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The development of a waterborne pathogen detection method that is rapid, multiplex, sensitive, and specific, would be of great assistance for water treatment facilities and would help protect water consumers from harmful pathogens. Here we have utilized surface enhanced Raman spectroscopy (SERS) in a sensitive multiplex pathogen detection method. Two strategies are proposed herein, one that utilizes SERS antibody labels and one that measures the intrinsic SERS signal of organisms. For the SERS label strategy, gold nanoparticles are conjugated with antibodies specific to Cryptosporidium parvum and Giardia lamblia and with organic dye molecules. The dye molecules, rhodamine B isothiocyanate (RBITC) and malachite green isothiocyanate (MGITC) were surface enhanced by the gold nanoparticles resulting in unique fingerprint SERS spectra. The SERS label method was successful in detecting G. lamblia and C. parvum simultaneously. The method was subsequently coupled with a filtration step to both concentrate and capture cysts on a flat surface for detection. Raman mapping across the filter membrane detected ~95% of the spiked cysts in the optimized system. In the second type of strategy, intrinsic virus SERS signals were detected with silver nanoparticles for enhancement. Principal component analysis performed on the spectra data set resulted in the successful differentiation of MS2 and PhiX174 species and also for the differentiation of viable virus samples and inactivated virus samples. / Ph. D.

Foodborne disease outbreaks associated with the protozoan parasite Cryptosporidium spp. are an emerging public health concern. Shellfish, including Eastern oysters (Crassostrea virginica) in Chesapeake Bay and other Atlantic coastal waters, have been identified as a potential source of Cryptosporidium parvum infection for humans. The inactivation of C. parvum and other pathogens in raw molluscan shellfish would provide increased food safety for normal and at-risk consumers. The objectives of this study were to evaluate the efficacy of three alternative food-processing treatments (e-beam irradiation, microwave energy, and high hydrostatic pressure processing) on the viability of C. parvum oocysts in Eastern oysters. Oysters were artificially infected with the Beltsville strain of C. parvum and subjected to the three treatments in separate experiments. The effects of the treatments were evaluated by inoculation of the processed oyster tissues using the neonatal mouse bioassay. E-beam radiation of in-shell and shucked oysters treated with doses of 1.0, 1.5 or 2.0 kGy produced significant reductions (P < 0.05) in C. parvum mouse infectivity. A dose of 2.0 kGy completely terminated the infectivity of C. parvum and did not adversely affect the visual appearance of the oysters. Microwave treatments of shucked oysters at time (temperature) exposures of 1 sec (43.2°C), 2 sec (54.0°C), and 3 sec (62.5°C) produced a reduction in C. parvum mouse infectivity of 26.7%, 33.3%, and 46.7%, respectively. Microwave treatments at 2 sec (54.0°C) and 3 sec (62.5°C) showed extensive changes in oyster meat texture and color. Thus due to lack of efficacy and unacceptable tissue changes, microwave treatment of oysters is not considered a viable food processing method. High pressure processing of shucked oysters at all pressures tested (305 MPa, 370 MPa, 400 MPa, 480 MPa, 550 MPa) significantly (P < 0.05) reduced infectivity of C. parvum oocysts as measured by the neonatal mouse bioassay. A treatment of 550 MPa at 180 sec produced the maximum decrease of C. parvum infectivity (93.3%). The results indicate that HPP (high pressure processing) can produce significant (P < 0.05) reductions in infectivity of C. parvum oocysts. Measurement of tristimulus color values of pressurized raw oysters at extended processing times from 120 sec to 360 sec at 550 MPa showed an increase (P < 0.05) in whiteness. One (e-beam irradiation) of the three food-processing treatments shows promise for commercial applications to reduce public health risks from cryptosporidiosis in Eastern oysters. / Ph. D.

[EN] Since the past two decades a new special interest in water works sector has become, related to the water quality safety quantification, and the new legislations will change in that direction, recommended by Wolrd Health Organization, with the Water Safety Plans. Microbiology is included in this safety, as logical. Generally, Microbiology present in drinking water is classified as no-resistent or as resistant to chemical disinfectants. The one that presents major risk to the systems is this second group. Since 1993, the main hazard over the water quality in the water supply systems from surface raw water is a protozoan, considered by World Health Organization, as the most dangerous emergent pathogen. Its name is cryptosporidium. It was the direct consequence of a waterborne outbreak that happened in Milwaukee, in April 1993, becoming more than four hundred thousand people ill, and almost one hundred people died, and the city was fully off. On the other hand, during more than fifty years, the "conventional treatment" has been the axis of drinking water production in most of the cities. In last years, more advanced treatments (membranes, electro-dialysis, etc) have been displacing conventional treatments. It is necessary to check if conventional treatments are capable to fight with these new hazards. So, in this scenario, it`s known that there is a new hazard and there're some classical treatments that, nobody knows exactly, its abilities against this hazard. In that point, this work, developing in detail a tool to quantify the risk for the operator making decision process, is interesting for water sector. This is a methodological tool able to quantify risk over the actual most important emergent pathogen, cryptosporidium, and applied in the most known, classical and extended treatment over the world, the conventional treatment In the first part of this Thesis a literature review is done, about emergent pathogens, the presence of cryptosporidium in raw water, the analysis reliability, and the quantification of elimination with different treatments. Then, models to calculate individual and social risk are reviewed. Models about investments in drinking water treatment plants are reviewed, too. In the second part of this Thesis, the model is developed, showing calculation structures and processes, as well as tools used in model making. The decision model making as a function of calculated risk is developed, too. The risk model is based on facility's simple on-line operational parameters. The results are the risk estimation for the served population, with the plant operating in this mode. The calculation process is done using Montecarlo simulation. The third part is an application to a case study, especially interesting and very illustrative. The model is applied into a real facility. With the results, it's possible to conclude interesting guidelines and policies about improving plant's operation mode. In the fourth part if this Thesis the general conclusions from the developed tasks are extracted, and also the future research ideas. The main conclusion of this Thesis is that conventional treatment is able to work as effective barrier against this hazard, but it is necessary to assess the risk of the plant while operating. Taking into account limitations of knowledge, risk estimation can get non tolerable levels. In that situation, the plant must make investments in the treatment o improving the operation, to get tolerable risk levels. It's quite probably that, if treatment parameter's on-line monitoring would have been integrated with a quantifying risk tool, like presented in this job, cryptosporidiosis waterborne outbreak in Milwaukee in 1993 due to water treatment plant's failure, may be, could be avoided or reduced. Finally, literature review references and the annexes. / [ES] En las últimas dos décadas se ha desarrollado un interés especial, en el sector de la potabilización del agua, relativo a la cuantificación de la seguridad de la calidad del agua suministrada. En líneas generales, la microbiología presente en el agua se diferencia en dos tipos: la no-resistente a desinfectantes químicos y la que sí lo es. La que presenta más riesgo a los sistemas, es precisamente, la del segundo grupo. Desde el año 1993, una especie del grupo de la microbiología resistente ha pasado a ser considerada la mayor amenaza sobre la calidad del agua de los abastecimientos que se alimentan de agua superficial. Se trata de un protozoo considerado por la Organización Mundial de la Salud como el patógeno emergente de más peligrosidad, llamado genéricamente cryptosporidium. Ello se debe a un brote epidemiológico que sucedió en Milwaukee (Wisconsin, Estados Unidos), en abril de 1993, enfermando a más de cuatrocientas mil personas, con casi un centenar de fallecidos y paralizando completamente la ciudad. Con lo cual, en este escenario, se sabe que existen nuevas amenazas en el agua bruta y que hay gran número de potabilizadoras de tratamiento convencional, de las que se desconocen sus capacidades frente a ellas. Precisamente por ello, este trabajo, consistente en desarrollar en detalle una metodología que permita cuantificar el riesgo para la toma de decisiones del operador, partiendo de parámetros operacionales de la planta potabilizadora, es interesante para el sector. En la primera parte de la tesis se realiza una revisión bibliográfica de la temática, de los patógenos emergentes, de la presencia del cryptosporidium en las aguas superficiales, de la fiabilidad del análisis de los mismos y de la cuantificación de la eliminación que la planta potabilizadora efectúa con diferentes tratamientos. Posteriormente se analizan los modelos que permiten estimar el riesgo individual y social por esta amenaza, y finalmente se revisan los modelos existentes de valoración de infraestructuras de potabilización. En la segunda parte de la Tesis, se desarrolla el modelo, presentando las estructuras y procesos de cálculo seguidos, así como las herramientas utilizadas en su construcción. Del modelo de riesgo desarrollado, partiendo de parámetros operacionales en línea de la planta potabilizadora, se obtiene como resultado la estimación del riesgo al que la planta está sometiendo a la población servida, funcionando en ese modo. Se ha desarrollado un proceso de cálculo en el que se utilizan técnicas de Montecarlo para la simulación. En la tercera parte se aplica un caso de estudio, especialmente interesante e ilustrativo. Se ha aplicado el modelo a una planta potabilizadora en servicio, obteniendo interesantes conclusiones y recomendaciones sobre cómo mejorar su modo de funcionamiento. En la cuarta parte de la tesis se extraen las conclusiones generales del trabajo desarrollado y las futuras líneas de investigación a seguir. Como principal conclusión del trabajo realizado, es que el tratamiento convencional de las potabilizadoras puede actuar de barrera efectiva frente a estas amenazas, pero es necesario evaluar en las plantas el riesgo de su modo de funcionamiento, pues aún con las limitaciones del conocimiento disponible, en la estimación del riesgo, se pueden alcanzar valores del mismo, no tolerables. En ese caso, en la planta deberán realizarse mejoras su instalación o en su operación para alcanzar unos niveles de riesgo tolerables. Muy probablemente, si la monitorización en línea del tratamiento hubiese estado integrada con una herramienta de cuantificación del riesgo como la que se desarrolla en el presente trabajo, el brote de cryptosporidiosis de Milwaukee en 1993, ocasionado por el fallo de una planta potabilizadora de tratamiento convencional, podría haberse evitado o, al menos, reducido en magnitud. Finalmente, se recogen las referencias bibliográficas y los ap / [CA] En les últimes dos dècades s'ha desenrotllat un interés especial en el sector la potabilització de l'aigua, relatiu a la quantificació de la seguretat de la qualitat de l'aigua subministrada i en eixe sentit s'espera precisament que canvien les pròximes legislacions nacionals per recomanacions de la Organització Mundial de la Salut, en el que es diu Plans de Seguretat de l'Aigua. Dins d'esta seguretat es considera inclosa la microbiologia present en l'aigua, com és lògic. És des de l'any 1993, quan canvia dràsticament el panorama en el sector. Això es deu a un brot epidemiològic que va succeir en Milwaukee, a l'abril de 1993, emmalaltint a més de quatrecentes mil persones, amb quasi un centenar de difunts i paralitzant completament la ciutat. De tota la potencial microbiología present en l'aigua, aquella que es considerava de major amenaça sobre la qualitat de l'aigua dels abastiments que s'alimenten d'aigua superficial, és un protozou considerat per la Organització Mundial de la Salut, com el patogen emergent de més perillositat, cridat genèricament cryptosporidium, que va ser el que va ocasionar el brot de Milwaukee de 1993. Amb tot això, és necessari veure si respecte a estes noves amenaces potencials com el cryptosporidium, els tractaments convencionals poden continuar sent capaços. És llavors quan este treball, consistent a desenrotllar en detall una metodologia que permeta quantificar el risc per a la presa de decisions de l'operador és interessant per al sector. Una ferramenta metodològica que siga capaç de quantificar el risc que se cern sobre el patogen emergent de més importància en l'actualitat. En la primera part del treball realitza una revisió bibliogràfica de la temàtica, dels patògens emergents, de la presència del cryptosporidium en les aigües superficials, de la fiabilitat de l'anàlisi dels mateixos i de la quantificació de l'eliminació que la planta potabilitzadora efectua amb diferents tractaments. Posteriorment s'analitzen els models que permeten estimar el risc individual i social per esta amenaça. A continuació es revisen els models existents de valoració d'infraestructures de potabilització. En la segona part del treball, es desenrotlla el model, presentant les estructures i processos de càlcul seguits, així com les ferramentes utilitzades en la seua construcció. També es desarolla el model de presa de decisions en funció del risc calculat. El model de risc, té com a començament sencills paràmetres operacionals de la planta potabilitzadora, obtenint com a resultat, l'estimació del risc que la planta està donant a la població, funcionant d'aquesta manera. Es desarrolla un procés de càlcul utilitzant tècniques de Montecarlo per a la simulació. En la tercera part s'aplica un cas d'estudi, especialment interessant i molt pròxim i il¿lustratiu. En la quarta part del treball s'extrauen les conclusions generals del treball desenrotllat i les futures línies d'investigació que s'ha de seguir. Com a principal conclusió del treball fet, és que el tractament convencional de las potabilitzadores hi pot fer-ne de barreta front a aquestes amenaçes, no obstant és necessari evaluar à les plantes el risc de funcionament, encara que amb les limitacions del coneiximent disponible en l'estimació del risc, es poden tindre valors de risc no tolerables. En eixe cas, à la planta es deuràn de fer-ne millores en la seua instal.lació ó en la seua operació fins a tindre uns nivels de risc tolerables. És molt probable que si la monitorització de la línia de tractament hi-haguera estat integrada amb una ferramenta de cuantificació del risc, com la desarrollada en el present treball, el brot de Milwaukee de 1993 ocasionat pel fallo de funcionament de la planta potabilitzadora, es podra haver evitat o reduït. Finalment, s'arrepleguen referències bibliogràfiques i apèndixs. / Macián Cervera, VJ. (2015). Desarrollo de una herramienta de análisis de riesgo microbiológico en plantas potabilizadoras de agua como soporte a la toma de decisiones de inversión y operación [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58770

Ionisierende Strahlung wird auf vielfältige Weise in Bereichen der Industrie, Landwirtschaft, Medizin und in der Forschung eingesetzt, z.B. zur Sterilisation von Oberflächen und Medizinprodukten, Polymermodifikation, Diagnostik oder in der Entwicklung von Impfstoffen. Im Rahmen dieser Arbeit wurden Prozesse zur automatisierten Bestrahlung von flüssigen Pathogen-Proben mit niederenergetischer Elektronenstrahlung (eng. Low-Energy Electron Irradiation, LEEI) etabliert. LEEI, wie auch andere Arten der ionisierenden Strahlung, erzielt Effekte in biologischen Proben vorrangig durch Schädigung von Nukleinsäuren. Proteinstrukturen, unter anderem essentielle Antigene für eine effektive Immunantwort, bleiben größtenteils intakt. Kernstück der Arbeit ist eine Prototyp-Anlage, die sich am Fraunhofer-Institut für Zelltherapie und Immunologie befindet, die erstmalig die Bestrahlung von Flüssigkeiten in einem automatisierten, skalierbaren und produktionsgeeigneten Prozess ermöglicht. In diese Anlage können verschiedene Module integriert werden, die dünne Flüssigkeitsfilme generieren, da der Einsatz von LEEI stark durch die niedrige Eindringtiefe (<200 µm) limitiert ist. Fokus der Arbeit liegt auf der Entwicklung von Prozessen zur Inaktivierung vom Früh-Sommer-Meningoenzephalitis-Virus (FSME bzw. engl. TBEV) und dem humanen respiratorischen Synzytial-Virus (RSV), sowie zur Attenuierung (Reduktion der Virulenz eines Erregers unter erhalt der Immunogenität) zweier Endoparasiten der Gruppe Apicomplexa, Toxoplasma gondii und Cryptosporidium parvum.

Titre de l'écran-titre (visionné le 12 janvier 2024) / Cryptosporidium sp. et Giardia sp., deux protozoaires parasites de l'homme et/ou de l'animal, sont des ennemis tenaces des usines de production d'eau potable partout à travers le monde. En effet, leur forme de dissémination les rend plus aisément dispersibles dans les étendues d'eau et plus robustes faces aux stress environnementaux en attendant leur ingestion par leur prochain hôte. Par ailleurs, cette forme les rend tous deux résistants au chlore, le composé le plus couramment utilisé dans la désinfection chimique de l'eau. D'autres moyens sont disponibles pour les éliminer, mais la performance de ceux-ci dépend de la concentration de parasites dans l'eau brute. Il est donc important de connaître la concentration de ces deux organismes dans les sources d'eau alimentant les usines de production d'eau potable et de cibler les conditions et/ou les moments de l'année durant laquelle chaque source est vulnérable à la contamination par ces agents pathogènes. Les objectifs du présent projet consistaient à 1) développer un outil biomoléculaire de détection spécifique à ces deux micro-organismes, 2) à optimiser le protocole de récupération de ceux-ci à partir d'eau brute, 3) à évaluer leur présence dans trois eaux de source du Québec d'un point de vue temporel ainsi que géographique et finalement, 4) à rechercher des corrélations entre le nombre de parasites et d'autres paramètres environnementaux. En cours de projet, il a été constaté que les méthodes de détection biomoléculaire actuellement disponibles manquent soit de sensibilité, soit de spécificité ou soit de reproductibilité pour être appliquées à la surveillance de Cryptosporidium sp. et de Giardia sp. dans l'eau environnementale. Les techniques de PCR et de LAMP ont été tentées avec plusieurs cibles génétiques différentes ainsi qu'avec différentes amorces sans permettre de trouver un protocole permettant de répondre à ces trois critères. Il a donc été décidé de se tourner vers le seul protocole standardisé actuellement disponible, soit la méthode 1623.1 de l'USEPA. Toutefois, compte tenu de ses pourcentages de récupération globalement faibles, des essais ont été entrepris pour tenter de modifier cette technique dans l'optique d'améliorer sa performance et/ou de diminuer les coûts requis pour son utilisation. Parmi les quatre approches mises à l'épreuve, le protocole original de la méthode 1623.1 a permis d'obtenir la meilleure récupération en ce qui concerne Giardia. Cependant, pour Cryptosporidium, les quatre protocoles testés ne produisaient pas de récupération statistiquement différente les uns des autres. Ultimement, il a été décidé d'utiliser le protocole original de la 1623.1 pour la suite du projet. Ensuite, une campagne d'échantillonnage d'un an a été menée entre mars 2022 et février 2023 au niveau de l'eau brute de trois usines de production d'eau potable du Québec (Québec, Charny et Lévis). Des prélèvements ont été faits de manière mensuelle avec des dates supplémentaires lors des périodes de transition saisonnière. Chaque échantillon était analysé avec la 1623.1 pour déterminer la concentration de parasites dans chacun des échantillons. Au final, un patron annuel a pu être dessiné pour Giardia avec de fortes concentrations durant les périodes froides et de transition saisonnière ainsi que des concentrations statistiquement plus faibles en période chaude. Pour sa part, la concentration de Cryptosporidium était davantage variable selon la saison et la source d'eau. Avec les données de la campagne d'échantillonnage de 2022-2023, des corrélations statistiques ont pu être établies entre la variation de la concentration d'(oo)cystes et celle de plusieurs paramètres environnementaux. Il a été rapidement constaté qu'aucun des paramètres environnementaux testés ne peut servir de proxy universel aux trois sites ni aux trois saisons pour la même source, pas même les micro-organismes indicateurs de pollution fécale conventionnels. Cependant, des corrélations fortes ont pu être trouvées pour chaque parasite à chaque site pour au moins une saison de l'année. Des modèles de régressions linéaires multiples ont également pu être établis pour chaque site et pour chaque parasite. Grâce aux travaux effectués durant ce projet, on peut conclure que chaque source d'eau possède ses caractéristiques et influences propres qui la rendent unique. Ainsi, les tendances observées à une source ne peuvent pas nécessairement être extrapolées aux autres sources, malgré leur proximité géographique. Par ailleurs, chaque année est unique en soi. Il est donc important d'effectuer une surveillance régulière au niveau de chaque source pour rester à l'affût de variations impromptues dans leur concentration. / Cryptosporidium sp. and Giardia sp., two protozoan parasites of humans and/or animals, are tenacious enemies of water treatment plants around the world. They adopt a dissemination conformation making them easily spread through watercourses and tougher against environmental stresses they undergo while waiting to be ingested by their next host. This conformation also makes them more resistant face to chlore-based treatments used as chemical disinfection in water treatment plants. Other means are available to get rid of them in raw water, but the use of these alternative treatments is concentration-dependent. It is therefore necessary to monitor the concentration of these parasites in water sources supplying treatment plants and to determine the conditions and/or the moments of the year where each water source is vulnerable to contamination by these pathogenic agents. The objectives of this project were to 1) develop a biomolecular tool for the detection of Cryptosporidium and Giardia, 2) optimize the protocol for recovery of these parasites from raw water, 3) evaluate the presence of these microorganisms in three water sources of Québec from a geographical and from a temporal angle, 4) seek statistical correlations between the parasite's concentration and other environmental parameters. Throughout the project, it was found that biomolecular methods available lack either sensitivity, specificity, or reproducibility to be applied for the monitoring of Cryptosporidium sp. and Giardia sp. in environmental water samples. PCR and LAMP experiments were performed targeting different genes and using different primers without being able to fulfill these three criteria. It was then decided to use the only standardized protocol available up to today, the USEPA Method 1623.1. However, knowing the low recovery percentages of this technique, tests were performed to try to improve this protocol and/or decrease the costs associated with it for a similar performance. Among the four approaches tests, the original protocol of the 1623.1 gave the highest recoveries of Giardia. On the other hand, the four protocols did not give statistically different recoveries when compared with each other for Cryptosporidium. Therefore, the original protocol of the 1623.1 was selected for the subsequent steps of this project.Afterwards, a sampling campaign of one year was performed between March 2022 and February 2023. Sample sites were three major water treatment plants of Québec (Québec, Charny and Lévis). Samples of raw water from each site were taken on a monthly basis with supplementary dates during transition periods (spring and fall). Each sample was analyzed with the 1623.1 protocol to determine the parasite concentration in each. Throughout this campaign, an annual pattern was found for Giardia with high concentrations during cold and transition months while concentrations were statistically lower during the warm season. Cryptosporidium's concentration had a much more variable behavior depending on the season and on the water source. With the data collected during the sampling campaign of 2022-2023, statistical correlations could be established between the variation in concentration of parasites and the variation of several environmental parameters. No proxy common to all sites and to all seasons could be found for either protozoan, not even the common fecal pollution indicator microorganisms. However, strong statistical correlations were found for each parasite in at least one season of the year. Multiple linear regression models could be made for each site and each parasite. With the work performed during this project, we can reach the conclusion that each water source has its own characteristics and influences which makes it unique. Therefore, patterns observed for a given source cannot necessarily be extrapolated to other sources, despite their geographical proximity. Moreover, each year has its own particularities, So, it is important to monitor regularly each water source and to stay alert face to unexpected variations in the concentration of these two protozoa.

MSc (Microbiology) / Department of Microbiology / Background: Diarrheal diseases constitute an important problem among children but also among HIV positive patients particularly in developing countries such as South Africa. Cryptosporidium infect humans and has been shown to be an important cause of infection among different types of animals. Because of its small size, Cryptosporidium can easily go through the water purification system and can easily become a cause of an epidemic. Previous studies have shown that Cryptosporidium is an important cause of diarrhea in Limpopo Province. However, very few studies have been conducted on the genetic diversity of these organisms in the region. Therefore, the aim of this study was to detect and identify the genetic diversity of Cryptosporidium species from humans and animals in Giyani situated in the northern part of South Africa and Pretoria situated in the central part of the country. Methodology: A total of 560 samples were collected from human and animals and were all screened by microscopy using modified Ziehl-Neelsen staining technique. All the samples were tested by Enzyme-Linked Immunosorbent Assay (ELISA) using the Cryptosporidium II kits from Techlab, Virginia, USA. Positive samples from microscopy and ELISA were examined by different PCR protocols including conventional PCR for amplification of Cryptosporidium oocyst wall protein (COWP) region; Real-time PCR employing SYBR Green detection format for amplification of 18S rRNA region; Real-time PCR employing Hydrolysis probes detection format for amplification of SSU rRNA region; Real-time PCR specific for amplification of C. hominis region and C. parvum region. Positive samples from real-time PCR that gave clear bands on gel electrophoresis were sent for sequencing. The sequences were analysed using Staden package software to edit the nucleotides, Bioedit and MEGA6 software were used to align sequences and draw phylogenetic trees. The SPSS software was used for statistical analysis. xiii Results: The overall prevalence of Cryptosporidium as detected by ELISA method from the samples collected from humans was 41.2% (239/580). The prevalence was higher from the rural area 73.0% (159/218) compared to the urban area 22.1% (80/362) and the difference was statistically significant (χ2 = 145.1; p = 0.0001). Due to the limited amount of samples, only 134 ELISA-positive samples were tested using real-time PCR. Of these samples, 35.8% (48/134) tested positive. Of 48 real-time positive samples 25 were successfully sequenced and two different species (C. hominis and C. muris) were identified. Of all the sequences obtained, one (4.0%) was C. muris and 20 (80%) were C. hominis isolated from rural area, whereas 16.0% (4/25) were also C. hominis isolated from samples obtained from urban area. Cryptosporidium was not associated with diarrhea in the present study. A total of 85 samples were collected from animals (52 from cattle and 33 from goats) and of these 4 (4.7%) were positive by microscopy and ELISA. All these samples were non diarrheal. Conventional PCR also detected a similar number. Of these 4 positive samples, 1 was from a male goat, while the 3 others were obtained from female adult goats. Real-time PCR detected 56.5% (48/85) positive samples. Only 12 of the 85 animal samples were diarrheal and of these 4 were positive for Cryptosporidium. The prevalence of Cryptosporidium infection was higher 68.4% (13/19) in male animals compared to female animals 53.0% (35/66). The prevalence rates in cattle and goats were 55.8% (29/52) and 60.6% (20/33) respectively. Of 48 real-time positive samples from animals, 12 (25.0%) were successfully sequenced and two species (C. parvum and C. andersoni) were identified. Of these 6 were from cattle and the other 6 were from goats. Out of the 12 samples 10 (83%) were C. parvum while 2 (17%) were C. andersoni. Of the two C. andersoni, one was from a goat and one was from a cow. Of the 10 C. parvum, 5 were from goats and 5 were from cattle. xiv In conclusion, microscopy remains the low sensitive tool for the detection of Cryptosporidium while real time PCR appeared to be far much more sensitive by detecting more samples than all the three other methods combined. Closer to the real time PCR was ELISA that detected also more samples compared to conventional PCR and microscopy. The present study identified C. muris from humans’ samples in our area for the first time. However, C. hominis remains the dominant species that infects humans in our area. Cryptosporidium species was mostly found in samples from asymptomatic individuals. In animals, C. parvum was the most commonly isolated organism while C. andersoni was identified in our region for the first time as well and occurred in both goats and cattle. Populations in the affected areas need to be made aware of the infections so that care should be taken to avoid the spread of infection in water sources or in immunocompromised individuals.

La multichimiorésistance est caractérisée par une résistance simultanée à des agents chimiothérapeutiques de structures diverses, induite notamment par l’efflux des substances actives hors des cellules. Les transporteurs ABC (ATP-Binding Cassette) sont des protéines transmembranaires impliquées dans cet efflux et qui participent à l’échec du traitement de certains cancers. Par ailleurs, ce mécanisme d’efflux a également été évoqué dans le cadre de la résistance de certains microorganismes aux antimicrobiens. Dans cette étude, nous avons conçu et synthétisé des dérivés susceptibles d’inhiber certains transporteurs ABC, en particulier, la glycoprotéine-P (Pgp) impliquée dans la multichimiorésistance des tumeurs humaines, et CpABC3, rencontré chez le parasite Cryptosporidium parvum. Dans un premier temps, nous avons synthétisé trois dérivés de type 4-alkyl-imidazo[4,5-b]pyridin-7-one, hétérocycles destinés à se fixer sur le site à ATP des transporteurs ABC. L’activité de ces composés a été évaluée vis-à-vis d’un fragment recombinant (H6-NBD1) de CpABC3, et un de ceux-ci a montré une liaison (faible) à ce fragment. Nous avons ensuite préparé dix-sept dérivés bivalents susceptibles d’inhiber la Pgp, constitués d’une molécule d’adénine (ciblant le site à ATP) reliée à la progestérone (ciblant le site aux stéroïdes) par un bras de géométrie variable. Ces dérivés ont été testés sur des lignées cellulaires K562/R7 surexprimant la Pgp, et un de ceux-ci a montré une activité supérieure à celle de la progestérone. Enfin, nous avons mis au point une synthèse de chaînes de type oligocyclohexylidène, qui sont de bons candidats pour constituer des bras espaceurs rigides / Multi-drug resistance (MDR) is characterized by a simultaneous resistance to a wide range of structurally unrelated chemotherapeutic agents, partly caused by the efflux of active compounds out of the cell. ABC transporters (ATP-Binding Cassette) are transmembrane proteins implicated in this efflux, and thus, they contribute to the failure of some cancer treatments. Furthermore, this mechanism was evoked in some microorganism resistances to antimicrobial agents. In this study, we designed and synthesized potential inhibitors of ABC transporters, especially P-glycoprotein (Pgp) implicated in human tumors multi-drug resistance, and CpABC3, a transporter found in a human parasite, Cryptosporidium parvum. First, we synthesized three 4-alkyl-imidazo[4,5-b]pyridin-7-one derivatives, targeting ATP-binding site of ABC transporters. Their biological activities were evaluated toward a recombinant fragment of the CpABC3 transporter (H6-NBD1 fragment). One of these compounds showed a weak-binding to this fragment. Next, we prepared seventeen progesterone-adenine hybrids as potential bivalent ligands which may bind simultaneously to the ATP-binding site and the steroid-binding region. We chose to synthesize derivatives with rather short-length linkers with different conformational flexibilities. These bivalent compounds were tested on K562/R7 human leukemic cells overexpressing Pgp. One of them has showed a better activity than progesterone. Finally, we optimized the synthesis of oligocyclohexylidene chains, which are good candidates to constitute rigid linkers

Esta pesquisa avaliou o uso de ozônio para inativar cistos de Giardia spp. e oocistos de Cryptosporidium parvum presentes na água filtrada obtida após a utilização da tecnologia de ciclo completo com flotação &#40;coagulação, floculação, flotação e filtração&#41; em escala de bancada, empregando o cloreto de polialumínio &#8211; PAC &#8211; como coagulante. O método de floculação em carbonato de cálcio &#8211; FCCa, sem e com a etapa de separação imunomagnética &#8211; IMS &#8211; foi utilizado na quantificação dos protozoários. A recuperação nos ensaios de qualidade utilizando o kit Easyseed&#174; de Giardia spp. foi de 8,4&#37; &#177; 97,4&#37; sem IMS e com IMS, com duas dissociações, o valor foi de 7,4&#37; &#177; 39,7&#37; e, com três dissociações, a recuperação alcançou 9,6&#37; &#177; 34,7&#37;, portanto, somente o ensaio com IMS e duas dissociações, não atendeu os padrões do Método 1623.1. A recuperação de Cryptosporidium parvum, obteve valor de 3,4&#37; &#177; 100&#37; em ensaio sem IMS e com IMS, o valor obtido foi de 1,0&#37; &#177; 70,0&#37; com duas dissociações e 1,8&#37; &#177; 44,4&#37; com três dissociações e nos três métodos apresentados, não houve conformidade com os critérios do Método 1623.1. Na etapa de desinfecção com ozônio, os ensaios realizados na Etapa 1, que se utilizou 5 mgO3L-1 e tempo de contato de 1 min sem IMS, as maiores inativações atingidas foram de 2,52 e 2,22 log para cistos de Giardia spp. e oocistos de Cryptosporidium parvum, respectivamente. Com o tempo de contato de 5 min, as maiores inativações foram de 2,52 e 2,92 log de cistos de Giardia spp. e oocistos de Cryptosporidium parvum, respectivamente. Na Etapa 2, com IMS, utilizando a mesma dosagem e tempo de contato de 1 min, obteve-se 2,27 e 0,21 log de inativação para cistos e oocistos, respectivamente. Comparando-se com o tempo de contato de 5 min, foram obtidos 2,9 e 2,3 log de inativação para cistos e oocistos, respectivamente. Na avaliação de custo, o método de FCCa sem IMS demonstrou ser o mais econômico em relação ao procedimento com IMS. A influência da inclusão da terceira dissociação ácida no método com IMS também foi analisada e este procedimento não resultou em diferenças estatísticas significativas nos resultados. / This research evaluated the use of ozone to inactivate Giardia spp. cysts and Cryptosporidium parvum oocysts present in the filtered water obtained after the use of the complete cycle of flotation technology &#40;coagulation, flocculation, flotation and filtration&#41; on a bench scale employing polyaluminium chloride - PAC as coagulant. The calcium carbonate flocculation method - FCCa, without and with immunomagnetic separation step - IMS has been used in the quantification of protozoa. The recovery in quality test using the kit Easyseed&#174; for Giardia spp. was 8.4&#37; &#177; 97.4&#37; non-IMS and IMS with two dissociations, the value was 7.4&#37; &#177;39.7&#37; and with three dissociations, the recovery reached 9.6&#37; &#177; 34,7&#37;, so only the test with IMS and two dissociations, did not meet the standards of method 1623.1. Recovery of Cryptosporidium parvum obtained value of 3.4&#37; &#177; 100&#37; in non-IMS and IMS testing, the value obtained was 1.0&#37; &#177; 70.0&#37; with two dissociations and 1.8&#37; &#177; 44.4&#37; with three dissociations and the three methods presented, there was non-compliance with the criteria of Method 1623.1. In step disinfection with ozone, tests performed in Step 1 was used 5 mgO3L-1 and contact time of 1 min without IMS, the major inactivation achieved were 2.52 and 2.22 log for Giardia spp. cysts and Cryptosporidium parvum oocysts, respectively. With 5 minutes of contact time, the greater inactivation were 2.52 and 2.92 log for Giardia spp. cysts and Cryptosporidium parvum oocysts, respectively. In Step 2, with IMS, using the same dose and 1 min contact time, there was obtained 2.27 and 0.21 log inactivation for cysts and oocysts, respectively. Compared with the 5 min of contact time, were obtained 2.9 and 2.3 log inactivation for cysts and oocysts, respectively. In evaluating cost, the FCCa method without IMS proved to be the most economical in relation to the procedure with IMS. The influence of inclusion of the third acid dissociation method in IMS was also analyzed and this procedure did not result in statistically significant differences in the results.

O lançamento de esgotos domésticos in natura, efluentes das estações de tratamento de esgoto e escoamento superficial, são relatados como importantes causas de poluição das águas superficiais. Sabe-se que a alteração da qualidade das águas dos rios restringe seus múltiplos usos e contribui para o aumento de doenças de veiculação hídrica, em decorrência da exposição oral a bioagentes patogênicos. Neste contexto, o objetivo do presente estudo foi identificar e quantificar bioagentes presentes na água do rio Pardo, Brasil, e estimar o risco de infecção e de doença por Cryptosporidium spp. e Giardia spp. para a população, devido ao uso do rio como fonte de abastecimento público e recreação de contato primário, por meio da abordagem da Avaliação Quantitativa de Risco Microbiológico (AQRM). Durante os anos de 2015 e 2016, foram realizadas seis coletas de amostras da água do rio Pardo (período chuvoso e período seco) em seis pontos, totalizando 36 amostras. Foram realizadas análises de identificação e quantificação de E. coli, Salmonella Não Tifóide, Cryptosporidium spp. e Giardia spp. Para estimativa de risco de infecção e de doença por Cryptosporidium spp. e Giardia spp. (AQRM), foram considerados diferentes populações (crianças e adultos), volumes de água ingerido, concentração de (oo)cistos e duração e frequência da exposição, de acordo com o cenário estabelecido. Os valores médios para E. coli variaram de 6,57 x 101 UFC/100 mL a 6,07 x 103 UFC/100 mL, apresentando diferenças estatisticamente significantes (p < 0,05) entre os períodos chuvoso e seco. As densidades de Salmonella Não Tifóide foram baixas (<0,6473 a 1,55 NMP/100 mL), com frequência de 13,9% das amostras positivas, evidenciando a circulação desse patógeno no ambiente. A concentração de (oo)cistos de Cryptosporidium spp. e Giardia spp. variou de <0,1 a 0,4 oocistos/L e <0,1 a 4,4 cistos/L, respectivamente. Para abordagem da AQRM devido a ingestão da água do rio Pardo usada para abastecimento público, a probabilidade anual de infecção por Cryptosporidium spp. e Giardia spp. foi maior para adultos do que para crianças, sendo que na maioria dos pontos apresentou resultados superiores ao risco anual tolerável pela USEPA (1 x 10-4). No que diz respeito ao uso da água do rio Pardo para recreação de contato primário, a probabilidade diária e anual de infecção, bem como a probabilidade de doenças, foi maior para crianças, seguida de adultos/homens e adultos/mulheres. A probabilidade de criptosporidiose e giardíase esteve abaixo do limite tolerável pela USEPA (3,6 x 10-2), exceto no Ponto 4, em que a estimativa de risco de doença por Giardia spp. para crianças esteve acima deste valor. A presença de bioagentes em amostras de água do rio Pardo pode estar relacionada à poluição das águas por fontes pontuais e difusas. Esses achados refletem a importância de priorizar os recursos para implantação e complementação das Estações de Tratamento de Esgoto na UGRHI 4, a fim de prevenir as doenças de veiculação hídrica em populações que utilizam a água do rio Pardo para abastecimento público e recreação de contato primário / The discharge of domestic sewage, effluents of wastewater treatment plants and surface runoff, are reported as important causes of surface water pollution. It is known that the alteration of river water quality restricts its multiple uses and contributes to the increase of waterborne diseases, due to oral exposure to pathogenic bioagents. In this context, the aim of the present study was to identify and quantify bioagents present in Pardo river water, Brazil, and to estimate the risk of infection and disease by Cryptosporidium spp. and Giardia spp. for the population, due to the use of the river as source of public supply and primary contact recreation, through the approach of Quantitative Microbial Risk Assessment (QMRA). During the years of 2015 and 2016, six samples of water from the Pardo river (rainy and dry season) were collected at six points, totaling 36 samples. Identification and quantification analyzes of E. coli, Non-typhoid Salmonella, Cryptosporidium spp. and Giardia spp. To estimate the risk of infection and disease by Cryptosporidium spp. and Giardia spp. (QMRA), different populations (children and adults), volumes of ingested water, concentration of (oo) cysts, duration and frequency of exposure were considered according to the established scenario. Mean values for E. coli varied from 6.57 x 101 CFU / 100 mL to 6.07 x 103 CFU / 100 mL, showing statistically significant differences (p <0.05) between the rainy and dry season. Non-typhoid Salmonella densities were low (<0.6473 at 1.55 MPN / 100 mL), with a frequency of 13.9% of the positive samples, evidencing the circulation of this pathogen in the environment. Cryptosporidium spp. and Giardia spp. concentration ranged from <0.1 to 0.4 oocysts / L and <0.1 to 4.4 cysts / L, respectively. In order to approach the QMRA due to the ingestion of Pardo river water used for public supply, the probability of annual infection by Cryptosporidium spp. and Giardia spp. was higher for adults than for children, and in most points presented results higher than the risk tolerable by USEPA (1 x 10-4). Regarding the use of Pardo river water for primary contact recreation, the daily and annual probability of infection, as well as the probability of illness, was higher for children, followed by adults / men and adults / women. The probability of cryptosporidiosis and giardiasis was below the limit tolerable by USEPA (3.6 x 10-2), except in Point 4, where the estimated risk of disease by Giardia spp. for children was above this value. The presence of bioagents in Pardo river water may be related to water pollution by point and diffuse sources. These findings reflect the importance of prioritizing the resources for implementation and complementation of wastewater treatment plants at UGRHI 4, in order to prevent waterborne diseases in populations that use Pardo river water for public supply and primary contact recreation