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.

犬内脏利什曼病（CVL）一直是几项研究的主题，因为狗在地方病地区是婴儿利什曼原虫病原体的天然贮藏库的重要性及其在传播CVL和人类内脏利什曼病（VL）中的作用。当前的CVL免疫诊断在准确性，特异性和敏感性方面存在局限性。因此，需要改进。rLiNTPDase2先前已被强调为来自婴儿乳杆菌的新重组抗原通过ELISA测定（rLiNTPDase2-ELISA）检测CVL。在这项研究中，我们旨在评估rLiNTPDase2-ELISA在II期研究中的651份狗血清样本，并将其与先前在CVL和VL的流行国家巴西建立并用于流行病学监测的方法进行比较。使用标准对照血清的rLiNTPDase2-ELISA具有区分阳性和阴性血清的高能力，灵敏度为92.6％，特异性为88.5％。该测试具有生殖能力，kappa统计判断为“基本一致”。rLiNTPDase2-ELISA与埃希氏菌病试剂血清无交叉反应。但是，我们证实了与恰加斯病试剂血清的15.3％的交叉反应性。使用来自接种狗的血清样品（Leish-Tec®）评估了rLiNTPDase2-ELISA的性能。结果显示与寄生虫学和PCR结果高度吻合（敏感性为100.0％，特异性为91.7％）。此外，我们比较了rLiNTPDase2-ELISA在来自地方性地区的CVL试剂血清样品中的性能，这些样品先前已使用其他CVL测试进行了诊断：免疫荧光（IFI-LVC-Bio-Manguinhos），IFI-LVC-Bio-Manguinhos与ELISA（EIE-LVC-Bio-Manguinhos）和Rapid Dual PathPlatform®（TR-DPP®-Bio-Manguinhos）与EIE-LVC-Bio-Manguinhos偶联。rLiNTPDase2-ELISA与IFI-LVC-Bio-Manguinhos（88.6％）以及与IIE-LVC-Bio-Manguinhos（82.9％）偶联的IFI-LVC-Bio-Manguinhos（82.9％）高度一致，但与TR-DPP®不一致Bio-Manguinhos与EIE-LVC-Bio-Manguinhos耦合（占33.3％），这对该最新测试的有效性产生了怀疑。此外，吸附在96孔板上的rLiNTPDase2抗原足够稳定，至少可以使用三个月。综上所述，我们的数据通过使用数百个样品的II期研究证实，rLiNTPDase2-ELISA的潜在潜力可在该领域用作CVL的新诊断方法。