Reactivity of various leishmanial antigens in a direct agglutination test and their value in differentiating post-kala azar dermal leishmaniasis from leprosy and other skin conditions

Abstract

A direct agglutination test (DAT) for the detection of post-kala azar dermal leishmaniasis (PKDL) was evaluated in conditions that simulate the disease clinically or immunologically. A reference strain of Leishmania donovani (LEM 1399), and antigen preparations from two Leishmania isolates from Bangladeshi patients with post-kala azar dermal leishmaniasis or visceral leismaniasis were used. A titre of at least 51200 was obtained in tests of patients with PKDL with all three antigens, whereas a maximum titre of 1600 was recorded in patients with cutaneous leishmaniasis, mucocutaneous leishmaniasis or leprosy. Antigens from dermal isolates of L. tropica (LV 140) and L. braziliensis (LV 65) yielded titres of 1600-6400 in patients with PKDL. The lowest titre recorded in 70 patients tested with the homologous PKDL antigen was 409 600. In patients with leprosy, cutaneous leishmaniasis, syphilis, onchocerciasis, tuberculosis, blastomycosis or vitiligo, titres ranged from 100 to 1600. The DAT is better than current parasitological and histopathological methods for the diagnosis of PKDL in areas in which leprosy is co-endemic.

Evaluation of Enzyme-Linked Immunosorbent Assay for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis with Crude or Recombinant k39 Antigen

“…In order to improve the sensitivity of the ELISA, we used promastigote antigen prepared with parasite isolates from dermal lesions of a PKDL patient. The indigenous PKDL antigen was found to give generally higher titers than those obtained with the reference strain (AG83), which is similar to the observation made during a direct agglutination test for PKDL in Sudan (9). Since Leishmania parasites are present in the amastigote form in the human host, the humoral immune response would be directed against antigens of the amastigote form.…”

“…The use of recombinant k39 (rk39) has been shown to overcome these limitations to a considerable extent (2,16,22,26,29). Antileishmanial antibodies of the immunoglobulin G (IgG) and IgM classes have been demonstrated in the sera from PKDL patients (8,20); however, limited studies have been conducted to develop serological methods for the diagnosis of PKDL (9). Increased sensitivity has been reported when the immunoperoxidase technique and PCR are used (10,15,21).…”

Potential of Direct Agglutination Test Based on Promastigote and Amastigote Antigens for Serodiagnosis of Post-Kala-Azar Dermal Leishmaniasis

“…The titer for geographic controls was relatively higher than for controls from areas where KA was not endemic, indicating the possibility of previous exposure of the subjects to the parasite (3). Earlier studies from Sudan had shown that a cutoff titer of 1:3,200 was suitable for KA diagnosis and 1:1,600 was suitable for PKDL diagnosis (6,7). Studies from India on DAT for KA used a cutoff titer of 1:800 (22), while no studies from India are available on DAT for the diagnosis of PKDL.…”

“…Promastigotes of L. donovani isolated from KA and PKDL patients and AG83 strain (MHOM/IN/AG/83) were mass cultured in M-199 with 10% fetal calf serum. The antigen was prepared by the method of Harith et al (6,7). Briefly, the promastigotes were harvested in logarithmic phase, washed repeatedly with cold Hanks balanced salt solution (HBSS) (Invitrogen Corporation, Grand Island, NY), treated with 0.4% trypsin (Sigma) in HBSS at 10 8 parasites/ml for 1 h at 37°C.…”

“…DAT. The DAT was performed essentially as described earlier (6,7). Serum samples were diluted in physiological saline (0.9% NaCl) containing 0.2% gelatin (Sigma) and 0.78% ␤-mercaptoethanol (Sigma).…”