Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/7305
Title: Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs
Authors: Solcà, Manuela da Silva
Guedes, Carlos Eduardo Sampaio
Nascimento, Eliane Gomes
Oliveira, Geraldo Gileno de Sá
dosSantos, Washington Luis Conrado
Fraga, Deborah Bittencourt Mothé
Veras, Patrícia Sampaio Tavares
Affilliation: Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil
Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil
Centro de Referência em Doenças Endêmicas Pirajá da Silva-PIEJ. Jequié, Ba, Brasil
Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil
Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil
Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil / Universidade Federal da Bahia. Escola de Medicina Veterinária. Departamento de Medicina Veterinária Preventiva. Salvador, BA, Brasil
Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil
Abstract: Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle
Keywords: Leishmania
Canine
PCR
Spleen
Diagnostic method
DeCS: Doenças do Cão/diagnóstico
Leishmania/genética
Leishmaniose/veterinária
Reação em Cadeia da Polimerase/veterinária
Baço/parasitologia
Animais
Primers do DNA/genética
Doenças do Cão/parasitologia
Cães
Leishmaniose/diagnóstico
Leishmaniose/parasitologia
Sensibilidade e Especificidade
Issue Date: 2012
Publisher: Elsevier
Citation: SOLCA, M. da S. et al. Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs. Veterinary Parasitology, v. 23, n. 2-4, p. 133-140, 2012.
DOI: 10.1016/j.vetpar.2011.08.026
ISSN: 0304-4017
Copyright: open access
Appears in Collections:BA - IGM - Artigos de Periódicos

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