Dissertations / Theses on the topic 'Microbiology diagnostics'
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1
Spichler, Anne, Bonnie L. Hurwitz, David G. Armstrong, and Benjamin A. Lipsky. "Microbiology of diabetic foot infections: from Louis Pasteur to 'crime scene investigation'." BioMed Central Ltd, 2015. http://hdl.handle.net/10150/610294.
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Were he alive today, would Louis Pasteur still champion culture methods he pioneered over 150 years ago for identifying bacterial pathogens? Or, might he suggest that new molecular techniques may prove a better way forward for quickly detecting the true microbial diversity of wounds? As modern clinicians faced with treating complex patients with diabetic foot infections (DFI), should we still request venerated and familiar culture and sensitivity methods, or is it time to ask for newer molecular tests, such as 16S rRNA gene sequencing? Or, are molecular techniques as yet too experimental, non-specific and expensive for current clinical use? While molecular techniques help us to identify more microorganisms from a DFI, can they tell us ‘who done it?', that is, which are the causative pathogens and which are merely colonizers? Furthermore, can molecular techniques provide clinically relevant, rapid information on the virulence of wound isolates and their antibiotic sensitivities? We herein review current knowledge on the microbiology of DFI, from standard culture methods to the current era of rapid and comprehensive ‘crime scene investigation' (CSI) techniques.
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Giordani, Federica. "New approaches to fluorescence-based diagnostics for human African trypanosomiasis." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2454/.
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In the absence of any vaccine, prophylactic drug and effective vector control, the fight against human African trypanosomiais (HAT) is based on the the combination of active case-finding and consequent drug treatment of identified positive cases. Unfortunately, low sensitivity and specificity of current diagnostic techniques often result in misdiagnosis, leaving infected patients without cure or exposing them to inappropriate chemotherapy protocols, which use dangerous and expensive drugs. The development of more efficient, simple, cheap and field-robust diagnostic tests is, therefore, urgently needed. In the field, direct observation by light microscopy of trypanosomes in human fluids (blood, lymph node aspirate, cerebrospinal fluid) is considered the ideal way of confirming HAT infection. However, in practice this approach is problematic, especially for the Gambian form of the disease, where patients may present with very low parasitaemia. Detection limits of parasitological techniques can be improved by adding a preliminary step of sample concentration, although this further increases the laboriousness of HAT diagnostic algorithm. Recent advances in fluorescence microscopy could be exploited to facilitate trypanosome detection. The introduction and implementation of fluorescence microscopy in HAT endemic countries would offer the advantages of an increased overall sensitivity of microscopical examination and a more rapid screening of the specimen. In contrast to traditional, expensive and fragile fluorescence microscopes, new LED-illuminated instruments are relatively cheap, very efficient and portable, lending themselves to utilisation in poorly equipped rural settings. In order to design a new diagnostic tool that exploits LED technology, however, selective and reliable fluorescent markers to label trypanosomes in human fluids are needed. The development of new tools to assist in the diagnosis of African trypanosomiasis by use of LED fluorescence microscopy was the overall objective of this project. The work was mainly focused on testing various fluorescent compounds for their ability to selectively stain trypanosomes. Fluorophores were otained from commercial and academic sources, or else directly synthesised during the project. An important requirement evaluated was the compounds’ compatibility with the currently available SMR LED Cytoscience fluorescence microscope, developed and kindly provided by our collaborator Prof. D. Jones (Philipps University, Marburg). The utility of a UV LED-driven microscope in performing the arsenical drug resistance test was also assessed. This assay, developed in our laboratory to detect trypanosome strains resistant to arsenical and diamidine compounds, could represent a useful tool for chemotherapeutic decision making in the field, where resistance to arsenical drugs is a rising problem.
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Yngve, Sara. "Optimization of PCR Sensitivity for Detection of Bacterial Species in Blood of Patients with Suspected Sepsis." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-12246.
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Sepsis is commonly caused by bacteria, fungi or both present in the blood stream during inflammation. In response, inflammatory cascades are released in multiple organ systems which if prolonged causes sepsis and can eventually lead to organ failure and death. The major diagnostic technique of sepsis is blood culturing. However, the technique is time consuming and lacks sensitivity; especially in patients under antimicrobial therapy. Molecular techniques particularly PCR could possibly become implemented in sepsis diagnostics in the future. The aim of the thesis was to compare the effect on PCR sensitivity by different PCR kits, with optimized PCR conditions to find an ideal Real-time PCR applicable for direct detection of rRNA or rDNA in whole blood, using the 16S rDNA gene. The study also surveyed the overall background flora of bacterial species circulating in the blood. During the optimization Haemophillus influenzae and Streptococcus pneumoniae were added to whole blood, rRNA or rDNA was isolated and extracted and subsequently processed by Real-time PCR. Four commercially available PCR kits were compared. Attempts using rRNA did not significantly increase the PCR sensitivity. LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics) used for rDNA, generated low cp-values, the cleanest sequences and the finest separation between amplification curves. Twenty whole blood and pre-cultured patient samples were processed by the optimized PCR. The effect on PCR sensitivity by pre-culturing patient blood samples was studied and no statistical difference was noted. Increased PCR sensitivity is essential for implementation of PCR techniques in sepsis diagnostics.
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Näslund, Jonas. "Rift Valley fever : development of diagnostics and vaccines." Doctoral thesis, Umeå universitet, Klinisk mikrobiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-30676.
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Rift Valley Fever virus (RVFV) causes an infection with severe impact on animal and human health. The disease is endemic throughout almost the entire African continent and large regions of the Arabian Peninsula. During epidemics, high mortality is observed in animals, especially among cattle, goats, and sheep. In humans, the symptoms vary from a benign influenza-like disease to a life-threatening hemorrhagic fever. Due to the devastating effect on communities in endemic regions and the possibility of further spread of this virus, there is an imperative need to improve and develop control measurements against this emerging disease. Therefore, this thesis focuses on diagnostics and vaccines against RVFV. RVFV infection kinetics was studied in a mouse model system by detection and quantification of viral genomes, using a developed quantitative real-time PCR (QRT-PCR) method. This novel QRT-PCR method proved to be reliable and serves as a supplement to standard diagnostics, direct virus isolation and serological methods. High levels of viral RNA were found in blood and liver samples from experimentally infected mice during the first days post infection. Thereafter the levels declined rapidly and dropped below detection limit approximately seven days post infection. The QRT-PCR technique was also used in a study aimed to improve diagnosis of RVFV from field samples collected on filter strips. Today, the available RVFV vaccines are only approved for animal use and these vaccines have several shortcomings. Since RVFV is a highly pathogenic organism requiring bio-safety level 3 laboratories, two different none-replicating vaccine approaches have been applied and evaluated using a mouse model. A DNA based vaccine, administered via gene-gun, and the use of virus-like particles (VLP), by the intra-peritoneal route. RVFV specific and neutralising antibodies were raised with both vaccine approaches. However, VLP vaccination against Rift valley Fever proved to be more promising as a future vaccine, since higher titres of neutralising antibodies and improved survival rate were found upon a lethal RVFV challenge in mice. In conclusion, a sensitive and specific method for quantifying RVFV infection and a promising vaccine candidate against RVFV were developed.
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Bourquin, Yannyk Parulian Julian. "Shaping surface waves for diagnostics." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/4167/.
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Infectious diseases continue to kill millions of people every year and are a significant burden on the socio-economic development of developing countries. After many years of international policy aimed at containing diseases, it has recently become an explicit aim to move towards elimination of infectious diseases. However, if this is to occur, it will be necessary to have highly eficacious diagnostic tools to ensure infected individuals are identified and treated. However, the diagnosis of infectious diseases in the developed and developing world requires the full integration of complex assays in easy-to-use platforms with robust analytical performances at low cost. Many relevant bioanalytical technologies have been developed for use in laboratories and clinics, including the current gold standard for the diagnosis of tuberculosis and malaria. The miniaturization and integration of complex functions into lab-on-a-chip (LOC)technologies using microfluidics have only had limited success in translating diagnosis assays out of a centralized laboratory to point-of-care (POC) settings, because they still remain constrained due to chip interconnection and they are either not likely to go out of research laboratories or are not appropriate for low resource settings. In this thesis, a new microfluidic platform was developed that reduced the dependency of the diagnostic procedure on large laboratory instruments providing simplicity of use, enabling the patient sample to be processed and diagnosed on a low cost, disposable biochip. Surface acoustic wave (SAW) devices, which are commonly used in mobile phone technologies, were adapted to provide controlled microfluidic functions by shaping the SAW using particular designs of electrodes and phononic structures. The control of lateral positioning of the SAW was demonstrated using a slanted finger interdigitated transducer (IDT) in a frequency tuneable manner allowing microfluidic functions such as mixing, moving and merging, sequentially performed using a single IDT both on the substrate and on a disposable chip. Alternatively, phononic bandgaps were designed to break the symmetry of the SAW in a tuneable manner and gradient index phononic crystals (GRIN-PC) lenses were designed to focus the SAW and successfully increased the amplitude of the wave by a factor 3 while the focal position could be tuned with the frequency. The potential of these techniques was demonstrated by controlling the amplitude and direction of water jet towers by the use of a phononic horn structure that allowed the enhancement of energy at defined positions and by propelling and directing a macrometer scale object in water using a slanted IDT. As proof of concepts of diagnostic devices for the developing world, an immunoassay for tuberculosis using only mobile phone technologies (SAW, light-emitting diode(LED) and complementary metaloxidesemiconductor (CMOS) camera) was demonstrated with a limit of detection of 1 pM, which is the limit required in an interferon-release assay. This limit of detection was only achievable because of the ability of SAW to increase the mixing and to reduce the non-specific binding. Furthermore, a method to enrich malaria infected cells, based on SAW and isopycnic gradient, was also demonstrated and showed an enrichment up to 100x in the equivalent of a fingerprick of blood in 3 seconds. This technique will allow to reduce the limit of detection of the current gold standard. This platform not only opens a clear road toward POC diagnostics due to its size, cost, versatility and ease in integration, but has also the potential to provide useful tools in laboratory settings for large scale, high throughput technologies.
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Vervier, Kevin. "Méthodes d’apprentissage structuré pour la microbiologie : spectrométrie de masse et séquençage haut-débit." Thesis, Paris, ENMP, 2015. http://www.theses.fr/2015ENMP0081/document.
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L'utilisation des technologies haut débit est en train de changer aussi bien les pratiques que le paysage scientifique en microbiologie. D'une part la spectrométrie de masse a d'ores et déjà fait son entrée avec succès dans les laboratoires de microbiologie clinique. D'autre part, l'avancée spectaculaire des technologies de séquençage au cours des dix dernières années permet désormais à moindre coût et dans un temps raisonnable de caractériser la diversité microbienne au sein d'échantillons cliniques complexes. Aussi ces deux technologies sont pressenties comme les piliers de futures solutions de diagnostic. L'objectif de cette thèse est de développer des méthodes d'apprentissage statistique innovantes et versatiles pour exploiter les données fournies par ces technologies haut-débit dans le domaine du diagnostic in vitro en microbiologie. Le domaine de l'apprentissage statistique fait partie intégrante des problématiques mentionnées ci-dessus, au travers notamment des questions de classification d'un spectre de masse ou d'un “read” de séquençage haut-débit dans une taxonomie bactérienne.Sur le plan méthodologique, ces données nécessitent des développements spécifiques afin de tirer au mieux avantage de leur structuration inhérente: une structuration en “entrée” lorsque l'on réalise une prédiction à partir d'un “read” de séquençage caractérisé par sa composition en nucléotides, et un structuration en “sortie” lorsque l'on veut associer un spectre de masse ou d'un “read” de séquençage à une structure hiérarchique de taxonomie bactérienneUsing high-throughput technologies is changing scientific practices and landscape in microbiology. On one hand, mass spectrometry is already used in clinical microbiology laboratories. On the other hand, the last ten years dramatic progress in sequencing technologies allows cheap and fast characterization of microbial diversity in complex clinical samples. Consequently, the two technologies are approached in future diagnostics solutions. This thesis aims to play a part in new in vitro diagnostics (IVD) systems based on high-throughput technologies, like mass spectrometry or next generation sequencing, and their applications in microbiology.Because of the volume of data generated by these new technologies and the complexity of measured parameters, we develop innovative and versatile statistical learning methods for applications in IVD and microbiology. Statistical learning field is well-suited for tasks relying on high-dimensional raw data that can hardly be used by medical experts, like mass-spectrum classification or affecting a sequencing read to the right organism. Here, we propose to use additional known structures in order to improve quality of the answer. For instance, we convert a sequencing read (raw data) into a vector in a nucleotide composition space and use it as a structuredinput for machine learning approaches. We also add prior information related to the hierarchical structure that organizes the reachable micro-organisms (structured output)
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Oliver, Lee D. Jr. "Development of Novel Chimeric Epitope Based Diagnostic Antigens and Vaccines for Lyme Disease." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3876.
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Lyme disease (LD) is the leading arthropod-borne disease in North America with 300-600,000 cases each year. There are currently no approved human LD vaccines. Outer surface protein C (OspC) has emerged as a leading vaccine candidate and an attractive diagnostic marker due to its antigenicity and expression early in infection. Several chimeric, epitope based OspC derived proteins were generated. The constructs were found to be highly immunogenic in mice and vaccination induced complement-dependent bactericidal antibodies. These results suggest that a broadly protective polyvalent OspC epitope based vaccine can be produced. Currently, LD diagnostic approaches are unreliable and unable to differentiate between early and late stage disease. An Ab response to OspE family proteins occurs later in infection. The two-Ag diagnostic assay using chimeric OspC proteins and a site-directed mutant of an OspE paralog, accurately differentiated between early and late infection in experimentally infected canines and humans.
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López, Siles Mireia. "Ecophysiology and philogeny of Faecalibacterium prausnitzii in healthy and diseased gut. Application in Inflamatory Bowel Disease diagnostics." Doctoral thesis, Universitat de Girona, 2016. http://hdl.handle.net/10803/369044.
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In this PhD thesis Faecalibacterium prausnitzii populations of patients with gut disease and healthy individuals have been characterized.
First, isolates from healthy volunteers have been phenotypically characterised, which has allowed to gain insight into the physiology of this species. A possible link between F. prausnitzii sensitivity to changes in gut physicochemical conditions and its disappearance in a diseased gut has been revealed.
Second, molecular studies on F. prausnitzii populations have allowed to define two phylogroups within this species, and to describe the diversity of phylotypes in healthy individuals and in patients with intestinal disease. The phylotypes specifically compromised in patients suffering some gut disorders have been identified.
Finally, new molecular tools for the detection and quantification of this species and its phylogroups have been designed. Their usefulness to be implemented as complementary molecular tools for the diagnosis and prognosis of intestinal diseases has been determined.En aquesta tesi doctoral s'ha estudiat la població de Faecalibacterium prausnitzii de pacients amb malalties intestinals i individus sans. En primer lloc, es va realitzar una caracterització fenotípica d'aíllats d'aqueta espècie obtinguts d'individus sans, el que ha permès adquirir coneixement sobre la fisiologia d'aquesta espècie. S'ha evidenciat una possible relació entre la sensibilitat de F.prausnitzii a canvis en les condicions fisicoquímiques de l'intestí i la seva desaparició en un intestí malalt. En segon lloc, s'han realitzat estudis moleculars de les poblacions de F. prausnitzii. Això ha permès definir dos filogrups dins d'aquesta espècie, i descriure la diversitat de filotips en individus sans i pacients amb malalties intestinals.Per primera vegada, s'han identificat els filotips especificament compromesos en pacients que pateixen determinades malalties intestinals. Per últim, s’han dissenyat eines moleculars per a la detecció i quantificació d'aquesta espècie i els seus filogrups. S’ha determinat la utilitat d’aquestes eines moleculars per al suport al diagnòstic o prognòstic de malalties intestinals.
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Wrightson, John M. "Pathogen identification in lower respiratory tract infection." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:30c757ec-99b7-492e-a12e-ff996581863a.
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Treatment of lower respiratory tract infection (pneumonia and pleural infection) relies on the use of empirical broad spectrum antibiotics, primarily because reliable pathogen identification occurs infrequently. Another consequence of poor rates of pathogen identification is that our understanding of the microbiology of these infections is incomplete. This thesis addresses some of these issues by combining the acquisition of high quality lower respiratory tract samples, free from nasooropharyngeal contamination, with novel molecular microbiological techniques in an attempt to increase rates of pathogen identification. Four main areas are examined: (i) The role of so-called ‘atypical pneumonia’ bacteria in causing pleural infection. These pathogens have been previously identified in the pleural space infrequently and routine culture usually fails to isolate such bacteria. High sensitivity nested polymerase chain reaction (PCR) is a culture-independent technique which is used to undertake a systematic evaluation for these pathogens in pleural infection samples. (ii) The role of Pneumocystis jirovecii in pleural infection, either as a co-infecting pathogen or in monomicrobial infection. This fungus causes severe pneumonia, particularly in the immunosuppressed, but is increasingly recognised as a co-pathogen in community-acquired pneumonia, and is frequently isolated in the upper and lower respiratory tract in health. A high sensitivity real-time PCR assay is used to examine for this fungus. (iii) Ultra-deep sequencing of the 16S rRNA gene is used to perform a comprehensive microbial survey in samples taken from the multi-centre MIST2 study of pleural infection. The techniques employed allow analysis of polymicrobial samples and give very high taxonomic resolution, whilst incorporating methods to control for potential contamination. Further, these techniques provide confirmation of the results from the ‘atypical’ bacteria nested PCR study. (iv) Bedside ultrasound-guided percutaneous transthoracic needle aspiration (TNA) of consolidated lung is undertaken in patients with pneumonia, as part of the PIPAP study. An evaluation is undertaken of the efficacy and acceptability of TNA. Aspirate samples acquired are also processed using ultra-deep sequencing of the 16S rRNA gene. Other samples obtained as part of the PIPAP study, such as ‘control’ lung aspirates and ‘control’ pleural fluid samples, are similarly processed to enable calculation of sensitivity and specificity of the sequencing methodology.
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Schuurman, Timothy. "Developments and clinical applications in diagnostic molecular microbiology." [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2008. http://hdl.handle.net/1765/13137.
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Ros, Bascuñana Carlos. "Diagnostic application of the polymerase chain reaction (PCR) in veterinary microbiology /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1997. http://epsilon.slu.se/avh/1997/91-576-5247-3.gif.
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Lowe, Jonathan. "Novel enzyme substrates and cell labelling reagents for use in diagnostic microbiology." Thesis, Northumbria University, 2016. http://nrl.northumbria.ac.uk/31602/.
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This thesis is divided into two parts; the first part describing the synthesis and evaluation of novel fluorogenic enzymatic substrates for microorganism detection, and the second part investigating the synthesis of thiophenes with extended conjugation as fluorescent labels with potential applications in antimicrobial susceptibility testing. ‘Spacer’ substrates were identified as a method of incorporating a phenolic fluorophore (ArOH) into an enzymatic substrate designed at targeting aminopeptidase activity. Substrates of the general structure ArO-spacer-AA were therefore synthesised (AA = amino acid). The action of aminopeptidases on these substrates would therefore result in fragmentation, liberating the phenolic fluorophore. The spacer group of choice was para-aminobenzyl alcohol (PABA), as it has been widely used as such for the synthesis of prodrugs. After condensation of the amine group of PABA with either Boc-L-alanine or pyroglutamic acid and subsequent chlorination of the benzylic alcohol giving the corresponding benzyl chloride, 2-(2-hydroxyphenyl)benzothiazole and 2-(2-hydroxyphenyl)benzoxazole were attached using a Williamson ether synthesis to produce the Boc-protected substrates. After removal of the Boc groups, the substrates produced strongly fluorescent colonies with Gram-negative bacteria. Also investigated were a series of fluorogenic substrates, both with and without ‘spacer’ methodology, designed for the detection of nitroreductase activity but little activity was observed with bacteria. Three BODIPY-derived substrates were also prepared and evaluated for detecting nitroreductase and esterase activity. These substrates showed good activity in agar media when the agar plates were post-treated with acid resulting in the generation of strong colour and fluorescence. The second section of this thesis focuses on antimicrobial susceptibility testing. Two types of substrates possessing highly conjugated thiophene cores were synthesised: hydrazides and D-amino acids. The hydrazides are expected to label dead cells by reacting with aldehyde groups that are produced by protein carbonylation. The D-amino acid series of compounds are expected to be incorporated into the cell walls of growing bacteria, and hence live cells can be labelled. The thiophene core structures were chosen to have excitation wavelengths of approximately 488 nm because this excitation wavelength is commonly used in flow cytometry. Results from fluorescence measurement testing indicate that both sets of cell labelling substrates synthesised have an excitation wavelength of roughly 450 – 460 nm with emission wavelengths ranging from 480 nm to as high as 520 nm.
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Milovic, Ana. "Novel Diagnostic approach for tuberculosis diagnosis." Master's thesis, University of Cape Town, 2010. http://hdl.handle.net/11427/2715.
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There is a clear need for a rapid, inexpensive point-of-care diagnostic test for tuberculosis (TB). The aim of this study was to analyze the performance of a novel rapid diagnostic test for TB, GeneXpert MTB/RIF, in symptomatic adults and to compare it to other commercially available nucleic acid amplification assays and to standard microbiological smear and culture. The GeneXpert system performs real time, nested PCR from sputum and provides a result within two hours of sampling. The result includes a semi-quantitative assessment of bacillary load in the sample and simultaneously detects rifampicin resistance. This study was part of a cross-sectional, multi-centre clinical trial. The Cape Town component of this study was conducted at three sites, one hospital based and other two community clinics, all with high TB/HIV coinfection rate. Among 43.2% of patients diagnosed with TB during the evaluation study, GeneXpert detected TB in 95.5% of all culture positive cases. In smear positive patients, sensitivity was 99.0% and in smear-negative, culture positive patients, 86.1%. Specificity in patients who were culture negative and clinically diagnosed as non-TB after follow up was 98.4%. Sensitivity and specificity of GeneXpert in detecting rifampicin resistance was 100% comparing to phenotypically detected drug resistance. GeneXpert is a highly promising novel tool for the rapid diagnosis of adult TB. Future studies are needed to establish the performance and impact of GeneXpert when performed at the level of the microscopy centre.
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Dionne, Natasha. "Développement d'une puce à ADN sur plate-forme microfluidique utile pour le diagnostic rapide des virus responsables des infections respiratoires." Thesis, Université Laval, 2009. http://www.theses.ulaval.ca/2009/26266/26266.pdf.
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Vernier, Arnaud. "Développement instrumental en spectrométrie de masse pour le diagnostic in vitro en microbiologie clinique." Phd thesis, Université Claude Bernard - Lyon I, 2014. http://tel.archives-ouvertes.fr/tel-00986856.
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La spectrométrie de masse, en particulier le couplage HPLC/MRM3, est un outil bien adapté au diagnostic in vitro, particulièrement en microbiologie clinique. L'utilisation en routine de cette technologie est cependant tributaire de sa sensibilité et de sa spécificité. Ce travail de thèse a pour objectif d'étudier la possibilité d'éjecter et de détecter simultanément et de façon sélective des ions de ratio masse/charge donnés, ceux-ci étant confinés dans un piège ionique quadrupolaire. Cette approche permet de supprimer les étapes de balayage en masse et d'intégration mathématique du signal en mode MRM3 ce qui permet de gagner à la fois en sensibilité et en spécificité (en diminuant le temps de cycle et en diminuant le rapport signal sur bruit). Cet objectif a été poursuivi premièrement par une étude théorique approfondie des équations du mouvement des ions dans un piège radiofréquence ; deuxièmement par une étude numérique de la stabilité de ces équations et enfin troisièmement par une validation expérimentale de ces résultats théoriques. La présentation de ces trois approches fait l'objet du présent mémoire
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Bagge, Joakim, Erik Hedman, Sabina Smedsrud, Cornelia Svärdström, Elisabeth Söderberg, and Fernando Valdés. "Utveckling av metodik för påvisning och typning av Listeria i livsmedelskedjan." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-324030.
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Sistek, Viridiana. "Identification des staphylocoques, streptocoques et entérocoques par des méthodes génotypiques." Thesis, Université Laval, 2010. http://www.theses.ulaval.ca/2010/27533/27533.pdf.
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McKenna, Paula Mary. "The study of the biology, immune response and diagnostic testing of Hantaviruses." Thesis, Queen's University Belfast, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317460.
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Barbut, Frédéric. "Infections digestives a clostridium difficile : diagnostic, epidemiologie clinique et moleculaire en milieu hospitalier (doctorat : microbiologie)." Paris 11, 2000. http://www.theses.fr/2000PA114817.
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Kerr, Paul Gerard. "Diagnostic application of monoclonal antibodies to surface antigens of enterohaemorrhagic and enteropathogenic Escherichia coli strains." Thesis, Queen's University Belfast, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246540.
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Rodriguez, Perez Mario Alberto. "Assessment and monitoring of onchocerciasis control in Mexico : application of novel immunological and molecular diagnostic tests." Thesis, University of Salford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301432.
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Tsim, Selina. "Diagnostic and prognostic biomarkers of malignant pleural mesothelioma." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/30687/.
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Malignant Pleural Mesothelioma (MPM) is an aggressive intrathoracic malignancy with an overall poor prognosis. MPM is associated with asbestos exposure but has a long latency period between exposure and disease development. Incidence of MPM in the UK is therefore still rising, predicted to reach a peak in 2020. The majority of patients with MPM present with breathlessness, frequently due to a pleural effusion and/or chest pain. Diagnosis of MPM can be difficult. Radiological detection of early stage MPM in particular can be challenging, as pleural tumour, nodularity or significant pleural thickening may not be evident. Diagnosis is further complicated by the low yield of pleural fluid cytology examination in MPM and pleural biopsy is therefore usually required to allow definitive diagnosis. This can be achieved under image guidance, at surgical thoracoscopy or at local anaesthetic thoracoscopy (LAT). A significant number of patients are either elderly or have co-morbidity precluding general anaesthesia and surgical thoracoscopy. Image-guided pleural biopsy is not always feasible, particularly in the absence of significant pleural thickening. LAT remains a limited resource in the UK. A non-invasive biomarker of MPM, which could be performed early in the patient’s presentation, and that could be available to most hospitals, would therefore be a major clinical advance, allowing clinicians to direct appropriate patients to specialist centres with access to LAT and specialist MDT input where MPM appears likely. There have been several potential blood biomarkers identified in the mesothelioma literature, including the most widely studied, Mesothelin, and more recently Fibulin-3 and SOMAscanTM. Unfortunately study results have been variably limited by retrospective study design, inconsistent sampling time points, inconsistent results and lack of external validation, therefore despite initial promising results, none of these biomarkers have entered routine clinical practice for diagnosis. Similarly, utility of imaging biomarkers such as perfusion Computed Tomography (CT), Positron Emission Tomography (PET) and Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) has been limited by high radiation dose, limited availability, and requirement for bulky (and therefore late stage) disease for assessment respectively. In chapter 2, study design, recruitment and preliminary results of the DIAPHRAGM (Diagnostic and Prognostic Biomarkers in the Rational Assessment of Mesothelioma) study are reported. A prospective, multi-centre study was designed, recruiting patients with suspected pleural malignancy (SPM) at initial presentation to secondary care services, from a mixture of academic and more clinical units in the UK and Ireland, in addition to asbestos-exposed control subjects. In one of the largest biomarker studies in mesothelioma to date, 639 patients with SPM and 113 asbestos-exposed control subjects were recruited over three years. Data cleaning is being finalised by the Cancer Research UK Clinical Trials Unit Glasgow at the time of writing. Preliminary results reveal that 26% (n=154) patients recruited to the SPM cohort were diagnosed with MPM, 33% (n=209) had secondary pleural malignancy and 34% (n=218) were diagnosed with benign pleural disease. A final diagnosis is awaited in 7% (n=47) at the time of writing. SOMAscanTM and Fibulin-3 biomarker analyses are ongoing and DIAPHRAGM will definitively answer the question of diagnostic utility of these blood biomarkers in routine clinical practice, in a ‘real-life’ MPM population, relative to that of Mesothelin. In chapter 3, contrast-enhanced MRI was performed in patients with suspected MPM and a novel MRI biomarker of pleural malignancy defined (Early Contrast Enhancement – ECE). ECE was defined as a peak in pleural signal intensity at or before 4.5 minutes after intravenous Gadobutrol administration. ECE assessment was successfully performed in all patients who underwent contrast-enhanced MRI. This included patients with pleural thickening < 10mm (49/58 (84%)), the mean pleural thickness of all patients was 5mm. ECE demonstrated good overall diagnostic performance for the detection of pleural malignancy (sensitivity 83% (95% CI 61 – 94), specificity 83% (95% CI 68 - 91%), positive predictive value 68% (95% CI 47 – 84%), negative predictive value 92% (95% CI 78 – 97%)), comparable to morphology assessment at CT morphology and MRI morphology by experienced thoracic radiologists. In addition, ECE demonstrated good reproducibility (inter-observer κ = 0.864), superior to subjective morphology assessment at CT and MRI. Mean signal intensity gradient (MSIG), a marker of patient’s contrast enhancement pattern, correlated with tumour Microvessel Density (MVD) using Factor VII immunostain (Spearman’s rho = 0.43, p=0.02). Additionally, a high MSIG (>0.533AU/s), indicative of high tumour vascularity, was associated with poor median overall survival (12 months vs. 20 months, p=0.047). Staging of MPM represents an additional challenge to clinicians. This is due to the complex morphology and often rind-like growth pattern of MPM. In addition, delineation of pleural disease from adjacent structures such as intercostal muscle and diaphragm can be difficult to assess, particularly at CT, which is the most commonly used imaging modality for diagnostic and staging assessment in MPM. Current clinical staging frequently underestimates extent of disease, with a significant proportion of patients being upstaged at time of surgery, and is limited by high inter-observer variability. Recent studies have reported the prognostic significance of CT-derived tumour volume; however, many of these studies have been limited by the laborious or complex nature of tumour segmentation, significant inter-observer variability or challenges encountered in separating pleural tumour from adjacent structures, which are often of similar density. MRI is superior to CT in the detection of invasion of the chest wall and diaphragm in MPM. In Chapter 4, MRI was used to quantitatively assess pleural tumour volume in 31 patients with MPM using novel semi-automated segmentation methodology. Four different segmentation methodologies, using Myrian® segmentation software were developed and examined. Optimum methodology was defined, based on the accuracy of volume estimates of an MRI phantom, visual-based analysis, intra-observer agreement and analysis time. Using the optimum methodology, there was acceptable error around the MRI phantom volume (3.6%), a reasonable analysis time (approximately 14 minutes), good intra-observer agreement (intra-class correlation coefficient (ICC) 0.875) and excellent inter-observer agreement (ICC 0.962). Patients with a high MRI-estimated tumour volume (≥300cm3) had a significantly poorer median overall survival (8.5 months vs. 20 months) and was a statistically significant prognostic variable on univariate (HR 2.273 (95% CI 1.162 – 4.446), p=0.016) and multi-variate Cox proportional hazards model (HR 2.114 (95% CI 1.046 – 4.270), p=0.037).
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Nougairede, Antoine. "Pandémie grippale A/H1N1 2009/2010 : Diagnostic et épidémiologie au laboratoire hospitalier de microbiologie clinique à Marseille." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM5000/document.
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Fin avril 2009, un nouveau virus grippal A/H1N1 d'origine porcine émerge dans le monde causant la première pandémie grippale du XXIème siècle. Les différents travaux présentés dans cette thèse retracent la gestion de cette situation au laboratoire de virologie des hôpitaux publics de Marseille. D'avril 2009 à avril 2010, nous avons analysé plus de 13 000 prélèvements issus de cas suspects. Nous avons dû adapter continuellement les moyens mis en œuvre pour effectuer le diagnostic et la mise en place d'une stratégie 'Point of Care' s'est avérée très utile. Nos résultats montrent que l'usage des tests rapides en complément de la RT-PCR en temps réel permet de réduire significativement le délai de rendu des résultats pour les patients infectés. Les données épidémiologiques sur les nombreux cas suspects dépistés ont également permis d'obtenir en temps réel des informations précieuses sur l'épidémiologie de cette pandémie comme l'estimation de l'incidence par classe d'âge, la proportion de patients hospitalisés et la mortalité. Enfin, nous avons réalisé une étude de séroprévalence qui montre qu'environ 12% de la population française a été infectée par ce nouveau virus en 2009-2010 et que les taux d'attaque les plus élevés ont été observés chez les enfants et les jeunes adultesIn late April 2009, a new swine-origin A/H1N1 Influenza virus emerged and spread rapidly worldwide causing the first influenza pandemic of the 21st century. This work describes how we coped with this emergency situation in the virology laboratory of Marseille public hospitals. From April 2009 to April 2010, we analyzed more than 13,000 samples from suspected cases. We needed to adapt continuously the organization to maintain diagnostic capacity and the implementation of a point of care strategy revealed very useful to achieve this goal. Our results support the use of rapid Influenza detection tests in combination with real-time RT-PCR because it reduces significantly the delay from sample to result for positive cases, thus giving the opportunity to improve patient management. Epidemiological data from all suspected cases tested allowed us to obtain timely precious information about the epidemiology of this pandemic as the estimation of (i) the incidence by age group, (ii) the rate of hospitalization and (iii) the mortality rate among tested patients. Finally, we set up a serological study and showed that around 12% of the French population had been infected by this new virus in 2009-2010 with higher attack rates observed in children and young adults
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Barbieri, Daniela <1985>. "Human papillomavirus (HPV) and associated diseases: between applied diagnostic and basic research." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5314/.
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Human Papillomavirus (HPV) is the cause of cervical cancers (among these, adenocarcinoma, AdCa) and is associated to a subgroup of oropharyngeal carcinomas (OPSCCs). Even if the risk for cancer development is linked to the infection by some viral genotypes, mainly HPV16 and 18, viral DNA alone seems not to be sufficient for diagnosis. Moreover, the role of the virus in OPSCCs has not been totally clarified yet.
In the first part of the thesis, the performances concerning viral genotyping in clinical cervical samples of a new pyrosequencing-based test and a well-known hybridization-based assay have been compared. Similar results between the methods have been obtained. However, the former showed advantages in detecting intratype variants, higher specificity and a broader spectrum of detectable HPV types.
The second part deals with the evaluation of virological markers (genotyping, viral oncoproteins expression, viral load, physical state and CpG methylation of HPV16 genome) in the diagnosis/prognosis of cervical AdCa and HPV-associated OPSCCs. HPV16 has been confirmed the most prevalent genotype in both the populations. Interestingly, the mean methylation frequency of viral DNA at the early promoter showed the tendency to be associated to invasion for cervical AdCa and to a worse prognosis for OPSCCs, suggesting a promising role as diagnostic/prognostic biomarker.
The experiments of the third part were performed at the DKFZ in Heidelberg (Germany) and dealt with the analysis of the response to IFN-k transfection in HPV16-positive cervical cancer and head&neck carcinoma cell lines to evaluate its potential role as new treatment. After 24h, we observed increased IFN-b expression which lead to the up-regulation of genes involved in the antigens presentation pathway (MHC class I and immunoproteasome) and antiviral response as well, in particular in cervical cancer cell lines. This fact suggested also the presence of different HPV-mediated carcinogenic pathways between the two anatomical districts.Il Papillomavirus umano (HPV) è causa dei carcinomi della cervice uterina (tra cui adenocarcinomi, AdCa) ed è associato ad un sottogruppo di tumori dell’orofaringe (OPSCCs). Nonostante il rischio di sviluppo di tumore sia associato all’infezione da parte di alcuni genotipi virali, principalmente HPV16 e 18, il DNA virale da solo sembra non essere sufficiente in campo diagnosico. Inoltre, per tumori orofaringei il ruolo del virus non è ancora del tutto chiaro. Nella prima parte della tesi, sono state confrontate le performance riguardo la genotipizzazione di HPV su campioni clinici cervicali di una tecnica innovativa, basata su amplificazione e pirosequenziamento, e una di routine, basata su amplificazione e ibridazione inversa. Lo studio ha evidenziato performance simili tra le due metodiche, sottolineando per il sequenziamento una maggiore specificità e capacità di rilevare varianti intratipo. Nella seconda parte sono stati analizzati marker virologici (genotipizzazione, espressione delle oncoproteine virali, carica virale, stato fisico e metilazione del genoma di HPV16) in funzione dei dati clinici disponibili, per un possibile impiego nella diagnosi/prognosi di AdCa cervicali e OPSCCs HPV-associati. HPV16 si è confermato il genotipo prevalente in entrambe le popolazioni. La frequenza di metilazione nel promotore precoce virale ha mostrato una tendenza ad essere associata ad invasione negli AdCa, e ad una prognosi peggiore negli OPSCCs, emergendo come il più promettente marker diagnostico/prognostico. La terza parte, svolta presso il DKFZ di Heidelberg (Germania), ha visto l’analisi della risposta alla transfezione di IFN-k in linee cellulari tumorali HPV16-positive della cervice uterina e della regione testa-collo, per valutarne l’impiego terapeutico. Dopo 24h, è stato osservato un incremento dell’espressione di IFN-b e, di conseguenza, una up-regolazione dei geni coinvolti nella presentazione antigenica (MHC classe I ed immunoproteasoma) e nella risposta antivirale, specialmente nelle cellule cervicali, suggerendo la presenza di diversi meccanismi patogenetici tra tumori HPV-positivi dei due distretti anatomici.
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HILALI, FARIDA. "Approches moleculaires pour le diagnostic et la caracterisation des bacteriemies chez les sujets atteints de cancer (doctorat : microbiologie)." Paris 11, 1998. http://www.theses.fr/1998PA114829.
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Alegre, Melissa Marie. "Thymidine Kinase 1: Diagnostic and Prognostic Significance in Malignancy." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4049.
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Thymidine kinase 1 (TK1) is a cancer biomarker which has diagnostic and prognostic potential in a variety of malignancies. TK1 is significantly elevated in the serum and tumor tissue of most malignancies. This increase in TK1 can be detected in the very early stages of malignancy, including in pre-malignant disease with an increased risk for progression. Several studies have demonstrated that elevated TK1 is found in serum months before any clinical symptoms of malignancy. It has also been demonstrated that TK1 is elevated months before clinical recurrence of malignancy. This work first sought to demonstrate the early nature of TK1 expression in breast tumor tissue and pre-malignant tissue. We found that TK1 is elevated in breast hyperplasia tissue and breast carcinoma tissue. In this study we also identified some cases of ‘normal’ tumor margins (considered normal by current pathological standards) which also had elevated TK1 expression. Conversely, true normal breast tissue from noncancerous individuals had no reported elevation in TK1 expression. This study illustrated that TK1 is elevated in pre-malignant breast hyperplasia tissue, as well as some 'normal' tumor margins. TK1 expression was significantly elevated in lung, prostate, colon, esophagus, stomach, liver, and kidney tissues. This work further investigated TK1 expression in a variety of malignant tissue including the two leading causes of cancer mortality in men: lung and prostate cancer. In our study, TK1 was significantly elevated in lung and prostate cancer but not significantly elevated in prostate hyperplasia tissue. TK1 expression also increased with increasing grade in prostate carcinoma tissue. Overall, this work demonstrated that TK1 is a good universal marker of malignancy and is elevated in early cancer development. Despite the potential for TK1 as both a screening and monitoring treatment tool, there have been significant challenges associated with developing a clinically relevant method of TK1 detection. This work proposes one clinically relevant method of detection, namely a TK1 ELISA. Using preoperable lung cancer patients and normal controls, we developed a sensitive and specific ELISA which shows highly statistically significant differences in serum TK1 levels between stage 1 and stage 2 lung cancer compared with normal controls. In fact, this TK1 ELISA is more sensitive and accurate than the traditional TK radioassay, which was unable to detect differences in TK1 between early stage lung cancer and normal patients. Although elevated TK1 is not lung cancer specific, we reported significantly elevated TK1 levels in lung cancer sputum. Screening of sputum and serum for TK1 may be one method for the early detection of lung cancer. Overall, we report TK1 has promising diagnostic potential in a variety of malignancies. We also propose one sensitive and specific method to detect TK1 levels which may easily be adapted to meet current clinical applications. We hope this work will help propel TK1 forward into clinical view in the coming years.
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VISCONTI, ALEXANDRE. "Diagnostic microbiologique et mortalite des pneumopathies nosocomiales." Aix-Marseille 2, 1994. http://www.theses.fr/1994AIX20174.
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Westman, Natalee. "Direktresistensbestämning för blododlingar : Volymoptimering och reproducerbarhet." Thesis, Jönköping University, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:hj:diva-53082.
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Direct antimicrobial susceptibility testing (dAST) is not a standardized method and there is no recommendations regarding the volume of blood culture media to be used. The aim of the study was to optimize the method of dAST by determining a volume of blood culture media in µL to be used and to test the reproducibility of the volume optimized method. The dASTs (n=160) were performed on blood culture bottles (n=40) inoculated with reference bacteria (n=4), using four test volumes (50 µL, 75 µL, 100 µL and 125 µL). The optimized method was implemented on frozen and fresh bacterial isolates (n=120) derived from blood cultures. Susceptibility tests according to EUCASTs method for disk diffusion was also performed. Deviations in SIR-categorical agreement was calculated. The optimal volume of blood culture media for dAST was 75 µL. All dASTs were approved for three out of four reference bacteria whereas for the fourth reference bacteria eight out of ten dASTs was approved. Testing the reproducibility, the optimized method showed a sensitivity and specificity of 100 %. No deviation in categorical agreement of SIR-categorization was observed. The result shows a possibility of implementing a standardized method for dAST regarding the volume of blood culture media.Vid direktresistensbestämning används blododlingsmedium för tillverkning av bakteriesuspensioner. Metoden är inte standardiserad och det finns ingen rekommenderad volym blododlingsmedium som bör användas. Syftet med studien var att optimera metoden för direktresistensbestämning genom att bestämma en volym blododlingsmedium i µL som används för tillverkning av bakteriesuspensioner. Den optimerade metodens prestanda utvärderades därefter på kliniska patientisolat. Metoden optimerades genom att direktresistensbestämningar (n=160) utfördes baserat på fyra volymer (50 µL, 75 µL, 100 µL och 125 µL) blododlingsmedium (n=40), som var inokulerade med fyra olika referensstammar. Den optimerade metodens reproducerbarhet testades på frysta och färska patientisolat (n=120) genom jämförelse av SIR-kategorisering mellan direktresistensbestämning samt resistensbestämning enligt EUCASTs metod för diskdiffusion. Den optimala volymen blododlingsmedium med kända referensstammar fastställdes vara 75 µL då samtliga direktresistensbestämningar godkändes för tre av fyra referensstammar, för den fjärde referensstammen godkändes åtta av tio. Då metoden implementerades på kliniska patientisolat från positiva blododlingar var känsligheten och specificiteten 100 % avseende kategorisk överensstämmelse enligt SIR-systemet. Inga avvikelser avseende SIR-kategorisering observerades. Resultaten visar att det är möjligt att optimera metoden avseende volymen blododlingsmedium som används för direktresistensbestämning på blododlingsflaskor. Då den optimerade metodens känslighet och specificitet var 100 %, är det möjligt att implementera metoden i rutinarbetet.
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Leo, Elisa <1981>. "Marker molecolari relativi all'infezione da Papillomavirus umani: significato diagnostico e prognostico in lesioni virus-correlate." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2010. http://amsdottorato.unibo.it/2297/.
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Introduzione: Il cervico-carcinoma è la seconda neoplasia maligna per incidenza e mortalità nelle donne in tutto il mondo dopo il cancro al seno. L’infezione persistente da Papillomavirus Umani (HPV) è causa necessaria dell’insorgenza del cervico-carcinoma e delle sue lesioni pre-cancerose.
L’infezione da HPV si associa anche ad altri carcinomi del distretto ano-genitale (a livello anale, vulvare, vaginale e del pene) e a circa il 25% dei carcinomi squamosi dell’orofaringe. I circa 40 genotipi di HPV che infettano la mucosa genitale vengono suddivisi in alto rischio (HR-HPV) e basso rischio (LR-HPV) oncogeno a seconda della alta e bassa associazione con la neoplasia cervicale. I 13 genotipi a più alto rischio oncogeno sono 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 e 68. Di questi, otto (16, 18, 31, 33, 35, 45, 52 e 58) sono associati alla maggior parte dei carcinomi cervicali (circa 89%) e i genotipi 16 e 18 da soli sono riscontrati nel 70% circa delle neoplasie. L’insorgenza e progressione delle lesioni preneoplastiche cervicali è, però, associata non solo alla presenza di HPV ad alto rischio, ma, soprattutto, alla persistenza virale (> 18 mesi), alla capacità di integrazione degli HPV ad alto rischio e alla conseguente sovraespressione delle oncoproteine E6/E7. Inoltre, l’integrazione è spesso favorita da un’alta carica virale soprattutto per quanto riguarda alcuni genotipi (HPV16 e 18).
L’infezione da HPV non interessa solo la cervice uterina ma tutto il distretto ano-genitale e quello testa-collo. L’HPV, in particolare il genotipo 16, è implicato, infatti, nell’insorgenza delle lesioni preneoplastiche della vulva (VIN) classificate come VIN classiche e nei carcinomi ad esse associati. L’incidenza del carcinoma vulvare in Europa è di 1.5/100.000 di cui circa il 45% è dovuto a HR-HPV (80% ad HPV16). Nonostante l’associazione tra HPV16 e carcinoma vulvare sia alta, ancora poco si conosce sul ruolo della carica virale e dell’integrazione in tali lesioni.
Le lesioni che possono presentarsi nella regione testa-collo possono essere sia di natura benigna che maligna. I genotipi più frequentemente riscontrati in associazione a lesioni benigne (papillomi) sono HPV 6 e 11, quelli associati a forme tumorali (HNSCC) sono il genotipo 18 ma soprattutto il 16.
Molti aspetti del coinvolgimento di HPV in queste patologie non sono ancora perfettamente conosciuti e spesso studi su tale argomento hanno mostrato risultati contraddittori, soprattutto perché vengono utilizzate metodiche con gradi diversi di sensibilità e specificità.
Recenti dati di letteratura hanno tuttavia messo in evidenza che i pazienti affetti da HNSCC positivi ad HPV hanno una elevata risposta al trattamento chemioradioterapico rispetto ai pazienti HPV-negativi con un notevole impatto sul controllo locale e sulla sopravvivenza ma soprattutto sulla qualità di vita di tali pazienti, evitando di sottoporli a chirurgia sicuramente demolitiva.
Scopo del lavoro: Sulla base di queste premesse, scopo di questo lavoro è stato quello di valutare l’importanza di marker quali la presenza/persistenza di HPV, la carica virale, la valutazione dello stato fisico del genoma virale e l’espressione degli mRNA oncogeni nella gestione di pazienti con lesioni preneoplastiche e neoplastiche di diverso grado, associate a papillomavirus mucosi.
Per la valutazione dei markers virologici di progressione neoplastica abbiamo sviluppato dei saggi di real time PCR qualitativi e quantitativi studiati in modo da poter fornire, contemporaneamente e a seconda delle esigenze, risposte specifiche non solo sulla presenza e persistenza dei diversi genotipi di HPV, ma anche sul rischio di insorgenza, progressione e recidiva delle lesioni mediante lo studio di markers virologici quali carica virale, integrazione ed espressione degli mRNA.
Abbiamo pertanto indirizzato la nostra attenzione verso tre popolazioni specifiche di pazienti:
- donne con lesioni vulvari preneoplastiche (VIN) e neoplastiche, allo scopo di comprendere i complessi meccanismi patogenetici di tali patologie non sempre associate ad infezione da HPV;
- pazienti con lesioni maligne a livello della regione testa-collo allo scopo di fornire informazioni utili all’elaborazione di un percorso terapeutico mirato (radiochemioterapico o chirurgico) a seconda o meno della presenza di infezione virale;
- donne con lesioni cervicali di alto grado, trattate chirurgicamente per la rimozione delle lesioni e seguite nel follow-up, per stabilire l’importanza di tali marker nella valutazione della persistenza virale al fine di prevenire recidive di malattia.
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Martiny, Delphine. "Contribution of MALDI-TOF mass spectrometry in the microbiological diagnosis and clinical management of patients suffering from infectious diseases." Doctoral thesis, Universite Libre de Bruxelles, 2013. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209372.
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In infected patients, the rapid identification of pathogens is critical. After a long period of slow technological improvement, the microbiology laboratory is now undergoing significant evolution. This work evaluated the contribution of recent MALDI-TOF MS technology in terms of the diagnosis and clinical management of patients and its implementation in the laboratory of tomorrow. The studies were conducted over a 3.5-year period, mostly in the iris public hospital network of Brussels. First, we confirmed the accurate performance of MALDI-TOF MS in the identification of routine isolates, regardless of whether the Biotyper (92.7% correct species identification) or VITEK MS (93.2%) (n=986) commercial system was used, and demonstrated the supremacy of this technology over conventional identification techniques for fastidious bacteria, including Campylobacter and related organisms (98.3%, 72.2% and 79.9% correct species identification by Biotyper, Vitek NH Card and API Campy, respectively; n=234).
Second, we showed that the direct MALDI-TOF MS identification of bacteria from positive blood cultures was not only feasible but also led to an 24-h reduction in the time-to-identification. In an adult population, more than 13% of the direct identifications from positive blood cultures resulted in the faster adaptation of the antimicrobial treatment.
Third, we demonstrated that MALDI-TOF MS could easily be implemented in a network, which was associated with significant cost savings and reduction in the time-to-identification. Finally, our promising Blastocystis subtyping results suggest that the number of MALDI-TOF MS applications may be increased.
In the future, automation of the technique will make its use in clinical laboratories even easier, eliminating the use of conventional identification techniques. Improvement of the preanalytical procedures is also important to make MALDI-TOF MS a suitable instrument for resistance and toxicity mechanism detection and subtyping.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished
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Fourmaux, Éric. "Diagnostic et traitement des endophtalmies bactériennes post-opératoires." Bordeaux 2, 1995. http://www.theses.fr/1995BOR23032.
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Ntuli, Sindile Venessa. "Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory." Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/27816.
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The laboratory diagnosis of fungal infection is complicated, based on the microscopic detection, culture isolation, and detection of serological response. In recent years, there has been an increase in the utilization of molecular diagnostic techniques for the detection and accurate identification of fungal pathogens. This was a laboratory accuracy study evaluating the performance of a selected pan-fungal PCR using 70 previously identified reference fungal isolates. The DNA yield and purity of three different DNA extraction methods was assessed, using 6 representative fungal isolates. The ZR Fungal/Bacterial DNA MicroPrep™ produced a median concentration of 17.28 ng/μl,which was significantly higher (p value = 0.0079) than the MagNA pure LC DNA Isolation Kit III and QIAamp DNA Mini Kit, which produced median yields of 11.08ng/μl and 3.54 ng/μl, respectively. The selected pan-fungal PCR was optimized PCR and successfully performed on 62 of the 70 reference isolates. A selection of 56 amplicons were submitted for DNA sequence determination. Of all the sequences queried on the NCBI and Ribosomal Development Project (RDP) databases, 95/111(86%) were concordant with the results obtained from the reference laboratory. Study findings have shown that the selected pan-fungal PCR, coupled with DNA sequence analysis is an excellent diagnostic tool for the identification of medically relevant fungi. This assay, in combination with conventional culture, is useful for the rapid and accurate identification of fungal isolates. Future work will involve evaluating the utility of this assay for the detection and identification of medically relevant fungi in deep tissue biopsies.
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Botha, Elizabeth Magdelena. "Molecular characterization of South African lineage II West Nile virus isolates ltime PCR assay." Pretoria : [s.n.], 2008. http://upetd.up.ac.za/thesis/available/etd-06122008-130924/.
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Olanié, Florian Amador del Valle Gilles. "Les tests biologiques en parodontologie." [S.l.] : [s.n.], 2008. http://castore.univ-nantes.fr/castore/GetOAIRef?idDoc=50576.
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Khumalo, Jermaine. "The use of molecular diagnostic methods to improve the detection of the common bacterial and viral causes of community acquired meningitis in children in South Africa." Master's thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/15539.
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With conventional methods of diagnosis, substantial overlaps are common due to an absence of the expected CSF findings clearly aligning to bacterial or viral infection. Reduced sensitivity is commonly observed chiefly due to empiric antibiotic treatment leading to bacterial culture negative results. This leads to costly hospital admissions and unnecessarily prolonged treatment for the unexplained aetiology, further compounded by routine viral diagnostics not being commonly implemented for meningitis diagnosis. We developed and validated in-house quantitative real-time (qPCR) multiplex assays to test for bacterial causes namely: Neisseria meningitidis (ctrA gene), Haemophilus influenzae (hpd gene) and Streptococcus pneumoniae (lytA gene) and viral causes namely: enterovirus (5' UTR), herpes simplex (UL30 gene) and mumps virus (Fusion protein gene). The qPCR assays were carried out on the Biorad CFX 96 real-time instrument. These validated assays were used to screen a cohort of suspected meningitis cases. The retrospective study included 300 paediatric patients aged from 60 days-12 years, over a 1-year period (November1, 2012 to November 30, 2013) with suspected meningitis presented to the outpatient departments of the Red Cross War Memorial Children‟s Hospital (RCCH) in Cape Town. Cerebrospinal fluid with abnormal chemistry and cell counts was selected and total nucleic acid was extracted with the QIAsymphony virus/bacterial DSP kit (QIAGEN, Valencia, CA). The median age of children was 19 months (IQR: 6-65 months). Among the screened 291 CSF samples, 7 (2.4%) cases Gram stain results were obtained along with relatively few cases with positive bacterial culture growth 4/291 (1.4%). Based on bacterial qPCR results, 8 (2.7%), 3 (1%) and 1 (0.3%) were positive for S. pneumoniae, N. meningitidis and H. influenzae respectively. A majority of cases were viral positives with enteroviruses being the dominant at 91/291 (31.3%) and mumps virus 3/291 (1%). No herpes simplex DNA was detected. The bacterial qPCR showed a sensitivity and specificity of 85.7% and 97.7% respectively when compared against a composite reference standard (CRS). We report an improvement with additional detected causes of bacterial meningitis and highlight the burden of the common viral causes. However, a large proportion of cases (63.6 %) have aetiology still unknown. PCR shows valuable in concluding viral aetiology in routine diagnosis.
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Campeão, Mariana Esteves. "Diversidade genômica e diagnostico fenotípico de vibrios." Laboratório Nacional de Computação Cientifica, 2014. https://tede.lncc.br/handle/tede/184.
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Previous issue date: 2014-04-25Vibrios sao bacterias amplamente distribuidas no meio aquatico e podem ser encontradas em associacao com organismos marinhos, tanto como causadores de doencas quanto como simbiontes. O advento das tecnicas de sequenciamento de nova geracao e de alto desempenho tem possibilitado o acesso cada vez mais amplo a dados genomicos microbianos, incluindo vibrios. Tal quantidade e disponibilidade de dados permitem analises in silico, que podem compreender desde caracteristicas genomicas ate fenotipicas. A taxonomia microbiana e fundamentada na abordagem polifasica, que mede as relacoes evolutivas a partir do uso de sequencias de genes, especialmente o RNAr 16S, similaridade genomica, por meio de hibridizacao de DNA, e ampla caracterizacao fenotipica. A caracterizacao fenotipica requer testes experimentais, que muitas vezes sao demorados, caros e requerem grande experiencia. Neste estudo propomos o uso de genomas para a analise da diversidade e identificacao fenotipica de vibrios. Para tanto, foram avaliadas caracteristicas basicas de vibrios (tais como tamanho do genoma, conteudo genico e posicao logenetica); analisaram-se genes unicos e suas possiveis funcoes ecologicas; e desenvolveu-se uma ferramenta prototipo para identificacao de fenotipos diagnosticos de vibrios, denominada vibriophenotyping 1.0. A logenia construida a partir do genoma minimo recuperou os diferentes generos e clados descritos na literatura para o grupo vibrio, bem como posicionou as especies consideradas irmas em relacao a um ancestral comum proximo. Os genes unicos, por sua vez, puderam ainda revelar peculiaridades entre especies irmas. Por m, o programa de identificacao fenotipica desenvolvido foi testado com genomas de linhagens tipo de vibrios e apresentou uma media de similaridade superior a 70% entre os fenotipos obtidos in vitro e in silico, sendo alcançada uma similaridade de 100% para genomas integros. Dessa forma, concluiu-se que analises do pangenoma permitem a recontrucao logenetica dentro do grupo vibrios e a identificacao de genes unicos relevantes para o papel ecologico da especie no ambiente de origem, e, ainda, que a identificacao fenotipica atraves da automatizacao por uma ferramenta computacional e possivel a partir da analise de genomas.
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Herthnek, David. "Molecular diagnostic methods for Mycobacterium avium subsp. paratuberculosis : more than a gut feeling /." Uppsala : Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, 2009. http://epsilon.slu.se/200920.pdf.
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Morellet, Isabelle. "Détection des chlamydia dans les prélèvements pathologiques par des méthodes de diagnostic mettant en œuvre des anticorps monoclonaux spécifiques du lipopolysaccharide et de la protéine majeure de la membrane externe." Tours, 1992. http://www.theses.fr/1992TOUR3803.
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Abbaszadegan, Morteza 1955. "Detection of Giardia cysts by cDNA probe and application to water samples." Diss., The University of Arizona, 1991. http://hdl.handle.net/10150/191163.
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Giardia is the most common human parasite infection in the United States causing a lengthy diarrhea. Transmission of Giardia is by the fecal-oral route and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia in drinking water through the "Surface Water Treatment Rule." Current methods for detection of Giardia in water rely primarily on microscopic observation of water concentrates by immunofluorescent techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 base pairs long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of M13 vector with insert was isolated from lysed host E. coli XL1- Blue and used for production of the cDNA probe by nick translation with ³²P-labeled nucleotides. Seven different protocols were tested for extracting nucleic acids from the cysts. Using the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates by dot-blot hybridization assays. Environmental concentrates from secondary and tertiary treated sewage or surface waters were screened for Giardia cysts by immunofluorescent and the genespecific probe. Positive signals were observed in sewage and surface water samples without floatation at ten fold greater dilutions than after floatation. It appeared that gene probe detection was slightly more sensitive than microscopic detection of Giardia cysts for wastewater samples. In six surface water samples and two sewage sample no positive results were found either by the cDNA probe or immunofluorescent. Usually, DNA probes are radiolabeled and the most commonly used is ³²P. ³²P is expensive, hazardous and has an extremely short half-life of 14.3 days, necessitating frequent preparation of the nucleic acid probes. Three non-radioactive labeling methods, chemiluminescence, enzyme-linked immunoassay and enhanced chemiluminescence were evaluated. The cDNA probe was labeled by nick translation for chemiluminescence method. Biotinylated deoxyuridine triphosphate was used in place of deoxythymidine triphosphate to produce biotinylated DNA strands. The result of hybridization was visualized by chemiluminescenct detection of DNA. The sensitivity of the chemiluminescent method and the 32P labeled probe was 0.1 pg of DNA in a slot-blot hybridization assay.
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Mescam, Frédérique. "Herpès oculaire : techniques diagnostiques, apport de l'amplification génique 7." Bordeaux 2, 1992. http://www.theses.fr/1992BOR23062.
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Souweine, Bertrand. "Contribution au diagnostic microbiologique des infections respiratoires acquises en reanimation (doctorat : reanimation medicale)." Clermont-Ferrand 1, 2000. http://www.theses.fr/2000CLF1MM03.
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Corraini, Priscila. "Perfis microbianos subgengivais e doenças periodontais em uma população isolada brasileira." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/23/23146/tde-11092012-154729/.
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OBJETIVOS: investigar a prevalência e a presença de distintos perfis microbianos no biofilme subgengival e avaliar o seu papel no diagnóstico e risco das doenças periodontais destrutivas em uma população isolada brasileira sem acesso à tratamento periodontal e tradição ao uso de métodos de higiene bucal. MATERIAL E MÉTODOS: A população-alvo consistiu de todos os indivíduos com 12 ou mais anos de idade (N= 264) residentes na microárea Cajaíba, identificados por meio de um censo. Estes indivíduos foram entrevistados por meio de um questionário estruturado e submetidos a um exame periodontal completo que consistiu na avaliação de 6 sítios por dente em toda a boca e na coleta de amostras do biofilme subgengival em 4 sítios por indivíduo. A detecção dos micro-organismos A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia e C. rectus, bem como a distribuição dos sorotipos e presença do clone JP2 do A. actinomycetemcomitans foram avaliadas por meio da reação em cadeia da polimerase (PCR). RESULTADOS: A. actinomycetemcomitans foi detectado em 25% dos indivíduos, enquanto que P. gingivalis T. forsythia, P.intermedia e C. rectus foram detectados em 64%, 59%, 38% e 90% dos indivíduos, respectivamente. Entre as amostras positivas para o A. actinomycetemcomitans (n=42), 18 (42%) representaram o sorotipo a, 2 (5%) o sorotipo b, 19 (46%) o sorotipo c, 1 (2%) o sorotipo e, e 4 (10%) foram não-sorotipáveis. O clone JP2 do A. actinomycetemcomitans não foi detectado em nenhum indivíduo desta população. Dois perfis microbianos subgengivais foram identificados: (perfil 1) nenhum dos microrganismos estudados, com exceção do C. rectus (n = 31), e (perfil 2) co-ocorrência de P. gingivalis e T. forsythia (n = 77). O perfil 1 demonstrou valores de sensibilidade extremamente baixos, enquanto que o perfil 2 apresentou valores de sensibilidade variados na identificação dos desfechos subrrogados periodontais avaliados, e valores de baixos a moderados para a especificidade. Os seguintes perfis subgengivais estiveram associados com a prevalência de perda clínica de inserção (NCI) e profundidade de sondagem (PS) nos modelos finais de regressão logística múltipla, ajustados para variáveis demográficas, biológicas e comportamentais: T. forsythia (PS e NCI 5 mm, e 7 mm), P. gingivalis (NCI 7 mm) e o perfil 2 (PS 5 mm e NCI 7 mm). CONCLUSÕES: Os micro-organismos periodontais estudados foram prevalentes nessa população isolada. Esta população apresentou predominância dos sorotipos a e c do A. actinomycetemcomitans. Dois perfis microbianos subgengivais puderam ser identificados nesta população isolada. Porém, eles não foram superiores ao diagnóstico de parâmetros clínicos periodontais específicos, quando adicionados à informação clínica tradicional. Perfis microbianos subgengivais apresentando T. forsythia como indicador de risco foram significativamente associados com o aumento da PS e do NCI nessa população isolada.AIMS: To investigate the prevalence and describe the subgingival microbial profiles of selected periodontal pathogens in the subgingival biofilm; and assess their role as possible diagnostic markers or risk indicators for destructive periodontal diseases in a periodontally untreated and isolated population from Brazil. MATERIAL AND METHODS: The target population consisted of all subjects aged 12 years (n=264) in an isolated Brazilian population. A full-mouth clinical examination was conducted, and pooled subgingival plaque samples were obtained from four sites per subject. PCR analyses were performed to identify the following microorganisms: A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia and C. rectus, as well as the A. actinomycetemcomitans serotype distribution and JP2 clone detection. RESULTS: A. actinomycetemcomitans was detected in 25% of the subjects, whereas P. gingivalis, T. forsythia, P.intermedia and C. rectus were detected in 64%, 59%, 38% and 90% of the subjects, respectively. From the A. actinomycetemcomitans positive isolates (n=42), 18 (42%) were serotype a, 2 (5%) b, 19 (46%) c, 1 (2%) e, and 4 (10%) were non-serotypeable. None of the strains belonged to the JP2 clone. Two specific subgingival microbial profiles were identified: (1) In one, only C. rectus could or not be present (n = 31), while in the other, (2) Co-occurrence of T. forsythia and P. gingivalis was observed (n = 77). Profile 1 showed very low sensitivity values, and profile 2 showed varying sensitivity values for the identification of the various periodontal states, and considerably low to moderate specificity values. The following subgingival profiles were significantly associated with the prevalence of periodontal attachment loss (CAL) and probing depth (PD) in the final multiple logistic regression models adjusted for demographic, biological and behavioral variables: T. forsythia (PD and CAL 5 mm and 7 mm), P. gingivalis (CAL 7 mm) and the profile 2 (PD 5 mm and CAL 7 mm). CONCLUSIONS: The five studied periodontal microorganisms were prevalent in this isolated population. The A. actinomycetemcomitans positive subjects consisted predominantly of a and c serotypes. Two specific microbial profiles could be identified in this isolated population. They did not result in significant superior diagnostic accuracy when compared totraditional clinical markers. Subgingival microbial profiles presenting T. forsythia as risk indicator were significantly associated with increased PD and CAL in this isolated population.
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Wai, Chi-wan, and 衛至韻. "Development of shell vial culture assay for the rapid diagnosis of respiratory viruses using the human colorectal adenocarcinoma (CaCo2) cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193551.
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Background: Respiratory diseases are common worldwide, which are caused by various respiratory viruses. As symptoms caused by these viruses are similar, laboratory diagnosis is essential to distinguish the virus. Conventionally, respiratory viruses are isolated by cell culture with a panel of cell lines. However, handling of several cell lines is labour intensive, and the turnaround time of conventional culture is long. In previous study, the use of human colon adeno-carcinoma (Caco-2) in conventional culture was investigated. The study has proven that Caco-2 is generally susceptible to the eight common respiratory viruses, i.e. Adenovirus, Influenza A and B, Respiratory Syncytial virus, Parainfluenza virus 1, 2,3 and 4. As turnaround time of conventional culture is long; therefore, in this study, rapid shell vial culture using Caco-2 cells were evaluated. Moreover, the application of Caco-2 shell vial culture on recovering human metapneumovirus (hMPV) was also investigated.
Materials and methods: This study consisted of four stages. First, recovery of viruses by conventional culture and shell vial culture of Caco-2 were compared. Specimens were added to conventional culture and shell vial simultaneously. For conventional culture, formation of CPE was examined daily and IF staining was performed when CPE was indicated; meanwhile, shell vial culture were incubated for seven days and stained with IF to detect infected cells. In stage two, the effect of incubating shell vial culture in rolling drum was investigated. Shell vials inoculated with the same specimen in duplicate were incubated in rolling drum and without rolling drum simultaneously. IF staining was performed in day 2, and results were obtained. For those which are IF negative in day 2, second shell vial was further incubated to seven days before harvest. In the next stage, a large batch of samples was used to evaluate on the use of Caco-2 shell vial culture in day 2 and day 7. Lastly, Caco-2 shell vial and conventional culture and LLC-MK2 conventional culture were tested for isolation of hMPV.
Results: Compared to Caco-2 conventional culture, recovery rate of shell vial culture was elevated slightly. When experimenting on the effect of incubation in rolling drum, results showed that recovery rate was raised in shell vial with rolling drum in day 2, moreover, the percentage of positive cells were increased significantly (p value < 0.05). Furthermore, in the evaluation of Caco-2 shell vial in day 2 and day 7, 75% of samples were isolated in day 2 while 85% were recovered in day 7. Lastly, in the investigation on recovery of hMPV, 53%, 42% and 17% hMPV positive cases were isolated by Caco-2 shell vial, Caco-2 conventional culture and LLC-MK2 conventional culture respectively.
Conclusion: First, although recovery rate by shell vial and conventional culture were similar, turnaround time was reduced from a week to a few days by shell vial culture. Therefore, Caco-2 shell vial culture is a more efficient than Caco-2 conventional culture in isolating respiratory viruses. The study also showed that incubation of shell vial in rolling drum able to increase the number of positive cells. Furthermore, in this study, Caco-2 cells were also shown to be more efficient in isolating hMPV when compare to LLC-MK2 cells.published_or_final_version
Microbiology
Master
Master of Medical Sciences
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BILLIOTTE, ANNE. "Apport de la microbiologie au diagnostic etiologique des meningites : a propos des observations du service des maladies infectieuses du c.h.u. de marseille hopital de la conception." Aix-Marseille 2, 1992. http://www.theses.fr/1992AIX20066.
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Bicudo, Cesar Fernanda. "RHODOCOCCUS EQUI IN THE FOAL – IMPROVING DIAGNOSTIC AND PREVENTION MEASURES." UKnowledge, 2018. https://uknowledge.uky.edu/gluck_etds/36.
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Although Rhodococcus equi (R. equi), previously known as Corynebacterium equi, was first isolated from pneumonic foals almost a century ago, it remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals. While the majority of foals exposed to R. equi develop a protective immune response (regressors), others exhibit a unique susceptibility to infection (progressors). The determinants for either outcome are not completely understood. Therefore, current diagnostic and preventive measures are suboptimal and require betterment. In light of this current need, we hypothesized that immunoglobulin G subisotype T [IgG(T)] against the virulence-associated protein A (VapA) of R. equi, and whole blood cytokine expression profile of foals predict the outcome of infection and can be used as diagnostic markers of clinical disease. Further, we hypothesized that the use of R. equi hyperimmune plasma (HIP) decreases severity of disease in naturally infected foals, playing an important role in disease prevention in the field. Lastly, we hypothesized that specific anti-Rhodococcus equi pili antibodies passively acquired by foals via colostrum after immunization of pregnant mares with a Rhodococcus equi pili-based candidate vaccine will confer protection against induced disease, and therefore have an immediate impact on R. equi pneumonia prophylaxis.
The objectives of this study were: (1) to describe the humoral immune response of progressor and regressor foals to R. equi following experimental challenge and natural infection, (2) to compare the cytokine and cell-marker expression profile in whole blood of progressor and regressor foals after challenge, (3) to evaluate the Vap-A specific IgG profile of a commercially available HIP product and its value as a prophylactic tool on an endemic farm, and (4) to evaluate the efficacy of a vaccine based on the Rhodococcus equi pili (Rpl).
Although the IgG(T) response of progressor foals after challenge or following natural infection tended to be more pronounced than that observed in regressor foals, its performance as a diagnostic test for predicting disease outcome was poor. Likewise, whole blood cell-marker and cytokine expression profiles of progressor and regressor foals were not significantly different, undermining its reliability as a diagnostic tool. Evaluation of the association of HIP VapA specific IgG profile and rhodococcal disease outcome in the field resulted in the conclusion that progressor foals received significantly less VapA specific IgG, suggesting that HIP may have provided some protection to regressor foals. Although HIP appeared to have provided some protection against clinical pneumonia, Rpl maternally-derived IgG failed to confer any advantage to foals born from vaccinated mares. The Rpl candidate vaccine failed to confer protection to foals after challenge, and did not decrease disease severity in comparison to a control group.
In summary, the results of this study do not support the use of VapA specific IgG(T) or whole blood cytokine expression profile as predictors of disease outcome. Further, our results suggest a positive effect of HIP on disease outcome. Lastly, the presence of systemic and local Rpl antibodies was not protective in foals.
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Seng, Piseth. "Application de la spectrométrie de masse MALDI-TOF en microbiologie clinique." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM5033/document.
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L'objectif de cette thèse est d'appliquer la méthode d'identification bactérienne par spectrométrie de masse MALDI-TOF pour une utilisation en routine dans un laboratoire de microbiologie clinique. Dans un 1er temps et de manière prospective, nous avons évalué la performance et le coût-efficacité de l'identification bactérienne de routine par MALDI-TOF par rapport aux techniques conventionnelles d'identification phénotypique. Durant la période des 16 semaines d'étude, nous avons comparé la performance de la technique par MALDI-TOF aux techniques conventionnelles d'identification phénotypique comprenant la coloration de Gram, la galerie API ANA et le Vitek 2. En cas de résultats discordants entre ces deux techniques, l'identification était réalisée par biologie moléculaire. Nous avons montré que le MALDI-TOF est un moyen efficace et rentable pour l'identification des bactéries de routine. Le MALDI-TOF peut être utilisée en 1ère intention dans l'identification bactérienne avant l'ensemble de techniques phénotypiques. Dans un 2ème temps, nous avons évalué rétrospectivement la performance et le coût-efficacité de l'utilisation exclusive de MALDI-TOF en diagnostic bactériologique de routine en comparaison avec les techniques conventionnelles. En analysant les données des 11 dernières années, nous avons montré que le MALDI-TOF est efficace et tout à fait adaptée pour l'identification d'espèce bactérienne en routine. Nous avons également prouvé que MALDI-TOF est un outil puissant pour identifier les espèces bactériennes rarement impliquées dans les infections humaines. Cette technique pourrait être une alternative aux méthodes moléculaires dans le laboratoire cliniqueThe objective of this thesis is to apply the method of bacterial identification by MALDI-TOF MS in daily practice in a routine clinical microbiological laboratory. Firstly, we prospectively evaluated the performance and the cost-effective of bacterial identification by MALDI-TOF in comparison with conventional phenotypic identification methods. During a 16-week study, we compared the performance of MALDI-TOF with conventional techniques of identification including Gram staining, API ANA identification strip and automated identification using the Vitek 2. The unmatched identifications between MALDI-TOF and conventional methods were resolved by molecular identification. In this study, we showed that MALDI-TOF was an effective tool and less expensive for the rapid identification of bacterial species in clinical microbiology laboratory. MALDI-TOF can be used in first intention for identification before Gram staining or other phenotypic identification techniques based on physicochemical properties of bacteria. Secondly, we retrospectively evaluated the performance and the cost-effectiveness of the exclusive use of MALDI-TOF in bacteriological diagnosis in comparison with conventional phenotypic identification. 11-year retrospective analysis of data showed that MALDI-TOF was efficient and completely adapted for the routine identification of bacterial species. We also showed that MALDI-TOF had capacity to identify bacterial species that were rarely involved in human diseases. This technique could be an alternative to molecular methods in the clinical laboratory
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Gantelius, Jesper. "Novel diagnostic microarray assay formats towards comprehensive on-site analysis." Doctoral thesis, Stockholm : Skolan för bioteknologi, Kungliga Tekniska högskolan, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11221.
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Gouriet, Frédérique. "Mise au point d'une nouvelle technique par microarray antigénique pour le diagnostic sérologique par syndrome en maladies infectieuses." Aix-Marseille 2, 2009. http://www.theses.fr/2009AIX20652.
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Gonçalves, Marcos Cesar. "Caracterização molecular do Sugarcane yellow leaf virus, desenvolvimento de um metodo de diagnostico altamente sensivel e aspectos moleculares da interação luteovirus/vetor." [s.n.], 2001. http://repositorio.unicamp.br/jspui/handle/REPOSIP/315200.
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Orientador: Jorge VegaTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O vírus do amarelecimento foliar da cana-de-açúcar, Sugarcane yellow leaf vírus (ScYL V) constitui atualmente um grande problema nos principais países produtores de cana-de-açúcar. Este vírus possui diversas características físicas e biológicas, como tamanho e morfologia da partícula, reações serológicas, alterações morfológicas no hospedeiro e aspectos de transmissão, comuns aos membros da família Luteovírídae. As informações da sequência genõmica do isolado brasileiro obtidas nesse trabalho, referentes às regiões codificantes para capa protéica do vírus (CP), proteína de movimento (17 kOa ou MP) e parte da RNA polimerase dependente de RNA (RdRp), mostram um alto grau de identidade e similaridade de aminoácidos com sequências de luteovirus conhecidos. Isso permite estabelecer o ScYLV como um membro definitivo da família Luteovíridae. Análises filogenéticas empregando as sequências deduzidas de aminoácidos da CP e da região C-terminal da RdRp do ScYL V sugerem diferentes afinidades taxonõmicas desse vírus dentro da família Luteovírídae, de maneira similar ao que acontece com o Soybean dwarf vírus (SOV). Análises comparativas de seqüência entre o isolado brasileiro e um isolado norte-americano revelaram diferenças de apenas dois nucleotídeos nas posições 4201(G por T) e 4232 (A por C) do genoma do ScYLV, correspondentesrespectivamente à região codificadora da proteína P17 e CP. Os dados de seqüência obtidos neste trabalho foram usados no desenvolvimento de um método sensível de diagnóstico baseado na combinação da técnica de amplificação isotérmica de ácidos nucléicos NASBA (nucleic acid sequence based amplification) e "molecular beacons", denominado AmpliOet RNA. Esse sistema de detecção foi altamente específico e ofereceu uma sensibilidade de aproximadamente 100 fg de vírus purificado, permitindo a detecção em plantas com baixos níveis de infecção viral e em um único pulgão virulífero. Os membros da família Luteoviridae são transmitidos por afídeos de uma maneira circulativa e não-propagativa. Após a aquisição, o vírus permanece no corpo do inseto por várias semanas, graças a associação com a proteína GroEL, produzida por um endossimbionte primário do pulgão. As partículas de vírus purificadas apresentam dois tipos de proteína, a proteína principal da capa protéica de 22 kDa e menores quantidades de outro componente do capsídeo, a proteína de transleitura ou "readthrough" (RTD) de 54 kDa. A protéina RTD contém determinantes responsáveis pela transmissão e acúmulo do vírus em plantas agroinfectadas. O Potato leafroll vírus (PLRV) é uma espécie do gênero Polerovirus dentro da família /,..uteoviridae. Clones infectivos de cDNA do PLRV e um mutante deletério da proteína RTD foram usados para estudar as interações moleculares entre esse luteoviruse seu afídeo vetor Myzus persicae. O mutante do PLRV, no qual a proteína RTD estava inteiramente ausente, não foi transmissível por M. persicae e não se ligou a proteína GroEL. Adicionalmente, esse mutante mostrou-se significativamente menos persistente na hemolinfa do afídeo do que as partículas não modificadas do vírus
Abstract: Sugarcane yellow leaf vírus (ScYL V) is widely distributed in Brazil and other sugarcane producing countries causing significant yield losses. This virus shares biological features typical of the luteovirids. Comparisons of the coat protein (CP), 17 kDa protein and C-terminus of the RNA-dependent RNA polymerase coding regions showed that the deduced amino acid sequences of the Brazilian isolate share a considerable degree of identity and similarity with corresponding sequences of known luteovirids, thus clearly establishing ScYL V as a member of the family Luteovíridae. Phylogenetic analyses also suggest that the 5' and 3' coding blocks of the ScYLV genome possess different taxonomic affinities within the Luteovíridae family, as for the genome of Soybean dwarf vírus (SDV). Our results were published simultaneously as the sequence of a North American strain of ScYL V. Comparative analyses between the deduced peptides of Brazilian and North American strains revealed two nucleotide substitutions at positions 4201 (G_ T) and 4232 (A_C) of the ScYLV genome, corresponding to P17 and CP proteins coding regions, respectively. Our sequence data were used to develop a highly sensitive detection method based on the combination of isothermic nucleic acid sequence based amplification (NASBA) and molecular beacons, named AmpliDet RNA. This system offered a sensitivity of about 100 fg of purified virus and could detect ScYL V in plant samples with low virus titer and in one single aphid. Members in the Luteovírídae are transmitted by aphids in a circulative, nonreplicative manner. After acquisiton, luteovirus particles persist in the aphid's hemolymph for several weeks in association with a GroEL homolog, produced by the primary endosymbiont of the aphid. Luteovirus purified particles contain two types of proteins; a major 22 kDa coat protein (CP) and the minor capsid component of 54 kDa, the readthrough protein (RTD). The RTD contains determinants responsible for virus transmission and accumulation in agroinfected plants. Potato leafroll virus (PLRV) is a member of the genus Po/erovírus in the family Luteovíridae. An infectious cDNA fuI! length clone of PLRV and a mutant devoid of the RTD were used to study and better understanding the molecular interactions between this luteovirus and its aphid vector Myzus persícae. The PLRV mutant lacking the entire RTD protein was not transmissible by M. persícae and did not bind to Buchnera GroEL. Furthermore, the mutant was significantly less persistent in the aphid's hemolymph than the wild type virus. These data corroborate previous observations with Beet westem yellow vírus (BWYV) and Bar/ey ye//ow dwarf vírus (BYDV) that the RTD domain is involvedin luteovirus transmission and persistence in the aphid's body
Doutorado
Doutor em Biologia Vegetal
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Correia, Letícia Borges Nunes [UNESP]. "Perfil microbilógico de diferentes tipos de saladas provenientes de cozinhas hospitalares." Universidade Estadual Paulista (UNESP), 2015. http://hdl.handle.net/11449/132071.
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000850005.pdf: 1236403 bytes, checksum: b1877b4be5ffb104086b39681ebd86a7 (MD5)As hortaliças são itens indispensáveis na dieta dos seres humanos, atuando como adjuvantes na prevenção e tratamento de doenças. Porém, destaca-se o aumento de surtos de Doenças Transmitidas por Alimentos (DTA) associados ao consumo de produtos hortícolas. O objetivo desse trabalho foi verificar o perfil microbiológico de diferentes tipos de saladas de cozinhas hospitalares. No período de 2010 a 2014, o Serviço de Orientação a Alimentação Pública (SOAP) recebeu 641 amostras de saladas provenientes de hospitais da região de Bauru e Botucatu, onde foram submetidas às análises microbiológicas para a determinação do número mais provável de coliformes a 35°C e 45°C, pesquisa de Salmonella spp e contagem de estafilococos coagulase positiva. Os resultados revelaram que em 30,56% das amostras a contagem de coliformes a 35°C foi maior que 1.100 NMP/g e 12,17% apresentaram coliformes a 45°C acima de 100 NMP/g, limite máximo estabelecido pela legislação brasileira. A prevalência de amostras contaminadas sem tratamento térmico foi de 97,44% e de 2,56% para amostras com tratamento térmico, cozidas ou refogadas. Todas as amostras foram negativas para presença de Salmonella spp e apresentaram contagem de estafilococos coagulase positiva < 1,0X102 UFC/g. Apesar das amostras de saladas não oferecerem risco microbiológico associado à patógenos, deve-se atentar para os micro-organismos indicadores, pois as refeições são servidas a indivíduos hospitalizados
Vegetables crops are indispensable items in human diet, acting as adjuvants in the prevention and treatment of diseases. However, the number of Foodborne Diseases outbreaks associated with their consumption increases. The aim of this study was determine the microbiological profile of different salads from hospital kitchens. In the period between 2010 and 2014 the Public feeding orientation service (SOAP) received 641 samples of salads from hospital kitchens in the region of Botucatu and Bauru. They, where subjected to microbiological analysis to determine the most probable number of coliforms at 35 ° C and 45 ° C, besides Salmonella spp presence and coagulase-positive staphylococci counts. The results reveal that 30.56% of the samples had counts for coliform at 35°C greater than 1.100 MPN/g and 12.17% had MPN/g of coliforms at 45°C above 100 MPN/g, the maximum limit established by Brazilian legislation. The prevalence of samples without heat treatment was 2.56% and 97.44% with heat treatment, cooked or braised. All samples were negative for Salmonella spp count and coagulase positive staphylococci < 1,0x102 UFC/g. Although most salads had an adequate microbiological quality, attention should be paid to the safety indicators as the meals are served to hospitalized individuals