Isolation and characterisation of Leishmania donovani protein antigens from urine of visceral leishmaniasis patients.

Tegwen Marlais,Bathsheba L Gardner,Shyam Sundar,Steven G Reed,Callum Pearson,Safiyyah Marhoon,Tapan Bhattacharyya,Michael A Miles,Stephanie Airs,Andrew K Falconar,Sayda El-Safi,Kiera Hayes,Om Prakash Singh

doi:10.1371/journal.pone.0238840

Abstract

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease.

Highlights

Visceral leishmaniasis (VL) is most commonly caused by Leishmania donovani in the Indian subcontinent and eastern Africa, whereas L. infantum is the agent in the Mediterranean, Middle East and South America
We identified four L. donovani proteins in Indian VL urine and two in Sudanese VL urine
A potential limitation to our technique is that while we have presented L. donovani protein identities here, there is no certainty that these proteins exist as whole proteins in VL urine as we can only identify short peptides by mass spectrometry

Summary

Introduction

Visceral leishmaniasis (VL) is most commonly caused by Leishmania donovani in the Indian subcontinent and eastern Africa, whereas L. infantum is the agent in the Mediterranean, Middle East and South America. Both species are transmitted by female phlebotomine sand flies. Leishmania antigens in visceral leishmaniasis urine partnership supported by the European Commission under the Health Cooperation Work Programme of the 7th Framework Programme The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Results

Discussion

Conclusion