Evaluation of enzyme-linked immunosorbent assay for diagnosis of post-kala-azar dermal leishmaniasis with crude or recombinant k39 antigen.

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.

Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis.

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.

Western blot analysis of humoral immune response to Leishmania donovani antigens in patients with post-kala-azar dermal leishmaniasis.

Sera from 32 Indian patients with post-kala-azar dermal leishmaniasis (PKDL) were examined for antibodies by immunoblot analysis using an antigen extract of Leishmania donovani. The study revealed that the humoral immune response in PKDL patients was quite distinct compared to that in kala-azar patients. Antibodies to 3 antigens of L. donovani (molecular sizes 110, 65 and 38-42 kDa) were predominant in a majority (78%) of PKDL patients. The most important finding was the consistent recognition of 2 parasite antigens (of 110 and 65 kDa) by PKDL sera; antibodies to the 110-kDa antigen were detectable in 97% of cases, while antibodies to the 65-kDa antigen were detectable in 100% of cases that were examined. None of the 18 cases of leprosy, 10 of vitiligo, or the 30 healthy persons included in the study showed antibodies to these 2 antigens. Thus Western blot analysis provided a highly sensitive test for PKDL patients. Further, it led to the identification of 2 parasite antigens (110 and 65 kDa) that elicit an antibody response in 97-100% of PKDL patients. Purified or recombinant versions of these proteins deserve consideration as potential target antigens in development of simpler, highly specific and sensitive serodiagnostic tests for PKDL.

Immunoblot analysis of the antibody response to antigens of Leishmania donovani in Indian kala-azar.

When infected with Leishmania donovani, patients develop specific antibodies that constitute the basis of serodiagnosis. Using immunoblot analysis, we examined the antibody response to antigens of L. donovani in 35 kala-azar (KA) patients and 67 controls. Sera from KA patients recognised numerous antigens with molecular weights ranging from 14-110 kDa. Antigens of 40 kDa, 55 kDa, 65 kDa, 70 kDa and 82 kDa were recognised most frequently. All KA patients produce an antibody response to one or more of these antigens. The majority (83%) of KA cases recognised at least four of these five parasite antigens. The 70 kDa antigen showed the greatest sensitivity for Indian KA, and produced a positive reaction in 94% of patients. This antigen gave 10% false-positive reactions in controls comprising patients with related diseases (i.e. tuberculosis, leprosy and malaria) and in healthy controls. Data indicated that the 70 kDa antigen may include a member of the heat shock protein 70 family. Studies with four clinical isolates of L. donovani showed that the 70 kDa component was expressed in all the strains examined. Immunoblot assay (Western blotting) provided a sensitive diagnostic test for KA patients, and identified the 70 kDa parasite antigen that is promising as a potential target antigen for the development of less complex serodiagnostic assays for KA.