Identification of a Novel Recombinant Protein for Improved Diagnosis of Visceral Leishmaniasis in Sudan

For effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study was to improve diagnosis of VL in East Africa by testing new antigens from an autochthonous Leishmania donovani strain. We cloned and expressed a new antigenic protein (designated rKLO8) of Leishmania donovani containing putative conserved domains of significant similarity with immunodominant kinesin proteins of several Leishmania strains. rKLO8 exhibited 93% amino acid identity with cloned kinesin proteins of Leishmania infantum (rK39) and 88% with Leishmania donovani (rKE16). Sequence analysis of rKLO8, rK39 and rKE16 revealed genetic heterogeneity within immunodominant epitopes of these antigens. Immunoreactivity of the purified recombinant protein rKLO8 was confirmed by Western blot and enzyme-linked immunosorbent assay (ELISA). Importantly, antibody reactivity against rKLO8 was detected in VL patients but not in healthy controls. We successfully developed a diagnostic ELISA based on rKLO8 which was evaluated with sera from VL patients originated from Sudan, India and France. Direct comparison between rKLO8– and rK39 ELISA revealed that our newly developed test system showed similar reactivity with sera of VL patients from France but increased with sera from Sudanese and Indian patients. Next, we compared the diagnostic performance of rKLO8- and rK39 ELISA with other commercially available tests, including rK39- and rKE16 rapid tests and direct agglutination test (DAT). Results showed that all tests performed very well in India but best sensitivity in all countries was observed with rKLO8- and rK39 ELISA. However in Sudan and France, the two rapid tests showed low sensitivity. DAT showed better sensitivity in Sudan and India than in France. The sensitivity of all tests was markedly reduced in VL patients co-infected with human immunodeficiency virus (HIV). Furthermore, the rKLO8 ELISA was also evaluated with sera of Leishmania-infected dogs from Portugal, Croatia and Brazil. The results showed that rKLO8 ELISA was similar to DAT but more sensitive than the routinely used immunofluorescent antibody test (IFAT). Thus, increased reactivity of sera from Sudanese VL patients with rKLO8 shows that this antigen is a potential candidate for improving VL diagnosis in Sudan and other regions of East Africa where similar strains of Leishmania donovani are endemic.