Aplicabilidade de anticorpos monoclonais e soros hiperiumunes, na técnica de imuno-histoquímica, para o diagnóstico da leishmaniose tegumentar

Submitted by Nuzia Santos ([email protected]) on 2020-02-21T16:22:17Z No. of bitstreams: 1 D_2019_Juliana Wike Saliba.pdf: 1476165 bytes, checksum: e421270ba8935395e469ce6cd44f766d (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2020-02-21T16:34:31Z (GMT) No. of bitstreams: 1 D_2019_Juliana Wike Saliba.pdf: 1476165 bytes, checksum: e421270ba8935395e469ce6cd44f766d (MD5)Made available in DSpace on 2020-02-21T16:34:31Z (GMT). No. of bitstreams: 1 D_2019_Juliana Wike Saliba.pdf: 1476165 bytes, checksum: e421270ba8935395e469ce6cd44f766d (MD5) Previous issue date: 2019Conselho Nacional de Desenvolvimento Científico e TecnológicoFundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Existem diferentes métodos descritos para o diagnóstico laboratorial da leishmaniose tegumentar (LT). A imuno-histoquímica (IHQ) é considerada uma técnica alternativa e complementar do exame histopatológico para o diagnóstico da doença. Entretanto, as evidências sobre a aplicabilidade, reprodutibilidade e o desempenho de anticorpos monoclonais e soros hiperimunes disponíveis para a técnica de IHQ são relativamente escassas. Por isso, foram selecionados, a partir de uma pesquisa de anticorpos comerciais ou registrados em base de dados, dois anticorpos monoclonais (AcMo), um anti-Leishmania A2 (AcMo-A2) e outro anti-Leishmania GP63 (AcMo-GP63), e incluídos dois soros hiperimunes, um produzido contra a proteína recombinante KMP-11 de L. braziliensis (SH-KMP-11) e outro obtido de um “pool” de soros de cães diagnosticados com leishmaniose visceral (SH), para a realização deste trabalho. A padronização da técnica de IHQ foi realizada em amostras de lesões de pele de hamsters infectados experimentalmente com L. braziliensis, L. amazonensis e L. guyanensis. O desempenho da técnica de IHQ, usando anticorpos monoclonais e soros hiperimunes foi avaliado em 72 amostras de lesões de pacientes com suspeita clínica de LT, atendidos no Centro de Referência em Leishmanioses do Instituto René Rachou/FIOCRUZ. Os resultados foram interpretados por três observadores independentes, de acordo com os critérios “Topografia”, “Localização” e “Forma”. A maior sensibilidade e área sob a curva (AUC) da técnica de IHQ foram de 66,1% e 0,761 com o uso do SH para diagnóstico da LT. Ao comparar os resultados da IHQ com outros exames de referência, foi observado que a IHQ e o exame direto (imprint) apresentaram uma proporção de 73,4% de casos corretos e a IHQ e a PCR convencional uma proporção de 70,8% de casos corretos. A concordância entre os observadores variou conforme o critério de análise morfológica e o propósito da interpretação da IHQ. Adicionalmente, os resultados da técnica de IHQ foram muito influenciados pelo tempo de evolução da lesão. A positividade da IHQ nas lesões recentes, com tempo menor ou igual a 75 dias, foi quatro vezes maior (Odds ratio = 4,04) que nas lesões antigas. Os resultados obtidos nesse estudo confirmam que a técnica de IHQ, usando soro hiperimune de cão, constitui uma importante ferramenta no diagnóstico laboratorial da LT.Different methods are described for the laboratory diagnosis of tegumentary leishmaniasis (TL). Immunohistochemistry (IHC) is considered an alternative and complementary technique of histopathological examination for the diagnosis of TL. However, evidences on the applicability, reproducibility and performance of available monoclonal antibodies and hyperimmune sera are relatively scarces. Therefore, two monoclonal antibodies (mAb), one anti-Leishmania A2 (AcMo-A2) and one anti-Leishmania GP63 (mAb-GP63) were selected from a commercial antibody database, and included two hyperimmune sera, one produced against L. braziliensis recombinant protein KMP-11 (HS KMP-11) and the other obtained from a pool of sera from dogs diagnosed with visceral leishmaniasis (SH) for the accomplishment of this work at the Reference Center on Leishmaniasis of the René Rachou / FIOCRUZ Institute. The standardization of the IHC technique was performed on samples of skin lesions of hamsters experimentally infected with L. braziliensis L. amazonensis and L. guyanensis. The performance of the IHC, using mAb or HS was evaluated in72 lesion samples from patients with clinical suspicion of TL and interpreted by three independent observers according to the “Topography”, “Location” and “Shape” criteria. The highest sensitivity and area under the curve (AUC) of the IHC technique were 66.1% and 0.761, respectively, with HS. When comparing the results of the IHC with other reference exams, it was observed that the IHC and the direct examination (imprint) had a proportion of 73.4% of correct cases and the IHC and the conventional PCR had a proportion of 70.8% of correct cases. The agreement between the observers varied according to the criterion of morphological analysis and the purpose of the interpretation of the IHC. In addition, the results of the IHC technique were strongly influenced by the time evolution of the lesion. The IHC positivity in the recent lesions, with time less than or equal to 75 days, was four times greater (Odds ratio = 4.04) than in the old lesions. The results obtained in this study confirm that the IHC technique, using hyperimmune dog sera, is an important tool in laboratory diagnosis of LT

A prototype of the direct agglutination test kit (DAT-Canis) for the serological diagnosis of canine visceral leishmaniasis

Submitted by Nuzia Santos ([email protected]) on 2016-10-17T15:32:51Z No. of bitstreams: 1 ve_Oliveira_Edward_A prototype_CPqRR_2016.pdf: 495335 bytes, checksum: 06e9887ee0126fabb84ec99ae9329805 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-10-18T13:29:45Z (GMT) No. of bitstreams: 1 ve_Oliveira_Edward_A prototype_CPqRR_2016.pdf: 495335 bytes, checksum: 06e9887ee0126fabb84ec99ae9329805 (MD5)Made available in DSpace on 2016-10-18T13:29:45Z (GMT). No. of bitstreams: 1 ve_Oliveira_Edward_A prototype_CPqRR_2016.pdf: 495335 bytes, checksum: 06e9887ee0126fabb84ec99ae9329805 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Laboratorio de Pesquisas Clinicas. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Laboratorio de Pesquisas Clinicas. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Laboratorio de Pesquisas Clinicas. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Laboratorio de Leishmanioses. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Laboratorio de Leishmanioses. Belo Horizonte, MG, BrasilThis report describes the stege I/II development of a new direct agglutination test (DAT) for the diagnosis of canine visceral leishmaniasis (CVL) using freeze-dried antigen produced Coomassie blue-stained Leish-mania (Leishmania) infantum promastigotes. In stage I, 16 canine serum samples, collected from eight dogs carrying CVL and eight healthy dogs, were assessed with the DAT using 2-mercaptoethanol (2-ME), N-acetyl-cysteine (NAC), kaolin or NAC plus urea (NAC + U) to improve the assay conditions. Stage II assessed the diagnostic accuracy with 100 serum samples collected from dogs with symptomatic CVL and clin-ically healthy dogs, comparing the four different sample diluents. The CVL-DAT prototype kit showed equivalent performances when 2-ME, NAC or NAC + U were used: 97.1% sensitivity (CI: 83–99.8%), 97% specificity (CI: 88.5–99.5%) and a 97% diagnostic accuracy (CI: 90.8–99.2). With kaolin, a 94.1% sensitivity (CI: 79–99%), 97% specificity (CI: 88.5–99.5%) and 96% diagnostic accuracy were observed (CI: 89.5–98.7), with no statistically significant differences among the four reagents (p = 1.0). The NAC plus urea in sample diluent decreased non-specific agglutination, promoted a better defined sharp-edged blue spot and was thus chosen as a component for the new DAT prototype to diagnose canine VL, designated DAT-Canis

Monitoring the Establishment of VOC Gamma in Minas Gerais, Brazil: A Retrospective Epidemiological and Genomic Surveillance Study

Since its first identification in Brazil, the variant of concern (VOC) Gamma has been associated with increased infection and transmission rates, hospitalizations, and deaths. Minas Gerais (MG), the second-largest populated Brazilian state with more than 20 million inhabitants, observed a peak of cases and deaths in March–April 2021. We conducted a surveillance study in 1240 COVID-19-positive samples from 305 municipalities distributed across MG’s 28 Regional Health Units (RHU) between 1 March to 27 April 2021. The most common variant was the VOC Gamma (71.2%), followed by the variant of interest (VOI) zeta (12.4%) and VOC alpha (9.6%). Although the predominance of Gamma was found in most of the RHUs, clusters of Zeta and Alpha variants were observed. One Alpha-clustered RHU has a history of high human mobility from countries with Alpha predominance. Other less frequent lineages, such as P.4, P.5, and P.7, were also identified. With our genomic characterization approach, we estimated the introduction of Gamma on 7 January 2021, at RHU Belo Horizonte. Differences in mortality between the Zeta, Gamma and Alpha variants were not observed. We reinforce the importance of vaccination programs to prevent severe cases and deaths during transmission peaks