Development and comparative evaluation of two antigen detection tests for Visceral Leishmaniasis

Abstract

BackgroundVisceral leishmaniasis (VL) can be fatal without timely diagnosis and treatment. Treatment efficacies vary due to drug resistance, drug toxicity and co-morbidities. It is important to monitor treatment responsiveness to confirm cure and curtail relapse. Currently, microscopy of spleen, bone marrow or lymph node biopsies is the only definitive method to evaluate cure. A less invasive test for treatment success is a high priority for VL management.MethodsIn this study, we describe the development of a capture ELISA based on detecting Leishmania donovani antigens in urine samples and comparison with the Leishmania Antigen ELISA, also developed for the same purpose. Both were developed as prototype kits and tested on patient urine samples from Sudan, Ethiopia, Bangladesh and Brazil, along with appropriate control samples from endemic and non-endemic regions. Sensitivity and specificity were assessed based on accurate detection of patients compared to control samples. One- Way ANOVA was used to assess the discrimination capacity of the tests and Cohen’s kappa was used to assess their correlation.ResultsThe Leishmania Antigen Detect™ ELISA demonstrated >90 % sensitivity on VL patient samples from Sudan, Bangladesh and Ethiopia and 88 % on samples from Brazil. The Leishmania Antigen ELISA was comparable in performance except for lower sensitivity on Sudanese samples. Both were highly specific. To confirm utility in monitoring treatment, urine samples were collected from VL patients at days 0, 30 and 180 post- treatment. For the Leishmania Antigen Detect™ ELISA, positivity was high at day 0 at 95 %, falling to 21 % at day 30. At day 180, all samples were negative, corresponding well with clinical cure. A similar trend was also seen for the Leishmania Antigen ELISA albeit; with lower positivity of 91 % at Day 0 and more patients, remaining positive at Days 30 and 180.DiscussionThe Leishmania Antigen Detect™ and the Leishmania Antigen ELISAs are standardized, user- friendly, quantitative and direct tests to detect Leishmania during acute VL as well as to monitor parasite clearance during treatment. They are a clear improvement over existing options.ConclusionThe ELISAs provide a non-invasive method to detect parasite antigens during acute infection and monitor its clearance upon cure, filling an unmet need in VL management. Further refinement of the tests with more samples from endemic regions will define their utility in monitoring treatment.

Novel Antigen Detection Assay to Monitor Therapeutic Efficacy of Visceral Leishmaniasis

“…This test sensitivity is 100 pg/mL, which is 40 times more sensitive than that described for the two recently antigen detection tests (about 4,000 pg of total Leishmania antigens per mL of urine). 10 In addition, the discrimination achieved between positive and negative samples of the current test is 9-fold, almost double to that of the other two tests. 10 These differences may be because the selected biomarkers described here were chosen because they were initially found in abundance in the urine of VL patients.…”

“…10 In addition, the discrimination achieved between positive and negative samples of the current test is 9-fold, almost double to that of the other two tests. 10 These differences may be because the selected biomarkers described here were chosen because they were initially found in abundance in the urine of VL patients. Hence, an assay that targets these previously selected proteins will be by definition more sensitive and specific than an assay that uses antibodies against the whole parasite antigenic repertoire.…”

“…2 Recently, two protein antigen detection tests have been reported for the diagnosis of VL. 10 Both methods used preparations of whole Leishmania donovani promastigotes to generate the anti-whole parasite promastigote antibodies used in the tests.…”

Identification and functional characterization of a bacterial homologue of Zeta toxin in Leishmania donovani

“…Promastigotes. Whole cell lysate of Promastigote was prepared by freeze-thaw cycles as described previously [24].…”

Visceral leishmaniasis: a forgotten epidemic

“…An RDT based on rK28 antigen seems promising 67. Recently two ELISAs based on antigen detection in urine were more sensitive and easier to use than the KAtex agglutination test that requires boiling of urine 68. Parasitological diagnosis is often still the method of choice 69–71.…”