Strategies for improving differential diagnosis and providing biomarkers for visceral leishmaniasis

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the Leishmania donovani complex. Without treatment, VL is fatal. Although diagnostic techniques, mainly based on the detection of anti-Leishmania antibodies are available, invasive procedures such as microscopy from spleen or bone marrow aspirates are still required for the diagnosis of seronegative VL suspects, for the detection of recurrent cases and to confirm cure after successful treatment. The rK39, the most widely used antigen for VL diagnosis has been an essential point-of-care tool for the detection of active VL cases, although it cannot be used as a test of cure due to antibody persistance (IgG) even after succeessful chemotherapy. Furthermore, this test has a decreased sensitivtiy in Eastern Africa, where the direct agglutination test (DAT) is still an intergral part of the algorithm to diagnose active VL cases. Previous investigations showed the potential of IgG1 as a biomarker of post-chemotherapeutic relapse for VL in rapid diagnostic tests (RDTs) sensitised with crude lysate antigen (CLA). We employed in silico tools to search for desired protein features in a large number of L. donovani antigens detected by human IgG1 in western blots. We then employed prediction algorithms to profile epitopes from the shortlisted proteins. We screened a panel of high-scoring peptide epitopes in a high-throughput manner using arrays, with low reagent consumption. The most reactive peptide was adapted to RDTs, showing promising results of both sensitivity and specificity. This peptide has the potential of replacing the CLAs in IgG1 RDTs. Using cure paired serum samples from India we evaluated the potential of IgG1 against the rK39 to monitor treatment outcome. ELISAs with rK39 and IgG1-specific conjugate gave a far more discriminative decrease in post-treatment antibody response when compared to IgG. Novel IgG1 rK39 RDTs provided strong evidence for decreased IgG1 response in patients who had successful treatment. Furthermore, both IgG1 rK39 RDTs and ELISAs showed a highly significant difference in test outcome between cured patients and those who relapsed. RDTs were more sensitive than corresponding ELISAs. Considering VL diagnosis in eastern Africa, we showed that the pool of Leishmania antigens located at the surface of the parasite (membrane proteins - MPs) represent an interesting target for additional investigations. This protein extract might contain the DAT antigen while the identification of specific antigens would enable the development of an RDT with improved sensitivity for active cases detection in eastern Africa.

Publication year:2019

Keywords:Doctoral thesis

Accessibility:Open