Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

Abstract

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmaniaderived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28-and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas

“…Comparing the sensitivity of the rKR95-ELISA (95.6%) in the group of VL samples, it was not significantly different from rK39-ELISA (94.3%) and rK28-ELISA (95.9%). Fujimori et al [ 30 ] obtained 95.9% sensitivity in the rKR95-ELISA to diagnose canine leishmaniasis, similar to our results in human infection. In the same study, plates sensitized with recombinant KR95 were evaluated for 180 days in storage at 4°C.…”

“…In the present study, we aimed to evaluate the performance of two scantily studied recombinant proteins, K18 and KR95, in the serological diagnosis of VL. In our previous study analyzing VL dog samples, rKR95 showed good performance in serological diagnosis [ 30 ]. In this study, using samples from a well-characterized panel, we achieved excellent results with the recombinant KR95 protein, with 95.6% sensitivity and 97.8% specificity, close to the results with the already-known rK28 and rK39 proteins.…”

“…In the same study, plates sensitized with recombinant KR95 were evaluated for 180 days in storage at 4°C. There was no significant decrease in reactivity, indicating that this protein is stable for diagnostic kits [ 30 ]. Searching for healthy individuals living in endemic areas, the rKR95-ELISA showed the highest specificity (96.4%).…”

“…ELISA standardization of the four recombinants was performed to maintain the same laboratory conditions and make a more reliable comparison. The standardization of the human ELISA protocol was based on the standardization performed by Fujimori [ 30 ] for canine samples.…”

Use of Antigen Combinations to Address Complex Leishmania-Seropositivity Patterns in Dogs Living in Canine Leishmaniosis Endemic Regions of Portugal

“…Crude promastigote antigens, the SPLA, have been described as highly sensitive for subclinical and clinical infections, but with a lower specificity [ 25 , 26 ]. The rK39, rK28, and rKDDR are all kinesin-derived recombinant proteins with proven value for the serodiagnosis of CanL [ 27 , 28 ]. LicTXNPx is a recombinant protein that demonstrated an increased capacity to detect subclinical infections [ 20 , 29 ].…”