Surto de pitiose equina e padronização de qPCR para rápida detecção de Pythium insidiosum
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Data
2020-10-27
Autores
Paz, Giselle Souza Da
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Universidade Estadual Paulista (Unesp)
Resumo
A pitiose é uma doença granulomatosa causada pelo oomiceto P. insidiosum, com curso clínico progressivo e frequentemente fatal em humanos e animais. Descrevemos o primeiro surto de pitiose equina envolvendo cinco animais, diagnosticados por cultivo microbiológico e nested-PCR em maio de 2018 em Botucatu e São Manuel-SP, após pastejarem nas margens do rio Tietê. A combinação de técnicas microbiológicas, moleculares e filogenéticas permitiram o isolamento de P. insidiosum de amostras de água do rio e a caraterização filogenética, com a primeira identificação do clado III de P. insidiosum no Brasil. Além disso, realizamos a padronização de qPCR para o diagnóstico de P. insidiosum com alvo molecular o gene ELI025. Para tanto foram utilizadas 23 isolados de P. insidiosum e 10 amostras clínicas (09 de kunkers e 01 de tecido) de animais com pitiose previamente comprovada por cultivo microbiológico ou nested-PCR. O limite de detecção foi de 1pg/μL de DNA, correspondente a 17 cópias do genoma, curvas de dissociação com valor médio de 88,7ºC e coeficiente de linearidade (R²) em 0,994. A sensibilidade, especificidade e acurácia da qPCR foram de 100% em comparação com a nested-PCR, com homologia de 99% dos produtos amplificados para o gene ELI025, específico de P. insidiosum. Concluímos que a descrição do primeiro surto de pitiose equina e a identificação da fonte de infecção podem auxiliar na implantação de medidas preventivas, e a padronização de qPCR com alvo no gene ELI025 auxiliará para o diagnóstico precoce da pitiose, contribuindo substancialmente para a melhora do prognóstico do paciente.
Pythiosis is a granulomatous disease, caused by the oomycete P. insidiosum, with a progressive and often fatal clinical course in humans and animals. We described a first outbreak of equine pythiosis involving five animals diagnosed by microbiological culture of the agent and nested-PCR in May 2018 in the cities of Botucatu and São Manuel-SP, São Paulo state, after grazing on the margins of the Tietê River. The combination of microbiological, molecular, and phylogenetic techniques allowed the isolation of P. insidiosum from water samples of the river and the phylogenetic characterization of isolates in clades I and III. We also standardized qPCR for the diagnosis of P. insidiosum targeting the ELI025 gene. For this, 23 isolates of P. insidiosum and 10 clinical samples (9 from kunkers and 1 from tissue) from animals previously confirmed with pythiosis by microbiological culture or nested-PCR were employed. The detection limit was 1pg/μl of DNA, corresponding to 17 copies of the genome, maximum quantification cycle of up to 35.3 cycles, dissociation curves with a mean value of 88.7ºC, and determination coefficient (R²) of 0.994. The sensitivity of the technique for clinical samples was 100%, with 99% homology for the ELI025 gene, which is specific to P. insidiosum. We concluded that the qPCR technique targeting the ELI025 gene helps in the early diagnosis and certainly will contribute to the significantly improvement of the prognosis of pythiosis.
Pythiosis is a granulomatous disease, caused by the oomycete P. insidiosum, with a progressive and often fatal clinical course in humans and animals. We described a first outbreak of equine pythiosis involving five animals diagnosed by microbiological culture of the agent and nested-PCR in May 2018 in the cities of Botucatu and São Manuel-SP, São Paulo state, after grazing on the margins of the Tietê River. The combination of microbiological, molecular, and phylogenetic techniques allowed the isolation of P. insidiosum from water samples of the river and the phylogenetic characterization of isolates in clades I and III. We also standardized qPCR for the diagnosis of P. insidiosum targeting the ELI025 gene. For this, 23 isolates of P. insidiosum and 10 clinical samples (9 from kunkers and 1 from tissue) from animals previously confirmed with pythiosis by microbiological culture or nested-PCR were employed. The detection limit was 1pg/μl of DNA, corresponding to 17 copies of the genome, maximum quantification cycle of up to 35.3 cycles, dissociation curves with a mean value of 88.7ºC, and determination coefficient (R²) of 0.994. The sensitivity of the technique for clinical samples was 100%, with 99% homology for the ELI025 gene, which is specific to P. insidiosum. We concluded that the qPCR technique targeting the ELI025 gene helps in the early diagnosis and certainly will contribute to the significantly improvement of the prognosis of pythiosis.
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Palavras-chave
Pitiose, Ambiental, Oomicetos, Elicitina, PCR em tempo real