Abstract
The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the Leishmania antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of L. donovani proteins in circulating antigens were further validated using anti-Ld actin and anti-α tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit.
Highlights
Human Visceral Leishmaniasis (VL), a lethal form of leishmaniasis caused by Leishmania donovani (L. donovani) contributes significantly to the annual burden of infectious diseases in India [1]
Formation of immune complexes occurs during the various scavenging process as a phenomenon of humoral immunity to remove antigen or pathogen [51]
Higher expression of the immune complex is associated with L. donovani induced inversion of the normal albumin to globulin ratio [52] and hypergamma-globulinemia in human VL subject [6, 17, 53, 54]
Summary
Human Visceral Leishmaniasis (VL), a lethal form of leishmaniasis caused by Leishmania donovani (L. donovani) contributes significantly to the annual burden of infectious diseases in India [1]. The conventional diagnostic test for VL is the microscopic demonstration of L. donovani amastigotes in aspirates from visceral organ [2]. PCR is capable of diagnosing relapse or re-infection cases of VL. It is complex and not suitable for mass screening in the field [3]. Were less invasive, but its reliability is not assured due to cross-reactivity in other disease conditions, co-infection cases and relapse [4]. RK-39 showed 100% sensitivity and 98% specificity respectively, but it has no prognostic values as well as it is not reliable for co-infection cases and relapse cases of VL. The previous reports have evidenced that the sensitivity of rK-39 is as low as 71% in HIV patients co-infected with Leishmania [4]. Is an another successful example of commercially available rapid antibody detection antigen based on membrane filtration technology [5]
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