Submitted by Bruno Giorno Eberhardt (bruno.giorno@agricultura.gov.br) on 2018-04-21T01:40:57Z No. of bitstreams: 1 TESE DOUTORADO - BRUNO GIORNO EBERHARDT.pdf: 3876871 bytes, checksum: 3c2c7eac21349ce401f54a1cb03fae4f (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-04-23T12:55:33Z (GMT) No. of bitstreams: 1 eberhardt_bg_dr_botfca.pdf: 2911225 bytes, checksum: 9f9ad5ea743405838741796482e9ab66 (MD5) / Made available in DSpace on 2018-04-23T12:55:33Z (GMT). No. of bitstreams: 1 eberhardt_bg_dr_botfca.pdf: 2911225 bytes, checksum: 9f9ad5ea743405838741796482e9ab66 (MD5) Previous issue date: 2018-03-01 / EBERHARDT, B.G. Avaliação de micro-organismos zoonóticos em filés de tilápia do Nilo (Oreochromis niloticus). Botucatu, 2018. 71p. Tese (Doutorado) – Faculdade de Medicina Veterinária e Zootecnia, Campus de Botucatu, Universidade Estadual Paulista. RESUMO Cinquenta filés de tilápia do Nilo (Oreochromis niloticus) obtidos em mercado de peixes no município de Ourinhos, Estado de São Paulo, foram analisados quanto à prevalência para Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium spp. e Cianobactérias. Amostras de músculo foram avaliadas por PCR para Aeromonas hydrophila, Edwardsiella tarda e Mycobacterium spp., enquanto que as amostras para cianobactérias foram analisadas por PCR em Tempo Real (qPCR). Os resultados obtidos demonstraram ausência de Aeromonas hydrophila e Edwardsiella tarda nas amostras de filés. A prevalência para Mycobacterium spp. foi de 100% (50/50). Realização posterior de sequenciamento revelou Mycobacterium gordonae. Esta bactéria é considerada um colonizador comum, normalmente não patogênico, porém, há relatos de literatura que demonstram risco de infecção em indivíduos imunossuprimidos e até mesmo imunocompetentes. A taxa de prevalência para cianobactérias foi de 48% (24/50). As cianobactérias (algas azuis) produzem grande quantidade de metabólitos bioativos ou mesmo tóxicos, incluindo toxinas associadas a problemas ambientais e de saúde pública. Considerando a natureza e o papel das cianobactérias como patógenos emergentes, a elevada prevalência deste organismo em um alimento popular como o filé de tilápia do Nilo desperta preocupação, uma vez que os métodos tradicionais de inspeção são incapazes de detectar o patógeno, pelo fato de não provocar alterações macroscópicas nos produtos, bem como pelo potencial de toxicidade para humanos. Entretanto, estudos adicionais são necessários a fim de entender se estes compostos tóxicos estão presentes nos peixes e, caso estejam, se podem sofrer bioacumulação em níveis suficientes para afetar a saúde humana. Palavras-chave: Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium spp, Mycobacterium gordonae, cianobactéria, cianotoxina, tilápia do Nilo, Oreochromis niloticus, diagnóstico. / Fifty fillets of Nile tilapia (Oreochromis niloticus) from a Ourinhos fish market, Sao Paulo State (Southeast Brazil) were analyzed for the prevalence of Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium spp. and cyanobacteria. Muscle samples were evaluated by PCR for Aeromonas hydrophila, Edwardsiella tarda and Mycobacterium spp. Samples for cyanobacteria were analyzed by real time PCR. Both Aeromonas hydrophila and Edwardsiella tarda were not present in fish samples. The prevalence of Mycobacterium spp. was 100% (50/50). Sequencing revealed Mycobacterium gordonae. This agent is usually a ubiquitous and commonly nonpathogenic colonizing organism, although many research publications have reported disease in immunocompromised or even in immunocompetent patients. Prevalence for cyanobacteria was 48% (24/50). Cyanobacteria (blue-green algae) produce a diversity of toxic or otherwise bioactive metabolites, including a number of toxins that have been associated with human and environmental health concerns. Considering the nature and role of cyanobacteria as a pathogen of emerging importance, the high prevalence of this organism in a popular food item such as Nile tilapia raises concern, since no macroscopic alterations can be detected through regular food inspection methods. However, further studies are necessary in order to understand whether these compounds are present in fish and, if so, if they could accumulate sufficiently to affect human health.

L'isolement de Mycobacterium tuberculosis par culture est la méthode de référence pour le diagnostic de la tuberculose. Le but de notre travail était d'améliorer et de faciliter le diagnostic par culture de la tuberculose. Dans un premier temps, nous avons produit une revue bibliographique en comparant les différentes techniques ou protocoles utilisés pour le diagnostic de la tuberculose. Ce travail nous a permis d'actualiser notre protocole de diagnostic, avec la mise en place d’un "kit-tuberculose" contenant des containers imprégnés de chlorhexidine pour la récupération et la décontamination directe d’échantillons cliniques non- invasifs, suivi par la culture sur un milieu solide à base d'oeuf, et détection des colonies par microscope inversé ou par un système d'imagerie en temps réel. Nous avons mis en place une méthode de décontamination par 0,7%-chlorhexidine et avons montré que cette méthode était plus efficace que la méthode de référence NALC-NaOH. Ensuite, nous avons développé un milieu de culture à base de sérum animal, le MOD9 dont nous avons montré par une étude comparative qu'il était supérieur au milieu solide LJ de référence. Une deuxième étude comparant un protocole de décontamination par la chlorhexidine et culture sur milieu MOD9 au protocole standard, NALC-NaOH/Bactec960 a montré une supériorité par rapport au protocole standard. Enfin, la mise en place d'un système de détection des micro-colonies de M. tuberculosis sur MOD9 par imagerie en temps réel Advencis-Biosystem a permis de réduire le temps de détection de M. tuberculosis à 3,2 jours avec le protocole chlorhexidine/MOD9/Advencis, avec un record mondial de détection en 25h. / Isolation of Mycobacterium tuberculosis by culture is the gold standard for the diagnosis of tuberculosis. The aim of my thesis work was to simplify and improve the culture diagnosis of tuberculosis. At first we started with a bibliographic study, comparing step by step the different techniques and protocols that have been used for the diagnosis of tuberculosis. This work has allowed us to update our tuberculosis diagnosis protocol, starting with the implementation of a "Tuberculosis-kit" consisting of chlorhexidine containing containers for the recovery and decontamination of non-invasive specimens, followed by culture on an egg-based medium, a micro- colonies detection using an inverted microscope or an automated real-time imaging incubator system and finally an identification using mass spectrometry. We established a new chlorhexidine- based decontamination method that we showed to be more efficient for the recovery and isolation of M. tuberculosis than the standard NALC-NaOH method. Than we developed a new serum-based culture medium, the MOD9 that we showed in a comparative study to be superior to the reference LJ medium for the recovery of M. tuberculosis. In a second study we proved that our chlorhexidine/MOD9 protocol was superior to the standard NALC-NaOH/Bactec 960 MGIT protocol for the isolation of M. tuberculosis. And finally the implementation of a real time imaging system for the detection of M. tuberculosis micro-colonies on MOD9 permits us to dramatically reduce the detection time from 15 days with the standard NALC-NaOH/Bactec 960 MGIT protocol to 3.2 days with our 0.7%-chlorhexidine/MOD9/Advencis-Biosystem protocol with a world record detection time of 25h.

Made available in DSpace on 2016-01-26T12:51:36Z (GMT). No. of bitstreams: 1 adrianafurini_dissert.pdf: 2158923 bytes, checksum: d683b1dc320b2c811cfcf58d6d1c21ac (MD5) Previous issue date: 2010-11-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Introduction. The diagnosis of paucibacillary forms of tuberculosis (TB), which mostly affects children, immunocompromised patients, transplanted and extra-pulmonary forms is limited by phenotypic techniques of smear and culture. Moreover, the diagnosis of mycobacteriosis, also has committed itself by the phenotypic methods. The polymerase chain reaction (PCR) and its variations, such as Nested-PCR (NPCR), has been described as promising techniques for rapid diagnosis of tuberculosis and mycobacteriosis. Objective. Evaluation of a NPCR protocol for detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary anatomic sites of patients with clinical suspicion of TB. Subjects and Methods. Were included, prospectively, 24 pulmonary samples of 85 extrapulmonary, collected from 49 individuals HIV-seropositive and 28 HIV-seronegative and submitted to the gold standard method and NPCR targeting the transposon IS6110. Results. Tuberculosis was diagnosed in 11 patients (14,3%), with 54,5% of extrapulmonary forms and 45,5% pulmonary. The NPCR was positive in all, while the culture was positive in only seven of them. Smear positivity among the clinical specimens was 9,2%. The culture allowed the isolation of seven strains of M. tuberculosis and two M. avium complex (8,25%). The molecular positivity was described in 23,85% of samples. NPCR's performance against the culture, for pulmonary (n=22) and extrapulmonary samples (n = 83) was similar (100% sensitivity and specificity of approximately 83%). Positivity by NPCR was significantly greater than the isolation by culture among the extrapulmonary samples (p = 0,0042). Conclusions. The results suggest to perform further studies to corroborate the potential of NPCR-IS6110 for detection of mycobacterial genome in extrapulmonary TB, especially among immunocompromised. Furthermore, the detection of mycobacteria remains as diagnostic confirmation and the opportunity of investigation of the sensitivity profile, promoting effective treatment for any age and immune status. / Introdução. O diagnóstico das formas paucibacilares da tuberculose (TB), que acomete principalmente crianças, imunocomprometidos, transplantados e formas extrapulmonares é limitado pelas técnicas fenotípicas de baciloscopia e cultura. Ademais, o diagnóstico das micobacterioses, também apresenta-se comprometido pelos métodos fenotípicos. A reação da polymerase em cadeia (PCR) e suas variações, tais como a Nested-PCR (NPCR), tem sido descritas como técnicas promissoras para o rápido diagnóstico da tuberculose e micobacterioses. Objetivos. Avaliação de um protocolo de NPCR para detecção do complexo Mycobacterium tuberculosis em sítios anatômicos pulmonares e extrapulmonares de indivíduos com suspeita clínica de TB. Casuística e Método. Foram incluídas, prospectivamente, 24 amostras de sítios pulmonares e 85 extrapulmonares, coletadas a partir de 49 indivíduos soropositivos para o HIV e 28 soronegativos e submetidas ao método gold standard e a NPCR tendo como alvo o transposon IS6110. Resultados. A tuberculose foi diagnosticada em 11 pacientes (14,3%), com 54,5% de formas extrapulmonares e 45,5% pulmonares. A NPCR foi positiva em todos, enquanto a cultura em apenas cinco deles. A positividade da baciloscopia entre os espécimes clínicos foi de 9.2%. A cultura permitiu o isolamento de sete cepas de M. tuberculosis e duas do Complexo M. avium (8,25%). A positividade molecular foi descrita em 23,85% das amostras. O desempenho da NPCR frente a cultura, para amostras pulmonares (n=22) e extrapulmonares (n=83) foi similar (100% de sensibilidade e aproximadamente 83% de especificidade). A positividade pela NPCR foi significantemente maior que o isolamento por cultura entre as amostras extrapulmonares (p=0.0042). Conclusões. Os resultados obtidos sugerem a realização de estudos mais amplos que corroborem o potencial da NPCR-IS6110 para a detecção do genoma micobacteriano na TB extrapulmonar, em especial entre imunocomprometidos. Ademais, a detecção da micobactéria permanece como confirmação diagnóstica e a oportunidade de investigação do perfil de sensibilidade, favorecendo o tratamento efetivo para qualquer faixa etária e status imunológico.