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UNIVERSIDAD NACIONAL DEL COMAHUE Estudio De La UNIVERSIDAD NACIONAL DEL COMAHUE Estudio de la variabilidad genética y fenotípica de especies y genotipos de Echinococcus granulosus sensu lato en hospedadores intermediarios de la provincia de Neuquén TRABAJO DE TESIS PARA OPTAR AL TÍTULO DE DOCTOR EN BIOLOGÍA Autora: Lic. María Florencia Debiaggi Directora: Prof. Dra. Nora B. Pierangeli Co-directora: Dra. Silvia Viviana Soriano Lugar de realización: Cátedra de Microbiología y Parasitología. Facultad de Ciencias Médicas. Universidad Nacional del Comahue. 2020 A mis padres AGRADECIMIENTOS En primer lugar quiero agradecer a Nora, mi directora, que sin conocerme me dio un lugar en su grupo de trabajo. Gracias por todo lo que me enseñaste y por tu apoyo incondicional y por permitirme crecer profesionalmente. Además, quiero agradecerte por ese trato especial que siempre me brindaste, muy familiar y afectuoso. A mi codirectora Vivi, gracias por compartir conmigo toda tu experiencia y conocimiento. Gracias por tu incondicional y permanente ayuda y por los buenos momentos compartidos. A Lore, mi compañera de laboratorio, por su alegría, gracias por ayudarme en lo experimental y por los buenos momentos compartidos. A Irene, por su ayuda en mis primeros años de CoproAg, por su compañía y largas charlas en nuestras mañanas de laboratorio. A Gaby, por ser tan compañera y por darme una mano siempre que lo necesitaba. A Mai, Seba, Barby y Agustín, que me ayudaron en el laboratorio. A Marcos Gómez, por ayudarme siempre que se lo pedía. A Luis, por capacitarme en el mundo de la biología molecular, por darme un lugar en el Laboratorio Central y por ayudarme siempre que lo necesitaba. A Meli, Mariano y Coti que me daban una mano cada vez que iba a la subse. A mi querida amiga Euge, por ayudarme en el laboratorio, por sus consejos y por nuestras largas charlas en el laboratorio y en nuestras caminatas. A los infectólogos y cirujanos del Hospital Castro Rendón y clínicas privadas, por su colaboración para la obtención de quistes hidatídicos de humanos y los datos clínicos de esos pacientes, sin los que no hubiera sido posible este trabajo. A Héctor Bergagna, por su colaboración para la obtención de quistes hidatídicos de ganado. También agradecer al Dr. Juan Basualdo por confiar en mí y ser mi director de CONICET. A Cristian Alvarez Rojas, por invitarme a realizar una pasantía en el instituto de parasitología de la Universidad de Zúrich, por compartir todo tu conocimiento y experiencia y por la buena onda. Al Dr. Peter Deplazes, por darme un lugar en su grupo de trabajo durante un mes y así poder realizar mi pasantía. A Christian Hidalgo, gracias por ayudarme en el laboratorio y por las charlas en el almuerzo y durante la espera de los termocicladores. A Sophie, por darme un lugar en su casa y hacer muy agradable mi estadía en Suiza. A mis amigos de siempre y mi familia, por apoyarme a lo largo de este trayecto. A Juli, mi hermana, por su apoyo incondicional. A mi mamá y mi papá, que siempre me apoyaron en todas las decisiones, por sus consejos y por ayudarme siempre con lo que necesitaba. A Esteban, por su amor, por su apoyo incondicional durante mi carrera, sobretodo en los últimos dos años, y por apoyarme siempre en todas mis decisiones. A todos, muchísimas gracias!!! ÍNDICE GENERAL Páginas RESUMEN i ABSTRACT iii ABREVIACIONES v 1. INTRODUCCIÓN ......................................................................................................... 1 1.1. El parásito ................................................................................................................ 2 1.2. Morfología y ciclo biológico de E. granulosus sensu lato ...................................... 3 1.2.1. Morfología del estadio adulto ............................................................................. 3 1.2.2. Morfología del huevo .......................................................................................... 4 1.2.3. Morfología del metacestodo ………………………………………………………………………….. 5 1.2.4. Ciclo biológico de E. granulosus sensu lato………………………………………………………. 8 1.3. Especies y genotipos de E. granulosus sensu lato………………………………………………… 10 1.3.1. Microdiversidad de los genotipos de E. granulosus sensu lato………………………….. 12 1.4. Patrones del ciclo de vida de E. granulosus sensu lato…………………………………………. 14 1.4.1. E. granulosus sensu stricto..…………………………………………………………………………….. 15 1.4.2. E. equinus………………………………………………………………………………………………………… 16 1.4.3. E. ortleppi………………………………………………………………………………………………………… 16 1.4.4. E. canadensis…………………………………………………………………………………………………… 17 1.4.5. E. felidis…………………………………………………………………………………………………………… 18 1.5. Distribución global de E. granulosus sensu lato…………………………………………………… 18 1.5.1. Epidemiología molecular en Europa………………………………………………………………… 19 1.5.2. Epidemiología molecular en Asia……………………………………………………………………… 21 1.5.3. Epidemiología molecular en Oceanía……………………………………………………………….. 22 1.5.4. Epidemiología molecular en África…………………………………………………………………… 22 1.5.5. Epidemiología molecular en América………………………………………………………………. 23 1.6. Echinococcosis quística en Argentina…………………………………………………………………. 26 1.7. Impacto socio-económico de la echinococcosis quística……………………………………… 29 1.8. Echinococcosis quística en humanos…………………………………………………………………… 30 1.8.1. Patología y clínica……………………………………………………………………………………………. 30 Páginas 1.8.2. Aspectos inmunológicos de la infección por E. granulosus sensu lato en humanos…………………………………………………………………………………………………………………… 34 1.8.3. Diagnóstico y seguimiento de la Echinococcosis quística…………………………………. 39 1.8.3.1. Técnicas de imagen………………………………………………………………………………………. 39 1.8.3.2. Técnicas inmunodiagnósticas……………………………………………………………………….. 40 1.8.3.3. Técnicas de detección histológica del parásito………………………………………………. 43 1.8.4. Tratamiento……………………………………………………………………………………………………. 43 1.8.4.1. Cirugía………………………………………………………………………………………………………….. 44 1.8.4.2. Drenaje percutáneo……………………………………………………………………………………… 45 1.8.4.3. Tratamiento farmacológico con benzimidazoles…………………………………………… 45 1.8.4.4. “Watch and wait” (observar y esperar)…………………………………………………………. 47 1.9. Situación de la Echinococcosis quística en Neuquén……………………………………………. 47 2. OBJETIVOS E HIPÓTESIS………………………………………………………………………………………… 52 2.1. Objetivo general…………………………………………………………………………………………………. 53 2.2. Objetivos específicos………………………………………………………………………………………….. 53 2.3. Hipótesis de trabajo……………………………………………………………………………………………. 54 3. MATERIALES Y MÉTODOS……………………………………………………………………………………… 56 3.1. Estudio 1: Caracterización molecular y biológica de quistes hidatídicos de caprinos, ovinos, bovinos y porcinos de la provincia de Neuquén………………………………. 57 3.1.1. Área y período de estudio………………………………………………………………………………… 57 3.1.2. Diseño del estudio…………………………………………………………………………………………… 58 3.1.3. Obtención y caracterización biológica del material parasitario…………………………. 58 3.1.4. Procesamiento de los quistes hidatídicos…………………………………………………………. 59 3.1.4.1. Separación de los elementos del quiste y recuento de protoescólices…………… 59 3.1.5. Determinación de las especies y genotipos de E. granulosus sensu lato……………. 59 3.1.5.1. Procesamiento del material parasitario………………………………………………………… 59 3.1.5.2. Extracción de ADN………………………………………………………………………………………… 60 3.1.5.3. Amplificación de ADN…………………………………………………………………………………… 61 3.1.5.4. Secuenciación………………………………………………………………………………………………. 61 Páginas 3.1.5.5. Análisis e interpretación de las secuencias……………………………………………………. 61 3.1.6. Evaluación de la fertilidad de quistes hidatídicos…………………………………………….. 62 3.1.7. Evaluación de la viabilidad de quistes hidatídicos……………………………………………. 63 3.1.8. Análisis estadístico de los datos……………………………………………………………………….. 63 3.2. Estudio 2: Caracterización molecular, biológica y clínica de quistes hidatídicos humanos de la provincia de Neuquén……………………………………………………………………….. 64 3.2.1. Área y período del estudio……………………………………………………………………………….. 64 3.2.2. Diseño del estudio…………………………………………………………………………………………… 64 3.2.3. Obtención y caracterización del material parasitario……………………………………….. 64 3.2.3.1. Características moleculares y biológicas del quiste……………………………………….. 65 3.2.3.2. Características clínicas del paciente………………………………………………………………. 65 3.2.3.3. Aspectos éticos…………………………………………………………………………………………….. 65 3.2.4. Determinación de haplotipos de E. granulosus sensu lato en humanos…………….. 66 3.2.4.1. Amplificación del gen completo cox1……………………………………………………………. 66 3.2.4.2. Secuenciación………………………………………………………………………………………………. 67 3.2.4.3. Construcción de redes de haplotipos……………………………………………………………. 68 3.2.5. Descripción de los ciclos de transmisión de E. granulosus sensu lato en ganado y humanos………………………………………………………………………………………………………………… 68 3.2.6. Análisis estadístico de los datos…………………………………………………………............... 68 3.3. Estudio 3: Aspectos inmunológicos de la infección por E. granulosus sensu lato en humanos……………………………………………………………………………………………………………… 70 3.3.1. Evaluación de la sensibilidad de diferentes métodos serológicos para la detección de anticuerpos IgG anti E. granulosus sensu lato……………………………………….. 70 3.3.2. Evaluación de la respuesta inmune de pacientes con hidatidosis en función de distintas variables……………………………………………………………………………………………………… 72 3.3.3. Obtención y procesamiento del líquido hidatídico……………………………………………. 72 3.3.3.1. Caracterización y fraccionamiento del LH……………………………………………………… 72 3.3.3.2. Concentración y conservación del LH……………………………………………………………. 72 3.3.4. Electroforesis de proteínas en geles de poliacrilamida……………………………………… 72 3.3.5. Caracterización antigénica del líquido hidatídico………………………………………………
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