global health. Considering that the resources for developing POC devices for low-resource settings are limited, during Chapter 2 we focused on setting health research priorities to aid test developers setting their targets to increase the impact of the technology. The criteria for prioritization considered were very broad and took into account the impact of a new test on the burden of disease, the availability and expense of disease treatments, the technological investment to develop a new device, and the bioethical principles. Chapter 3 describes the development of a medical device that can be easily manufactured in limited resources laboratories: paper diagnostic chemical dipsticks to detect biomarkers present in biological fluids produced with domestic inkjet printers and simple ink preparation recipes. This fabrication technique for diagnostic strips was tested for the detection of iodine deficiency, a severe global health problem worldwide. In this chapter we present successful experiments for chemical inks preparation, printing on paper, detection of iodine in the concentrations present in the urine, and guidelines for new ink development to target other disease biomarkers. This simple and versatile manufacturing process for diagnostic tests would allow hospitals and laboratories with limited infrastructure to design diagnostics for relevant diseases in a format and quantity adapted to each community needs. Unfortunately, not all diseases can be diagnosed using simple chemical dipstick assays and more complex diagnostic devices are required. Chapter 4 is focused on the development of a low-cost, real-time, point-of-care PCR and RT-PCR systems for quantitative detection of DNA and RNA-based pathogens. Our systems are based on continuous-flow PCR which maintains fixed temperatures zones and pushes the PCR solution between heated areas allowing for faster heat transfer and as a result, faster PCRs. Both PCR and RT-PCR systems were built around disposable microfluidic chips designed to be economically produced industrially by roll-to-roll embossing methods. The optical system allows for pathogen detection via real-time fluorescence measurements. To demonstrate the function of the chips, two infectious bacteria and one viral target were selected: Chlamydia trachomatis, Escherichia coli O157:H7, and Ebola virus. For the three pathogens, different flow velocities were tested, the limit of detection of the system was determined, and PCR efficiencies were calculated. Our successful results, and the versatility of our system, make it promising for the detection of other DNA and RNA-based pathogens such as Zika or chikungunya, which constitute global health threats worldwide. The two in vitro diagnostic tests presented in this thesis are good examples of promising POC diagnostic devices appropriate for global health.

Thesis: S.M. in Technology and Policy, Massachusetts Institute of Technology, Engineering Systems Division, Technology and Policy Program, 2015.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 94-99).
Pediatric Pneumonia (PNA) is the single leading cause of death in children under five, accounting for 19% of all childhood deaths worldwide. Due to severe resource constraints on healthcare, the global burden of the disease in children is disproportionately shared by developing countries. In particular, India, having the highest incidence rate of PNA, accounts for more than 30% of the world's neonatal deaths from pneumonia every year. The three-tier referral systems, shared by many other developing countries, has introduced inefficiencies into delivering appropriate healthcare to patients in need. Point-of-care (POC) diagnostics is a type of tool used to assist physicians to make clinical decisions. Its key advantage include the quick turnaround of results, low cost and high diagnostic power could potentially improve India's pressing situation due to pneumonia. Since the disease progresses quickly in infants and babies and transportation of patients within the healthcare system is time consuming, POC diagnostic is crucial in lowering both the mortality of children with pneumonia and the cost of treating PNA. To this effect, we investigate the potential impact of POC diagnostics when implemented in a three-tier referral system. Using India as a case country, I construct a decision tree model that evaluates cost, mortality, and the combined cost-effectiveness in Tree-Age software as a framework which evaluates five implementation strategies of a POC diagnostic for PNA within the Indian public healthcare system. The strategies reflect various prescription decisions and referral patterns in current medical practice in India. I concluded that (1) the diagnostic will result in both higher cost and mortality in areas where the practice is to provide all patients antibiotics and thus not recommended, (2) the diagnostic is very likely to achieve lower cost and mortality when patients do not always receive antibiotics and sometimes are given only symptom-relieving drugs and thus recommended, (3) the diagnostic has great potential in generating savings by limiting patients from being transported to urban hospitals, (4) when dual diagnostic is employed combining POC diagnostics and X-ray, confirming positive cases from the diagnostic by X-ray results in lower cost and confirming negative cases results in lower mortality and (5) the diagnostic can save resources and benefit health outcomes and should be implemented in places where X-ray is not available.
by Biao Zhang.
S.M. in Technology and Policy

Cells often exist in heterogeneous mixtures. Density provides a property to separate several types of cells from the mixed sample in which they originate. Density-based separation methods provide a standard method to quickly separate or enrich specific populations of cells, such as lymphocytes from whole blood. This dissertation explores the use of aqueous multiphase systems (AMPS) as self-forming step-gradients in density for the separation of cells. AMPS were first discovered over a hundred years ago as aqueous two-phase systems. Density as a tool to separate cells is at least as old. Despite this long history, the work in this thesis is the first work to use AMPS to perform density-based separations on cells. This combination provides a powerful technique to separate cells and enable new testing at the point-of-care. Chapter 1 provides a short overview of aqueous multiphase systems and density-based separations of cells. Chapter 2 describes the process of taking technology, including AMPS, from a demonstration in a laboratory to a large scale evaluation in a field setting. In Chapter 3 and Appendix I, AMPS provide a means to enrich reticulocytes from whole blood as a means to grow malaria parasites. Chapter 4 and Appendix II describe the development and proof-of-prinicple of a density-based diagnostic test for sickle cell disease (SCD) using AMPS. Chapter 5 and Appendix III detail the results of a large scale field evaluation of a rapid test for SCD using AMPS in Zambia. Demonstrations of AMPS for density- and size-based separations are provided in Appendices IV and V. Appendix VI demonstrates the general usefulness of density to separate crystal polymorphs with another density-based separation method: magnetic levitation in a paramagnetic fluid. Beyond density, novel combinations of technology, such as electrochemistry and telecommunications provide opportunities for enabling global health (Appendix VII).
Engineering and Applied Sciences

Thesis (S.M. in Health Sciences and Technology)--Harvard-MIT Program in Health Sciences and Technology, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 92-95).
Disruptive models of innovation are starting to appear in healthcare. In the US, for instance, retail medicine clinics are changing the way in which patients satisfy their basic medical needs. In Mexico, similar retail medicine models (e.g. Farmacias Similares) are also disrupting healthcare delivery for basic medical needs. Disruptive innovations, however, are not limited to healthcare delivery, but also change the face of devices and diagnostics markets. A low CD4+ T cell count is the primary clinical indicator for HIV/AIDS disease progression, and thus is used as the primary trigger to initiate antiretroviral therapy. An entire diagnostic industry has emerged around CD4+ T cell counts for the management and treatment of HIV/AIDS patients. The diagnostic gold standards of CD4+ counts are flow cytometers. These large, capital intensive devices are commonly located in central laboratory settings, typically in urban areas. In developing nations, particularly, suburban and rural regions have no access to flow cytometers and typically face logistical problems of blood sample transportation and loss to follow-up of patients. Point-of-Care (POC) diagnostics promise disruptive models in diagnostics that will increase access, enhance care, and help better allocate healthcare resources. The concept of POC embodies the trade-off of lower "quality" (usually in the form of lower specificity and sensitivity) in exchange for higher "convenience" (i.e. better accessibility and portability, and significantly lower cost). POC diagnostics promise typical low-end and new-market disruptions in medical diagnostics and devices. Cambridge-based Daktari Diagnostics is one of such companies focused in POC diagnostics. It has developed a CD4+ T cell count diagnostic device for the management and treatment of HIV/AIDS patients. It is hypothesized in this thesis that there exists a relevant unmet medical need for POC CD4 count diagnostics in the Mexican HIV/AIDS market. In order to evaluate this hypothesis, secondary sources were reviewed, as well as primary interviews conducted across the Mexican HIV/AIDS healthcare landscape. While this hypothesis was evaluated on a preliminary basis only, responses suggested a relevant, albeit not urgent, medical need for POC CD4 count diagnostics. This primary hypothesis evaluation is extended by and complemented with market size estimations, and competitive dynamic discussions, that arrive at the following preliminary conclusions: the current market opportunity in Mexico ranges from baseline of ~100,000 tests per year to an upper bound potential of ~200,000 tests per year. In the context of this potential opportunity, Daktari's CD4 count diagnostic device is well positioned, as defined by diagnostic quality, technological characteristics, and competitive offering, to obtain a portion of this estimated market opportunity in Mexico.
by Mauricio Camargo Támara.
S.M.in Health Sciences and Technology

Thesis advisor: Thomas C. Chiles
Rapid diagnosis of infectious disease at the site of the patient is critical for preventing the escalation of an outbreak into an epidemic. This is particularly true for cholera, a disease known to spread swiftly within resource-limited populations. A device suited to point-of- care (POC) diagnosis of cholera must not only demonstrate laboratory levels of sensitivity and specificity, but it must do so in a highly portable, low-cost manner, with a simplistic readout. Here, we report novel proof-of-concept lab-on-a-chip (LOC) electrochemical immunosensors for the detection of cholera toxin subunit B (CTX), based on two nanostructured architectures: the gold dendritic array, and the extended core coax (ECC). The dendritic array has an ~18x greater surface area than a planar gold counterpart, per electrochemical measurements, allowing for a higher level of diagnostic sensitivity. An electrochemical enzyme-linked immunosorbant assay (ELISA) for CTX performed via differential pulse voltammetry (DPV) on the dendritic sensor demonstrated a limit-of detection of 1 ng/mL, per a signal-to-noise ratio of 2.6, which was more sensitive than a simple planar gold electrode (100 ng/mL). This sensitivity also matches a currently available diagnostic standard, the optical ELISA, but on a miniaturized platform with simple electrical readout. The ECC was optimized and explored, undergoing several changes in design to facilitate sensitive LOC electrochemical detection. The ECC matched the off-chip sensitivity towards CTX demonstrated by a previous non-extended core coaxial iteration, which was comparable to a standard optical ELISA. In contrast to the previous coaxial architecture, the ECC is amenable to functionalization of the gold core, allowing for LOC detection. ECCs were functionalized using a thiolated protein G, and CTX was detected via an electrochemical ELISA. While this work is ongoing, the ECC shows promise as a platform for LOC electrochemical ELISA. The ability to potentially meet POC demands makes biofunctionalized gold dendrites and ECCs promising architectures for further development as LOC sensors for the detection of infectious disease biomarkers
Thesis (PhD) — Boston College, 2018
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

This study investigates the history of venereal diseases in Sweden in the period from 1785 to 1903. Medical and political perceptions of these diseases as well as the patients and their continued lives have been studied. Venereal diseases were considered a significant threat to the growth of the population throughout the period. They were recognised through the dramatic sores that they produced on the body of the patient, and were frequently cured with mercurial therapies. In the late nineteenth century, syphilis and gonorrhoea became the two most significant sexually transmitted diseases. They were believed to cause paralysis, mental illness, infant mortality and infertility. Sweden fought venereal diseases with a network of State-controlled health measures. County hospitals that contained special wards for patients diagnosed with venereal diseases were established in the late eighteenth century. These hospitals were financed by mandatory revenue after 1817. Medical care was mandatory and ministers, law officers and heads of households could inform the provincial physicians about the incidence of venereal disease. During the nineteenth century, the regulation of prostitution was enforced which implied that women were blamed for the spread of these diseases. Patients with venereal disease belonged to a cross section of contemporary Swedish society. Most of them were from the lower- or working-classes. They suffered higher age-specific mortality in the first half of the century, and high infant mortality throughout the period. It appears, however, that the constructed image of a patient with venereal disease had little impact upon their lives. Contemporary poverty and societal problems, such as unemployment and poor housing, probably played a larger part in their lives.
digitalisering@umu

Tuberculosis is one of the deadliest communicable diseases in the world, and consequently remains one of the biggest global healthcare challenges. Tuberculosis is treatable and curable. However, within many low resource settings, underdeveloped medical infrastructure limits the effectiveness and accuracy of existing diagnostics. These limitations severely impede the timely diagnosis of the disease, and thus contribute to the disease spreading, developing drug resistance, and killing more individuals. There is an urgent need for an inexpensive, portable, rapid, easy-to-use point of care diagnostic that can function outside of the laboratory at the community level. Currently, there is a wide range of available tuberculosis diagnostics ranging from sputum smear microscopy to nucleic acid amplification tests. Yet, none have met every standard of the ideal point of care diagnostic. Since the World Health Organization’s endorsement of Xpert MTB/RIF in 2010, there has been a resurgence of interest in point of care diagnostic development. This investigation reviewed diagnostic development projects funded by the National Institutes of Health in 2008 and 2014 in order to examine the technologies being developed, how researchers in industrial and academic sectors are addressing this problem, and what challenges still need to be overcome. More projects in 2014 were expected to rely on sample types other than sputum and be funded than those in 2008. The results of this investigation confirm this hypothesis, and that the development of a point of care device is a multi-faceted challenge with numerous underlying issues that need to be addressed before such a device can be successfully implemented.

Thesis (Ph.D.)--University of Cincinnati, 2008.
Advisor: David Klotzkin (Committee Chair), Ian Papautsky (Committee Member), Marc Cahay (Committee Member), Fred Beyette (Committee Member), Paul Bishop (Committee Member). Title from electronic thesis title page (viewed Jan. 18, 2009). Keywords: OLED; OPD; microfluidic; lab-on-a-chip; on-chip fluorescence detector; MEMS; thin films; organic electronics. Includes abstract. Includes bibliographical references.

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FACEPE
A infecção pelo vírus da Hepatite B (HBV) é considerada uma enfermidade de alta morbimortalidade, apresentando diagnóstico complexo e quadro de persistência, fatores que dificultam a detecção, terapêutica e cura. Relatos variados têm apontado os imunossensores como importantes ferramentas de auxílio no diagnóstico de doenças, definido como um dispositivo que converte respostas de eventos biológicos a partir da interação antígenoanticorpo em sinal elétrico. No presente estudo foram desenvolvidos biossensores para detecção de anticorpos contra o nucleocapsídeo do HBV (Anti-HBc) mais perene apresentado no diagnóstico da doença. Recentemente, o emprego de nanomateriais no desenvolvimento de tais dispositivos tem despertado interesse devido às propriedades destes materiais. Particularmente, os nanotubos de carbono (NTCs) têm oferecido aos imunossensores melhoria na condutividade, aumento na velocidade de transferência de carga, aumento da área eletródica com maior possibilidade de imobilização de biomoléculas. Nesta tese, foram empregados o ácido hialurônico e o náfion como suporte para forte interação com os NTC funcionalizados em eletrodos de carbono vítreo e de ouro fabricado sobre folha de acetato. Os dispositivos foram caracterizados por técnicas de imagem (microscopia de força atômica) e eletroquímicas (voltametrias de onda quadrada e cíclica), as quais demonstraram a estabilidade da plataforma, imobilização eficaz e sensibilidade. O primeiro protótipo em eletrodos de carbono vítreo modificado com filme de ácido hialurônico associado a nanotubos funcionalizados apresentou resposta linear de 1 a 6ng/ml com limite de detcção de 0,03ng/ml. No segundo protótipo com eletrodos impressos de ouro modificado com filme etanólico de náfion associado a nanotubos funcionalizados, o imunossensor apresentou resposta linear de 0,5 até 2ng/ml, com limite de detecção de 0,15 ng/ml de anti-HBc. Os protótipos desenvolvidos apresentam-se como potenciais para diagóstico da HBV.
Infection with hepatitis B virus (HBV) is considered a high mortality disease, with complex diagnosis and persistence framework, factors that hinder detection, therapy and cure. various reports have pointed out the immunosensors as important aid tools in the diagnosis of disease, defined as a device that converts biological events of answers from the electrical signal in antigen-antibody interaction. In the present study biosensors have been developed for the detection of antibodies to the HBV nucleocapsid (anti-HBc) Perennial presented in the diagnosis of disease. Recently, the use of nanomaterials in the development of such devices has aroused interest because of the properties of these materials. Particularly, carbon nanotubes (CNTs) have offered immunosensors improvement in conductivity, increased charge transfer speed, increased electrodic area with the highest possibility of immobilization of biomolecules. In this thesis, we employed hyaluronic acid and nafion as support for strong interaction with the NTC functionalized glassy carbon electrodes and manufactured gold on acetate sheet. The devices were characterized by imaging techniques (atomic force microscopy) and electrochemical (cyclic and square wave voltammetry), which demonstrated the platform stability, effective restraint and sensitivity. The first prototype on glassy carbon electrode modified with hyaluronic acid film associated with functionalized nanotubes showed a linear response of 1 to 6ng/ ml with detction limit 0,03ng / ml. In the second prototype printed gold electrodes modified with ethanolic nafion film associated with functionalized nanotubes, the immunosensor showed a linear response of 0.5 to 2 ng / ml, the detection limit of 0.15 ng / ml of anti-HBc. The developed prototype is present as diagnostic potential for HBV.

This dissertation discusses the development of inexpensive, easy-to-use, and field-deployable diagnostic techniques and devices for the early detection of various pathogens, commonly found in clinical samples and contaminated food and water. Infectious diseases account for about 90% of world health problems, killing approximately 14 million people annually, the majority of which reside in developing countries. In 2012, the World Health Organization (WHO) published data on the top 10 causes of death across the globe. Although communicable disease is a prevalent cause of fatality, both low-income and high-income countries, pathogen species and transmission are very different. Nearly 60% of deaths in developing countries are caused by food, water, air or blood-borne pathogens. The most prevalent illnesses are diarrheal disease, malaria, and HIV/AIDS. By contrast, the leading causes of death in developed countries (heart disease, cancer, and stroke) are not communicable and are often preventable. However, there is an increasing need for the development of rapid and accurate methods for pathogen identification in clinical samples, due to the growing prevalence of antibiotic-resistant strains. Incorrect, or unneeded antibiotic therapies result in the evolution of extremely aggressive nosocomial (hospital-acquired) infections, such as methicillin- (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The implementation of rapid, easy to use and cost-effective diagnostics will reduce the frequency of pathogen-related deaths in underdeveloped countries, and improve targeted antibiotic treatment in hospital settings, thus decreasing the potential development of more treatment-resistant "super bugs". This research includes novel techniques utilizing two major sensing modalities: serological (i.e. immunological), and nucleic acid amplification testing (NAATs). We first developed a highly sensitive (limit-of-detection = 100 CFU mL-1) particle immunoassay that takes advantage of elastic and inelastic light scatter phenomena, for optical detection of target antigens. This assay is performed upon a unique nanofibrous substrate that promotes multiplexing on a user-friendly platform. We then developed a novel technique, termed emulsion loop-mediated isothermal amplification (eLAMP), in which the target amplicon is detected in real-time, again utilizing light scattering detection and quantification. Both techniques require no sample pre-treatments, and can be combined with smartphone imaging for detection of targets in under 15 minutes. These methods have the potential to improve the speed and sensitivity of early pathogenic identification, thus leading to a reduction in preventative deaths and a decrease in global economic costs associated with infectious disease in clinical and other settings.

Yes
Rapid point of care tests for bacterial infection diagnosis are of great importance to reduce the misuse of antibiotics and burden of antimicrobial resistance. Here, we have successfully combined a new class of non-biological binder molecules with electrochemical impedance spectroscopy (EIS)-based sensor detection for direct, label-free detection of Gram-positive bacteria making use of the specific coil-to-globule conformation change of the vancomycin-modified highly branched polymers immobilized on the surface of gold screen-printed electrodes upon binding to Gram-positive bacteria. Staphylococcus carnosus was detected after just 20 min incubation of the sample solution with the polymer-functionalized electrodes. The polymer conformation change was quantified with two simple 1 min EIS tests before and after incubation with the sample. Tests revealed a concentration dependent signal change within an OD600 range of Staphylococcus carnosus from 0.002 to 0.1 and a clear discrimination between Gram-positive Staphylococcus carnosus and Gram-negative Escherichia coli bacteria. This exhibits a clear advancement in terms of simplified test complexity compared to existing bacteria detection tests. In addition, the polymer-functionalized electrodes showed good storage and operational stability.

År 2020 listade Världshälsoorganisationen (WHO) cancer som den globalt ledande dödsorsaken med över 10 miljoner dödsfall årligen. Av dessa 10 miljoner fall förekommer nästan 70% i låg- till medelinkomstländer - en siffra som på grund av den låga prioriteringen av cancerbehandling- och diagnostik förväntas öka till 85% redan år 2030. Att utveckla enkla, specifika och prisvärda verktyg för diagnostik kommer därför att bli avgörande för förebyggandet av cancer på en global nivå. För att komma ett steg närmare denna utveckling optimerades och testades i denna studie ett mikrofluidiskt system, utvecklat genom layer-bylayer- metoden, baserat på cellulosa nanofibriller med förmågan att isolera och fånga cirkulerande tumörceller. För att uppnå en termodynamisk jämvikt optimerades systemets hydrodynamiska parametrar optimerades för att uppnå en homogen fördelning med hög densitet av det cellulosa-baserade systemet i det mikrofluidiska chippet. Då jämvikt är grundläggande för att maximera det efterföljande beläggningen av antikroppar, och därmed hur effektivt celler isoleras, modifierades parametrar såsom koncentration, flödeshastighet, inkubationstid med fler tills att önskad effekt uppnåtts. Således koncepttestades systemet genom att fånga celler spetsade i blod och därmed demonstrera att systemet kan användas i syfte att isolera cancerceller från blodprov. Detta öppnar upp för utveckling av liknande diagnostiska verktyg som kan användas för att isolera lågfrekventa celler direkt från blod.
In 2020, the World Health Organization (WHO) listed cancer as the leading cause of death worldwide, reaching a staggering number of 10 million cancer-related deaths annually. Of these 10 million deaths, nearly 70% occurred in low- and middle-income countries; a number that is expected to increase to 85% by 2030 due to the lack of resources as well as low priority of the development of cancer treatment and diagnosis. Hence, the development of a sophisticated, specific and affordable diagnostic tool will be crucial for global cancer prevention and control. In this study, a cellulose nanofibril-based Layer-by-Layer system for immuno-capture of tumour cells in a microfluidic device was optimized and tested for the development of a simple and cost-effective diagnostic tool for use in resource-limited areas. In the pursuit of a thermodynamic equilibrium, the hydrodynamic parameters of the system were optimized to achieve a homogeneous distribution with a high surface density of the cellulose-based system across the microfluidic channels. Since an equilibrated system is essential to maximize the antibody coating, and thereby cell capture efficiency, parameters including but not limited to concentration, flow rate and incubation time were altered until a desired effect had been achieved. Thus, as proof-of-concept, the system was tested by capturing cancer cells spiked into whole blood, thereby demonstrating that the system can be utilized for the purpose of isolating cancer cells from blood samples. This paves the way for the development of similar clinical diagnostic tools for the isolation of rare cells directly from whole blood.

UA Open Access Publishing Fund
Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

This research set out to develop one or more conceptual models of diagnostic radiography based on the ways in which clinical radiographers themselves viewed their skills. The report analyses the historical relationship between radiographers and radiologists and the limited attempts at theory development prior to this research. This context is set against selected literature from nursing metatheory. This comparison is made since radiography had in part developed from the nursing profession and the historical and gendered background of both professions may be seen to be similar. •The methodology is largely qualitative and the use of computers for qualitative data analysis is discussed in some detail. The data collection was completed in three major phases- a diagnostic phase; a theory development phase and an applications phase, utilising several brainstorming groups and two questionnaires as well as action research in the third phase. In the diagnostic (first) phase of the research categories representing parts of the radiographic process were derived. Two models (or theoretical frameworks) were developed and their various concepts were explored and refined. In the second phase of theory development the research was extended to develop a statement of the role of the diagnostic radiographer and several concepts from the model were explored. In the final phase, the concept of holism was explored together with the relevance of the models for the education of student radiographers. In the final sections of the report there is a detailed evaluation of the potential impact of the models including a reflexive analysis. The conclusions are that the models provide a valid conceptual representation of the radiographic process and philosophy and that they have a role to play in education of student radiographers.

Les maladies infectieuses sont des pathologies dont le diagnostic étiologique est souvent complexe. Le clinicien doit baser son diagnostic sur ses observations cliniques et les relier aux mesures biologiques du patient. Plusieurs groupes recherchent activement de nouveaux marqueurs biologiques pour préciser ce diagnostic. C’est dans cette optique que la cytométrie en flux a été utilisée et optimisée pour comparer l’expression de nouveaux biomarqueurs sur les cellules du sang des patients infectés ou des sujets sains. La caractérisation des mécanismes d’expression des marqueurs montre que l’expression du CD64 sur les neutrophiles est amplifiée chez les patients infectés par une bactérie via l’interféron γ, alors que l’expression du CD169 sur les monocytes est amplifiée chez les patients infectés par un virus via la famille des interférons de type I (α, β, ω). De plus, l’expression de l’HLA-DR sur les monocytes semble aider à l’identification étiologique de l’infection. Les travaux suggèrent que le dosage de ces trois biomarqueurs par la technique de cytométrie en flux optimisée pourrait être un candidat intéressant dans les études sur le diagnostic des infections bactériennes et virales
Infectious diseases are pathologies whose etiological diagnosis is often complex. The clinician must base his diagnosis on his clinical observations and link them to the patient's biological measurements. Several groups are actively seeking new biomarkers to clarify this diagnosis. It is for this purpose that flow cytometry has been used and optimized to compare the expression of new biomarkers on blood cells of infected patients or healthy subjects. Characterization of the expression mechanisms of the markers shows that the expression of CD64 on neutrophils is amplified in patients infected by a bacterium via interferon γ, whereas the expression of CD169 on monocytes is amplified in patients infected with a virus via the type I interferon family (α, β, ω). In addition, the expression of HLA-DR on monocytes seems to help the etiological identification of the infection. The work suggests that the assay of these three biomarkers combined into an optimized flow cytometry technique could be an interesting candidate in studies on the diagnosis of bacterial and viral infections

Inledning: För patienter som behöver akut sjukvård är tiden till diagnos och behandling en viktig faktor. Röntgensjuksköterskornas roll är betydelsefull vid akut diagnostik då de ansvarar för att genomföra röntgenundersökningar. Idag är mobila röntgentjänster, främst konventionell röntgen, tillgängliga för till exempel instabila patienter. Detta bidrar till snabb diagnostik, men många akuta frågeställningar kräver en datortomografiundersökning (DT). Syfte: Syftet med studien var att sammanställa för- och nackdelar med mobil DT vid akut diagnostik. Metod: Studien genomfördes som en litteraturöversikt. Det gjordes en systematisk litteratursökning i tre medicinska databaser. Totalt kvalitetsgranskades och analyserades tio kvantitativa artiklar. Resultat: Det har visat sig i resultatet att fördelarna med en mobil DT är flera, som till exempel minskad tid till diagnos och behandling samt minskad arbetsbelastning för vårdpersonal. En mobil DT motverkar även transportrelaterade komplikationer hos högrisk-patienter. Det framgår dock även att nackdelarna, som till exempel ökad stråldos, sämre bildkvalitet och ökade kostnader, bör beaktas. Slutsats: Den mobila DT:n har visat sig höja patientsäkerheten, vilket är en viktig faktor inom vården. Denna enhet har, trots sina nackdelar, stor potential för att underlätta diagnostik hos patienter i kritiska situationer. Förhoppningsvis kommer denna enhet att utvecklas vidare för att användas i mer vardagligt bruk.
Introduction: For patients in need of urgent medical care, the time to diagnosis and treatment is an important factor. The role of the X-ray nurse is important in emergency diagnostics as they are responsible for conducting X-ray examinations. Today, mobile X-ray services, primarily conventional X-ray examinations, are for instance available for unstable patients. These services offer rapid diagnosis, but many urgent issues require examinations using a computed tomography (CT). Aim: The aim of this study was to compile the pros and cons of mobile CT in acute care diagnostics. Method: The study was conducted as a literature review. A systematic literature search was conducted in three medical databases. In total, ten quantitative articles were quality-reviewed and analyzed. Results: It appears in the results that the advantages of a mobile CT are several, such as reduced time for the diagnosis and treatment and reduced workload for the healthcare personnel. A mobile CT also counteracts transport-related complications in high-risk patients. However, it also states that the disadvantages, such as increased radiation dose, inferior image quality and increased costs, should be taken into account. Conclusion: The mobile CT has been shown to increase patient safety, which is an important factor in healthcare. The device, despite its drawbacks, has a great potential to facilitate diagnostics in patients in critical situations. Hopefully, this device will be further developed to be used regularly.