ELISA plasmônica na detecção de anticorpos IgG anti-leishmania sp.
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Data
2019-06-10
Autores
Maciel, Marilene Oliveira dos Santos
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O cão tem sido alvo do controle da Leishmaniose Visceral (LV), pois são reservatórios potenciais de Leishmania infantum e desempenham um papel fundamental na cadeia epidemiológica da doença no homem. Portanto, o diagnóstico da leishmaniose canina (Lcan) no Brasil tem sido um desafio para os órgãos de controle de endemias, uma vez que apresentam limitações quanto à sensibilidade e especificidade em áreas endêmicas. Nesta perspectiva a presente pesquisa objetivou desenvolver e validar um ELISA plasmônica indireto rK28 (pELISA) para o diagnóstica da Lcan. Para o desenvolvimento do pELISA, foram realizados diferentes ensaios de otimização, determinação das concentrações ideais de peróxido de hidrogênio, íons ouro, anticorpo IgG anti-dog biotinilado e também do soro. Para a validação do ensaio, 170 amostras de soro de cães de área endêmica para Lcan e 26 amostras de cães saudáveis de área não endêmica para a doença foram testadas pelo pELISA e comparadas com ELISA indireto rk28 e com o teste imunocromatográfico (Dual Path Platform, TR_DPP®) usando como teste padrão-ouro o qPCR em amostras de sangue e/ou swab de subconjuntival. O ensaio foi padronizado com as concentrações de 250 μM de peróxido de hidrogênio, 0,30 mM de íons ouro e a melhor diluição do conjugado de estreptavidina-catalase foi de 1/50. O TR_DPP®, ELISA indireto rK28 e pELISA apresentaram sensibilidade de 79,0%, 89,5% e 94,7% e especificidade de 90,1%, 91,4% e 100,0%, respectivamente. Os maiores valores preditivos positivos (100,0%), negativos (99,3%) e de precisão (99,4%) foram observados no pELISA. O coeficiente Kappa entre o pELISA e a qPCR mostrou excelente concordância (0,970), diferentemente do ELISA indireto rK28 e do TR_DPP®, que mostraram um boa concordância (0,645 e 0,551, respectivamente). O resultados revelaram que o pELISA melhorou a sensibilidade e apresentou alta especificidade em relação ao método oficial recomendado pelo Ministério da Saúde no Brasil e pode aumentar a praticidade do diagnóstico em países com recursos limitados, pois não requer instrumentos sofisticados para leitura, sugerindo que este método pode ser usado como uma ferramenta adicional para o diagnóstico de Lcan nessas áreas.
Dogs have been the target of control of Visceral Leishmaniasis (VL) in humans, as they are potential reservoirs of Leishmania infantum and play a key role in the epidemiological chain of the disease. Therefore, the diagnosis of Canine Leishmaniasis (CanL) in Brazil has been a challenge for endemic control organs, since they have limitations on sensitivity and specificity in endemic areas. In this perspective the present research aimed to develop and validate an indirect plasmonic ELISA rK28 (pELISA) for the diagnosis of CanL. For the development of pELISA, different concentrations of hydrogen peroxide, gold ions, biotinylated anti-dog IgG antibody and serum were tested in order to establish ideal values to each parameter. For the validation of the assay, 170 dog serum samples from endemic area to CanL and 26 healthy dog samples from an area nonendemic to the disease were tested by pELISA and compared with indirect ELISA rk28 and the imunocromatografic test (Dual Path Platform, TR_DPP®) using as gold standard assay the real-time PCR in blood samples and/or subconjunctival swab. The assay was standardized with the concentrations of 250 μM hydrogen peroxide, 0.30 mM gold ions, and dilution of the streptavidin-catalase conjugate of 1/50. The TR_DPP®, indirect ELISA rK28 and pELISA presented sensitivity of 79.0%, 89.5% and 94.7% and specificity of 90.1%, 91.4% and 100%, respectively. The highest predictive positive (100%), negative (99.3%) and accuracy (99.4%) values were observed in pELISA. Kappa coefficient between pELISA with real-time PCR showed excellent agreement (0.970), differently of indirect ELISA rK28 and TR_DPP®, which showed good agreement (0.645 and 0.551 respectively). The results revealed that the pELISA improved sensitivity and presented higher specificity compared to official method recommended by the Ministry of Health in Brazil and may increasing the practicality of diagnosis in resource-constrained countries, because it does not require sophisticated instruments to read, suggesting that this method can be used as an additional tool for the diagnosis of CanL in these areas.
Dogs have been the target of control of Visceral Leishmaniasis (VL) in humans, as they are potential reservoirs of Leishmania infantum and play a key role in the epidemiological chain of the disease. Therefore, the diagnosis of Canine Leishmaniasis (CanL) in Brazil has been a challenge for endemic control organs, since they have limitations on sensitivity and specificity in endemic areas. In this perspective the present research aimed to develop and validate an indirect plasmonic ELISA rK28 (pELISA) for the diagnosis of CanL. For the development of pELISA, different concentrations of hydrogen peroxide, gold ions, biotinylated anti-dog IgG antibody and serum were tested in order to establish ideal values to each parameter. For the validation of the assay, 170 dog serum samples from endemic area to CanL and 26 healthy dog samples from an area nonendemic to the disease were tested by pELISA and compared with indirect ELISA rk28 and the imunocromatografic test (Dual Path Platform, TR_DPP®) using as gold standard assay the real-time PCR in blood samples and/or subconjunctival swab. The assay was standardized with the concentrations of 250 μM hydrogen peroxide, 0.30 mM gold ions, and dilution of the streptavidin-catalase conjugate of 1/50. The TR_DPP®, indirect ELISA rK28 and pELISA presented sensitivity of 79.0%, 89.5% and 94.7% and specificity of 90.1%, 91.4% and 100%, respectively. The highest predictive positive (100%), negative (99.3%) and accuracy (99.4%) values were observed in pELISA. Kappa coefficient between pELISA with real-time PCR showed excellent agreement (0.970), differently of indirect ELISA rK28 and TR_DPP®, which showed good agreement (0.645 and 0.551 respectively). The results revealed that the pELISA improved sensitivity and presented higher specificity compared to official method recommended by the Ministry of Health in Brazil and may increasing the practicality of diagnosis in resource-constrained countries, because it does not require sophisticated instruments to read, suggesting that this method can be used as an additional tool for the diagnosis of CanL in these areas.
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Palavras-chave
Cães, Diagnóstico, Leishmaniose Visceral, Nanopartículas metálicas, Dogs, Diagnosis, Leishmaniasis Visceral, Metal nanoparticles