Enzyme-Linked Immunosorbent Assay for Recombinant K39 Antigen in Diagnosis and Prognosis of Indian Visceral Leishmaniasis

Abstract

The recombinant product (rK39) of the 39-amino-acid repeats encoded by a kinesin-like protein-encoding gene of Leishmania chagasi was evaluated by enzyme-linked immunosorbent assay (ELISA) for diagnostic potential and the ability to predict the response to therapy in Indian kala-azar or visceral leishmaniasis (VL); we also compared its performance with that of crude soluble antigen (CSA). At the diagnosis of VL, the anti-rK39 antibody titer was 59-fold higher than the anti-CSA antibody titer. With successful therapy, antibody titers declined steeply at the end of treatment and during follow-up. In contrast, patients who relapsed showed increased titers of antibodies to rK39. The extremely high levels of anti-rK39 antibodies in VL cases suggest the application of rK39 for sensitive and specific serodiagnosis, and rK39 ELISA is also valuable in monitoring drug therapy and detecting relapse of the disease.Indian kala-azar or visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania donovani. It is endemic in eastern parts of India and often turns epidemic (22,29). Definitive diagnosis of this disease continues to require demonstration of parasites in splenic or bone marrow aspirates through invasive procedures. Recent efforts to improve the diagnosis of Indian VL and post-kala-azar dermal leishmaniasis (PKDL) have been made by detecting anti-parasite antibodies (9, 11) via indirect hemagglutination (22,29), indirect immunofluorescence (6), direct agglutination (26), latex agglutination (8), and enzyme-linked immunosorbent assay (ELISA) (5, 10).Recently, a kinesin-related protein-encoding gene has been discovered in Leishmania chagasi that contains a repetitive 117-bp sequence encoding 39 amino acid residues (K39) conserved at the C-terminal end in all of the VL-causing isolates examined so far (4). The recombinant product of K39 (rK39) has proven to be a very sensitive and specific antigen in an ELISA for the serodiagnosis of VL from the endemic foci in Brazil, China, Pakistan, and Sudan (4,18). In the present study, we evaluated the ability of titers of antibodies against rK39 to diagnose active disease and predict either a successful response to therapy or a relapse of the disease and compared its performance with that of crude soluble lysate of L. donovani promastigotes. The crude soluble antigens (CSA) used in this study were largely L. donovani whole promastigotes or their soluble lysates. MATERIALS AND METHODSPatients. The Ethical Committee of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, approved this study. The first study group consisted of sera from 43 patients with parasitologically proven VL that were tested by ELISA using the rK39 antigen (kind gift of Steven G. Reed, Corixa Corporation, Seattle, Wash.), as well as by crude soluble lysate. The second study group consisted of 17 L. donovani-infected patients who were under going therapy. Titers of antibodies to rK39 and crude soluble lysate were determined for these patients at sequential time points...

Diagnosis of leishmaniasis

“…Cross-reactivity with sera from patients with tuberculosis, trypanosomiasis, and toxoplasmosis has been reported [47][48][49][50][51]. When selective antigenic molecules were used (with molecular weights of 116 kDa, 72 kDa, and 66 kDa), the specificity approached 100%, while the sensitivity was very low (37%) [31,52].…”

Detection of LD body in Peripheral Blood Buffy-coat from Suspected Kala-azar Cases of Bangladesh

“…Leishmaniasis prevalence is estimated to be 12 million people, and, in developing countries, approximately for antibodies in patients with VL caused by members of the L. donovani complex 12 . Another important facet of anti-rK39 antibody is that the titer correlates directly with the disease activity, indicating its potential for use in predicting response to chemotherapy 13 .…”

Compararative evaluation of rK9, rK26 and rK39 antigens in the serodiagnosis of Indian visceral leishmaniasis

“…The tests were performed following the method described by Kumar et al [16]. Briefly, 96-well microtitre plates were coated with rK9 (25 ng) or rK26 (25 ng) or rK39 (25 ng) or 5 µg of CSA per well.…”