title: PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.
abstract: In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.
authors.affiliation: Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Estela Gallucci
authors.initials: EG
authors.lastname: Lopes
authors.affiliation: Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Carlos Alberto
authors.initials: CA
authors.lastname: Geraldo Junior
authors.affiliation: Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Arlei
authors.initials: A
authors.lastname: Marcili
authors.affiliation: Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Ricardo Duarte
authors.initials: RD
authors.lastname: Silva
authors.affiliation: Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brasil.
authors.forename: Lara Borges
authors.initials: LB
authors.lastname: Keid
authors.affiliation: Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brasil.
authors.forename: Trícia Maria Ferreira da Silva
authors.initials: TM
authors.lastname: Oliveira
authors.affiliation: Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil.
authors.forename: Rodrigo Martins
authors.initials: RM
authors.lastname: Soares
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D003576:Electron Transport Complex IV; D050259:Genes, Mitochondrial; D007891:Leishmania / Q000235:genetics* / Q000302:isolation & purification; D007896:Leishmaniasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis; D016133:Polymerase Chain Reaction / Q000662:veterinary*
chemical_list: D017931:DNA Primers; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV