Project Lead(s): Greg Matlashewski
Visceral leishmaniasis (VL) is one of the major neglected parasitic diseases caused by Leishmania donovani in Asia and Africa, and is fatal if left untreated.
Although assays based on detection of antibodies against the parasite (such as the rK39 test) have been developed, these tests cannot distinguish active disease from asymptomatic infections because they cannot directly detect the presence of the parasite in the blood.
Definitive diagnosis of VL still relies on non-specific clinical symptoms, such as fever and enlarged spleen, combined with a positive rK39 serological test.
Development of a simple assay that can determine Leishmania levels in the blood is urgently needed for VL diagnosis, monitoring, treatment and to determine which asymptomatic-infected individuals can transmit or develop disease.
The project aimed to develop a diagnostic test that could detect Leishmania parasites in the blood.
Based on the assumption that Leishmania proteins that are highly abundant would be easier to detect than low-abundance proteins, they raised antibodies against several recombinant Leishmania proteins that are highly abundant and used these antibodies to develop an Enzyme-Linked Immunosorbent Assay (ELISA).
The genes encoding these abundant Leishmania proteins were cloned from Leishmania donovani genomic DNA, then expressed and purified from bacteria using standard molecular biology approaches. Antibodies were raised by immunizing rabbits against these proteins.
The antibodies were purified and labeled with biotin for developing the prototype ELISA.
The sensitivity and specificity of these ELISA assays were determined and compared with Leishmania promastigote lysates, and blood taken from visceral leishmaniasis patients from Bihar, India.
The ELISA targeting the Leishmania ribosomal protein S12 was able to detect 1 pg of purified protein and was also able to detect about 500 promastigotes when mixed with 500,000 peripheral blood mononuclear cells (PBMCs), demonstrating that the assay did not lose sensitivity or specificity in the presence of human blood cells.
Next, the ELISA assay was used on PBMCs derived from 20 Visceral Leishmaniasis patients from Bihar, India, and compared to PBMCs from 12 healthy controls.
The key achievement of the project was the development of an ELISA assay that can detect Leishmania donovani in the blood of VL patients from Bihar, India.
These results suggest that this ELISA may be used in the field for confirming VL diagnosis, monitoring treatment progress, confirming relapse infections and possibly to identify those asymptomatic infection cases that will develop Visceral Leishmaniasis. Through a planned collaboration with the Indian Council of Medical Research (ICMR), the team proposes to optimize the assay under field conditions in highly endemic regions in India.