High Performance of ELISA test using recombinant rLiNTPDase2 from Leishmania infantum: a Phase II diagnosis of Canine Visceral Leishmaniasis.

Canine visceral leishmaniasis (CVL) has been the theme of several studies given the importance of dog as natural reservoir of the pathogen Leishmania infantum in endemic regions and its role on dissemination of CVL and human visceral Lesihmaniasis (VL). The current immunodiagnosis of CVL has limitations concerning accuracy, specificity and sensitivity. Therefore, improvements are required. rLiNTPDase2 has been previously highlighted as a new recombinant antigen from L. infantum to the CVL diagnosis by ELISA assay (rLiNTPDase2-ELISA). In this study, we aimed to evaluate rLiNTPDase2-ELISA in a Phase II study with 651 dog sera samples, also comparing it with methodologies previously established and used in epidemiology surveillance in Brazil, an endemic country of CVL and VL. The rLiNTPDase2-ELISA using standard control sera showed high capability to distinguish between positive and negative sera, sensitivity of 92.6% and specificity of 88.5%. The test was reproductive and the kappa statistics judgement "substantial agreement". rLiNTPDase2-ELISA does not show cross-reactivity with ehrlichiosis-reagent sera. However, we verified 15.3% of cross-reactivity with Chagas disease-reagent sera. The performance of rLiNTPDase2-ELISA was evaluated using sera samples from vaccinated dogs (Leish-Tec®). The results showed high agreement with parasitological and PCR results (sensitivity of 100.0% and specificity of 91.7%). Furthermore, we compared the performance of rLiNTPDase2-ELISA in CVL-reagent sera samples from endemic areas, which were previously diagnosed using other tests for CVL: immunofluorescent (IFI-LVC-Bio-Manguinhos), IFI-LVC-Bio-Manguinhos coupled to ELISA (EIE-LVC-Bio-Manguinhos) and the Rapid Dual Path Platform® (TR-DPP®-Bio-Manguinhos) coupled to EIE-LVC-Bio-Manguinhos. rLiNTPDase2-ELISA showed high level of concordance with IFI-LVC-Bio-Manguinhos (88.6%) and with IFI-LVC-Bio-Manguinhos coupled to EIE-LVC-Bio-Manguinhos (82.9%) but not with TR-DPP® -Bio-Manguinhos coupled to EIE-LVC-Bio-Manguinhos (33.3%), which casts doubts on the effectiveness of this latest test. In addition, the rLiNTPDase2 antigen adsorbed in 96-well plate was stable enough to be used at least for three months. Taken together, our data confirmed, by Phase II study using hundreds samples, the good potential of rLiNTPDase2-ELISA to be used in the field as a new diagnostic assay for CVL.