title: Molecular diagnosis of canine visceral leishmaniasis: a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection.
abstract: Polymerase chain reaction (PCR) and its variations represent highly sensitive and specific methods for Leishmania DNA detection and subsequent canine visceral leishmaniasis (CVL) diagnosis. The aim of this work was to compare three different molecular diagnosis techniques (conventional PCR [cPCR], seminested PCR [snPCR], and quantitative PCR [qPCR]) in samples of skin and spleen from 60 seropositive dogs by immunofluorescence antibody test and enzyme-linked immunosorbent assay. Parasitological analysis was conducted by culture of bone marrow aspirate and optical microscopic assessment of ear skin and spleen samples stained with Giemsa, the standard tests for CVL diagnosis. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR, and snPCR and qPCR were performed using the DNA polymerase gene (DNA pol α) primers from Leishmania infantum. The parasitological analysis revealed parasites in 61.7% of the samples. Sensitivities were 89.2%, 86.5%, and 97.3% in the skin and 81.1%, 94.6%, and 100.0% in spleen samples used for cPCR, snPCR, and qPCR, respectively. We demonstrated that the qPCR method was the best technique to detect L. infantum in both skin and spleen samples. However, we recommend the use of skin due to the high sensitivity and sampling being less invasive.
authors: Laboratório de Pesquisas Clínicas, Programa de Pós-graduação em Ciências Farmacêuticas/CiPharma, Escola de Farmácia, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil; Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais - INCT-DT, CNPq, Brazil.
authors: Levi Eduardo Soares
authors: LE
authors: Reis
authors: Wendel
authors: W
authors: Coura-Vital
authors: Bruno Mendes
authors: BM
authors: Roatt
authors: Leoneide Érica Maduro
authors: LÉ
authors: Bouillet
authors: Henrique Gama
authors: HG
authors: Ker
authors: Rory Cristiane
authors: RC
authors: Fortes de Brito
authors: Daniela de Melo
authors: Dde M
authors: Resende
authors: Mariângela
authors: M
authors: Carneiro
authors: Rodolfo Cordeiro
authors: RC
authors: Giunchetti
authors: Marcos José
authors: MJ
authors: Marques
authors: Cláudia Martins
authors: CM
authors: Carneiro
authors: Alexandre Barbosa
authors: AB
authors: Reis
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000175:diagnosis / Q000276:immunology*; D004285:Dogs; D007898:Leishmaniasis, Visceral / Q000175:diagnosis / Q000276:immunology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012867:Skin / Q000469:parasitology*; D013154:Spleen / Q000469:parasitology*
keywords: Canine visceral leishmaniasis; Leishmania infantum; Molecular diagnostic; PCR; Skin; Spleen; qPCR; snPCR