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Journal of Clinical Microbiology, March 2005, p. 1269-1277, Vol. 43, No. 3
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.3.1269-1277.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Samiran Saha,^1, Tuhina Mazumdar,^1,, Khairul Anam,^1, Rajesh Ravindran,^1,¶ Bibhas Bairagi,² Bibhuti Saha,² Ramapada Goswami,² Netai Pramanik,² Subhashis K. Guha,² Sourjya Kar,² Dwijadas Banerjee,² and Nahid Ali^1*

Infectious Diseases Group, Indian Institute of Chemical Biology,¹ Department of Tropical Medicine, School of Tropical Medicine, Calcutta, India²

Received 13 April 2004/ Returned for modification 29 May 2004/ Accepted 17 October 2004

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.

S. S. and T. M. contributed equally to this work.

Present address: Pulmonary Division, Department of Medicine, Baylor College of Medicine, Houston, TX 77030.

Present address: WIDDK, Navy Transplantation Autoimmunity Branch, National Institutes of Health, Bethesda, MD 20889.

^¶ Present address: Division of Immunology, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06034-4035.

This article has been cited by other articles:

• Saha, S., Mondal, S., Ravindran, R., Bhowmick, S., Modak, D., Mallick, S., Rahman, M., Kar, S., Goswami, R., Guha, S. K., Pramanik, N., Saha, B., Ali, N. (2007). IL-10- and TGF-beta-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. J. Immunol. 179: 5592-5603 [Abstract] [Full Text]