LeishmaniaPromastigote Membrane Antigen-Based Enzyme-Linked Immunosorbent Assay and Immunoblotting for Differential Diagnosis of Indian Post-Kala-Azar Dermal Leishmaniasis

Abstract

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani, is difficult, as the dermal lesions are of several types and resemble those caused by other skin diseases, especially leprosy. Since the disease generally appears very late after the clinical cure of kala-azar in India, it is also difficult to correlate PKDL with a previous exposure to L. donovani. Very few attempts have been made so far to diagnose PKDL serologically, and the diagnostic methods vary in their sensitivities and specificities. Diagnosis of PKDL through sophisticated PCR methods, although highly sensitive, has limited practical use. We have developed a serodiagnostic method using an enzyme-linked immunosorbent assay to detect specific immunoglobulin (Ig) isotypes and IgG subclass antibodies in the sera of Indian PKDL patients. Our assay, which uses L. donovani promastigote membrane antigens, was 100% sensitive for the detection of IgG and 96.7% specific for the detection of IgG and IgG1. Optical density values for individual patients, however, demonstrated wide variations. Western blot analysis based on IgG reactivity could differentiate patients with PKDL from control subjects, which included patients with leprosy, patients from areas where kala-azar is endemic, and healthy subjects, by the detection of polypeptides of 67, 72, and 120 kDa. The recognition patterns of the majority of serum samples from patients with PKDL were also distinct from those of the serum samples from patients with visceral leishmaniasis (VL), at least for a 31-kDa polypeptide. To further differentiate patients with PKDL from those with active and cured VL, we analyzed the specific titers of the Ig isotypes and IgG subclasses. High levels of IgG, IgG1, IgG2, and IgG3 antibodies significantly differentiated patients with PKDL from patients cured of VL. The absence of antileishmanial IgE and IgG4 in patients with PKDL differentiated these patients from those with active VL. These results imply intrinsic differences in the antibodies generated in the sera from patients with PKDL and VL.

Comparative study of amplification systems in immunoenzyme assays for the diagnosis of American tegumentary leishmaniasis

“…This result agrees with previous studies that also demonstrated an increased sensitivity of the assays when homologous instead of heterologous antigens were used (25)(26)(27).…”

IL-10- and TGF-β-Mediated Susceptibility in Kala-azar and Post-kala-azar Dermal Leishmaniasis: The Significance of Amphotericin B in the Control of Leishmania donovani Infection in India

“…Our preliminary observation of a significant elevation of the percentage of the population of CD4 ϩ CD25 ϩ T cells in VL in comparison to healthy controls, its decline at cure, and further significant elevation in PKDL patients indicate that in vivo expansion and suppressive activity of CD4 ϩ CD25 ϩ T cells is probably influenced by the production of IL-10 and TGF-␤. More interestingly, IgA production in the different PKDL patients followed the pattern of enhanced IL-10 and TGF-␤ production with disease severity and appears to confirm our earlier speculation that the elevation of IgA in some PKDL patients might be induced by IL-10 with help from TGF-␤ (46).…”

“…IL-10 and TGF-␤ are involved in enhancing IgA production in human cells (59 -61). Earlier, we reported an up-regulation of IgA in some of the PKDL patients (46) and postulated that this up-regulation might be correlated with enhanced IL-10 production. In this study we show that PKDL patients with up-regulated serum IgA in fact produce high amounts of IL-10 in vitro under stimulation with LAg ( Fig.…”

“…43 and 44, respectively) in vitro lymphoproliferation showed that, of the three Ags, LAg induced the least nonspecific response in healthy controls and active VL patients and the highest Ag-specific response in cured kala-azar patients (data not shown). Earlier, we reported the better diagnostic ability of LAg (extracted from the promastigote membrane) over whole cell lysate in the diagnosis of VL (45) and described the ability of LAg for differential serodiagnosis of VL and PKDL (46). Moreover, we also investigated the modulation of humoral immune responses in human VL in the course of SAG and AmB treatment (31) using LAg.…”

Systematic Review of Biomarkers To Monitor Therapeutic Response in Leishmaniasis

“…Though not very sensitive (44%), Leishmania-specific immunoglobulin E (L-IgE) has been suggested to be a specific (98.3%) marker of active VL disease (L. chagasi), although it is undetectable at subclinical levels in VL patients, Chagas disease patients, and healthy controls (143)(144)(145). Moreover, increased L-IgE concentrations were demonstrated to regress to normal values during the time span of treatment (143,145,146). In cases of atypical cutaneous leishmaniasis, IgE levels were not significantly different from those of asymptomatic or healthy controls (147).…”