Article
Visceral leishmaniasis: a practical strategy for quantitative molecular diagnosis in naturally infected dogs
Registro en:
CALDAS, Sérgio et al. Visceral leishmaniasis: a practical strategy for quantitative molecular diagnosis in naturally infected dogs. Parasitology Research, v. 119, n. 5, p. 1683-1690, 2020.
0932-0113
10.1007/s00436-020-06654-y
Autor
Caldas, Sérgio
Marcelino, Andreza P.
Faria, Gilson
Silva, Fernanda de Oliveira
Ataide, Ana Caroline Zampiroli
Cunha, Lucas Maciel
Bahia, Maria Terezinha
Paz, Gustavo Fontes
Gontijo, Célia M. F.
Resumen
The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.