High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil – A Phase II study

Abstract

BackgroundCanine Visceral leishmaniasis (CVL) is a serious public health problem, thus for its control, the Ministry of Health in Brazil recommends the rapid diagnosis and euthanasia of seropositive dogs in endemic areas. Therefore, our group had previously selected six recombinant proteins (rLci1, rLci2, rLci4, rLci5, rLci8, and rLci12) due to their high potential for CVL diagnostic testing. The present study aims to produce an immunodiagnostic test using the aforementioned antigens, to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health.Methodology/Principal findingsTo evaluate the recombinant proteins in the serological assays, positive and negative samples were selected based on parasitological test (culture) and molecular test (qPCR) of splenic aspirate. Initially, we selected 135 dog serum samples, 73 positives (symptomatic and asymptomatic) and 62 negatives to screen recombinant proteins on ELISA platform. Then, for rLci5 ELISA validation, 361 serum samples collected in a cross-sectional study were selected, being 183 positives (symptomatic and asymptomatic) and 178 negatives. In the screening of the recombinant proteins, rLci5 was the only protein to present a performance statistically higher than the performance presented by EIE-LVC test, presenting 96% (IC 95%; 85–99%) vs. 83% (IC 95%; 69–92%) of sensitivity for symptomatic dogs, 71% (IC 95%; 49–97%) vs. 54% (IC 95%; 33–74%) for asymptomatic dogs and 94% (IC 95%; 83–99%) vs, 88% (IC 95%; 76–95% of specificity. Thus, the rLci5 protein was selected to compose a final ELISA test. Validation of rLci5 ELISA showed 87% (IC 81–91%) of sensitivity, 94% (IC 95%; 90–97%) of specificity and 90% accuracy. Testing the EIE-LVC with the same validation panel, we observed a lower performance when compared to ELISA rLci5 (sensitivity of 67% (IC 95%; 59–74%), specificity of 87% (IC 95%; 81–92%), and accuracy of 77%). Finally, the performance of current CVL diagnostic protocol recommended by Brazilian Ministry of Health, using DPP-LVC as screening test and EIE-LVC as confirmatory test, was compared with a modified protocol, replacing EIE-LVC by rLci5 ELISA. The current protocol presented a sensitivity of 59% (IC 95%; 52–66%), specificity of 98% (IC 95%; 95–99%) and accuracy of 80% (IC 95%; 76–84%), while the modified protocol presented a sensitivity of 71% (IC 95%; 63–77%), specificity of 99% (IC 95%; 97–100%) and accuracy of 86% (IC 95%; 83–89%).ConclusionThus, we concluded that rLci5 ELISA is a promising test to replace EIE-LVC test and increase the diagnostic performance of CVL in Brazil.

Leishmania infantum-Derived Glycoinositolphospholipids in the Immunodiagnosis of Subclinically Infected Dogs

“…GIPL-ELISA was able to detect 80% of the subclinically infected dogs. This significant sensitivity result for asymptomatic dogs was higher than those previously reported for immunoassays using L. infantum total lysate antigens and recombinant proteins (11,(21)(22)(23). Like LPGs, GIPLs exhibited false negative results in the G2 and G3 groups (11).…”

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

“…Considering the fundamental role of dogs in the VL epidemiology, diagnostic algorithms are proposed for routine surveillance to detect L. infantum -infected dogs in control programs. Previous studies show unsatisfactory performance 9 , 35 of the currently used assays in the diagnostic routine in Brazil. This study evaluated ELISA using four different recombinant antigens, rK39, rK28, rKR95 and rK18, hypothesizing that these could provide better diagnostic performance relative to the current strategy.…”

Early detection and persistent positivity of anti-Leishmania antibodies using a recombinant protein-based ELISA in naturally infected dogs in Brazil

“…Recombinant protein rLci5 previously demonstrated promise in CVL serodiagnosis, showing high sensitivity and specificity [ 13 ]. The present study confirmed the accuracy of rLci5 ELISA in CVL serodiagnosis, as this test presented the highest detection rate among the diagnostic tests evaluated.…”

“…All serum samples previously obtained from the original cohort study were subsequently assessed using rLci5 ELISA as previously described by Borja et al [ 13 ]. Other diagnostic tests were originally employed during the cohort study, including EIE-LVC and DPP-LVC, which were performed in accordance with manufacturer’s specifications.…”

“…Our group previously identified recombinant antigens for use in canine visceral leishmaniasis (CVL) serodiagnosis [ 11 , 12 ]. Among these antigens, the recombinant protein rLci5 demonstrated promising results when used in an enzyme-linked immunosorbent assay (ELISA), offering 87% sensitivity and 94% specificity, as well as 90% diagnostic accuracy [ 13 ]. However, in addition to accuracy, it is also important to assess the ability of diagnostic assays to detect antibodies at early times of infection, as well as to evaluate the persistence of these antibodies in infected dogs, which allows clinicians to perform diagnosis in different phases of infection or stages of disease.…”