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Gastroenteritis Aguda Por Rotavirus En La Población Infantil Atendida En El Hospital Universitario Virgen De La Arrixaca D. Pedro Francisco Sánchez López 2017

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Gastroenteritis Aguda Por Rotavirus En La Población Infantil Atendida En El Hospital Universitario Virgen De La Arrixaca D. Pedro Francisco Sánchez López 2017 UNIVERSIDAD DE MURCIA FACULTAD DE BIOLOGÍA Gastroenteritis Aguda por Rotavirus en la Población Infantil Atendida en el Hospital Universitario Virgen de la Arrixaca D. Pedro Francisco Sánchez López 2017 AGRADECIMIENTOS En primer lugar, quiero expresar mi máximo agradecimiento a los directores de esta tesis doctoral, al Dr. Manuel Segovia Hernández, Jefe del Servicio de Microbiología del Hospital Universitario Virgen de la Arrixaca y Catedrático de Microbiología de la Universidad de Murcia, por haberme recibido en su laboratorio desde un ámbito profesional aparentemente distante, y haber confiado en el entusiasmo que le mostré; y a la Dra. Carme Salvador García, facultativo adjunto del Servicio de Microbiología y profesora asociada de la Universidad de Murcia, por su constante apoyo durante todos estos años. También me siento profundamente agradecido a la Prof. Miren Iturriza-Gómara, del Institute of Infection and Global Health de la Universidad de Liverpool, por acogerme en su laboratorio para realizar las secuenciaciones masivas de algunas cepas de rotavirus interesantes y permitirme vivir una experiencia tan enriquecedora a su lado. Gracias también al Dr. Khuzwayo Jere, que con ese carácter tan afable me enseñó los secretos de la secuenciación masiva. Gracias a la Dra. Elisa Gómez Campoy, Jefe del Servicio de Sanidad Ambiental de la Consejería de Salud, por su apoyo y su aliento continuos. Gracias al Dr. Javier Buesa Gómez, médico adjunto del Hospital Clínico Universitario de Valencia y Catedrático de Microbiología de la Universidad de Valencia, por ese primer empujón en las técnicas de genotipado de los rotavirus. Gracias a Pedro José Bernal González, del Servicio de Prevención y Protección de la Salud de la Consejería de Salud, por su ayuda con los datos de vacunación. Gracias a los estadísticos de la Consejería de Salud, Mónica Ballesta Ruiz y Diego Salmerón Martínez, por su ayuda con el análisis estadístico de los datos. Gracias también a todos los facultativos adjuntos y residentes, y al resto de personal del Servicio de Microbiología del Hospital, con los que he coincidido durante mi trabajo de laboratorio y que siempre me han ayudado. Y en el ámbito personal, quiero dar las gracias a mi familia, que es el pilar que ha sustentado mi fortaleza; a mi pareja, que es el pilar que mantiene viva mi ilusión; y a mis amigos, que son quienes han mantenido a flote mi cordura. ____________________________________________________________________________________ Abreviaturas ABREVIATURAS ADN Ácido desoxirribonucleico ARN Ácido ribonucleico BSA Albúmina de suero bovino EIA Enzimoinmunoanálisis GEA Gastroenteritis aguda HUVA Hospital Universitario Virgen de la Arrixaca ICTV International Commitee on Taxonomy of Viruses MMOH Hidróxido de metilmercurio OMS Organización Mundial de la Salud PBS Tampón fosfato salino PEG Polietilenglicol PCR Reacción en cadena de la polimerasa RPM Revoluciones por minuto TBE Tris-borato-EDTA VIH Virus de la inmunodeficiencia humana I II ___________________________________________________________________________________ Índice general ÍNDICE GENERAL I - INTRODUCCIÓN Y OBJETIVOS DE ESTA TESIS ............................................................................ 1 1. Antecedentes e identificación del problema. ................................................................... 3 2. Motivación y fundamentos del estudio. ........................................................................... 3 3. Objetivo principal y objetivos específicos. ........................................................................ 4 4. Diseño del estudio. ............................................................................................................ 4 II- REVISIÓN BIBLIOGRÁFICA ......................................................................................................... 7 1. Breve reseña histórica ....................................................................................................... 9 2. Filogenia del rotavirus. .................................................................................................... 12 3. Estructura del virión ........................................................................................................ 15 4. Genoma ........................................................................................................................... 18 5. Ciclo replicativo ............................................................................................................... 21 6. Clasificación y nomenclatura de rotavirus ...................................................................... 24 7. Evolución de rotavirus ..................................................................................................... 28 8. Detección y caracterización de rotavirus ........................................................................ 30 9. Patogénesis ..................................................................................................................... 35 10. Clínica del rotavirus. .................................................................................................... 38 11. Epidemiología de la infección por rotavirus ................................................................ 39 12. Tratamiento y prevención ........................................................................................... 45 13. Vacunas ....................................................................................................................... 46 14. Microbioma y viroma .................................................................................................. 52 III- MATERIAL Y MÉTODOS .......................................................................................................... 55 Población de estudio ............................................................................................................... 57 Toma de muestras ................................................................................................................... 57 Análisis estadístico .................................................................................................................. 58 1. Incidencia de gastroenteritis por rotavirus entre 2010-2013 ......................................... 58 1.1. Recogida de información sobre fecha de recogida de la muestra, edad y sexo del paciente ............................................................................................................................... 58 1.2. Detección de antígeno de rotavirus A en heces mediante el test rápido VIKIA ® Rota- Adeno (Biomerieux, Francia) ............................................................................................... 59 1.2.1. Fundamento ........................................................................................................ 59 1.2.2. Realización del ensayo ........................................................................................ 59 1.2.3. Controles de calidad ............................................................................................ 59 I Gastroenteritis aguda por rotavirus en la población infantil atendida en el Hospital Universitario Virgen de la Arrixaca 1.2.4. Interpretación de resultados ............................................................................... 60 2. Características clínico-epidemiológicas de las gastroenteritis por rotavirus durante 2012 60 2.1. Recogida de información clínico-epidemiológica de los casos de gastroenteritis positivos a antígeno de rotavirus A ..................................................................................... 60 2.1.1. Origen de la infección .......................................................................................... 60 2.1.2. Procedencia de las muestras en los casos comunitarios .................................... 60 2.1.3. Tiempo en desarrollar una gastroenteritis por rotavirus de adquisición nosocomial .......................................................................................................................... 61 2.1.4. Duración de la hospitalización y número de ingresos en urgencias ................... 61 2.1.5. Procedencia de los casos nosocomiales .............................................................. 61 2.1.6. Motivo de la hospitalización en los casos nosocomiales .................................... 61 2.1.7. Sintomatología de los casos comunitarios .......................................................... 61 2.1.8. Severidad de las gastroenteritis en los casos comunitarios ................................ 62 2.2. Estudio de genotipado de cepas de rotavirus ......................................................... 63 2.2.1. Fundamento ........................................................................................................ 63 2.2.2. Extracción y purificación del ARN de las muestras de heces .............................. 66 2.2.3. Reacción de transcripción inversa para síntesis de ADN copia y amplificación de la secuencia consenso VP7 ó VP4........................................................................................ 67 2.2.4. Segunda PCR para la amplificación de productos específicos de genotipo ........ 68 2.2.5. Visualización de los amplicones .......................................................................... 71 2.2.6. Controles de calidad ............................................................................................ 71 2.2.7. Interpretación de resultados ..............................................................................
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