Urine-based antigen detection assay for diagnosis of visceral leishmaniasis using monoclonal antibodies specific for six protein biomarkers of Leishmania infantum / Leishmania donovani.

The development of an accurate protein-based antigen detection assay for diagnosis of active visceral leishmaniasis (VL) would represent a major clinical advance. VL is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard confirmatory diagnostic test for VL is the demonstration of parasites or their DNA from aspirates from spleen, lymph node, and bone marrow or from blood buffy coats. Here we describe the production and use of monoclonal antibodies (mAbs) for the development of a sensitive and specific antigen detection capture ELISA for VL diagnosis. This test simultaneously detects six leishmania protein biomarkers that we have previously described (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1). The initial clinical validation of this new mAb-based multiplexed capture ELISA showed a sensitivity of >= 93%. The test was negative with 35 urine samples from healthy control subjects as well as with 30 patients with confirmed non-VL tropical diseases (cutaneous leishmaniasis, n = 6; Chagas disease, n = 6; schistosomiasis, n = 6; and tuberculosis, n = 12). These results strongly support the possible utility of this mAb-based multiplexed capture ELISA as a promising diagnostic test for active VL as well as for monitoring the treatment efficacy of this disease. The test is ready for upscaling and validation for clinical use. Author summary Visceral leishmaniasis (VL) or kala-azar is a systemic parasitic disease that is endemic in 75 countries with more than 500 million people at risk of infection. VL is caused primarily by the parasites known as Leishmania infantum and Leishmania donovani. It is estimated that VL affects 50,000-90,000 people each year, with 90% of the cases occurring in India, Kenya, Somalia, Sudan, South Sudan and Brazil. VL is a fatal disease if not treated. Laboratory diagnosis of this disease can be performed using several different approaches. However, there is still a need for a simple, non-expensive, sensitive and specific test that can be used not only for the accurate diagnosis of this serious disease but also to monitor the efficacy of its treatment. Here we describe a new test that can circumvent most of the drawbacks of existing approaches. This new test detects six leishmanial proteins or biomarkers that are eliminated in the urine of patients with VL. The test is a conventional ELISA assembled with monoclonal antibodies (mAbs) specific for each of the six different biomarkers and it is performed in urine samples of suspected patients. An initial clinical validation demonstrated that the test has a sensitivity of >= 93% and specificity of 100%. This new and accurate mAb-based multiplexed assay could be a useful resource to diagnose most clinical forms and/or the severity of the disease e.g., active VL, asymptomatic VL, post kala-azar dermal leishmaniasis, VL/HIV co-infection, etc.