Utilização da LAMP para diagnóstico e avaliação da eficácia terapêutica do cloridrato de isometamidium em bovinos naturalmente infectados com Trypanosoma vivax
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Data
2018-08-31
Autores
Castilho Neto, Kayo José Garcia de Almeida
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Universidade Estadual Paulista (Unesp)
Resumo
Tripanossomíases são doenças causadas por agentes do gênero Trypanosoma, que infectam humanos, animais domésticos e silvestres nos continentes Africano, Asiático, Américas do Sul e Central. Os sinais clínicos da enfermidade, como anemia e emagrecimento, são genéricos e também devido à presença de intervalos aparasitêmicos, o diagnóstico da doença torna-se desafiador. Quimioterápicos como aceturato de diminazeno (AD) e cloridrato de isometamidium (ISM) estão disponíveis para o tratamento da doença no Brasil, muito embora existam relatos e indícios de resistência aos princípios. Este trabalho teve como objetivo utilizar a amplificação circular isotérmica do DNA (LAMP) no diagnóstico e avaliar a cura parasitológica de bovinos naturalmente infectados por Trypanosoma vivax após o tratamento com ISM. Foram utilizados 30 bovinos naturalmente infectados com T. vivax, que foram tratados com duas aplicações de ISM, na dosagem de 1,0 mg/Kg por via intramuscular profunda nos dias 0 e 150. Amostras de sangue e soro colhidas nos 0, 7, 15, 30, 60, 90, 120, 150, 180, 210 e 240 dias após o tratamento, foram submetidas ao diagnóstico de T. vivax pelas técnicas da reação em cadeia da polimerase (PCR) objetivando o gene alvo CatL-Like, LAMP visando o gene alvo DNA satélite e ensaio de imunoabsorção enzimático (ELISA) com antígeno bruto solúvel de T. vivax. Também, foi realizada uma PCR adicional para diagnóstico de infecção por T. theileri direcionado ao gene CATL. Houve persistência na detecção de DNA de T. vivax na PCR e LAMP bem como detecção contínua de anticorpos anti-T. vivax pelo método de ELISA após o tratamento instituído, sugerindo a presença de resistência do parasita ao ISM. Todas as amostras testadas para o diagnóstico de T. theileri pela PCR foram negativas. A LAMP se mostrou de grande utilidade no diagnóstico de animais naturalmente infectados, porém a combinação dos testes LAMP e ELISA podem evitar falsos diagnósticos, contribuindo para um melhor controle da doença. Os dados indicam que a PCR não é indicada para diagnóstico de T. vivax em animais apresentando baixa parasitemia, sendo, portanto não recomendada para promover o acompanhamento de animais após o tratamento com drogas tripanocidas.
Trypanosomiasis is disease caused by agents of the Trypanosoma genus, which infect humans, domestic and wild animals on the African, Asian, South and Central Americans continents. The clinical signs of the disease, such as anemia and weight loss, are generic and also due to the presence of parasitic intervals, the diagnosis of the disease becomes challenging. Chemotherapics such as diminazine aceturate (AD) and isomethemidum hydrochloride (ISM) are available for the treatment of the disease in Brazil, although there are reports and indications of resistance to the principles. This work aimed to use loop-mediated isothermal amplification (LAMP) in the diagnosis and to evaluate the parasitological cure of cattle naturally infected by Trypanosoma vivax after treatment with ISM. Thirty bovines naturally infected with T. vivax were treated with two ISM applications at a dose of 1.0 mg / kg intramuscularly on days 0 and 150. Blood and serum samples were collected on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210 and 240 days after treatment and were submitted to T. vivax diagnosis by polymerase chain reaction (PCR) targeting the CatL-Like, LAMP targeting the satellite DNA gene and enzyme immunoabsorption assay (ELISA) with soluble T. vivax antigen. Also, an additional PCR for diagnosis of T. theileri infection directed to the CATL gene was performed. There was persistence in the detection of T. vivax DNA in PCR and LAMP as well as continuous detection of anti-T. vivax antibodies by the ELISA method after the treatment, suggesting the presence of resistance of the parasite to the ISM. All samples tested for the diagnosis of T. theileri by PCR were negative. LAMP proved to be very useful in the diagnosis of naturally infected animals, but the combination of the LAMP and ELISA tests can avoid false diagnoses, contributing to a better control of the disease. The data indicate that PCR is not indicated for diagnosis in animals with low parasitemia, therefore is not recommended to promote the follow-up of animals after treatment with trypanocidal drugs.
Trypanosomiasis is disease caused by agents of the Trypanosoma genus, which infect humans, domestic and wild animals on the African, Asian, South and Central Americans continents. The clinical signs of the disease, such as anemia and weight loss, are generic and also due to the presence of parasitic intervals, the diagnosis of the disease becomes challenging. Chemotherapics such as diminazine aceturate (AD) and isomethemidum hydrochloride (ISM) are available for the treatment of the disease in Brazil, although there are reports and indications of resistance to the principles. This work aimed to use loop-mediated isothermal amplification (LAMP) in the diagnosis and to evaluate the parasitological cure of cattle naturally infected by Trypanosoma vivax after treatment with ISM. Thirty bovines naturally infected with T. vivax were treated with two ISM applications at a dose of 1.0 mg / kg intramuscularly on days 0 and 150. Blood and serum samples were collected on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210 and 240 days after treatment and were submitted to T. vivax diagnosis by polymerase chain reaction (PCR) targeting the CatL-Like, LAMP targeting the satellite DNA gene and enzyme immunoabsorption assay (ELISA) with soluble T. vivax antigen. Also, an additional PCR for diagnosis of T. theileri infection directed to the CATL gene was performed. There was persistence in the detection of T. vivax DNA in PCR and LAMP as well as continuous detection of anti-T. vivax antibodies by the ELISA method after the treatment, suggesting the presence of resistance of the parasite to the ISM. All samples tested for the diagnosis of T. theileri by PCR were negative. LAMP proved to be very useful in the diagnosis of naturally infected animals, but the combination of the LAMP and ELISA tests can avoid false diagnoses, contributing to a better control of the disease. The data indicate that PCR is not indicated for diagnosis in animals with low parasitemia, therefore is not recommended to promote the follow-up of animals after treatment with trypanocidal drugs.
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Palavras-chave
ELISA, Métodos moleculares, Tratamento, Tripanossomíases