Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

Abstract

A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia.

Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection

“…Molecular techniques have exploited multiple genes to detect Leishmania , most of which are high-copy-number genes, including cysteine protease B [ 36 ], gp63 [ 37 ], internal transcribed spacer 1 (ITS1) [ 38 ], 18S ribosomal RNA [ 26 , 31 ], and minicircle kinetoplast DNA [ 24 ]. As expected, comparative studies have shown that the minicircles, which have the highest copy number, yield the highest sensitivity [ 39 , 40 ].…”

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

“…Whole genomic DNA was extracted from the biological material collected from dogs (blood [n = 43] and ocular/gum swabs [n = 52]) as well as organ samples and biological liquids of Meriones (n = 45) and hedgehogs (n = 31) (Additional file 2 : Table S2). All extractions were realized by the conventional phenol/chloroform method [ 21 ] and all DNA was suspended in 10 mM Tris-1 mM EDTA p H8 solution and conserved at +4 °C. Extracted DNA was quantified using a Qubit Fluorometer (Invitrogen, France).…”

Canine visceral leishmaniasis: Diagnosis and management of the reservoir living among us

“…The DNA minicircles contained in Leishmania kinetoplast DNA (kDNA) has made this molecular target the basis for the majority of molecular tests aimed at detecting Leishmania species, although other targets have been utilized such as Internal Transcribed Spacer-1 (ITS-1), 18S rRNA, and glycoprotein-63 (gp-63) ( S1 Table ) [ 10 , 11 ]. The specificity of molecular tests is regularly 95% to 100% [ 8 , 12 – 14 ].…”