The diploma thesis "Determination of aflatoxin in selected products" deals mainly with aflatoxin, its occurrence in raw materials of plant origin and methods of decontamination. The studied toxin is produced by fibrous micromycetes primarily from genus Aspergillus is their secondary metabolites. It is very widespread and is a frequent cause of retention of raw materials at the border. Factors that affect production of aflatoxin are, among other things, water activity and pH and are also determined in samples and mentioned in theoretical part. A total of 30 samples were tested for aflatoxin B1, including batch of apricot jam, two batches of Svatojánské ořechy and two batches of Jarní hustopečské mandle. A heterogeneous competitive enzyme immunoassay (ELISA) was used for the analysis. Detectable amounts of AFB1 in the range of 1,26 - 1,66 μg∙kg-1 showed 26,67 % of samples. These values were determined in samples of Svatojánské ořechy and Jarní hustopečské mandle, the apricot jam did not contain measurable amount of AFB1.

Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of ∼30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.

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Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:48:40Z No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:48:50Z (GMT) No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) / Made available in DSpace on 2015-05-12T13:48:50Z (GMT). No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A dengue é atualmente um dos principais problemas de saúde pública do mundo e segundo a Organização Mundial de Saúde (OMS) é a doença que mais acomete o homem na atualidade. Sua incidência vem aumentando e estima-se que 50-100 milhões de pessoas desenvolvam a doença a cada ano no mundo. O diagnóstico laboratorial da dengue é realizado por diferentes tipos de testes, entre eles estão o isolamento viral, o RT-PCR, e a detecção por ELISA ou por meio de testes rápidos do antígeno viral NS1 e de anticorpos IgM específicos contra o vírus. A fim de contribuir para um melhor entendimento sobre a validade destes testes em diferentes circunstâncias, o objetivo principal deste trabalho foi avaliar a validade dos diferentes métodos laboratoriais no diagnóstico da dengue. A sensibilidade dos testes diagnósticos, ELISA IgM, ELISA NS1 e RT-PCR, foi avaliada de forma individual e de forma combinada utilizando amostras de soro de 623 pacientes incluídos em um estudo prospectivo de vigilância de base populacional entre fevereiro e julho de 2010. A sensibilidade destes testes também foi avaliada de acordo com a duração dos sintomas, com o tipo de infecção (primária vs secundária) e, por sorotipo infectante. A especificidade de cada método foi avaliada em um grupo de amostras de pacientes com diagnóstico laboratorial de leptospirose, hepatite, doadores de sangue e indivíduos sadios. Os resultados encontrados mostraram que 240 (38%) dos pacientes com doença febril aguda apresentaram dengue no período do estudo sendo que 194 (81%) dos pacientes com dengue representavam pacientes com infecções secundárias, o sorotipo predominante foi o DENV-2 (70%). As sensibilidades do RT-PCR, do ELISA NS1 e do ELISA IgM na amostra de fase aguda foram de 83,3%, 31,7% e 30%, respectivamente. O uso combinado do teste RT-PCR e do teste ELISA IgM em uma amostra de fase convalescente foi capaz de identificar 100% dos casos confirmados de dengue. As especificidades encontradas variaram de 97% a 100% para o ELISA NS1 e de 55% a 85% para o ELISA IgM. Os resultados indicam que na fase aguda da doença o RT-PCR é mais sensível a detecção de anticorpos IgM e do antígeno NS1 por ELISA, entretanto, o uso de métodos diagnósticos adicionais pode ser necessário em pacientes com uma suspeita da doença e resultado negativo do RT-PCR. / Dengue is currently one of the main problems of public health and the world according to the World Health Organization (WHO) is the disease that affects more men today. Its incidence is increasing and it is estimated 50-100 million people develop the disease each year worldwide. Laboratory diagnosis of dengue is done by testing different types, which include viral isolation, RT-PCR, and detection by ELISA or by rapid viral tests NS1 antigen and specific IgM antibodies against the virus. In order to contribute to a better understanding of the validity of these tests in different circumstances, the aim of this study was to evaluate the validity of different laboratory methods for diagnosis of dengue. The sensitivity of diagnostic tests, IgM ELISA, ELISA NS1 and RT-PCR was evaluated individually and in combination form using serum samples from 623 patients enrolled in a prospective population-based study of surveillance between February and July 2010. The sensitivity of these tests was also evaluated according to the duration of symptoms of infection with the type (primary versus secondary), and the infecting serotype. The specificity of each method was evaluated in a group of samples from patients with laboratory diagnosis of leptospirosis, hepatitis, blood donors and healthy individuals. The results showed that 240 (38%) of patients with acute febrile disease had dengue during the study period of which 194 (81%) of patients with dengue represented patients with secondary infections, the predominant serotype was DENV-2 (70% ). The sensitivity of the RT-PCR of NS1 and IgM ELISA ELISA in the acute phase of the sample were 83.3%, 31.7% and 30%, respectively. The combined use of RT-PCR and ELISA IgM in a sample convalescent phase was able to identify 100% of confirmed cases of dengue fever. The specificities found varied from 97% to 100% for ELISA NS1 and 55% to 85% for the IgM ELISA. The results indicate that the acute phase of the disease the RT-PCR is more sensitive detection of IgM antibodies and NS1 antigen by ELISA, however, the use of additional diagnostic methods may be necessary in patients with a suspicion of disease and negative outcome of RT-PCR.

Clostridium difficile est un pathogène entérique pouvant causer des diarrhées allant de modérées à sévères, des colites pseudomembraneuses et même la mort. Les traitements actuels contre la bactérie ont des taux de rechutes élevés. De plus, il n'existe pas à ce jour de vaccin permettant de prévenir l'infection. Cette étude épidémiologique porte sur la protection contre la colonisation et/ou la maladie conférée par la présence d'anticorps sériques naturels spécifiques aux antigènes de la bactérie. Nous avons développé un test ELISA (Enzyme-Linked ImmunoSorbent Assay) pour la détection des anticorps contre les toxines A et B de C. difficile et contre les protéines du flagelle (flagellines) dans des échantillons de sérum. Notre test ELISA servant à détecter les anticorps dirigés contre la toxine B a été calibré de façon à obtenir la meilleure corrélation possible avec le test de neutralisation de la cytotoxicité de la toxine B. Les paramètres du test ELISA ainsi mis au point ont été appliqués aux autres antigènes (toxine A et flagellines), pour lesquels un test de référence n'existe pas. Par la suite, nous avons tenté de définir le rôle de la réponse immunitaire humorale de l'hôte en corrélant les résultats de l'analyse des sérums de deux groupes de patients hospitalisés avec les informations sur le suivi des symptômes et la recherche de la présence de la bactérie dans les selles. Le premier groupe de patients nous a permis d'étudier la relation entre la survenue de la colonisation ou de l'infection par C. difficile et la production d'anticorps. Nos résultats montrent que les anticorps sériques ne semblent pas protéger le patient de l'implantation de la bactérie au tube digestif mais que suite à l'infection, une réponse humorale est mise en place contre les toxines du pathogène. Les patients du deuxième groupe, des patients hospitalisés infectés par C. difficile, ont été surveillés pour la survenue de symptômes sévères, du décès ou de rechutes suite à un épisode de maladie. Nous avons observé une plus grande prévalence d'anticorps sériques dirigés contre la toxine B et contre certaines flagellines chez les patients ayant eu une infection simple, sans complications ni rechutes. Les résultats de nos travaux indiquent donc que certains patients développent une réponse humorale contre les antigènes de C. difficile et que les anticorps produits, particulièrement ceux dirigés contre la toxine B, semblent impliqués dans la défense du patient contre la survenue de complications et de rechutes. Nos données laissent toutefois sous-entendre que d'autres caractéristiques de l'hôte contribuent de façon importante à la défense contre la colonisation et l'infection par C. difficile. Des travaux supplémentaires sont nécessaires afin de définir les paramètres qui permettront d'élaborer un protocole de vaccination optimal contre C. difficile.

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Memoria para optar al Titulo Profesional de Médico Veterinario / Las enfermedades asociadas a circovirus porcino tipo 2 (PCV-2) son consideradas el problema productivo y económico más grande de la industria porcina mundial. Además de mejorar la gestión y las prácticas de manejo (mejor higiene, menor hacinamiento y mejor ventilación), la disponibilidad de vacunas anti-PCV-2 representa la opción inmunológica más eficaz para paliar el impacto de estas enfermedades. Para el presente estudio y con la finalidad de estudiar el patrón de seroconversión contra PCV-2 entre cerdos vacunados con una vacuna comercial versus cerdos infectados naturalmente, se montó un ensayo de ELISA indirecto, usando como sustrato específico una versión recombinante de la subunidad proteica involucrada con el ensamblaje de la cápside viral (Cap). De estos análisis se concluyó, en primera instancia, que dicha estructura pudo ser reconocida en forma específica tanto por los sueros de los animales vacunados como no vacunados infectados naturalmente, analizados a distintos periodos de tiempo: 30, 50, 70, 90, 110 y 130 días, siendo posible analizar el patrón de seroconversión IgM/IgG anti-PCV-2 en dichos animales. En este estudio se concluye que el efecto diferenciador de la prueba montada de ELISA indirecto entre animales vacunados o no vacunados, depende si es que el animal cuenta o no con inmunidad de base materna anti-PCV-2, pudiendo ser esta una condición interferente con la vacunación temprana. Así, fue posible organizar y dividir los datos del patrón de seroconversión IgM/IgG anti-PCV-2 bajo cuatro escenarios distintos de resultados: 1, animales vacunados con baja inmunidad de base; 2, animales vacunados con alta inmunidad de base; 3, animales no vacunados con alta inmunidad de base y 4, animales no vacunados con baja inmunidad de base. Si bien en el presente estudio la determinación de la presencia de anticuerpos totales contra PCV-2, no resultó ser útil para diferenciar anticuerpos neutralizantes, los resultados presentados y discutidos en esta memoria son de utilidad para evaluar el status de seroconversión general contra PCV-2 de un plantel nacional de producción porcina / Fondecyt 11110135