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Previous issue date: 2013-03-27 / Abdominal angiostrongyliasis is a zoonotic infection caused by Angiostrongylus costaricensis, a nematode with intravascular location in the mesentery. Humans become accidentally infected by ingesting food or water contaminated with third stage larvae present in mucus secreted by intermediate hosts, terrestrial mollusks. The confirmed diagnosis of human infection is made only through histopathology of biopsies or mesenteric tissues removed during surgical treatment. There are many cases in which the pathology is very suggestive however there is no evidence of parasite structures in the sections. Thus, the goal of this study was to standardize the extraction of DNA from formalin fixed paraffin embedded (FFPE) tissues from mice experimentally infected with Angiostrongylus costaricensis for DNA detection and future study of human angiostrongyliasis suspected cases. Materials and methods: 45 samples of each tissue of liver, lung, intestine and mesentery, totalizing 180 samples from 15 infected mice with A.costaricensis. Worms of Angiostrongylus cantonensis were separately embedded in paraffin as controls. From embedded tissues, histological sections of 3 μm were performed and stained with HE to confirm the presence of parasite structures under the microscope. Approximately 24 to 31 sections of 10 μm were used for DNA extraction. Worms were embedded in paraffin under different conditions such as fixative chemicals, time of fixation and types of paraffin. Specific DNA of Angiostrongylus was detected by Polymerase Chain Reaction (PCR) to amplify a sequence of 232bp, previously designed for detection in human serum assays. There was a positive amplification in 33 samples from liver, 12 from lung, 45 from mesentery and 36 from intestine wall. For different conditions of embedment, the amplification of the DNA with the buffered formalin fixative was more efficient compared to the unbuffered one; the time of fixation and the commercial brand of wax did not affect the detection of nucleic acids, however, the ratio of paraffin to tissue appears to influence the performance of the method since samples with residual paraffin no amplification was obtained. The 232bp segment also was not visualized in samples where there was no evidence of presence of parasite structures, suggesting that histopathology findings should guide the choice of the samples for DNA extraction, and those most likely to have parasite structures surrounding the lesion, even if degraded. The description of the performance of PCR in paraffin embedded tissues as a diagnostic tool in human abdominal angiostrongyliasis depends on the detailed evaluation criteria in histopathology and their association with a positive amplification of the specific probe. / Angiostrongil?ase abdominal ? uma infec??o zoon?tica causada por Angiostrongylus costaricensis, um nemat?deo com localiza??o intravascular no mesent?rio. O homem se infecta acidentalmente ao ingerir alimentos ou ?gua contaminados com larvas de terceiro est?gio presentes no muco secretado por moluscos terrestres, hospedeiros intermedi?rios. O diagn?stico confirmado da infec??o humana ? feito pelo exame histopatol?gico de bi?psias ou segmentos ressecados durante o tratamento cir?rgico de casos complicados. H? muitos casos em que a histopatologia ? muito sugestiva, por?m sem evidenciarem-se estruturas do parasito nos cortes. Desse modo, o objetivo foi padronizar a extra??o de DNA em tecidos embebidos em parafina em modelo murino visando ? detec??o de ?cidos nucl?icos e posterior utiliza??o no estudo de casos suspeitos a partir do exame histopatol?gico em humanos. Materiais e M?todos: 45 amostras de cada tecido de f?gado, pulm?o, mesent?rio e intestino, totalizando 180 amostras de 15 camundongos infectados com Angiostrongylus costaricensis; e vermes de Angiostrongylus cantonensis foram embebidos separadamente em parafina e nesses blocos contendo os tecidos foram realizados cortes histol?gicos de 3 μm para colora??o por Hematoxilina-Eosina, e confirmado a presen?a de estruturas parasit?rias, foram coletados de 24 a 31 cortes de 10 μm em um microtubo para a extra??o de DNA.
Para a padroniza??o do m?todo, vermes foram embebidos em parafina em diferentes condi??es tais como o tipo de fixador, tempo de fixa??o e marcas comerciais de parafina. A partir dos cortes foram realizadas as extra??es de DNA com o Kit Quiagen DNEasy tissue. Para a detec??o de DNA espec?fico de Angiostrongylus foi utilizado a Rea??o em Cadeia da Polimerase (PCR), para amplifica??o de uma sequ?ncia de 232pb, previamente desenhada para ensaios de detec??o em soro humano. Em 33 amostras de f?gado, 12 de pulm?o, 45 de mesent?rio e 36 da parede intestinal houve amplifica??o do segmento de 232pb. Para as diferentes condi??es de emblocamento, as amplifica??es do DNA das amostras a partir do fixador formalina tamponada foram mais frequentes quando comparado com a formalina n?o tamponada; o tempo de fixa??o e o tipo de parafina n?o interferiram na detec??o de ?cidos nucl?icos, entretanto, a quantidade de parafina no tecido parece influenciar no desempenho do m?todo, pois nas amostras onde havia excesso de parafina n?o houve amplifica??o. O segmento de 232pb tamb?m n?o foi visualizado nas amostras onde n?o foi evidenciada a presen?a de estruturas parasit?rias, sugerindo que a informa??o provinda da histopatologia deve orientar a retirada da amostra para extra??o de DNA, como aquelas de maior probabilidade de possuir estruturas parasit?rias nas proximidades da les?o, mesmo que degradadas. A descri??o do desempenho da PCR como recurso diagn?stico da infec??o humana, em material embebido em parafina, depende da avalia??o detalhada dos crit?rios de suspeita na histopatologia correlacionada com a frequ?ncia e reprodutibilidade do resultado da extra??o de DNA e sua amplifica??o.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/5462 |
| Date | 27 March 2013 |
| Creators | Alves, B?rbara Rodrigues |
| Contributors | Graeff-teixeira, Carlos |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Biologia Celular e Molecular, PUCRS, BR, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 8198246930096637360, 600, 600, 36528317262667714 |
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