Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg, May 2013 / Cystic echinococcosis is a neglected parasitic zoonosis that is regarded as an emerging disease worldwide. Effective control of the disease is based on understanding the variability of Echinococcus granulosus (sensu lato), as genotypic characteristics may influence lifecycle patterns, development rate, and transmission. No molecular epidemiological research has previously been conducted to shed light on genotypes responsible for the disease in South Africa. To identify strains circulating in the country, parasite material was collected from patients between August 2010 and September 2012 and analyzed by PCR/RFLP methods. A total of 32 samples was characterized as E. granulosus (G1) (81%), E. canadensis (G6/7) (16%) and E. ortleppi (G5) (3%). Furthermore, two co-amplifying G6/7 genotypes were confirmed as G7 by sequencing. This is the first report on genotyping of Echinococcus species in South Africa, and, to our knowledge, the first report of the G5 and G7 genotypes from humans in Africa.

No description available.

No description available.

Note:

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

No description available.

Le parasite Echinococcus multilocularis cause l’échinococcose alvéolaire (EA), infection fatale si non prise en charge. Le traitement médical, pour les patients inopérables, est uniquement parasitostatique, et présente des effets secondaires. Néanmoins, chez certains patients, la viabilité du parasite régresserait suffisamment pour envisager un arrêt de ce traitement. Actuellement, les biomarqueurs pour estimer la viabilité parasitaire ne sont qu’indirects, évaluant la réponse immunitaire du patient. Trois études ont été menées, visant à évaluer des biomarqueurs, innovants ou déjà disponibles sur le marché.Nous avons étudié la présence d’ADN libre circulant (ADNlc), au moment du diagnostic, mais aussi quelques mois après la mise en place du traitement. Notre étude valide pour la première fois la présence d’ADNlc dans les cas d’EA, sur modèle animal puis sur des échantillons de patients. Même si la méthode n’est pas encore utilisable en diagnostic ou en suivi, c’est un point de départ vers l’utilisation de l’ADNlc pour la prise en charge de l’EA.De plus, nous avons mené une étude exploratoire sur des lésions parasitaires chez la souris et chez un patient ayant reçu un traitement médicamenteux très court. A partir d’un même échantillon, nous avons analysé l’ADN, afin d’estimer la proportion de cellules parasitaires, et nous avons quantifié différents transcrits parasitaires, afin d’estimer la viabilité du parasite de manière directe. Cet axe a permis de choisir la cible la plus transcrite : elle pourrait être utilisée sur une cohorte plus large, puis corrélée avec les biomarqueurs indirects utilisés aujourd’hui.Enfin, nous avons évalué un test de diagnostic rapide de l’échinococcose kystique, présentant de fortes réactions croisées en cas d’EA.Ces travaux ouvrent de nouvelles perspectives, principalement pour améliorer le suivi des patients atteints d’EA. / The parasite Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), a fatal infection if not adequately managed. Medical treatment, for inoperable patients, is only parasitostatic, with many side effects. Nevertheless, in some patients, the viability of the parasite could regress sufficiently for treatment to be stopped. Currently, biomarkers for estimating parasite viability are only indirect, evaluating the immune response of the patient. Three studies were conducted to evaluate biomarkers that are innovative or already available on the market.We investigated the presence of circulating cell-free DNA (ccfDNA) at diagnosis, and after a few months of treatment. For the first time, our study identified the presence of ccfDNA in cases of AE, in an animal model and in human samples. Although the method cannot yet be used for diagnosis or follow-up, it is a starting-point for the use of ccfDNA in the management of AE.In addition, we conducted an exploratory study of parasitic lesions in mice, and in a patient who had only received medical treatment for a very short time. We analyzed DNA to estimate the proportion of parasite cells present in each sample. We then quantified different parasite transcripts from each sample, in order to directly estimate parasite viability. This approach allowed us to identify the most abundantly transcribed gene, which could potentially be used to study a larger cohort, in order to correlate results with the indirect biomarkers used today.Finally, we evaluated a rapid diagnostic test for cystic echinococcosis, which produces strong cross-reactions in the case of AE infection.This work opens new perspectives, mainly to improve the follow-up of patients with AE.

L'échinococcose alvéolaire (EA) humaine est une maladie parasitaire, rare et grave, due à un cestode parasite, Echinococcus multilocularis, dont le cycle sauvage, en France, passe généralement par le renard (hôte définitif) et le campagnol (hôte intermédiaire). La moitié des cas européens recensés sont regroupés en France, et se concentrent surtout dans les zones rurales de Franche-Comté et Lorraine. Malgré de nombreuses études, la répartition des malades et les facteurs de susceptibilité ou de protection de certaines personnes face à la maladie sont encore mal connus.Depuis 1997, il existe un réseau de surveillance épidémiologique de l’échinococcose alvéolaire humaine, qui a constitué un registre des cas français d’EA, collectant des données sur la pathologie mais aussi sur les lieux et habitudes de vie des patients. L’objectif de ce travail était :- d’étudier les facteurs de risque de l’EA humaine en France, en particulier les facteurs comportementaux impliqués,- d’étudier l’impact du climat et de l’environnement sur l’incidence de la maladie- de déterminer les principaux facteurs utiles pour la prévention de l'infection, ainsi que les populations-cibles pour d'éventuelles campagnes de prévention. / Human alveolar echinococcois (AE), a rare and serious parasitic disease, is caused by a cestode, Echinococcus multilocularis. In France, foxes and water voles are the main hosts (respectively definitive and intermediate). Half of the European human cases are French, mainly living in rural areas in Franche-Comté and Lorraine. Despite numerous studies, human distribution and factors affecting human vulnerability remain poorly understood.Created in 1997, the French epidemiological surveillance network for human AE collects data on the disease characteristics and also on lifelong locations and behaviors of the patients.The objectives of this study were:- to determine individual risk factors for human AE in France, and especially at-risk behaviors,- to study the role of the environment on human AE incidence,- to identify key messages and target populations for potential prevention campaigns.

Einführung Die Prävalenz der alveolären (AE) und zystischen Echinokokkose (CE) in Deutschland nimmt zu, aber die Gesamtzahl der Fälle in den nördlichen und östlichen Bundesländern ist noch immer gering. Betroffene Patienten werden überwiegend in wenigen spezialisierten Zentren in Süddeutschland behandelt, sodass in anderen Regionen Deutschlands bislang nur relativ wenig klinische Erfahrung mit AE und CE vorhanden ist. Ziele Ziel unserer Studie war es, das derzeitige Management von Patienten mit Verdacht auf oder bestätigter Echinokokkose an einem Zentrum der tertiären Versorgungsstufe in einer Niedrigprävalenzregion zu bewerten, um eine Verbesserung des Vorgehens bei Verdacht auf AE/CE bzw. bereits gesicherter Diagnose zu erreichen. Methodik Alle Patienten, die am Universitätsklinikum Leipzig zwischen dem 01.01.2004 und dem 30.09.2018 infektionsserologisch auf Echinokokken untersucht wurden oder die Diagnose „AE“ bzw. „CE“ erhalten hatten, wurden in eine retrospektive Kohortenstudie eingeschlossen. Klinischer Verlauf, bildgebende sowie histopathologische Befunde, Therapiecharakteristika, relevante Begleiterkrankungen und mögliche Risikofaktoren für AE bzw. CE wurden bewertet. Die Datenanalyse umfasste weiter eine zeitlich gestaffelte Prävalenzabschätzung für unser Zentrum sowie Sensitivitäts- und Spezifitätsberechnungen für die durchgeführten serologischen Tests. Ergebnisse Insgesamt wurden 382 Patienten in die Analyse einbezogen. 11 AE- und 7 CE-Fälle wurden identifiziert. Die mittlere Prävalenzrate der AE in dieser Kohorte lag bei 2,9 % und die der CE bei 1,8 %. Bei 4 Patienten war die Diagnose bereits auswärtig gesichert worden und eine gezielte Überweisung zur spezialisierten Behandlung erfolgt. 56 % der Patienten wiesen bekannte Risikofaktoren für CE bzw. AE auf. Mit Ausnahme eines Falls waren alle CE-Fälle Migranten mit ausländischer Herkunft. Die durchgeführten serologischen Tests zeigten für beide Erkrankungen eine Sensitivität von 86 % und eine Spezifität von 91 %. 2 Patienten mit zunächst falsch negativer Diagnose (einer mit AE und einer mit CE) wurden durch ultraschallgestützte Biopsie korrekt diagnostiziert. Alle CE-Fälle und 5 AE-Fälle (45 %) wurden unter Albendazolschutz chirurgisch versorgt. Die restlichen 6 AE-Fälle erhielten eine Langzeitbehandlung mit Albendazol. Es wurden keine Todesfälle infolge Echinokokkose beobachtet. Schlussfolgerungen Sowohl AE als auch CE sind im Großraum Leipzig seltene Erkrankungen, wenngleich ein Anstieg der Fallzahlen an unserem Zentrum zu verzeichnen ist. Aufgrund unterschiedlicher begünstigender Faktoren wie z. B. Zunahme der Migration ist in Zukunftmit einemweiteren Anstieg zu rechnen. Diagnosestellung und Therapiemanagement stellen sowohl für die AE als auch für die CE eine Herausforderung dar und sollten von einem Expertenteam begleitet werden, darunter erfahrene Infektiologen, Bildgebungsspezialisten und versierte hepatobiliäre Chirurgen. Der Aufbau eines deutschen Echinokokkosenetzwerks wäre sinnvoll.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cystic echinococcosis (CE) is a zoonotic disease caused by Echinococcus genus. The larval stage (hydatid cyst) of the parasite causes serious public health problems, and affects various species of animals, causing economic losses to the meat industry. This study aimed to identify samples of fertile hydatid cysts in cattle and characterize them molecularly, analyzing in vitro and ex vivo Melaleuca alternifolia oil activity (tea tree oil - TTO) and its formulation in nanoemulsion (NE-TTO) and saris an (terpinen 4-ol) front to protoscolices of Echinococcus spp. Two thousand three hundred and ninety-six samples with cysts derived from an abattoir in the central region of the RS were analyzed, where 295 samples were classified as hydatid cysts by the presence of protoscolices. In PCR reactions for the 12S gene, 251 positive samples were identified for the group of genotypes G5/G6/G7 (E. ortleppi/ E. canadensis) and 40 samples for the G1 genotype (E. granulosus sensu stricto). In PCR subsequently, 250 samples were positive for G5 (E. ortleppi) and one sample for G7 (E. canadensis). A sample (n=12) of PCR products using the region of cox I gene, which was sequenced and compared with samples deposited in Genbank. Phylogenetic analysis identified three distinct groups E1, E2 and E3, which were grouped in sequences with greater similarity. The organ most affected by echinococcosis in cattle was the lung. After confirming the fertility of the cyst, protoscolices were removed and evaluated for viability using eosin 0.1%. For in vitro testing, concentrations of 2.5, 5 and 10 mg/mL of TTO, 10mg/mL of NE-TTO and 1, 1.5 and 2 mg/mL of terpinen-4-ol were analyzed front to protoscolices of E. ortleppi previously identified, in times of 5, 10, 15 and 30 minutes. For ex vivo test, TTO 10mg/mL and 20mg/mL was injected directly into hydatid cysts of E. ortleppi (n = 20) at times of 5, 15 and 30 minutes, and for each time one aliquot of protoscolices was removed for feasibility detection and implementation of PCR to confirm the species involved in the parasitism. Results of this in vitro study showed the protoscolicidal effect of M. alternifolia in all tested formulations and concentrations. Terpinen-4-ol (2mg/mL) had a better action when compared with the highest concentration of pure oil (TTO 10mg/mL). Nanoemulsion formulations (10mg/mL) showed a good activity front to the protoscolices. In ex vivo analysis, TTO (20mg/mL) showed 100% protoscolicidal action in hydatid cyst in 15 minutes. This study is pioneer in Brazil in identifying the potent effect of protoscolicidal M. alternifolia front protoscolices of E. ortleppi, being this kind of Echinococcus genus responsible for most fertile hydatid cysts analyzed in cattle in RS. / A equinococose cística (EC) é uma infecção zoonótica causada por parasito do gênero Echinococcus. O estágio larvário (cisto hidático) do parasito causa sérios problemas de saúde pública, além de acometer várias espécies de animais, ocasionando prejuízos econômicos à indústria de carnes. Neste contexto, o objetivo desse estudo foi identificar amostras de cistos hidáticos férteis em bovinos e caracterizá-las molecularmente, além de analisar in vitro e ex vivo a atividade anti-helmíntica do óleo de Melaleuca alternifolia (TTO), bem como sua formulação em nanoemulsão (NE-TTO) e seu componente majoritário (terpinen 4-ol) frente aos protoescóleces de Echinococcus spp. Foram analisadas 2396 amostras de cistos oriundos de bovinos abatidos em frigorífico da região central do RS, onde 295 amostras foram classificadas como cistos hidáticos pela presença de protoescóleces. Nas reações de PCR para o gene 12S, pode-se identificar 251 amostras positivas para o grupamento de genótipos G5/G6/G7 (E. ortleppi/ E. canadensis) e 40 amostras para o genótipo G1 (E. granulosus sensu stricto). Em subsequente PCR foram identificadas 250 amostras positivas para G5 (E. ortleppi) e uma amostra positiva para G7 (E. canadensis). Uma amostragem (n=12) dos produtos de PCR utilizando o gene da região cox I, foi sequenciada e comparada com amostras depositadas no Genbank. A análise filogenética identificou três grupos distintos E1, E2 e E3, onde agruparam-se sequências com maior similaridade. O órgão mais acometido pela parasitose em bovinos foi o pulmão. Após a confirmação da fertilidade do cisto, os protoescóleces foram retirados e avaliados quanto à viabilidade utilizando eosina 0.1%. Para o teste in vitro, concentrações de 2.5, 5 e 10mg/ml de TTO, 10mg/ml de NE-TTO e 1, 1.5 e 2mg/ml de Terpinen 4-ol foram analisadas frente aos protoescóleces de E. ortleppi previamente identificados, nos tempos de 5, 10, 15 e 30 minutos. Para o teste ex vivo, TTO 10mg/ml e 20mg/ml foi injetado diretamente em cistos hidáticos de E. ortleppi (n=20) nos tempos de 5, 15 e 30 minutos, para cada tempo foi retirada uma alíquota de protoescóleces para detecção da viabilidade e realização do PCR para confirmação da espécie envolvida na parasitose. Os resultados deste estudo in vitro indicaram efeito protoescolicida de M. alternifolia em todas as formulações e concentrações testadas. O Terpinen 4-ol (2mg/ml) obteve uma melhor ação quando comparado com a maior concentração testada do óleo puro (TTO 10mg/ml). As formulações em nanoemulsão (10mg/ml) demonstraram atividade frente aos protoescóleces. Na análise ex vivo, TTO (20mg/ml) apresentou 100% de ação protoescolicida no cisto hidático em 15 minutos. O presente estudo torna-se pioneiro no Brasil a identificar o potente efeito protoescolicida de M. alternifolia frente aos protoescóleces de E. ortleppi, sendo essa espécie do gênero Echinococcus a responsável pela maior parte dos cistos hidáticos férteis analisados em bovinos no RS.