title: 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.
authors.affiliation: Laboratory of Molecular Epidemiology and Experimental Pathology, Pasteur Institute of Tunis, Université de Tunis el Manar, Tunis, Tunisia; Laboratory of Epidemiology and Ecology of Parasitic Diseases, Pasteur Institute of Tunis, Tunis, Tunisia.
authors.forename: Souheila
authors.initials: S
authors.lastname: Guerbouj
authors.affiliation: Laboratory of Epidemiology and Ecology of Parasitic Diseases, Pasteur Institute of Tunis, Tunis, Tunisia.
authors.forename: Fattouma
authors.initials: F
authors.lastname: Djilani
authors.affiliation: Laboratory of Medical Epidemiology, Pasteur Institute of Tunis, Tunis, Tunisia.
authors.forename: Jihene
authors.initials: J
authors.lastname: Bettaieb
authors.affiliation: Molteno Institute for Parasitology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Bronwen
authors.initials: B
authors.lastname: Lambson
authors.affiliation: Laboratory of Epidemiology and Ecology of Parasitic Diseases, Pasteur Institute of Tunis, Tunis, Tunisia.
authors.forename: Mohamed Fethi
authors.initials: MF
authors.lastname: Diouani
authors.affiliation: Laboratory of Medical Epidemiology, Pasteur Institute of Tunis, Tunis, Tunisia.
authors.forename: Afif
authors.initials: A
authors.lastname: Ben Salah
authors.affiliation: Laboratory of Epidemiology and Ecology of Parasitic Diseases, Pasteur Institute of Tunis, Tunis, Tunisia.
authors.forename: Riadh
authors.initials: R
authors.lastname: Ben Ismail
authors.affiliation: Laboratory of Molecular Epidemiology and Experimental Pathology, Pasteur Institute of Tunis, Université de Tunis el Manar, Tunis, Tunisia; Laboratory of Epidemiology and Ecology of Parasitic Diseases, Pasteur Institute of Tunis, Tunis, Tunisia.
authors.forename: Ikram
authors.initials: I
authors.lastname: Guizani
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D018314:Leishmania infantum; D007896:Leishmaniasis / Q000175:diagnosis / Q000378:metabolism / Q000662:veterinary*; D057089:Pathology, Molecular / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014416:Tunisia