title: Evaluation of 18S rDNA PCR assay using skin fragments as a diagnostic test for Trypanosoma caninum.
abstract: Trypanosoma caninum is a new species that has been recently identified in Brazil and infects domestic dogs. To date, no accurate diagnostic assays for this parasite have been established; thus, our aim was to evaluate more than one type of PCR for the diagnosis and molecular screening of T. caninum in 229 dogs living in Rio de Janeiro state. The tests were based on the amplification and sequencing of the 18S ribosomal DNA (rDNA) gene using healthy skin fragments. Additionally, PCR amplification of the kDNA minicircles region specific to the Leishmania genus was performed. The PCR results were compared with those of culture-based analysis performed with the same specimen. Using cultures, T. caninum and Leishmania chagasi were isolated from 11 and 12 dogs, respectively, whereas the 18S rDNA PCR assay detected parasitic infection in 35 dogs. Among these, 25 dogs showed an amplification pattern similar to T. caninum and 10 showed a pattern similar to L. chagasi; these results were confirmed by sequencing analysis. The kDNA PCR analysis showed that 14 dogs were positive for Leishmania infection. Of these, 2 dogs showed negative culture results and 12 were positive for L. chagasi, including 4 with negative 18S rDNA PCR results. Thus far, culture-based testing has been the only tool used successfully for T. caninum diagnosis. Our results demonstrate that 18S rDNA PCR-based test should be a useful diagnostic tool, particularly for distinguishing between T. caninum and L. chagasi infections in areas where these 2 parasites co-exist.
authors.affiliation: Programa de pós-graduação em Pesquisa Clínica em Doenças Infecciosas, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil; Laboratório de Vigilância em Leishmanioses, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil.
authors.forename: A G S
authors.initials: AG
authors.lastname: Pinto
authors.affiliation: Laboratório de Diagnóstico Molecular e Hematologia, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Rio de Janeiro, RJ, Brazil.
authors.forename: H K
authors.initials: HK
authors.lastname: Toma
authors.affiliation: Laboratório de Pesquisa Clínica em Dermatozoonoses de Animais Domésticos, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil.
authors.forename: F B
authors.initials: FB
authors.lastname: Figueiredo
authors.affiliation: Laboratório de Vigilância em Leishmanioses, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil. Electronic address: fatima.madeira@ipec.fiocruz.br.
authors.forename: M F
authors.initials: MF
authors.lastname: Madeira
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D007891:Leishmania / Q000235:genetics / Q000302:isolation & purification; D007896:Leishmaniasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012867:Skin / Q000469:parasitology; D014345:Trypanosoma / Q000235:genetics / Q000302:isolation & purification*; D014352:Trypanosomiasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S
keywords: Dog; Leishmaniasis; PCR; Trypanosoma caninum