title: Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples.
abstract: Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites.
authors: Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil.
authors: Monica
authors: M
authors: Losada-Barragán
authors: Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil.
authors: Amanda
authors: A
authors: Cavalcanti
authors: Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias, Departamento de Química, Grupo de Investigación en Hormonas, Bogotá, Colombia.
authors: Adriana
authors: A
authors: Umaña-Pérez
authors: Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil.
authors: Renato
authors: R
authors: Porrozzi
authors: Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias, Departamento de Química, Grupo de Investigación en Hormonas, Bogotá, Colombia.
authors: Sergio
authors: S
authors: Cuervo-Escobar
authors: Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias, Departamento de Química, Grupo de Investigación en Hormonas, Bogotá, Colombia.
authors: Andrés Felipe
authors: AF
authors: Vallejo
authors: Universidad Nacional de Colombia, Sede Bogotá, Facultad de Ciencias, Departamento de Química, Grupo de Investigación en Hormonas, Bogotá, Colombia.
authors: Myriam
authors: M
authors: Sánchez-Gómez
authors: Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil. Electronic address: pcuervo@fiocruz.br.
authors: Patricia
authors: P
authors: Cuervo
mesh_terms: D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry; D004283:Dog Diseases / Q000469:parasitology*; D004285:Dogs; D018314:Leishmania infantum / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000469:parasitology / Q000662:veterinary*; D008099:Liver / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013154:Spleen / Q000469:parasitology
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan