<p>Uvod: Virusni gastrointestinalni sindrom je aktuelni zdravstveni problem u celom svetu. To važi kako u razvijenim zemljama, tako i u zemljama u razvoju, a posebno u nerazvijenim zemljama, gde je drugi po redu uzrok mortaliteta. Nagli početak bolesti, praćen pojavom velikog broja tečnih stolica, mukom, povraćanjem, bolovima u stomaku, temperaturom, malaksalo&scaron;ću, ima za posledicu dehidrataciju. U svim starosnim grupama obolelih, a naročito kod sasvim male dece, starih i imunodeficitarnih osoba može da dođe do smrtnog ishoda, ukoliko se brzo ne postavi tačna etiolo&scaron;ka dijagnoza bolesti i ne pristupi se odmah nadoknadi vode i elektrolita, kao i primeni svih ostalih mera simptomatske terapije. Brzo postavljena tačna dijagnoza, &scaron;to se najbolje postiže real-time PCR testom, sprečava pojavu komplikacija, pa i fatalnog ishoda bolesti. Istovremeno, omogućava primenu odgovarajućih epidemiolo&scaron;kih mera da se spreči nastanak epidemija i njihovo &scaron;irenje. Cilj ovog istraživanja bio je da se tačno utvrdi incidenca virusnog gastrointestinalnog sindroma u Vojvodini i učestalost pojave epidemijskog i sporadičnog javljanja ove bolesti. Cilj je bio i postavljanje algoritma za primenu real-time PCR testa u dijagnostici virusnog gastrointestinalnog sindroma u budućem radu. Isto tako, cilj je bio da se molekularnom analizom, sekvenciranjem delova genoma pozitivnih uzoraka stolice, izvr&scaron;i genetska tipizacija i odredi filogenetska pripadnost virusa. Materijal i metode: Tokom petogodi&scaron;njeg istraživanja molekularnim real-time PCR testom pregledane su 1003 obolele osobe sa simptomima virusnog dijarealnog sindroma, starosti od mesec dana do preko 90 godina. Pregledani su na rota, noro, astro i enterične adenoviruse. Na osnovu podataka iz anketnih upitnika i istorija bolesti, detaljno su analizirani svi klinički pokazatelji (javljanje bolesti tokom godine, trajanje bolesti, simptomi). Procena težine kliničke slike vr&scaron;ena je prema Vesikari skali. Svi podaci su upoređivani prema vrsti virusnog uzročnika, prema starosti obolelih, godinama trajanja istraživanja i epidemijskom i sporadičnom javljanju oboljenja. Dobijeni podaci su statistički obrađeni, tabelarno i grafički prikazani. Rezultati: U petogodi&scaron;njem periodu real-time PCR testom pregledan je uzorak od 1003 obolele osobe različite starosti na 4 virusna uzročnika dijarealnog sindroma (rota, noro, astro i enterične adenoviruse). Virusni dijarealni sindrom dokazan je kod 709 obolelih (70,69%). Najče&scaron;će su dokazane rotavirusne infekcije u 28,81%. Statistički značajno najče&scaron;će rotavirusi su bili utvrđeni kod dece do 5 godina (38,90%), ali u visokom procentu i kod dece uzrasta 6 do 14 godina (24,83%). Deca mlađa od 5 godina imala su statistički značajno najtežu kliničku sliku, bila su če&scaron;će hospitalizovana i imala su statistički značajno vi&scaron;u temperaturu. Pored vi&scaron;e temperature kod obolelih od rotavirusa, klinička slika je kod ovih bolesnika bila teža i bolest je duže trajala nego kod obolelih od drugih virusa. Norovirusna infekcija je dokazana u 23,03% obolelih i to statistički značajno če&scaron;će kod odraslih osoba, starijih od 20 godina. Od kliničkih simptoma kod ovih bolesnika statistički značajno če&scaron;će su dokazani muka, povraćanje i bolovi u stomaku, nego kod obolelih od drugih virusa. Norovirusi su značajno če&scaron;će bili uzročnici epidemijskog javljanja bolesti. Astrovirus je dokazan kod znatno manjeg broja obolelih (u 2,29%) i to samo kod dece do 5 godina i dece uzrasta 6 do 14 godina. Infekcija izazvana enteričnim adenovirusima dokazana je kod 13,36% bolesnika. Njače&scaron;će je utvrđena kod dece uzrsta do 5 godina i 6 do 14 godina. Oboleli od adenovirusa imali su statistički značajno blažu kliničku sliku bolesti. Dva virusna uzročnika u uzorku stolice dokazana su u 3,19% osoba, obično u toku epidemijskog javljanja bolesti. Ovi bolesnici su imali bitno težu kliničku sliku. Najvi&scaron;e obolelih od dijarealnog sindroma bilo je u hladnim mesecima, mada su dijagnostikovani i tokom cele godine. U petogodi&scaron;njem periodu utvrđene su 22 epidemije u kolektivima i 9 porodičnih epidemija. Epidemijsko javljanje bolesti bilo je statistički značajno najče&scaron;će kod najstarijih bolesnika (starijih od 50 godina), a sporadično javljanje bilo je statističko značajno najče&scaron;će kod dece. U cilju potvrde tačnosti dijagnostike virusa u ispitivanim uzorcima real-time PCR testom, genotipizacije, kao i detaljnije molekularne analize, izabrani su reprezentativni uzorci pozitivni na rota, noro, astro ili adenoviruse. Delovi genoma ovih uzoraka su amplifikovani, a zatim sekvencirani. Sekvencirani izolati rotavirusa pripadali su grupi A i tipovima G1P[8], G2P[4], G3P[8] i G9P[8]. Sekvencirani izolati norovirusa pripadali su genogrupi I tipu 2, zatim genogrupi II tipovima 1, 2, 4 i 17. Sekvencirani izolati astrovirusa pripadali su grupi klasičnih astrovirusa i tipovima 1, 4 i 5. Sekvencirani izolati adenovirusa pripadali su grupi F i tipovima 40 i 41, kao i grupi C tipu 2. Pripadnost dobijenih sekvenci u ovom istraživanju, dodatno je potvrđena izradom filogenetskog stabla za sekvence pozitivne na rota, noro, astro ili adenoviruse. Zaključak: Incidenca virusnog dijarealnog sindroma u Vojvodini (70,69%) vrlo je visoka i vi&scaron;a je nego &scaron;to je bilo pretpostavljeno prilikom prijave teze (u hipotezi). Real-time PCR test treba da bude redovno kori&scaron;ćen u budućem dijagnostičkom radu, jer dovodi do brze dijagnostike, čak i ako su virusi prisutni u malom broju u uzorcima tečnih stolica, &scaron;to je utvrđeno tokom ovog dijagnostičkog rada. Ispitivani virusi treba da budu redovno dijagnostikovani kod obolelih od dijarealnog sindroma i to u svim starosnim grupama, tokom epidemijskog i sporadičnog javljanja oboljenja.</p> / <p>Introduction: Viral gastrointestinal syndrome is a current ongoing health problem worldwide. This is true of both developed and developing countries, especially underdeveloped ones where it is the second leading cause of mortality. Sudden onset of the disease&mdash;accompanied by the occurrence of large numbers of liquid stools, nausea, vomiting, abdominal pain, fever, and exhaustion&mdash;leads to dehydration. A fatal outcome can occur in all age groups of patients, especially very young children, the elderly, and the immuno-deficient, unless an accurate etiological diagnosis of the disease is quickly established, followed by a prompt institution of fluid and electrolyte placement, and implementation of other symptomatic therapy measures. Quick establishment of an accurate diagnosis, which is best achieved using the real-time PCR test, prevents the onset of complications, including a potentially fatal outcome of the disease. Simultaneously, it enables the implementation of appropriate epidemiological measures to prevent epidemic outbreaks and their spread. The aim of this study was to accurately determine the incidence of viral gastrointestinal syndrome in Vojvodina and the frequency of epidemic and sporadic occurrence of this disease. The aim was also to set up an algorithm for the application of the real-time PCR test in diagnostics of viral gastrointestinal syndrome in future work. Likewise, the aim was to carry out genetic typing and determine phylogenetic affiliation of the virus using molecular analysis and sequencing of parts of genomes from positive stool samples. Material and Methods: During a five-year study, 1003 patients with symptoms of viral diarrheal syndrome, aged from one month to more than 90 years old, were examined using molecular real-time PCR test. They were screened for rota, noro, astro, and enteric adenoviruses. Based on the data from survey questionnaires and medical case history, all clinical indicators were meticulously analyzed (disease occurrence during the year, disease duration, symptoms). The assessment of the clinical severity was carried out according to the Vesikari Clinical Severity Scoring scale. All data were compared according to the type of the viral causing agent, age of the patients, duration of research in years, and epidemic and sporadic occurrence of the disease. Obtained data were statistically analyzed, tabulated, and graphically displayed. Results: In a five-year period, a sample of 1003 patients of different ages was screened for four different viral causing agents of diarrheal syndrome (rota, noro, astro, and enteric adenoviruses) using the real-time PCR test. Viral diarrheal syndrome was confirmed in 709 patients (70.69%). The most commonly found were rotavirus infections in 28.81% of the cases. Rotaviruses were statistically significantly most common in children younger than 5 years old (38.90%), but were also found in high percentage in children aged 6-14 years old (24.83%). Children under 5 years of age had statistically significantly highest clinical severity and fever, and were more frequently hospitalized. In addition to higher fever in patients with rotavirus, clinical severity in these patients was also higher, and the disease lasted longer than in patients with other viruses. Norovirus infections were reported in 23.03% of the subjects, statistically significantly more frequently in adults over 20 years of age. Regarding the clinical symptoms in these patients, nausea, vomiting, and abdominal pain were statistically significantly more common than in patients with other viruses. Noroviruses were significantly more common as causing agents of epidemic disease outbreaks. Astrovirus was found in a significantly smaller number of patients (in 2.29%), and only in children under 5 years of age and children aged 6-14 years old. Enteric adenovirus infections were reported in 13.36% of the subjects. They were most commonly found in children younger than 5, and those aged 6- 14 years old. Adenovirus sufferers had statistically significantly milder clinical disease. Two viral causing agents in the stool sample were found in 3.19% of the subjects, usually during an epidemic disease outbreak. These patients had a significantly more severe clinical disease. Highest numbers of sufferers from diarrheal syndrome occurred during the cold months, although they were diagnosed throughout the year. In a five-year period, 22epidemics in collective groups and 9 family epidemics were identified. Epidemic outbreaks of the disease were statistically significantly most frequent in the elderly patients (older than 50), while sporadic occurrences were statistically significantly most frequent in children. Representative samples positive for rota, noro, astro, or adenoviruses were selected in order to confirm the accuracy of virus diagnostics in samples tested by the real-time PCR test, and perform genotyping as well as more detailed molecular analyses. Parts of the genomes of these samples were amplified and then sequenced. Sequenced rotavirus isolates belonged to group A and types G1P[8], G2P[4], G3P[8], and G9P[8]. Sequenced norovirus isolates belonged to genogroup I type 2, and genogroup II types 1, 2, 4, and 17. Sequenced astrovirus isolates belonged to the group of classical astroviruses and types 1, 4, and 5. Sequenced adenovirus isolates belonged to group F and types 40 and 41, as well as group C type 2. The affiliation of the obtained sequences in this study was further confirmed by creating a phylogenetic tree for sequences positive for rota, noro, astro, or adenoviruses. Conclusion: The incidence of viral diarrheal syndrome in Vojvodina (70.69%) is very high&mdash;higher than what was assumed at the time of the thesis submission (in the hypothesis). The real-time PCR test should be regularly used in future diagnostic work, since it leads to rapid diagnostics even if viruses are present in small numbers in liquid stool samples, as determined in the course of this diagnostic study. The investigated viruses should be regularly tested in patients with diarrheal syndrome belonging to all age groups during both epidemic and sporadic occurrences of the disease.</p>

Rotavirus is responsible for the death of many children annually, and current vaccines have lower efficiency in developing countries. A reverse translated consensus gene sequence of the rotavirus VP6 cloned into a pET-28a(+) plasmid was used to transform BL21 and KRX Escherichia coli cells. Optimal expression of soluble protein was induced in KRX cells by adding 0.05% L-rhamnose and 0.0001 M IPTG, with an incubation temperature of 25ºC for 6 h. VP6 was purified by combining anion exchange chromatography followed by affinity chromatography. Far-UV circular dichroism and intrinsic fluorescence were used as probes to assess the native structure of VP6 and structural in the presence of a denaturant, high sodium chloride concentrations and varying temperatures. The 0.2 M sodium chloride had an impact on the VP6’s tertiary structure and also influenced the proteins conformational changes as detected during thermal unfolding to 90ºC. Although treatment with 3 M urea showed tertiary structural changes no secondary structural loss occurred due to the presence of a denaturant. / Life Sciences / M. Sc. (Life Sciences)

Rotavirus infection causes acute gastroenteritis in children younger than five years of age, and commonly occurring human rotavirus strains include G1 - G4 and G9 associated with P[4], P[6] and P[8]. In this study, of 6050 stool samples collected from a Private Pathology Practice in Pretoria, March 2010 - August 2011, 664 tested positive using Coris test-strips. Of these samples, 752 were retested using EIA and, results showed: Coris sensitivity was 93,7% and specificity 99,8%; the winter epidemic peaked in July of both years; more males and children under 30 months of age were particularly vulnerable to infections. Rotavirus-positive samples from Trichardt, Rustenburg and Middelburg were analysed by PAGE and RT-PCR showing circulating strains as mainly G8P[4] (60%) with short electropherotypes, G12P[8] (66%) with long electropherotypes, and G1P[8] at low incidence in the 2010/2011 seasons. These results suggest additional research to monitor the impacts of recently introduced rotavirus vaccines on changing strain profiles in South African communities / Life and Consumer Sciences / M.Sc. (Life Sciences)

L'évaluation médico-économique (EME) joue un rôle de plus en plus important dans le développement des recommandations cliniques et les décisions de prix et remboursement des produits de santé, notamment des vaccins. L'EME des vaccins fait l'objet de procédures et de recommandations méthodologiques spécifiques, distinctes des médicaments, dans de nombreux pays. Cette thèse illustre et répertorie les différentes questions méthodologiques concernant l'EME des programmes de vaccination sur la base de six études : estimation de la morbidité, mortalité et coûts liés aux infections à cytomégalovirus chez les receveurs de greffe d'organe solide ; description de l'état de santé subjectif et qualité de vie liée à la santé chez les femmes atteintes de candidose vulvovaginale récurrente (CVVR) ; revue critique des EME de la vaccination de rappel contre la coqueluche ; revue critique des EME de la vaccination contre le rotavirus ; analyse coût-efficacité de la vaccination antigrippale chez les personnes de 50 à 64 ans ; analyse coût-efficacité d'un vaccin antigrippal quadrivalent en Ontario. L'EME des programmes de vaccination nécessite de prédire l'effet d'un vaccin dans la vie réelle à partir d'essais cliniques, ce qui est particulièrement difficile pour plusieurs raisons : l'épidémiologie d'une infection peut varier dans le temps et l'espace, la réduction du risque d'infection après vaccination est différente de celle du risque de maladie, et la vaccination peut conduire à une augmentation ou diminution du risque chez les personnes non-vaccinées. De plus, la mesure et la valorisation des effets sur la qualité de vie soulèvent des questions méthodologiques et requièrent des choix normatifs liés aux faits que de nombreux vaccins ciblent les enfants, et que la réduction du risque peut améliorer la qualité de vie en dehors des périodes de maladie. Nous établissons finalement des recommandations pour les futures EME de programmes de vaccination, concernant la définition des stratégies à comparer, le choix de structure de modèle, l'estimation des paramètres cliniques et épidémiologiques, et la mesure et la valorisation de la qualité de vie et des coûts / Economic evaluation plays an increasingly important role in the development of clinical recommendations and pricing and reimbursement decisions for healthcare interventions, and particularly for vaccination. Specific processes and methodological recommendations have been developed for the economic evaluation of vaccines in many countries. This thesis identifies and illustrates different methodological questions about the economic evaluation of vaccination programs based on six studies: estimation of morbidity, mortality and costs associated with cytomegalovirus infections among receivers of solid organ transplant; description of subjective health state and quality of life among women with recurrent vulvovaginal candidosis; critical review of economic evaluations of pertussis booster vaccination; critical review of economic evaluations of rotavirus vaccination; cost-effectiveness analysis of influenza vaccination for people aged 50 to 64 years; cost-effectiveness analysis of a quadrivalent influenza vaccine in Ontario. The economic evaluation of vaccines requires predicting the effectiveness of vaccination based on clinical trial data, which is particularly difficult for several reasons: the epidemiology of an infection may vary over time and space, the effectiveness against infection may differ from effectiveness against disease, and vaccination may lead to an increase or decrease in the burden of disease among non-vaccinated persons. In addition, the measurement and valuation of effects of vaccination on quality of life raises methodological questions and requires normative choices related to the facts that many vaccines target children and that the reduction in risk may improve quality of life outside illness periods. We finally establish recommendations for future economic evaluations of vaccination programs, related to the definition of vaccination strategies to compare, the choice of model structure, the estimation of clinical and epidemiological parameters, and the measure and valuation of quality of life and costs

No description available.

In der Veterinärmedizin verursachen Rotaviren als Jungtiererkrankung vor allem in der Nutztierpraxis hohe ökonomische Verluste. Über die Prävalenz von Rotavirusinfektionen bei Hunden und Katzen ist sehr wenig bekannt, obwohl von den in der Literatur als wechselseitig zwischen Mensch und Tier übertragbaren Viren ein nicht zu unterschätzendes Risiko ausgehen kann. Zunächst wurden retrospektiv Prävalenzdaten über den Nachweis von Rotaviren bei Hunden und Katzen mit Durchfall im Vergleich zu Coronaviren und Parvoviren erhoben. Dazu wurden Kotproben von 2055 Hunden und 1481 Katzen quantitativ auf das Vorhandensein von Rota-, Corona- und Parvovirus untersucht. Desweiteren wurden Aspekte der geographischen Verteilung, der Altersverteilung, mögliche Rasseprädispositionen und das Auftreten saisonaler Erkrankungsgipfel untersucht und ausgewertet. Für Rotavirusinfektionen beträgt die statistische Prävalenz 7% bei Hunden und 8% bei Katzen. Bei Hunden und Katzen konnten signifikant häufiger Dreifachinfektionen nachgewiesen werden. Bei einer Infektion mit Rota- und Coronavirus liegt beim Hund zu 100% auch eine Infektion mit Parvovirus vor. Zweifachinfektionen kamen weniger häufig vor als Monoinfektionen. Alle drei Virusinfektionen kamen bei Hunden statistisch signifikant häufiger in der Altersgruppe ≤ 1 Jahr vor. Ein statistisch signifikant häufiger Rotavirusnachweis konnte bei der Katzenrasse Siam nachgewiesen werden, während keine Hunderasse besonders hervortrat. Im Postleitzahlengebiet 3 konnten im Beobachtungszeitraum von 2000 bis 2006 statistisch signifikant häufiger Rotavirusinfektionen bei Hunden nachvollzogen werden. Es konnte sowohl für Hunde, als auch für Katzen der Trend belegt werden, dass bei steigenden Lufttemperaturen, die Anzahl der Rotavirusinfektionen sinkt. Es kann somit von einer bedingten Saisonalität ausgegangen werden. Im zweiten Teil der Arbeit wurde die Empfänglichkeit von Rotaviren gegenüber kommerziell erhältlichem Typ I Interferon (rFeIFN-ω) in vitro getestet. Zunächst wurde zum Nachweis der Aktivität der Typ I Interferone (rFeIFN-ω, rBoIFN-α, rHuIFN-α) die Expression des Mx Proteins auf Zelllinien felinen, caninen, bovinen und humanen Ursprungs, sowie auf Affenzelllinien untersucht. Es konnte gezeigt werden, dass rBoIFN-α ausschließlich auf Zellen bovinen Ursprungs eine konzentrationsabhängige Expression des Mx Proteins induziert. Das rFeIFN-ω induziert auf Zellen felinen und bei höheren Konzentrationen auch auf Zellen caninen Ursprungs die Expression des Mx Proteins. Das rHuIFN-α zeigt eine konzentrationsabhängige Induktion des Mx Proteins in Zellen humanen, caninen, felinen und bovinen Ursprunges, sowie in Affenzelllinien. Somit konnte in vitro eine Kreuz-Speziesspezifität für rekombinantes humanes Interferon nachgewiesen werden. Zum Nachweis einer immunmodulatorischen Wirkung wurde die Expression der MHC I Oberflächenrezeptoren nach Behandlung mit rFeIFN-ω und rHuIFN-α untersucht. Die Behandlung mit rFeIFN-ω führte ausschließlich in felinen Zellen zu einer konzentrationsabhängigen signifikanten Erhöhung der Rezeptordichte. Die Behandlung mit rHuIFN-α führte zu einer konzentrationsabhängigen signifikanten Erhöhung der Rezeptordichte auf felinen Zellen und in der Affenzelllinie MA104. Die Empfänglichkeit von Rotaviren gegenüber rFeIFN-ω wurde auf der embryonalen felinen Fibroblastenzelllinie (KE-R) und auf der embryonalen felinen Gehirnzelllinine (KG-R) unter steigender Interferonkonzentration (101-104 Einheiten/ml) untersucht. Beide Zelllinien zeigten eine deutliche Reduktion der infizierten Zellen bei steigender Interferonkonzentration. Die antivirale Wirkung war in KE-R Zellen deutlicher ausgeprägt. Dort konnten bereits bei einer Interferonkonzentration von 103 Einheiten/ml keine sichtbar infizierten Zellen mehr nachgewiesen werden, während KG-R Zellen erst bei einer Konzentration von 104 Einheiten/ml keine sichtbar infizierten Zellen mehr nachzuweisen waren. Abschließend wird deutlich, dass Infektionen mit Rotaviren ein vielmals vernachlässigtes Problem in der Veterinärmedizin darstellt, vor allem, wenn man von einer Vergesellschaftung mit den für Hund und Katze pathogenen Viren Corona- und Parvovirus ausgeht. Mit dem rFeIFN-ω steht in vitro eine wirksame antivirale Substanz gegen Rotavirusinfektionen zur Verfügung.

Segundo a OMS as gastroenterites agudas são a segunda maior causa de morte de crianças no mundo. Este cenário é mais grave em países em desenvolvimento. Presume-se que a grande maioria das hospitalizações e mortes por gastroenterites agudas são causadas pela rotavirose. No ano de 2009 a OMS recomendou a vacinação internacional de crianças de 0-5 anos devido ao sucesso das campanhas de vacinação em países que adotaram esta política pública. Atualmente há uma variedade de vacinas para o rotavírus, tornando a avaliação do custobenefício destas vacinas desejável. O objetivo deste trabalho é adaptar um modelo da dinâmica da transmissão que possa contribuir com estas avaliações de custo-benefício. Para tanto foi adotada a abordagem ampla com ênfase na análise quantitativa da dinâmica da doença. O método consistiu em adaptar um modelo compartimental de referência da literatura internacional sobre modelagem de doenças com protocolo de vacinação. Este modelo de referência foi estudado e simulado para diferentes valores para posteriormente se imputar os parâmetros do modelo com os valores estimados para a rotavirose no Estado de São Paulo. Os resultados foram comparados com os valores obtidos dos dados do Datasus. Como resultado foram estimados alguns parâmetros da infecção e de seu comportamento dinâmico com informações da literatura. Conclui-se que são necessários mais estudos que possam caracterizar melhor a infecção no Estado de São Paulo, para que com isto se possa estimar melhor a infecção / According to WHO, acute gastroenteritis is the second leading cause of death for children in the world. This scenario is more serious in developing countries. It is assumed that the vast majority of hospitalizations and deaths from acute gastroenteritis are caused by rotavirus. In 2009, WHO recommended the international vaccination of children aged 0-5 due to the success of vaccination campaigns in countries that adopted this public policy. Currently, there are a variety of rotavirus vaccines, making the cost-benefit assessment of these vaccines desirable. The purpose of this paper is to contribute to these cost-benefit assessments. For that, a broad approach was adopted with emphasis on the quantitative analysis of the dynamics of the disease. The method consisted of adopting a compartmental model of the international literature on disease modelling. This reference model was studied, simulated for different values to later assign the parameters of the model to the estimated values for rotavirus in the State of São Paulo. The results were compared with the data obtained from Datasus. As a result of this work, some parameters of the infection have been estimated and it was studied the dynamic behaviour of the disease using the information available in the literature. It is concluded that further studies are needed to better characterize the infection in the State of São Paulo so that it is possible to better estimate the infection

Les gastroentérites aiguës (GEA) représentent un lourd fardeau pour la population pédiatrique. Elles sont responsables d’une mortalité importante, particulièrement chez les enfants de moins de 5 ans dans les pays à faibles revenus, et ont un impact socio-économique non négligeable dans les pays à hauts revenus. La prévalence des différents entéropathogènes potentiellement impliqués dans les GEA varie selon les études, dépendamment de la technique diagnostique utilisée, de la population étudiée – âge des patients, co-morbidités, situation géographique et socio-économique – et du moment où l’étude a été réalisée.Campylobacter est l’un des pathogènes entériques majeurs dans les pays à hauts revenus. Les espèces Campylobacter jejuni et coli sont les plus fréquemment retrouvées par les méthodes de culture sur des milieux sélectifs utilisées en routine dans la plupart des laboratoires de microbiologie. Cependant l’utilisation d’autres méthodes, comme la technique « de filtration » ou les techniques de PCR, permet de mettre en évidence d’autres campylobacters tels que Campylobacter concisus dont le rôle dans les GEA est sujet à controverse.Dans ce contexte, l’objectif général de ce travail de thèse est l’amélioration de la prise en charge diagnostique des GEA en pédiatrie à Bruxelles. Cet objectif se détaille en deux sousobjectifs: d’abord une étude de prévalence des entéropathogènes - et potentiels entéropathogènes -, ensuite une amélioration de techniques diagnostiques. Ces éléments sont détaillés ci-dessous.La première partie de ce travail nous a permis de recruter deux groupes de patients :l’un atteint de GEA (185 cas) et l’autre asymptomatique (179 témoins), mais comparables notamment en termes d’âge, de fréquentation de la crèche ou de l’école, de vaccination contre rotavirus, et de traitement par antibiotique. Au vu des techniques diagnostiques utilisées dans notre étude, Campylobacter jejuni-coli était le principal reponsable de GEA dans notre population (14% des cas), suivi de rotavirus (11% des cas). Seules 6 souches de C. concisus ont pu être retrouvées parmi les cas et 4 parmi les contrôles, ceci ne nous permettant pas de tirer de conclusions quant à un éventuel rôle de ce germe.Malgré une couverture vaccinale satisfaisante de plus de 80% parmi les cas et les témoins recrutés, rotavirus reste le deuxième entéropathogène en termes d’importance avec 11% des cas infectés dans la population étudiée. Si le calcul de l’efficacité vaccinale réalisé dans la seconde partie de ce travail n’a pas montré de résultat statistiquement significatif, les enfants de moins de 12 mois comptaient significativement plus de cas de GEA à rotavirus chez les non vaccinés que chez les vaccinés. La couverture vaccinale pourrait donc encore être améliorée afin de mieux couvrir cet âge à risque de GEA compliquée.Le faible nombre de souches de C. concisus retrouvé dans la première partie du travail nous a poussé à tenter d’améliorer la technique de culture dite « de filtration » utilisée au LHUB-ULB pour la mise en évidence des campylobacters, particulièrement des « non jejuni-coli ». En trois étapes, nous avons pu montrer la supériorité de la combinaison comprenant la gélose Columbia contenant 5% de sang de mouton, avec des filtres en polycarbonate comportant des pores de 0,60 μm de diamètre et une mise en culture dans une atmosphère microaérophile enrichie en hydrogène (7%) afin d’obtenir une meilleure sensibilité de la technique de filtration. Ces améliorations ont fait passer C. concisus en première position en termes de fréquence de Campylobacter, devant C. jejuni.Cette proposition de standardisation de la méthode permettra de faciliter la comparaison de futures études sur le sujet et d’augmenter le nombre de souches de C. concisus isolées afin de tester les hypothèses proposées de génotypes potentiellement pathogènes et de facteurs devirulence sur un échantillon plus large de souches.Parmi les techniques diagnostiques actuelles en microbiologie, les méthodes basées sur l’amplification d’acides nucléiques sont passées sur le devant de la scène, attrayantes par leur rapidité de résultats et par leur haut taux de réponse positive. L’une de ces techniques, le Luminex xTAG GPP a pu être testé sur les échantillons des cas et des témoins. Les résultats soulèvent quelques questions concernant l’utilité de cette technique pour la prise en charge clinique des patients au vu des hauts taux de positivité chez cas et témoins impliquant une réserve dans l’interprétation des résultats, particulièrement pour Salmonella. Certains faux négatifs gênent également l’implémentation en routine de ce test :certaines bactéries retrouvées en culture (Shigella, Yersinia, Campylobacter) ne sont pas détectées par le Luminex. Son intérêt est donc faible en clinique dans l’état actuel de la méthode.Ce travail permet de poser des balises pour l’interprétation des tests microbiologiques effectués et d’attirer l’attention des cliniciens sur l’importance de rester critique en ce qui concerne les résutats obtenus :un résultat positif n’indique pas systématiquement que l’entéropathogène détecté est responsable de la clinique présentée et un négatif ne l’absout pas pour autant. Pareillement, les microbiologistes doivent connaître les besoins des cliniciens afin de proposer des tests qui peuvent y répondre, tant en réduisant le délai de réponse qu’en améliorant la pertinence de celle-ci, selon le contexte de la demande :un individu malade, une prise en charge d’épidémie, une étude épidémiologique. Autant de situations où une discussion et une réflexion sont nécessaires afin d’améliorer la prise en charge des patients et d’éliminer les tests inadéquats générant des coûts inutiles pour le patient, l’hôpital et la société. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

A criação industrial de avestruzes no Brasil tem crescido nos últimos anos, mas avestruzes podem ser reservatórios de agentes com potencial zoonótico, como é o caso do vírus da influenza, rotavírus, vírus da encefalomielite eqüina do leste (EEEV) e do oeste (WEEV), salmonela, leptospira e micoplasma. O objetivo deste trabalho foi detectar rotavírus nas fezes de filhotes, anticorpos contra rotavírus, EEEV e WEEV, influenza A , leptospira, salmonela e micoplasma a partir do soro de reprodutores e efetuar o isolamento de Salmonella sp. a partir de de suabe cloacal. Para a detecção de rotavírus nas fezes utilizaram-se as técnicas de PAGE, isolamento viral e genotipagem pela RT-PCR. As técnicas de contraimunoeletroosmoforese, soroneutralização em cultura de células, inibição da hemaglutinação e aglutinação em placa foram utilizadas nos levantamentos sorológicos e a cultura bacteriológica foi utilizada para detecção de Salmonella spp. em suabes de cloaca. Rotavírus do grupo A com genotipos G[6], G[10], P[1] e P[7] foram detectados nas fezes de avestruzes e anticorpos anti-rotavírus em 10 amostras (10/182) de soros colhidos de avestruzes reprodutores. Foram detectados anticorpos para vários sorovares de Leptospira spp. (19/128), Salmonella pullorum (17/182), Mycoplasma gallisepticum (17/182) e Mycoplasma synoviae (47/182). Não foram detectados anticorpos anti-EEEV e WEEV e anti-vírus da Influenza A H3, bem como amostras de Salmonella spp. em suabes de cloaca. Estes resultados permitem sugerir o papel do avestruz na cadeia epidemiológica das rotaviroses e da leptospirose e dão bases para o aprimoramento sanitário do plantel brasileiro desta ave. / The industrial ostrich breeding in Brazil has grown in the last few years, but ostrich may be reservoirs of potentially zoonotic agents such as influenzavirus, rotavirus, eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV), Salmonella, Leptospira and Mycoplasma. The aim of the present study was to detect rotavirus in fecal samples of young ostriches, antibodies against rotavirus, EEEV and WEEV, influenza A , Leptospira, Sallmonela and Mycoplasma in sera from breeders and carry out Salmonella isolation in cloacal swabs. For the rotavirus detection, PAGE, viral isolation and genotyping by RT-PCR were used. Counterimmunoelectroosmophoresis, virus neutralisation assay in cell culture, hemagglutination inhibition and plate agglutination were used in the serological surveys and bacteriological culture was used to detetc Salmonella spp. in the cloacal swabs. Group A rotavirus with genotypes G[6], G[10], P[1] and P[7] was detected in the fecal samples and anti-rotavirus antibodies were detected in ten samples (10/182) from breeder ostriches. Antibodies to different serovars of Leptospira spp. (19/128), Salmonella pullorum (17/182), Mycoplasma gallisepticum (17/182) and Mycoplasma synoviae (47/182) were detected. Antibodies against EEEV, WEEV and influenzavirus A H3 were not detected, as well as Salmonella spp. in swab samples. These results allow one to suggest a role to ostriches in the epidemiological chain of rotavirus diseases and leptospirosis and give basis to the improvement of the sanitary condition of the Brazilian flocks.

A diarréia neonatal é o principal problema sanitário que acomete os bezerros nas primeiras semanas de vida, causando grandes prejuízos devido à morbidade, mortalidade, custos com tratamento e atraso no desenvolvimento. Os rotavírus são os mais importantes agentes virais causadores de gastroenterites em crianças e diarreia em diferentes espécies animais. Além do seu impacto econômico na produção animal, os bovinos podem atuar como reservatórios da diversidade genética e antigênica para humanos. Assim, o diagnóstico deste agente é fundamental para o desenvolvimento de medidas profiláticas mais especificas visando o seu controle. O objetivo deste trabalho foi desenvolver um método de PCR em Tempo Real para a detecção de rotavírus bovinos em sistema Taqman&reg; tendo como alvo, a proteína não-estrutural 5 (NSP5). Para isso, 113 amostras de fezes bovinas foram coletadas de reabanhos do estado de São Paulo, e previamente testadas por PCR convencional. Para a padronização da PCR em Tempo Real, a estirpe padrão foi clonada, gerando um plasmídeo com 3x1010 cópias/reação. Como controle exógeno foi utilizado &#946;-actina. O limite de detecção foi determinado por diluições seriadas na base 10, detectando até 6x101 cópias/&micro;l. A curva padrão da PCR em Temo Real para a detecção do segmento codificador da proteína NSP5 teve como resultados, uma eficiência de 100,47%; com slope igual a -3,18 e R2 de 1,0. De um total de 113 amostras testadas pela PCR convencional 63 delas (55,7%) foram consideradas positivas para rotavírus. Dessas mesmas amostras testadas, 5 não amplificaram para &#946;-actina e não foram incluídas nas análises posteriores. Para a PCR em Tempo Real o limite de detecção foi considerado o valor de 6x101 cópias/reação, sendo definido o ponto de corte o ciclo (Ct) de número 36 para o teste com a amostra viral a partir do DNA ligado em vetor plasmidial. Considerando-se o ponto de corte de 60 (6x101) cópias/reação, das 108 amostras testadas, 63 (58,3%) foram consideradas positivas ao teste. O valor de concordância obtido através do teste Kappa, a um intervalo de confiança de 95%, a partir dos resultados gerados entre os testes de PCR convencional e PCR em foi de 0.279 (baixa concordância). Os resultados obtidos nesse estudo demonstrou que o teste foi eficiente podendo ser utilizado para um diagnóstico rápido e eficiente do rotavirus do grupo A, aumentando assim o repertório dos testes já estabelecidos / Neonatal diarrhea is the main health problem affecting the calves in the first weeks of life, causing major losses due to morbidity, mortality, treatment costs and delayed development. Rotaviruses are the most important causative agents of viral gastroenteritis in children and in different animal species. In addition to its economic impact on livestock, cattle can act as reservoirs for genetic and antigenic diversity for human samples. Therefore, the diagnosis of this agent is critical to the development of more specific preventive measures for their control. The objective of this work is to develop a method of PCR for rotavirus detection in cattle using TaqMan&reg; system targeting the 5 nonstructural protein (NSP5). For this, 113 samples of cattle feces were collected from farms of São Paulo, and previously tested by convencional PCR. To PCR standardization, the standard sample was cloned, generating plasmid 3x1010 copies/reaction. The &#946;-actin was usede as exogenous control. The limit of detection was determined by serial dilutions, to detect 6 x101 copies/&micro;l. The standard curve of PCR to encoding segment detection NSP5 protein had as a result, an efficiency of 108.5%; with slope equal to -3.18 and R2 equal 1.. From a total of 113 samples tested by conventional PCR 63 (55.7%) were positive for rotavirus. From these samples 5 not amplifyed for &#946;-actin gene and were not included in subsequent analyzes. For detection limit of Real-Time PCR was considered the amount of 6x101 copies / reaction, the cut-off being defined cycle (Ct) number 36 to the test with a viral sample from the DNA ligated into plasmid vector. Considering the cut-off 60 (6x101) copies/reaction of the 108 samples tested, 63 (58.3%) were positive to the test. The correlation value obtained by the Kappa test, at a 95% confidence interval, based on results generated between the conventional and PCR testing PCR was 0.279 (low agreement). The results of this study demonstrated that the probe can be efficiently used for a fast and efficient diagnosis of rotavirus of group A, thereby enhancing the repertoire of the established tests