Implicações em saúde pública para diferentes técnicas diagnósticas e análise genotípica para Leishmania spp.
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Data
2022-10-10
Autores
Palmeira, Aghata Regina de Oliveira Alves
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As leishmanioses são zoonoses causadas por protozoários do gênero Leishmania, consideradas doenças negligenciadas e um grave problema de saúde pública, causando prejuízos sociais e econômicos em países tropicais e subtropicais. Este estudo teve como objetivo investigar a ocorrência de Leishmania spp. em cães e animais silvestres, provenientes do estado de São Paulo por meio do crescimento primário e isolamento do parasito na cultura; Teste rápido imunocromatográfico (TR-DDP); ensaio imunoenzimático (ELISA); Reação em Cadeia da Polimerase convencional (cPCR); Reação em Cadeia da Polimerase em tempo real (qPCR); análises de dissociação de DNA em alta resolução (HRM) e posteriormente sequenciamento genético para identificação das espécies não discriminadas no HRM. De um total de 191 amostras biológicas, 180 (94,2%) eram procedentes de cães e 11 (5,8%) de animais silvestres. Das 180 amostras biológicas caninas, 32 (17,8%) foram cultivadas em meio NNN-LIT, tendo-se observado uma (3,1%) amostra de aspirado de linfonodo positiva. Todas as amostras caninas foram reagentes no TR-DDP e ELISA. A detecção molecular na técnica cPCR, utilizando os primers LINR4/LIN19, amplificou 151/191 (79,1%) amostras, já a qPCR amplificou 72/191 (37,7%) amostras utilizando primers HSP70F/R. Por meio da técnica de HRM, utilizando o amplicon 1, 55/191 (28,8%) amostras foram discriminadas como Leishmania infantum. Os resultados dessa pesquisa evidenciaram a identificação da espécie Leishmania infantum em 53/180 (29,4%) amostras procedentes de cães domésticos, e 2/11 (18,2%) amostras de animais silvestres. Desse modo, o estabelecimento de protocolos otimizados de detecção e identificação dos agentes causadores das leishmanioses torna-se extremamente útil para os estudos epidemiológicos.
Visceral Leishmaniasis is a zoonosis infection caused by Leishmania genus, is a neglected disease and a serious public health problem that causes social and economic damage in tropical and subtropical countries. This study aimed to investigate the occurrence of Leishmania spp. in dogs and wild animals from the state of São Paulo through primary growth and isolation of the parasite in blood cultures; Rapid immunochromatographic test (TR-DDP); enzyme immunoassay (ELISA); Conventional Polymerase Chain Reaction (cPCR); Real-time Polymerase Chain Reaction (qPCR); high resolution DNA dissociation analysis (HRM) and later genetic sequencing to identify species not discriminated in the HRM. From a total of 191 biological samples, 180 (94,2%) were from dogs and 11 (5,8%) from wild animals. Of the 180 canine biological samples, 32 (17,8%) were cultured in NNN LIT medium, with one (3,1%) positive lymph node aspirate sample. All canine samples were reactive in TR and ELISA. Molecular detection in the cPCR technique using LINR4/LIN19 primer, amplified 151/191 (79,1%) samples, while qPCR amplified 72/191 (37,7%) samples using HSP70F/R primer. By means of the HRM technique using amplicon 1, 55/191 (28,8%) samples were discriminated as Leishmania infantum. The results of this research showed the identification of the Leishmania infantum species in 53/180 (29,8%) samples from domestic dogs, and 2/11 (18,2%) samples from wild animals. Thus, the establishment of optimized protocols for the detection and identification of the causative agents of leishmaniasis becomes extremely useful for epidemiological studies.
Visceral Leishmaniasis is a zoonosis infection caused by Leishmania genus, is a neglected disease and a serious public health problem that causes social and economic damage in tropical and subtropical countries. This study aimed to investigate the occurrence of Leishmania spp. in dogs and wild animals from the state of São Paulo through primary growth and isolation of the parasite in blood cultures; Rapid immunochromatographic test (TR-DDP); enzyme immunoassay (ELISA); Conventional Polymerase Chain Reaction (cPCR); Real-time Polymerase Chain Reaction (qPCR); high resolution DNA dissociation analysis (HRM) and later genetic sequencing to identify species not discriminated in the HRM. From a total of 191 biological samples, 180 (94,2%) were from dogs and 11 (5,8%) from wild animals. Of the 180 canine biological samples, 32 (17,8%) were cultured in NNN LIT medium, with one (3,1%) positive lymph node aspirate sample. All canine samples were reactive in TR and ELISA. Molecular detection in the cPCR technique using LINR4/LIN19 primer, amplified 151/191 (79,1%) samples, while qPCR amplified 72/191 (37,7%) samples using HSP70F/R primer. By means of the HRM technique using amplicon 1, 55/191 (28,8%) samples were discriminated as Leishmania infantum. The results of this research showed the identification of the Leishmania infantum species in 53/180 (29,8%) samples from domestic dogs, and 2/11 (18,2%) samples from wild animals. Thus, the establishment of optimized protocols for the detection and identification of the causative agents of leishmaniasis becomes extremely useful for epidemiological studies.
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Palavras-chave
Leishmaniose, Diagnóstico molecular, High Resolution Melting, Leishmaniasis, Molecular diagnosis, Leishmaniasis, Molecular diagnosis