Dissertations / Theses on the topic 'PCR diagnostic'

La leishmaniose viscerale (lv) s'etend depuis quelques annees en plusieurs regions du monde a la suite de changements epidemiologiques tels que l'urbanisation ou les mouvements de populations. Dans le meme temps, des coinfections leishmania/virus de l'immunodeficience humaine (vih) sont de plus en plus souvent rapportees, principalement sur le pourtour du bassin mediterraneen. L'amelioration du diagnostic biologique de la lv et l'evaluation de nouveaux traitements antileishmaniens ont constitue les objectifs de notre travail. Une technique de pcr pour le diagnostic des leishmanioses et plus particulierement pour le diagnostic de l. Infantum dans le cadre de l'association au vih a ete developpee. Des mesures physiques (locaux et materiels adaptes) et chimiques (decontamination enzymatique par emploi de l'uracyl-d-glycosylase) ont ete prises de maniere a eviter les faux positifs. Un controle interne de type heterologue, permettant la detection des inhibiteurs de la reaction, a ete developpe de maniere a eviter les faux negatifs. La technique a ete optimisee avec detection des produits d'amplification par technique elisa ce qui a permis d'augmenter la sensibilite et de controler la specificite de l'amplification. La pcr diagnostique a ete evaluee par comparaison avec les techniques classiques de diagnostic de leishmaniose : - aucun faux positif par pcr n'a ete observe pour les 77 sujets non atteints de leishmaniose au moment du prelevement. Ces sujets n'ont pas declare de leishmaniose dans les 12 mois ayant suivi le prelevement. - les prelevements positifs a l'examen direct et/ou a la culture ont toujours ete positifs par pcr a l'exception d'un cas. - la pcr a ete positive pour 6 prelevements provenant de patients leishmaniens connus alors que examens directs et cultures etaient negatifs. La vectorisation consiste a masquer les molecules actives au sein d'une particule capable de les conduire jusqu'a leur cible et de ne les liberer qu'a ce moment. Les phenomenes de toxicite sont donc normalement diminues, accroissant l'index therapeutique de la molecule vectorisee. Les principaux vecteurs synthetises au cours de ce travail ont ete des nanoparticules de polymethacrylate, des nanoparticules d'acide poly (d,l-lactique) et des liposomes. L'activite antileishmanienne des nanoparticules chargees par la pentamidine et des liposomes charges par l'amphotericine b (amb) a ete evaluee au moyen d'un modele original associant la souris balb/c et l. Infantum. La plus forte augmentation d'activite antileishmanienne apportee par la vectorisation a ete observee pour les nanoparticules de polymethacrylate chargees par la pentamidine (activite multipliee par 6). La faible biodegradabilite de ces nanoparticules constitue cependant un handicap par rapport aux autres vecteurs evalues dans ce travail. Les nanoparticules d'acide (d,l) lactique, liant la pentamidine de maniere plus labile, ont multiplie l'activite de la pentamidine par 3. L'ultrastructure des leishmanies observee apres traitement par la pentamidine chargee sur des nanoparticules de polymethacrylate n'a pas montre d'alterations specifiques ou plus importantes que celles observees avec la pentamidine libre.

ABSTRACT The aim of this study is develop and test an inexpensive molecular tool kit to be used for diagnostic PCR for diseases such as Leber hereditary optic neuropathy (LHON) and Cystic fibrosis(CF). By developing and optimizing recombinant Taq polymerase and making a DNA size ladder from plasmids pPSU1 and pPSU2 the financial cost for the tool kit would be reduced significantly compared to the commercial components. With an inhouse method both the recombinant Taq polymerase and the pPSU1 and pPSU2 plasmids were purified from the E.coil strain DH5-α. Thereafter to analyse the components of the tool kit both conventional PCR and Real-time PCR to make sure that the tool kit would work for both types of PCRs.     The homemade Taq polymerase proved to be able to sustain in room temperature for at least 24 h and the polymerase also showed that it works with different primers such as LHON, CF and Beta-globin in both endpoint and probe base real-time PCR. The homemade size marker produced a reliable in agarose gel electrophoresis but requires optimization for continued usage for smaller PCR products.     In conclusion the homemade Taq polymerase will be used in future PCR analysis in the laboratory and the recombinant production process as well. Meanwhile the homemade size marker did not work sufficiency enough to be continuously used with gel electrophoresis in the laboratory without being further modified.

La tuberculose continue d’être une cause majeure de morbidité et de mortalité dans le monde, principalement dans les pays en voie de développement, bien qu’elle soit une maladie curable. Le diagnostic rapide et précis de la TB active est essentiel pour l’initiation rapide du traitement et le contrôle de la maladie. Le développement de nouveaux tests rapides de diagnostic de TB active représente un véritable challenge pour l’optimisation du diagnostic.L’objectif principal de nos travaux de thèse était de développer et d’évaluer des approches de PCR en temps réel ciblant la séquence d’insertion IS6110 pour la détection de l’ADN de MTB dans les expectorations.Nous avons tout d’abord développé une PCR en temps réel ciblant la séquence répétée IS6110 pour la quantification de l’ADN de MTB. L’évaluation des étapes d’optimisation de la sensibilité de la PCR IS6110 a permis de préciser les performances analytiques et le gain de sensibilité comparativement à une PCR ciblant le gène unique senX3. Au terme d’une comparaison de six protocoles de lyse/ extraction la méthode Chelex® s’est avérée être la plus efficace dans la récupération de l’ADN. La performance diagnostique de la PCR optimisée a été évaluée et comparée avec la PCR automatisée Xpert MTB/RIF sur un panel de 62 échantillons respiratoires.Dans un deuxième temps, nous avons comparé la performance diagnostique de la PCR IS6110 optimisée, le test Xpert MTB/RIF et la version ultrasensible récemment commercialisée du test PCR leader Xpert MTB/RIF Ultra pour la détection de l’ADN de MTB dans des expectorations ayant une faible charge bacillaire.Enfin, à partir de 203 LCR collectés dans le cadre du diagnostic de méningites aseptiques au Burkina Faso, nous avons évalué la performance de la PCR en temps réel multiplexe (IS6110, HSV1, HSV2) combinée à l’extraction par la méthode Chelex® pour la détection de l’ADN de MTB et d’Herpès
TTuberculosis continues to be a major cause of morbidity and mortality worldwide, mainly in developing countries, despite being a curable disease. The rapid and accurate diagnosis of active TB is essential for rapid initiation of treatment and disease control. The development of new rapid diagnostic tests for active TB represents a real challenge for the optimization of the diagnosis.The main objective of our thesis work was to develop and evaluate real-time PCR approaches targeting the IS6110 insertion sequence for the detection of sputum MTB DNA. We first developed a real-time PCR targeting the IS6110 repeat sequence for the quantification of MTB DNA. The evaluation of the sensitivity optimization steps of the IS6110 PCR made it possible to specify the analytical performances and the sensitivity gain compared to a PCR targeting the single gene senX3. After a comparison of six lysis / extraction protocols, the Chelex® method proved to be the most efficient in the recovery of DNA. The diagnostic performance of optimized PCR was evaluated and compared with automated Xpert MTB / RIF PCR on a panel of 62 respiratory specimens.In a second step, we compared the diagnostic performance of the optimized IS6110 PCR, the Xpert MTB / RIF test and the highly marketed ultra-sensitive version of the Xpert MTB / RIF Ultra leader PCR assay for the detection of sputum MTB DNA. having a low bacillary load.Finally, from 203 LCR collected in the context of the diagnosis of aseptic meningitis in Burkina Faso, we evaluated the performance of the multiplexed real-time PCR (IS6110, HSV1, HSV2) combined with extraction by the Chelex® method for the detection of MTB and Herpes DNA

La toxoplasmose est une parasitose ubiquitaire occupant une large place en médecine humaine et vétérinaire. Cette affection parasitaire très fréquente en France est le plus souvent bénigne voire asymptomatique. Pourtant, elle reste redoutable en situation de transmission congénitale. De même, sa survenue chez les malades immunodéprimés a radicalement changé la conception de cette maladie. Pour compenser les insuffisances du sérodiagnostic et du diagnostic parasitologique, nous avons mis au point un diagnostic permettant un dépistage très précoce face à la suspicion d'une toxoplasmose congénitale ou cérébrale. Ce diagnostic repose sur l'amplification spécifique d'un gène répété 35 fois de Toxoplasma gondii (gène B1) associé à un système de révélation par chimioluminescence. A partir de prélèvements comparables aux liquides amniotiques humains, nous sommes capables de détecter spécifiquement un tachyzoïte. Nous avons également mis au point un modèle de PCR quantitative sur ADN utilisant un standard interne compétitif et spécifique des gènes SAGl, B1 et ARN 18S. Ce modèle est applicable à tout modèle d'étude expérimental où la quantification d'une charge parasitaire est nécessaire. Nos premiers essais ont porté sur la quantification d'une charge parasitaire cérébrale (kyste, souche Prugniaud) dans un modèle murin (BALB/C et C57 BI/6) d'encéphalite toxoplasmique. Afin de documenter une éventuelle corrélation entre l'expression des gènes de granules denses et la virulence des différentes souches du parasite, nous avons élaboré l'ensemble des outils techniques d'un modèle de RT-PCR quantitative pour le gène GRA2 (gène reconnu être impliqué dans le pouvoir pathogène). Ce système utilise une molécule standard interne compétitive, différente de 4 paires de bases comparée à la cible génomique. Les produits PCR marqués à la fluorescéine par nested-PCR, seront quantifiés à l'aide d'un analyseur automatique d'ADN. Les variations de rendement d'amplification seront quant à elles, corrigées par la quantification d'un gène rapporteur (bêta-tubuline).

Les tests moléculaires sont fréquemment demandés pour le diagnostic et le suivi des maladies infectieuses dans les pays développés. Cependant, sa disponibilité reste limitée dans les pays à ressources limitées dû à des contraintes de coûts, des technologies et des ressources humaines. Dans les régions éloignées, un accès limité aux installations de laboratoire constitue également un problème majeur. Le développement de méthodes de PCR sur plateformes ouvertes dans des laboratoires de référence tels que le Centre MURAZ au Burkina-Faso et l'utilisation d’échantillons de sang total capillaires sur DBS peuvent faciliter l'accès aux tests moléculaires.Selon la directive de l'OMS, la quantification de l'ARN du VIH à l'aide de DBS peut être une alternative dans les contextes d’accès difficile au laboratoire. Une préoccupation majeure est la spécificité sous-optimale de l’utilisation des DBS en raison de l'interférence de l'ADN du VIH archivé dans des cellules infectées sur la charge virale ARN du VIH. Dans une première étude, nous avons déterminé le niveau d'ADN du VIH-1 qui entravait la fiabilité de la quantification de l'ARN du VIH-1 sur des DBS. Une détection d'ARN du VIH-1 faussement positif (22/62, 35%) a été associée à des niveaux élevés d'ADN de VIH-1. Nos résultats indiquent que la spécificité des tests d'ARN du VIH-1 sur les DBS devrait être évaluée suivant les protocoles du fabricant sur les échantillons avec des niveaux d'ADN de VIH-1 de ≥1 000 copies / 106 cellules mononuclées du sang périphérique.Outre les infections fréquemment diagnostiquées, il est urgent d'intensifier la recherche d’infections négligées comme la leptospirose. Dans une seconde étude, nous avons exploré la leptospirose par des tests sérologiques et moléculaires comme cause négligée de maladie chez les patients atteints d’ictère fébrile au Burkina Faso. Les résultats ont montré pour la première fois que la leptospirose est une cause insoupçonnée de maladie fébrile aiguë dans ce pays semi-aride.Dans la dernière partie du doctorat, nous avons développé un test de PCR multiplex pour le diagnostic de la méningite à herpès simplex virus (HSV) et à Mycobacterium tuberculosis (MTB) chez des patients suspects de méningite aseptique. Cette qPCR qui a permis de tester dans un seul essai HSV 1/2 et MTB était très spécifique, sensible et reproductible. La concentration d'ADN la plus faible donnant 100% de signal de détection a été estimée à 2,12 copies / μl pour HSV1, 1,76 pour HSV2 et 2,15 copies / μl pour MTB. Parmi les 202 échantillons de LCR inclus dans cette étude, 5 (2,46%) étaient positifs: 2 (0,99%) pour HSV et 3 (1,47%) pour MTB. Ce test peut être particulièrement utile dans les cas de méningite / encéphalite avec un faible nombre de globules blancs dans le CSF.Notre projet a montré l'importance de la mise en œuvre de nouvelles méthodes moléculaires pour fournir des données préliminaires sur le fardeau des maladies infectieuses, y compris la leptospirose, la tuberculose et la méningite à HSV au Burkina Faso. Le DBS est un spécimen alternatif qui facilite l'accès aux tests moléculaires mais nécessite des études de validation. Les approches syndromiques doivent être testées et mises en œuvre dans les pays à ressources limitées en se basant sur l'expertise locale et la mise en œuvre de méthodes moléculaires dans les laboratoires de référence
Molecular assays are frequently requested for the diagnosis and monitoring of infectious diseases. While nucleic acid testing is the standard of care in developed countries, its availability remains limited and constrained by cost, technologies, and human resources in many settings, including West Africa. In remote areas, limited access to laboratory facilities is also a main issue. The development of PCR methods on open polyvalent platform in reference laboratories such as the Centre Muraz in Burkina-Faso and the use of capillary whole blood collected on DBS specimens can facilitate access to nucleic acid testing.According to WHO guideline HIV-RNA quantification using DBS can be considered in settings where there is a lack of access to sites or nearby laboratory facilities for nucleic acid test. A major concern is the suboptimal lower specificity of DBS due to the interference of HIV-DNA copies archived in infected cells with HIV-RNA copies. In the first study we determined the HIV-1 DNA level that interfered with the reliability of HIV-1 RNA quantification on DBS specimens used for therapeutic monitoring (1). False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Our results indicate that the specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/106 peripheral blood mononuclear cells.Beside infections frequently tested by nucleic acid tests there is an urgent need to scale up assay for neglected infection such leptospirosis. In the second study we explored leptospirosis by serological and molecular testing as a neglected cause of disease among patients with febrile icteric illness in Burkina Faso. The results showed for the first time that leptospirosis is an unsuspected cause of acute febrile illness in this semi-arid country.In the last part of the PhD, we developed a multiplex PCR assay for the diagnosis of tuberculosis and Herpes Simplex (HSV) meningitis among patients with suspected aseptic meningitis. This qPCR which allowed to test in a single run HSV 1/2 and M. tuberculosis was highly specific, sensitive and reproducible. The lowest DNA concentration giving 100% detection signal was estimated at 2.12 copies/µl for HSV1, 1.76 for HSV2 and 2.15 copies/µl for M. tuberculosis. Of the 202 CSF specimens included in this study, 5 (2.46%) were tested positive: 2 (0.99%) for HSV and 3 (1.47%) for M. tuberculosis. This assay may be especially useful in cases of meningitis/encephalitis with a low number of white blood cells count in the CSF.Our project stresses the importance of the implementation of news molecular methods to provide preliminary data about the burden of infectious diseases including leptospirosis, tuberculosis and HSV meningitis in Burkina Faso. DBS is an alternative specimen that facilitates access to nucleic acid tests but requires validation studies. Syndromic approach need to be tested and implemented in West Africa and should be based on local expertise and implementation of molecular methods in reference laboratories

BACKGROUND: Diagnostic molecular testing in colorectal cancer (CRC) offers a number of benefits including predicting prognosis, directing targeted therapies and screening for hereditary cancer syndromes. Molecular testing however is expensive, requires specialist facilities and staff and is time consuming, limiting its widespread availability. The Idylla System is an automated testing platform that could overcome these issues. AIMS: To appraise the suitability of the Idylla System for use in clinical practice by evaluating the system’s accuracy and financial impact. HYPOTHESIS: The Idylla System has high accuracy for detecting mutations in BRAF, KRAS and NRAS genes in CRC resection tissue and is a cost-effective alternative to current testing platforms. METHODS: Ethical approval was granted by Oxfordshire Research and Ethics Committee A (reference: 04/Q1604/21). Diagnostic accuracy was determined for the Idylla System in detecting BRAF and KRAS mutations with a comparison against conventional polymerase chain reaction (PCR). Further validations were also performed for BRAF, KRAS and NRAS mutation testing against NGS and IHC methods. An audit of the molecular diagnostics workload was carried out and a cost-analysis performed. RESULTS: The Idylla system had a sensitivity of 100.0% (95% CI: 88.3% to 100.0%) and a specificity of up to 100.0% (95% CI: 94.7% to 100.0%) for detecting BRAF mutations and a sensitivity of 100.0% (95% CI: 79.6% to 100.0%) and a specificity of up to 92.9% (95% CI: 68.5% to 98.7%) for detecting KRAS Mutations. There was 100% concordance for NRAS testing. A cost-analysis estimated that the Idylla System could save from around £12,000 to anywhere up to £40,000 per year in some centres. CONCLUSIONS: The results support the hypothesis that the Idylla System is an accurate system for detecting relevant mutations in CRC and demonstrate the system to be cost-effective. The Idylla system is therefore suitable for use in routine clinical practice.

Cette thèse présentait comme objectif mettre en évidence le potentiel apport de l’immuno-PCR dans le diagnostic des maladies infectieuses à travers 3 exemples. L’application de l’iPCR dans le diagnostic précoce de la fièvre Q aiguë via la détection des IgM Phase II anti Coxiella burnetii a permis de détecter 90% des sérums prélevés dans les 2 premières semaines après l’apparition des symptômes contre 55% détectés par PCR, 38% par ELISA et 35% par l’IF la technique de référence. De plus une spécificité de 92% a été retrouvée par iPCR, 100% par PCR et l’IF et 90% par ELISA. L’application de l’iPCR à la fièvre Q aiguë constitue un exemple d’application de la technique plus globalement pour tous types d’infections aiguës. D’autre part, dans le cadre de la mise au point d’un modèle expérimental murin d’infection à Tropheryma whipplei, l’iPCR a servi à mesurer la réponse immunitaire mucosale via la détection des IgA anti T. whipplei dans les selles de souris. Les résultats obtenus ont permis de confirmer le rôle de la bactérie comme agent de gastroentérite via entre autre la détection d’IgA anti T. whipplei dans les selles lorsque des atteintes intestinales étaient provoquées. Enfin l’’utilisation de l’iPCR pour la détection de l’antigène Y. pestis dans des dents anciennes a permis de confirmer Y. pestis comme agent étiologique de la peste noire dans 5 charniers à travers la France et l’Italie. Une sensibilité de 41% a été retrouvée par iPCR contre 32% par PCR et 10% par ELISA. Nos résultats suggèrent que la détection des antigènes et la détection de l’ADN du pathogène semblent être 2 approches complémentaires permettant de confirmer le rôle de Y. pestis dans les différentes pandémies de peste et de mettre fin aux controverses suscitées par la seule utilisation des techniques de biologie moléculaire.Globalement, les résultats que nous avons obtenus au cours de cette thèse démontrent le potentiel énorme de l’iPCR comme technique de détection des anticorps et d’antigènes dans le domaine des maladies infectieuses tant au niveau de la sensibilité de détection qu’au niveau de son adaptabilité à différentes applications
The objective of this thesis was to highlight the potential contribution of immuno-PCR in the diagnosis of infectious disease through 3 examples of infection. The iPCR was adapted for the early diagnosis of acute Q fever by the detection of IgM anti phase II Coxiella burnetii in patient’s sera. The results that we obtained show that iPCR could allow an early diagnosis of acute Q fever since 90% of early sera of patients with acute Q fever collected during the 2 first weeks after the onset of symptoms are detected against 55% by PCR, 38% by ELISA and 35% by IFA the gold standard. In addition, a specificity of 92% was found by iPCR, 90% by ELISA and 100% by PCR and IF. Application of iPCR to the diagnosis of acute Q fever is an example of application of the technique more generally for all types of acute infections. In a second time, the use of iPCR for the detection of Yersinia pestis antigen in ancient teeth allowed a confirmation of its role as the etiologic agent of plague in five mass graves across France and Italy. A sensitivity of 41% was recovered by IPCR against 32% by PCR and 10% by ELISA. Our results suggest that antigen and DNA detection of pathogen in ancient samples are 2 complementary approaches allowing the confirmation of the role of Y. pestis in different plague pandemic. Finally, as part of the development of an experimental murine infection with Tropheryma whippleii, the iPCR was used to measure the mucosal immune response via the detection of IgA anti T. whippleii in mouse stools. The results have confirmed the role of the bacterium as an agent of gastroenteritis via the detection of IgA anti T. whipleii in the stool when intestinal damages were caused.Overall, the results that we obtained during my thesis demonstrate the enormous potential of iPCR as a diagnostic tool of infectious disease by the ultrasensitive detection of antigens and antibodies

Dissertação de Mestrado Integrado em Medicina Veterinária
Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis are three hemoplasmas responsible for feline hemolytic anemias. The diagnosis of the infection can be performed by observation of the agents on blood smears or by their identification and quantification using PCR techniques, presently considered the gold standard method. A recombinant antigen for Mhf (rDnaK) has been identified, characterized, produced and then successfully applied in Western blot and ELISA assays (rELISA) to detect antibodies in samples from experimentally induced infections. To evaluate this application under field conditions, this rELISA was used in samples collected from naturally infected cats, in parallel with quantitative PCR and cytologic examination. Within the scope of the TNR program implemented by the Lisbon City Council, blood samples were collected from 104 cats. After sample collection, blood was immediately used to prepare blood smears which were then stained with Giemsa (Parasitology Lab, FMV/Lisbon University). The remaining blood was split into two aliquots: 1 – Plasma separation, shipped to Vetsuisse Faculty, Zurich University, for antibody testing with rELISA; 2 – Total DNA extraction for identification and quantification of the three species of hemoplasma using qPCR (Virology Lab, FMV/Lisbon University). Out of 22.1% (N=23) samples where the microorganism was identified by qPCR, mycoplasmas were also identified on the blood smears of 15.4% (N=16). 4.8% (N=5) of samples tested as seropositive and 2.9% (N=3) revealed bordeline results. From 77.9% (N=81) of samples that were negative by qPCR, only 44.2% (N=46) were also negative on cytologic examination. 60.6% (N=63) of samples were considered seronegative and 6.7% (N=7) were borderline. Sensitivity for cytology was 69.6% and specificity was 56.8%. Sensibility of rELISA was 25% and its specificity was 85.1%. Cohen’s Kappa (k) was calculated to assess agreement between PCR-Cytology (k=0.1836) and PCR-rELISA (k=0.1093). Given the low agreement, PCR was found to be the most appropriate diagnostic method. Further studies are necessary to characterize the response of the immune system and the role of different antigens in these infections in order to improve the suitability of rELISA in a clinical setting
RESUMO - HEMOPLASMAS FELINOS: AVALIAÇÃO DE ANTICORPOS ESPECÍFICOS E CORRESPONDÊNCIA COM O DIAGNÓSTICO MOLECULAR E CITOLÓGICO - Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum e Candidatus Mycoplasma turicensis são três hemoplasmas responsáveis por anemias hemolíticas em felinos. O diagnóstico desta infecção pode ser feito pela observação dos agentes em esfregaços de sangue ou através da sua identificação e quantificação pelas técnicas de PCR, sendo este ultimo considerado o método gold standard. Um antigénio recombinante do Mhf (rDnaK) foi identificado, caracterizado, produzido e posteriormente aplicado com sucesso, em técnicas de Western blot e rELISA, para a deteção de anticorpos em amostras colhidas de gatos infetados experimentalmente. De forma a avaliar a sua aplicação numa realidade clínica, esta rELISA foi utilizada em amostras colhidas de gatos naturalmente infectados, em paralelo com o PCR quantitativo e com o exame citológico. No âmbito do programa CED efetuado pela Câmara Municipal de Lisboa, foi realizada a recolha de sangue a 104 gatos. Após a colheita das amostras, o sangue foi utilizado para preparar esfregaços, corados de seguida com coloração Giemsa (Laboratório de Parasitologia, FMV/ULisboa), e dividido em duas alíquotas: 1- Separação do plasma, que foi enviado para a Faculdade Vetsuisse, Universidade de Zurique, onde realizaram a pesquisa de anticorpos com a rELISA; 2 – Extracção do ADN total para a identificação e quantificação das três espécies de hemoplasmas por qPCR (Laboratório de Virologia, FMV/ULisboa). Das 23 (22.1%) amostras PCR positivas, foram igualmente encontrados micoplasmas em 16 esfregaços (15.4%). 5 (4.8%) amostras foram consideradas seropositivas e 3 (2.9%) apresentaram resultados inconclusivos. Das 81 (77.9%) amostras negativas ao PCR, apenas 46 (44.2%) mostraram-se negativas ao exame citológico. 63 (60.6%) amostras foram consideradas seronegativas e 7 (6.7%) apresentaram valores não conclusivos. A sensibilidade da citologia foi de 69.6% e a especificidade foi de 56.8%. Por sua vez, a rELISA apresentou uma sensibilidade de 25% e uma especificidade de 85.1%. O Cohen’s Kappa (k) foi calculado para avaliar a concordância entre o PCR e a Citologia (k=0.1836) e o PCR e a rELISA (k=0.1093). Tendo em conta as baixas concordâncias, o nosso estudo confirma que o PCR é o método de diagnóstico mais adequado. Mais estudos são necessários para caracterizar a resposta imunitária e o papel dos vários antigénios destes agentes, de forma a melhorar a sua aplicação no rELISA e esta poder ser usada no contexto clínico.
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Les travaux présentés dans cette thèse font suite à celle de Melle LE GOFF. Ils se concentrent sur la technologie HIFI brevetée et développée pendant ses travaux. Une première partie du travail présenté dans ce manuscrit concerne le test HIFI Blood 96™ et plus particulièrement les améliorations et les évolutions apportées au test afin d'en faire un véritable outil de génotypage, multiparamétrique et haut-débit pouvant être installé dans les banques de sang dans le but de constituer des inventaires de sang génotypé de façon étendue, participant ainsi à améliorer la sécurité transfusionnelle. Il permet de caractériser 96 échantillons sur 15 polymorphismes (divisés en deux panels) associés aux groupes sanguins en approximativement 4h30. Cette plateforme a fait l'objet d'une étude de validation à moyenne échelle sur 583 donneurs pour le panel 1 et 190 donneurs pour le panel 2. La deuxième partie des travaux décrit l'adaptation de la technologie HIFI appliquée au diagnostic des pathologies respiratoires, avec le développement d'une autre plateforme, ReSynPlex, en partenariat avec 3 équipes de recherche de Grenoble
The work reported in this thesis follows the one undertaken by Ms LE GOFF. It is focused on HIFI technology, which is patented and developed during her thesis. The first part of this work concerns the HIFI Blood 96™ test, and particularly the improvements and developments adduced to the test to make it a real diagnostic tool, multiparametric and high-throughput which can be implemented in blood banks in order to constitute negative antigen inventories, thus contributing to improve blood safety. It allows to characterize 96 samples on 15 polymorphisms (divided in two panels) associated to blood group systems in approximately 4.5 hours. A mesoscale validation study has been conducted on 583 samples for panel 1 and 190 samples for panel 2. The second part of this work describes the adaptation of HIFI technology applied to diagnosis of respiratory tract infections, with the development of another platform, ReSynPlex, in partnership with 3 research teams in Grenoble

Tularemia is a zoonosis caused by the small, fastidious, gram-negative rod Francisella tularensis that appears over almost the entire Northern Hemisphere. In Sweden, tularemia has appeared mainly in restricted areas in northern parts of central Sweden.

The disease can be transmitted through several routes: direct contact with infected animals, by vectors, through contaminated food or water or through inhalation of aerosolized bacteria. Distinct clinical forms of the disease are seen, depending on the route of transmission. During the last years, tularemia has emerged in new areas in central Sweden, south of the endemic area. The emergence of tularemia in the County of Örebro prompted the investigations presented in this thesis.

We performed a case-control study, using a mailed questionnaire, to identify risk factors for acquiring tularemia in Sweden (Paper I). After multivariate analysis, mosquito bites and cat ownership could be associated with tularemia in all studied areas while farming appeared as a risk factor only in endemic areas.

In Paper II, we evaluated a PCR analysis, targeting the tul4 gene, used on samples from primary lesions in patients with ulceroglandular tularemia. The method performed well, with a sensitivity of 78% and a specifi city of 96%. The clinical characteristics of tularemia in an emergent area in Sweden were studied Paper III), using case fi les and a questionnaire. Of 278 cases of tularemia reported during the years 2000 to 2004, 234 had been in contact with a doctor from the Department of Infectious Diseases at Örebro University Hospital, and were thus included. The ulceroglandular form of the disease was seen in 89% of the cases, with the primary lesion, in most cases, on the lower leg. An overwhelming majority of cases occurred during late summer and early autumn, further supporting transmission by mosquitoes. Erythemas overlying the affected lymph node areas were seen in 19% of patients with forms of tularemia affecting peripheral lymph nodes. Late skin manifestations, of various appearances, were seen in 30% of the cases, predominantly in women. A raised awareness of tularemia among physicians in the county during the course of the outbreak was found, as documented by the development of shorter doctor’s delay and less prescription of antibiotics inappropriate in tularemia.

Finally, we developed a simplifi ed whole-blood lymphocyte stimulation test, as a diagnostic tool in tularemia (Paper IV). The level of IFN-γ, as a proxy for lymphocyte proliferation, was measured after 24-h stimulation. Additionally, a tularemia ELISA with ultra-purifi ed LPS as the antigen was evaluated, showing a high sensitivity. The lymphocyte stimulation test, when performed on consecutive samples from subjects with ongoing tularemia was able to detect the disease earlier in the course of the disease than both the new ELISA and the tube agglutination test. Furthermore, all tularemia cases became positive in the lymphocyte stimulation test within 12 days of disease. In conclusion, this thesis describes risk factors for acquiring tularemia as well as the clinical characteristics of the disease in Sweden. Additionally, a Francisella PCR analysis and a tularemia ELISA based on highly purifi ed LPS is evaluated, and a simplified lymphocyte stimulation test, for early confirmation of the disease, is developed.

Henrik Eliasson, Department of Infectious Diseases,

Örebro University Hospital, SE-701 85 Örebro, Sweden,

henrik.eliasson@orebroll.se

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Universidade Federal da Bahia. Faculdade de Medicina da Bahia. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
No Brasil, os cães são considerados como o principal reservatório doméstico para Leishmania infantum (sin. L. chagasi). Desta forma, o diagnóstico da leishmaniose visceral canina (LVC) deve ser rápido e preciso. Este trabalho visa comparar a performance da reação em cadeia da polimerase (PCR) em detectar o DNA do parasito em diferentes tecidos para diagnóstico da LVC. Com este intuito, na primeira parte do estudo, foi padronizado um protocolo de PCR convencional (cPCR), para detecção do DNA do minicírculo do cinetoplasto (kDNA) de Leishmania sp., em 45 fragmentos esplênicos caninos. As mesmas amostras foram avaliadas utilizando-se um protocolo de PCR quantitativa (qPCR) tendo como alvo o gene da subunidade do RNA ribossomal (SSU rRNA) de Leishmania sp. Também foi comparada a eficácia do diagnóstico para LVC pelas técnicas moleculares e convencionais como a cultura e o ELISA. A cPCR apresentou sensibilidade mais elevada para detecção de DNA de Leishmania sp. (88,9%), comparada à qPCR (83,3%). Possivelmente o melhor desempenho da cPCR foi devido ao maior números de cópias do kDNA no genoma da Leishmania sp. Diante dos promissores resultados apresentados pela cPCR tendo o kDNA como alvo, na segunda parte do estudo foi padronizado um novo protocolo de qPCR com este mesmo alvo, objetivando-se aumentar a sensibilidade da técnica. Foram selecionados aleatoriamente 61 cães errantes e classificados de acordo com o número de sinais clínicos associados à LVC apresentados. Todos os cães foram eutanasiados, e durante a necropsia, foram coletados fragmentos de linfonodo, aspirado esplênico, medula óssea e sangue. Também foram realizadas culturas esplênicas e ELISA. A qPCR foi empregada para a avalição da taxa de detecção do DNA do parasito e carga parasitária nos diferentes tecidos. As diferenças entre a carga parasitária de cada tecido foram avaliadas pelo teste de Friedman (p ≤ 0,05). Para inclusão dos tecidos nas análises dos resultados de qPCR, foi avaliada a integridade do material genético de cada amostra. Desta forma, 52 animais apresentaram resultados que atendiam aos critérios de seleção para baço, sangue e linfonodo, e destes, 24 animais também atendiam aos critérios para medula óssea. Utilizando-se a qPCR e considerando pelo menos um dos tecidos avaliados, foi detectado o DNA do parasito em todos os cães. A qPCR detectou DNA do parasito em 98,1% dos aspirados esplênicos, 80,8% das amostras sanguíneas, 53,8% dos linfonodos e 41,7% dos aspirados de medula óssea. A carga parasitária foi melhor detectada nos aspirados esplênicos, em relação ao linfonodo, nos animais oligossintomáticos e polissintomáticos (p ≤ 0,05). O aspirado esplênico foi o tecido com maior taxa de detecção do DNA de Leishmania sp. pela qPCR. No entanto, não foi achada diferença estatística entre a carga parasitária do aspirado esplênico e do sangue. Desta forma, a amostra sanguínea, por ser a segunda amostra de melhor taxa de detecção, e por necessitar uma coleta menos invasiva, foi considerada como uma amostra alternativa válida para a detecção do DNA de Leishmania sp. em cães sintomáticos, utilizando a qPCR.
Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn. L. chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The aim of the present study was to compare the performance of the Polymerase Chain Reaction (PCR) to detect parasite DNA in different clinical sample for CVL diagnosis. For this purpose, in the first stage of the study, a conventional PCR (cPCR) protocol was standardize to detect the presence of Leishmania sp. kinetoplast minicircle DNA (kDNA) in 45 canine spleen fragments. The same samples were evaluated using a quantitative PCR (qPCR) technique targeting the Leishmania sp. sub-unit of the ribossomal RNA (SSU rRNA) gene. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. The cPCR presented the highest sensitivity for Leishmania sp. DNA detection (88,9%), when compared to qPCR was (83.3%). Possibly the cPCR best performance was due to a higher copies number of the kDNA in the Leishmania sp. genome. Given the promising results presented by the cPCR targeting the kDNA, a new qPCR protocol with the same target was standardized in the second stage of the study, aiming increase the technique sensitivity. Sixty-one stray dogs were randomly selected and classified according to the number of clinical signs of CVL. All dogs were euthanized and lymph node fragments and splenic, bone marrow and blood aspirates were obtained during necropsies. ELISA and parasite culture of spleen aspirates were performed to confirm parasite infection. The qPCR was used to determine the parasite DNA detection rate and the parasite load in the clinical samples. Differences between parasite loads of each tissue were evaluated using Friedman test (p ≤ 0.05). In order to include the samples in the qPCR data analysis, the DNA integrity of each sample was analyzed. This way, 52 dogs fulfilled the selection criteria for DNA results for spleen, blood and lymph nodes, with 24 of these dogs also fulfilling the criteria for bone marrow results. Using qPCR, all the 52 dogs showed positivity, considering at least one of the tissues evaluated. Positivity in qPCR was detected in 98.1%8 of the splenic aspirates, 80.8% of blood samples, 53.8% of lymph node fragments and 41.7% of bone marrow samples. Using qPCR, parasite DNA was better detected in splenic aspirates in comparison with lymph node in both polysymptomatic and oligosymptomatic (p ≤ 0.05) dogs. Splenic aspirates have shown to be the tissue with highest Leishmania sp. DNA detection rate using qPCR, however no statistical difference was found between blood and splenic aspirate to detect. Thus, the blood sample, being the second sample with best DNA detection rate and requiring a less invasive collection, was considered a valid alternative sample for Leishmania sp. DNA detection in symptomatic dogs using the qPCR.

Notre objectif est d’évaluer la sérologie, la biologie moléculaire et la culture pour le diagnostic des infections liées aux bactéries intracellulaires.La sérologie occupe une place importante dans le dépistage, le suivi et le monitoring des patients présentant une infection cardiovasculaire à C. burnetii ou Bartonella. Cependant, nous avons montré quelques limites aux seuils précédemment établis pour le diagnostic d’endocardite. Ce travail suggère que les faibles titres d’anticorps n'excluent pas le diagnostic de l'infection cardiovasculaire chez les patients ayant des facteurs prédisposant et qu'une valeur de seuil sérologique ne peut fournir une VPP de 100%.La qPCR réalisée sur des prélèvements cardiovasculaires pour le diagnostic d’endocardite à C. burnetii et Bartonella est plus sensible que la culture et l’immunohistochimie. Toutefois, des qPCR négatives ont été obtenues chez des patients présentant une endocardite avec de fort titre d’anticorps, par conséquent une qPCR négative ne doit pas définitivement exclure le diagnostic. Nous avons montré que l’ADN est capable de persister dans les prélèvements, malgré un traitement antibiotique préalable. Nous avons alors développé un nouvel outil pour évaluer la viabilité bactérienne en quantifiant la transcription de l'ARNr 16S de C. burnetii.La culture des bactéries intracellulaires reste nécessaire pour permettre la caractérisation des bactéries et faciliter le développement d'outils diagnostiques. Au cours de cette thèse, nous avons mis au point une technique innovante de plage de lyse pour mettre en évidence un effet délétère des antibiotiques sur les cellules infectées par R. conorii
The aim of our study is to evaluate serology, molecular biology and culture for the diagnosis of intracellular bacteria.Serology plays an important role in the detection of Q fever and Bartonella infections and for the follow up and monitoring of patients with cardiovascular infection. However, we have shown some limits to the use of serological thresholds previously established for the diagnosis of endocarditis. In 2 series of Q fever and Bartonella endocarditis, we diagnosed patients with a definite cardiovascular infection associated with low antibody levels (<800). This work suggests that low antibody titers do not exclude the diagnosis of cardiovascular infection in patients with predisposing factors and a value of serological threshold cannot provide a positive predictive value of 100%.qPCR performed on cardiovascular samples for the diagnosis of C. burnetii and Bartonella endocarditis is more sensitive than the amplification of the 16S rRNA gene, culture and immunohistochemistry. Nevertheless, negative qPCR were obtained for patients presenting endocarditis with high antibody titer, therefore a negative qPCR should not definitively exclude the diagnosis. On the other hand, we have shown that DNA can persist in clinical specimens, despite previous antibiotic treatment. We developed a new tool to assess bacterial viability by quantifying the transcription of the 16S rRNA of C. burnetii.Culture of intracellular bacteria is necessary to enable the characterization of bacteria and facilitate the development of diagnostic tools. We developed an innovative technique of plaque assay to highlight a deleterious effect of antibiotics on infected cells by R. conorii

Les virus du genre Begomovirus (famille Geminiviridae) sont fréquemment détectés en association avec des ADN satellites appelées alphasatellite et betasatellite qui font la moitié de la taille du génome viral. L’alphasatellite est autonome pour sa réplication et dépend du virus pour son mouvement et son encapsidation tandis que le betasatellite est dépendant de ces fonctions virales. L’alphasatellite a rarement été montré comme ayant un impact sur le virus assistant, contrairement au betasatellite qui augmente la virulence de son virus assistant. En dehors des bégomovirus tels que le Cotton leaf curl virus (CLCuV) qui ont besoin d’un betasatellite pour initier une infection symptomatique dans leur hôte naturel, la plupart des bégomovirus peuvent causer des symptômes, même sans les satellites avec lesquels ils sont parfois détectés. Le Tomato yellow leaf curl virus (TYLCV), un des virus les plus dommageables dans le monde a rarement été détecté associé à des ADN satellites. Les souches méditerranéennes qui sont aussi les plus invasives, n’ont jamais été détectées avec des ADN satellites, bien qu’elles soient capables en conditions artificielles de les assister avec pour conséquence une considérable augmentation de la virulence en cas de co-inoculation avec un betasatellite. Le risque potentiel d’association de satellites avec le TYLCV-Mld a été évalué en testant divers facteurs potentiellement impliqués dans le maintien de l’association TYLCV-satellite: (i) l'accumulation relative intra-plante du TYLCV et des satellites, (ii) la fréquence de co-infection au niveau cellulaire du TYLCV et des satellites, et (iii) l'efficacité de transmission des satellites par le vecteur Bemisia tabaci. Trois satellites précédemment isolés sur coton au Burkina Faso ont été montrés comme pouvant être assistés par le TYLCV dans des plantes de tomate: Cotton leaf curl Gezira betasatellite (CLCuGB), Cotton leaf curl Gezira alphasatellite (CLCuGA) et Okra leaf curl Burkina Faso alphasatellite (OLCBFA). La quantification par PCR quantitative des ADN du TYLCV et des trois satellites entre 11 et 150 jours après inoculation (dpi) révèle qu’en général, les satellites ont une accumulation supérieure à celle du virus, et que, contrairement aux alphasatellites qui n’ont aucun impact, le betasatellite affecte l’accumulation du TYLCV-Mld. Bien que le rapport des quantités de virus/satellites varie au cours du temps, les satellites sont maintenus avec le TYLCV-Mld au temps tardif de 150 dpi et sont transmis par B. tabaci à 32 et 150 dpi. Le TYLCV-IL interagit différemment avec le CLCuGB car son accumulation n’est pas affectée dans les plantes coinfectées.L’estimation par la technique FISH à 18 et 32 dpi de la fréquence d’association des molécules au niveau cellulaire montre que plus de la moitié des cellules infectées sont coinfectées par le TYLCV et un satellite. Ce résultat est cohérent avec la fréquence observée d’ADN satellite dans les plantes. Cependant, on observe de manière inattendue un nombre important de cellules ne semblant contenir que le betasatellite, ce qui pose des questions sur le fonctionnement des associations virus/satellites. Comme la multiplicité d'infection (MOI) des bégomovirus et des satellites est attendue pour être un facteur déterminant de l’efficacité de la co-infection cellulaire, deux variants équi-competitifs de TYLCV ont été préparés afin de déterminer ce paramètre. Enfin, des amorces PCR permettant la détection générique de betasatellites ont été dessinées pour être utilisées dans le diagnostic par l'Agence française pour l'alimentation, l'environnement et la santé et sécurité au travail (ANSES). Outre les conséquences agronomiques d’un maintien possible des satellites avec le TYLCV, les résultats de cette étude donnent un aperçu novateur sur les interactions entre les bégomovirus et les satellites, au niveau de la plante, au niveau cellulaire et moléculaire
Begomoviruses (family Geminiviridae) are frequently detected with half genome sized defective virus DNAs, and for some of them with satellite DNAs of similar size, i.e. alphasatellite and betasatellite. Both molecules rely on the virus for maintenance in plant. The alphasatellite was rarely proved to have an impact on the helper virus but the betasatellite was often shown to increase its virulence. Except some begomoviruses, like Cotton leaf curl virus (CLCuV) which rely on a betasatellite for a full symptomatic infection in its natural host plant, most of the begomoviruses which were frequently detected with satellites do not rely on them for infectivity. Tomato yellow leaf curl virus (TYLCV) is one of the most damaging begomovirus worldwide. The Mediterranean IL and Mld strains, the most invasive ones, were never detected in association with satellites, although they were experimentally proved to readily assist them for replication and movement in plant. This was particularly true for betasatellites and resulted in a dramatic increase in the virulence of TYLCV.The potential of a TYLCV-satellite association was assessed by testing various factors involved in the maintenance of both molecules in tomato plants: (i) the relative intra-plant accumulation of TYLCV and satellites, (ii) the frequency of host cells co-infected with TYLCV and satellites, and (iii) the transmission efficiency of satellites by the natural whitefly vector of TYLCV, Bemisia tabaci. Three satellites previously isolated from okra in Burkina Faso, were shown here to be assisted by TYLCV in tomato plants: Cotton leaf curl Gezira betasatellite (CLCuGB), Cotton leaf curl Gezira alphasatellite (CLCuGA) and Okra leaf curl Burkina Faso alphasatellite (OLCBFA). The dynamic of TYLCV and satellite DNAs monitored between 11 and 150 days post-inoculation (dpi) by quantitative PCR revealed that satellites accumulated at a higher level than the virus, and that, in contrast with alphasatellites which have no impact, betasatellites affected TYLCV-Mld accumulation. Although the ratio of virus/satellite amounts varies over time, satellites were maintained in all test plants up to 150 dpi and were readily transmitted at 32 and 150 dpi. TYLCV-IL interacts differentially with CLCuGB as its accumulation was not affected in the coinfected plants.At 32 dpi, the TYLCV/satellite infection status of plant cells was determined by FISH and more than 50% of the monitored infected cells were co-infected with TYLCV and a satellite. The infection status was consistent with the frequency of satellite DNA in plants. Unexpectedly a substantial number of cells were positive only for betasatellite, suggesting that the coinfection with the virus could be dispensable for replication. This observation raises question on the functioning of virus/satellite association or multipartite viruses. As the multiplicity of infection (MOI) of begomoviruses and satellites is expected to be a determinant of the efficiency of virus/satellite cell coinfection, two equi-competitive TYLCV variants were prepared to determine this parameter for TYLCV. Finally, PCR primers designed for the generic detection of betasatellites were designed to be used as a diagnostic tool by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES).Besides the agronomic concern of the possible maintenance of DNA satellites with TYLCV, the results of our study are expected to provide a new insight on the interactions between begomovirus and satellites, at the plant, cellular and molecular levels

A Síndrome de Martin-Bell é uma forma hereditária de retardo mental determinada pela perda da expressão do gene FMR1 que está associado com a expansão da repetição dos tri-nucleotídeos citosina-guanina¬-guanina (CGG). Os indivíduos com um alto grau dessa expansão apresentam o silenciamento da expressão desse gene, com conseqüente alteração no desenvolvimento do sistema nervoso do embrião, causando danos neurológicos irreparáveis. A citogenética é uma metodologia clássica que contribui para o diagnóstico da síndrome em casos sem esclarecimentos, porém sua sensibilidade isoladamente em muitos casos é insuficiente para um diagnóstico positivo mesmo diante de características clínicas evidentes. Na busca de uma alternativa para suprir esta necessidade de definir exatamente as alterações encontradas em cada caso propôs-se a otimização de uma PCR de alta fidelidade no diagnóstico diferencial da Síndrome e simultaneamente comparar com os dados da citogenética clássica. Para tanto, foram coletadas amostras de sangue periférico de 102 pacientes e avaliou-se 100 a 150 metáfases para cada indivíduo pela citogenética clássica e para a PCR duplex. Os resultados demonstram pelo teste de Kruskal-Wallis que a PCR com a citogenética não apresentou diferenças significativas (p>0,05). Porém quando avaliadados pelo índice de Kappa sugere-se que os dois critéiros de diagnósticos devem ser utilizados simultaneamente com caracteristicas clínicas do paciente. A PCR mostrou-se rápida e com custo relativamente menor, sendo portanto, aplicável no auxílio ao aconselhamento genético dos indivíduos e suas famílias, bem como para um acompanhamento psico-pedagógico adequado.
The Martin-Bell Syndrome is a heredity mental retard form determined by the loss of FMR1 gene expression which is associated with the tri-nucleotides cytosine-guanine-guanine (CGG) repetition expansion. The individuals showing a high degree of this expansion present the silencing of this gene expression, with a consequent alteration of the embryo nervous system evolution, causing irreparable neurological damage The cytogenetics is a classical methodology that contributes for diagnosing the syndrome in cases without clarification, thus its sensitivity isolated in many cases is insufficient for a positive diagnosis even in front of evident clinical characteristics. On searching for an alternative to fulfill this need to define the found alterations in each case, an optimization of a high fidelity PCR to Syndrome diagnosis and simultaneously to compare with classical cytogenetics. Peripheral blood samples were collected from 102 patients for evaluating 100 to 150 metaphases evaluation by classical cytogenetics for each sample and to duplex PCR. The results showed by the Kruskal-Wallis test that the PCR with the cytogenetics have not presented significant differences (p>0,05). However, when evaluated by the Kappa index it was suggested that the two diagnosis criteria should be used simultaneously with patient s clinical characteristics. The PCR showed itself quick and with a relatively lower cost , and in this way, applicable in helping genetically counseling individuals and their families, as well as sending them to a more adequate psycho-pedagogical treatment.

Lyme borreliosis, more commonly referred to as Lyme disease, is the fastest growing zoonotic disease in North America with approximately 30,000 confirmed cases and 300,000 estimated infections per year. In nature, the causative agent of Lyme disease, the bacterium Borrelia burgdorferi, cycles between Ixodes sp. ticks and small mammals. Humans become infected with Lyme disease after being bitten by an infected tick. The primary indicator of a Borrelia burgdorferi infection is a bull's eye rash typically followed by flu-like symptoms with treatment consisting of a 2-4 week course of antibiotics. If not treated, later stages of the disease can result in arthritis, cardiovascular and neurological symptoms. Diagnosis of Lyme disease is challenging and currently requires a complex laboratory diagnostic using indirect detection of host-generated antibodies by a two-tiered approach consisting of an enzyme linked immunosorbent assay (ELISA) followed by IgM and IgG immunoblots. Although two-tier testing has provided an adequate approach for Lyme disease diagnosis, it has weaknesses including subjective analysis, complex protocols and lack of reagent standardization. Immuno-PCR (iPCR) is a method that combines ELISA-based detection specificity with the sensitivity of PCR signal amplification and has demonstrated increased sensitivity for many applications such as detection of disease biomarkers but has yet to be applied for diagnosis of Lyme disease. Herein, using iPCR and recombinant B. burgdorferi antigens, an assay for both the direct and the indirect detection of Lyme disease was developed and demonstrated improved sensitivity for detection of B. burgdorferi antibodies using a murine model. Moreover, we present evidence using human Lyme disease patient serum samples that iPCR using both multiple antigens and a unique single hybrid antigen is capable of achieving increased sensitivity and specificity compared to existing methodology. These data represent the first demonstration of iPCR for Lyme disease diagnosis and support the replacement of two-tier testing with a more simplified and objective approach.
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences

Salmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.

Les techniques de biologie moléculaire ont pris au cours des 20 dernières années une place importante dans le diagnostic direct des pathogènes viraux. Notre travail a porté sur la mise en place et le développement d’une plate-forme de biologie moléculaire, au sein du laboratoire de virologie de l’hôpital de la Timone, pour répondre aux demandes et contraintes du diagnostic en milieu hospitalier. L’organisation de cette plate-forme a nécessité plusieurs étapes : la prévention des risques de contamination, l’aliquotage et le stockage des réactifs, l’automatisation des techniques d’extraction des acides nucléiques, la mise au point de témoins positifs synthétiques et de témoins internes et l’optimisation des protocoles de PCR. Cette approche optimisée du diagnostic moléculaire des infections virales a été appliqué notamment à la détection de la grippe pandémique A/H1N1v dans les laboratoires de routine hospitalière et d’urgence « Point Of Care ». La mise en place de cette plate-forme a fait progresser de manière considérable le diagnostic moléculaire du laboratoire. Elle nous permet actuellement de détecter un grand nombre de pathogènes (>80) et de réaliser des tests dans un format à haut débit (≈40 000 tests/an). Au total, cette plateforme est au coeur de la capacité du laboratoire pour réagir de manière rapide aux évènements d'émergence en mettant en place rapidement des procédures diagnostiques standardisées. Ces techniques ont été transférées à de nombreux autres laboratoires de virologie partenaires nationaux et internationaux. Nous envisageons maintenant son utilisation dans une approche syndromique avec notamment, le développement du diagnostic des virus respiratoires
Molecular biology techniques have taken an important role in the direct diagnosis of viral pathogens over the last 20 years. Our work focused on establishing and developing a platform for molecular diagnosis in the laboratory of Virology (Timone Hospital) to meet the demands and constraints of diagnosis in hospitals. The organization of this platform required several steps: prevention of contamination risks, aliquoting and storage of reagents, automation techniques of nucleic acid extraction, development of synthetic positive controls and internal controls and optimization of PCR protocols. This optimized approach of the molecular diagnosis of viral infections has particularly been applied to the detection of pandemic influenza A/H1N1v in hospital laboratories for routine and emergency "Point Of Care." The implementation of this platform has significantly improved molecular diagnosis in our laboratory. It currently allows us to detect a large number of pathogens (> 80) and perform tests in a high-throughput (≈ 40,000 tests per year). In total, this platform is at the heart of the laboratory capacity to react quickly to emerging events by rapidly implementing standardized procedures. These techniques have been transferred to many other partners’ laboratories nationally and internationally. We are now considering its use in a syndromic approach including the development of the diagnosis of respiratory viruses

Beak and Feather Disease Virus (BFDV) is a circovirus which causes ill-thrift, feather loss and immunosuppression leading to secondary infections and eventually death in psittacine birds. The development of standardised reagents for the detection and characterisation of BFDV infections and for the production of protective vaccines has been difficult as no cell culture system has yet been found to grow the virus successfully in vitro. However, the development of consistent and effective diagnostic tests and vaccines is now more practical through the application of nucleic acid-based detection methods and recombinant technology. A quantitative real-time PCR assay for the detection of BFDV DNA was developed, using primers designed to amplify a conserved 81 bp fragment of ORFV1 and SYTO9, a fluorescent intercalating dye, with assays run on a Corbett RotorGene 3000. A synthetic oligonucleotide was used to establish standard curves for the quantitation of viral load in both blood and feather preparations. The assay was very sensitive, with a detection limit of 50 copies/ìL. The assay was developed using BFDV-positive DNA extracts from the feathers of 10 different species of birds and validated with blood and feather samples from corellas vaccinated with an experimental BFDV vaccine, then challenged with live virus. Viral DNA was reliably detected in the blood of all control (non-vaccinated) birds and in some vaccinated birds. Contamination of the environment with the feather dander of BFDV-infected birds meant that HA feather preparations were unreliable for the detection and quantitation of viral excretion. Nonetheless, the assay should prove to be a useful and sensitive test for the detection of viral DNA in a range of samples in future investigations. A recombinant BFDV capsid protein was also produced and a specific monoclonal antibody developed against it. The behaviour of the protein in haemagglutination (HA) assays and the behaviour of the monoclonal antibody in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays were characterised. The protein had the ability to agglutinate galah erythrocytes as per the wild-type virus and this agglutination was successfully inhibited by antibodies to wild-type BFDV from naturally immune psittacine birds. Furthermore, the protein self-assembled into virus-like particles as determined by electron microscopy. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from 3 genera of psittacine birds, including the recently described cockatiel BFDV isolate. A novel “blocking” (or “competitive”) ELISA (bELISA) for the detection of anti- BFDV antibodies in psittacine sera (Ab-bELISA) was also developed and validated with 166 samples from eastern long-billed corellas vaccinated with the recombinant capsid protein and challenged with live virus. The bELISA was found to be both sensitive and specific and correlated strongly with the HI test, thus it should have wide application for the serodiagnosis of BFDV. A survey of cockatiels (n=88) housed at commercial aviaries was conducted to investigate whether BFDV infection occurs in cockatiels. All birds were diagnosed as being virus-free by PCR and HA and had no detectable antibody titre by HI assay. In addition to this, the genomes of two BFDV isolates obtained from diseased cockatiel feathers were sequenced and cross-reactivity assays performed using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first report of an antigenically distinct BFDV in psittacine birds. Since the Ab-bELISA has a lower limit of detection than the HI assay, it was used to repeat the cockatiel sero-survey. No antibodies were detectable in any of the cockatiels tested and thus questions about the real prevalence of BFDV infection in cockatiels and the possible existence of a novel BFDV serotype adapted to cockatiels remain unanswered. The successful control of PBFD in both pet and wild birds depends on the development of vaccines that incite a strong specific immune response and can be efficiently produced in large quantities. Recombinant BFDV capsid proteins have recently been considered as candidate vaccines against BFDV and recombinant techniques allow the development of other candidate vaccines, including DNA vaccines. In order to examine the potential of DNA vaccination as a strategy for the prevention and control of BFDV, two DNA vaccines, based on the nucleotide sequence encoding the capsid protein of BFDV, were developed using the mammalian expression vector pVAX1. The vaccine constructs encoding both the full length and NLS-truncated capsid protein resulted in protein expression both in vitro and in vivo. Protein was detected in COS-7 cells transfected with the constructs with an indirect immunocytochemistry assay using the monoclonal antibody described in Chapter 5. Protein was present in the nucleus of cells transfected with the vaccine encoding the full-length nucleotide sequence and in the cytoplasm of cells transfected with the vaccine encoding the NLS-truncated sequence as expected. Both DNA vaccine constructs induced detectable levels of anti-BFDV antibodies in vaccinated birds, determined using the Ab-bELISA described in Chapter 5. Thus, DNA vaccines similar to those presented here may have application in the prevention and control of BFDV and some options for the further development of these vaccines into effective methods for the control of BFDV are discussed.

O Brasil é omaior exportador e o terceiro maior produtor de carne de aves, atrás apenas de Estados Unidos e China. Para que o país se mantenha neste patamar e continue a crescer como produtor de frango, é fundamental que o setor avícola brasileiro priorize as questões sanitárias. Dentre as doenças que acometem as aves, um dos grupos mais importantes são aquelas que afetam o trato digestivo. As infecções intestinais em aves podem ser determinadas por bactérias, protozoários, micotoxinas e vírus. Os vírus entéricos das galinhas, incluindo Adenovírus, Astrovírus, Reovírus e Rotavírus, têm sido descritos como possíveis causadores da síndrome da má absorção (runting stunting syndrome), caracterizada principalmente por deficiência no crescimento, desenvolvimento retardado das penase diarréia. Neste estudo foram avaliadas amostras provenientes de aves de produção de diferentes origens (frangos de corte, reprodutoras e poedeiras). O conteúdo intestinal de aves oriundas de lotes com sinais clínicos entéricos e também lotes com ausência destes sinais foi submetido ao diagnóstico diferencial por PCR para quatro agentes virais: Adenovírus, Astrovírus, Reovírus e Rotavírus. Das 162 amostras testadas, 65 amostras foram positivas (40,1%) para um ou mais vírus e 97 amostras foram negativas (59,9%),sendo 2 amostras (1,2%) positivas para Adenovírus, 50 (30,9%) amostras positivas para Astrovírus, 19 (11,7%) amostras positivas para Reovírus e 12 (7,4%) foram positivas para Rotavírus.Astrovírus, Reovírus e Rotavírus parecem desempenhar papel importante como agentes causadores de problemas entéricos em aves de produção, especialmente em frangos de corte. Entretanto, mais estudos direcionados à detecção, isolamento, inoculação experimental, possíveis interações e caracterização molecular relacionados aos vírus entéricos são fundamentais para melhor esclarecimento da participação destes como agentes causadores de doença em aves.
Brazil is the largest exporter and the world\'s third largest poultry produces, behind USA and China. It is essential that Brazil priorizes healthy issuesin order to maintain this position and keep growing as poultry producer. Diseases that affect the digestive system of chickens are one of the most important group of diseases in poultry flocks.There are different causes of Eenteric infections in chickens may be caused by, such as bacterias, protozoans, mycotoxins and viruses.Enteric viruses, like adenovirus, astrovirus, reovirus e rotavírus, have been described as possible cause of Runting Stunting Syndrome (RSS) in poultry, characterized by growth problems, poor feathering and diarrhea. In this study were evaluated samples from laying hens, breeders and broilers. The intestinal content from healthy and affected flocks were tested by PCR for four enteric viruses from chickens: adenovirus, astrovirus, reovirus e rotavirus. From the 162 samples tested, 65 (40,1%) were positive for one or more viruses and 97 (59,9%) were negative. Two samples (1,2%) were positive for adenovirus, 50 (30,9%) were positive for astrovirus, 19 (11,7%) were positive for reovirus and 12 (7,4%) were positive for rotavírus. Astrovirus, reovirus and rotavírusseem to play a important role as causative agent of enterical disorders in chickens, mainly in commercial broilers. However, more studies about detection, viral growth, molecular characterization, experimental inoculation and possible interaction with other agents related to enteric viruses in chickens are necessary to elucidate the participation of these viruses as causative agent of diseases in chickens.

Bovine genital campylobacteriosis is a disease of difficult diagnostic because of the microaerophilic nature of its etiologic agent, Campylobacter fetus, thereby causing the prevalence related to this disease to be underestimated. The purpose of this study was to evaluate the utilization of PCR for the diagnosis of genital campylobacteriosis, using samples obtained from bull prepuce aspirate, cow cervical mucus, and abomasum contents obtained from aborted fetuses, collected in transport and enrichment medium. Five different DNA extraction protocols were tested: thermal extraction, lyses with proteinase K, lyses with guanidine isothiocyanate, lyses with DNAzol? , and lyses with CTAB. The specificity, sensitivity and technical application of PCR assay were also evaluated with clinical samples. The PCR performance was compared to the culture technique for bacterial isolation. The CTAB was the most efficient extraction protocol; the pair of primers used was shown to be specific; and the limit of detection was of 63 CFU of Campylobacter fetus. PCR demonstrated that 24% (68/277) of the clinical samples were positive for Campylobacter fetus, while only 2.8% (8/277) of samples were positive by culture techinque. These results indicate that the PCR technique is specific and sensitivy, and is superior to the culture for the diagnosis of bovine genital campylobacteriosis.
A campilobacteriose genital bovina, caracterizada principalmente por infertilidade, é uma doença de difícil diagnóstico, devido à natureza microaerófila do seu agente etiológico, o Campylobacter fetus, tendo assim sua prevalência subestimada. Este trabalho teve por objetivo avaliar a utilização da técnica de PCR para o diagnóstico da campilobacteriose genital bovina, utilizando amostras de aspirado prepucial, muco cervical e conteúdo abomasal de fetos abortados, coletadas em meio de transporte e enriquecimento; bem como comparar seu desempenho com a técnica do isolamento bacteriano. Foram testados cinco diferentes protocolos de extração de DNA de Campylobacter fetus: termo extração, lise com proteinase K, lise com isotiocianato de guanidina, lise com DNAzol? e lise com brometo de cetiltrimetilamônio (CTAB). Também foram avaliadas a especificidade, a sensibilidade e a aplicação da técnica da PCR em amostras clínicas. Os resultados indicaram que o CTAB foi o protocolo de extração mais eficiente; o par de primers utilizado mostrou-se específico; e o limite de detecção foi 63 unidades formadoras de colônias (UFC) de C. fetus. A PCR encontrou 24% (68/277) das amostras clínicas positivas para C. fetus, enquanto a cultura encontrou 2,8% (8/277). A técnica da PCR é específica e sensível, e mostra-se superior a cultura no diagnóstico da campilobacteriose genital bovina.

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

Namao virus (NV) was associated with mortality in lake sturgeon Acipenser fulvescens reared as part of a conservation stocking program for this endangered species in Manitoba, Canada. The virus itself was large, doubly encapsidated and icosahedral-shaped. Phylogenetic analyses using the major capsid protein showed that NV and other epitheliotropic sturgeon nucleo-cytoplasmic large DNA viruses shared a common evolutionary past and formed a distinct evolutionary lineage within Megavirales. Three PCR tests were developed and their analytical performance was validated for detection of these viruses. Testing of wild sturgeon revealed that NV is endemic in the Nelson River water basin in Manitoba. Bath exposure resulted in transmission of NV to healthy sturgeon. The gills appeared to be the initial site of infection with virus persisting in the head skin tissue for up to 62 days. The molecular tests will be useful tools for disease management in sturgeon conservation stocking programs.
October 2015

Le développement d’un nouveau concept d’analyse biologique permettant de suivre en temps réel la réplication de séquences cibles d’ADN lors d’une PCR constitue l’objectif de ce travail. Nous avons envisagé le suivi de la PCR par des mesures électrochimiques, lesquelles présentent un certain nombre d’avantages (robustesse, faible coût, miniaturisation aisée,. . . ) contrairement aux méthodes commercialisées, basées sur des approches optiques. Un état de l’art sur l’amplification et la détection de fragments d’ADN sera d’abord décrit, en particulier la PCR en temps réel. Diverses méthodes non optiques de détection de l’ADN, notamment électrochimiques, seront également proposées. La première approche envisagée repose sur l’oxydation catalytique d’une base de l’ADN (dGTP) par un médiateur redox : Ru(bpy)32+. Les mesures obtenues à partir de ce couple, bien que peu reproductibles, font cependant la preuve de la faisabilité du concept. L’utilisation d’un nouveau couple : Os(bpy)32+/7-deaza-dGTP, dont le pic d’oxydation se situe à un potentiel plus bas, a permis d’améliorer les capacités de la méthode et d’atteindre une limite de détection de 30 aM. Toutefois celle-ci est environ 200 fois moins performante qu’une technique commerciale utilisant des sondes TaqMan™. Une nouvelle approche basée sur l’intercalation d’un complexe d’osmium dans le double brin de l’ADN a permis d’améliorer grandement les performances de notre PCR pour atteindre des résultats proches des techniques commerciales tant en termes de limite de détection que de sensibilité. Ces résultats démontrent ainsi l’intérêt de l’électrochimie comme nouvel outil de mesure de fragments d’ADN par PCR en temps réel
The aim of this work is to develop a new method for detection of the DNA amplification during the PCR. This technique based on the electrochemical signal gives to the method advantages as reliability, low cost and amenable for miniaturization in contrast to the fluorescent techniques usually used, which are expensive, bulky and fragile. First of all, we present a state of the art concerning different methods used to replicate specific sequences of DNA by enzymatic reactions which are based on fluorescent methods. In this chapter, we pay particular attention to the real time PCR and other non-optic methods of DNA detection, especially electrochemical one. The first approach developed in this work is based on the catalytic oxidation of a triphosphate nucleoside by a redox mediator. The weak reproducibility of the measures made with the couple mediator/base, Ru(bpy)32+/dGTP, is due to the high potential of oxidation of the dGTP. Nevertheless, the proof of concept is done. Another couple: Os(bpy)32+/7-deaza-dGTP, with a lower potential of oxidation, improve the performances of the method. So it is possible to detect 30 aM of viral DNA but this detection limit is about 200 times worse than the commercial kit using fluorescent probes as TaqMan™. Then a new concept was proposed based on the utilisation of an osmium-complex DNA intercalator. Results obtained showed good performances compared with TaqMan™ probes in terms as detection limit or sensitivity. These results emphasize electrochemistry as a new tool to detect DNA by real-time PCR

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

La présence de Leishmania infantum et Toxoplasma gondii en Provence Alpes Côte d’Azur (PACA) est connue depuis plus d’un siècle. Depuis, leur distribution évolue, l'environnement change, les populations touchées se déplacent, et de nouveaux outils techniques et statistiques permettent de mieux les saisir. Une réactualisation de nos connaissances paraissait donc nécessaire. Nous avons d’abord mené une revue de la littérature sur les leishmanioses viscérales. Ensuite, nous avons montré que la leishmaniose muqueuse à L. infantum est marquée par un probable sous-diagnostic, un caractère peu invasif localement et un risque de viscéralisation significatif. Puis une étude éco-épidémiologique a montré que les deux foyers de leishmaniose en PACA impliquaient des biotopes différents, avec une transmission en zone urbanisée dans le foyer marseillais. Enfin, une étude entomologique a confirmé cette transmission urbaine.Nous avons ensuite étudié la toxoplasmose congénitale. D’abord, nous avons essayé d'améliorer les performances techniques du dépistage en montrant l’intérêt pour le diagnostic moléculaire anténatal d’une extraction optimisée de l’ADN parasitaire sur liquide amniotique en utilisant NucliSENS easyMAG plutôt qu’une extraction manuelle utilisant QIAamp DNA minikit. Nous avons également montré l’apport pour le diagnostic néonatal de la toxoplasmose congénitale des IgM ciblant des antigènes de haut poids moléculaire lors de la comparaison des sera des mères et des enfants par Western Blot. Enfin, nous avons rapporté l’évolution sur 16 ans de 127 patients traités pour toxoplasmose congénitale et montré que 19% des enfants présentaient une choriorétinite au cours du suivi
The epidemiology of Leishmania infantum and Toxoplasma gondii in the Mediterranean basin has been studied for more than a century. Yet, our understanding of these diseases must be updated because ongoing environmental modifications impact their distribution, because affected population change, and because new technical and statistical tools have become available. We first reviewed scientific literature about visceral leishmaniasis. Then, we conducted a clinical study about autochtonous mucosal leishmaniasis due to L. infantum: we showed that this disease was characterized by underrecognition, low local invasiveness, and risk of visceral spreading. Afterwards, an eco-epidemiological study showed that foci of leishmanisis involved different biotopes in South-Eastern France: we specifically highlighted a urban transmission in the Marseille focus. Finally, an entomological survey confirmed this urban transmission and addressed cocirculation with phleboviruses.Then, we studied congenital toxoplasmosis. We contributed to improve technical performances of current screening strategy: we first showed that an optimized extraction of Toxoplasma DNA from amniotic fluid using NucliSENS easyMAG proved superior to manual extraction using QIAamp DNA minikit. Then, we found that comparison of mother and child antibodies that target high-molecular-mass Toxoplasma gondii antigens by immunoblotting improves neonatal diagnosis. Finally, we reported the 16-year long evolution of 127 children congenitally infected with T. gondii and showed that despite early treatment 19% of children finally developed chorioretinitis