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.

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 Leishmania isolates from Bangladeshi patients with post-kala azar dermal leishmaniasis or visceral leishmaniasis were used. A titre of at least 51,200 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. Tha DAT is better than current parasitological and histopathological methods for the diagnosis of PKDL in areas in which leprosy is co-endemic.

Evaluation of cleaving agents other than trypsin in direct agglutination test for further improving diagnosis of visceral leishmaniasis.

Trypsin treatment of Leishmania promastigote antigen has proved to be indispensible in the direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). In the present study four antigen batches were prepared with pronase (400 micrograms/ml), lipase (0.45% [wt/vol]), pancreatin (0.3% [wt/vol]), or 2-mercaptoethanol (2-ME) (1.2% [vol/vol]) at a ratio of 20:1 versus promastigote packed cell volume or a density of 10(8)/ml. Batches prepared in this way performed satisfactorily when compared with the performance of the initial trypsinated antigen. Even higher was the sensitivity and specificity of the 2-ME-processed antigen, scoring a minimum DAT titer of 1:102,400 in the VL and CVL group and a maximum of 1:400 in the negative control group. Corresponding titers ranging from 1:6,400 to 1:12,800 and 1:800 to 1:1,600 were obtained with the antigen variants processed with pronase, lipase, pancreatin, or trypsin. By combining the use of indigenous Leishmania donovani subspecies from Sudan, Bangladesh, or Morocco and incorporating 2-ME instead of trypsin in the antigen processing step, a threefold increase in titer was attained in sera from the respective areas where VL is endemic. 2-ME-processed antigen suspensions maintained stability at 4 degrees C for up to 9 months, as evidenced by the absence of autoagglutination and the reproducibility of DAT readings with standard sera. The specificity of DAT was further improved by supplementation of the sample diluent with 0.03 M urea and incubation of the test plates at 37 degrees C for 1 h. Titers ranging from 1:200 to 1:12,800 in the sera of patients and laboratory animals infected with various trypanosoma species were significantly reduce (/=1:51,200) against 2-ME-processed antigen, despite the incorporation of urea into the DAT.

Applicability of direct agglutination test (DAT) at a rural health setting in Bangladesh and feasibility of local antigen production.

As part of a large-scale sero-epidemiological survey on visceral leishmaniasis (VL) carried out in Mymensingh district of Bangladesh, applicability of DAT was assessed at the level of a rural health setting in Trishal (upazila) subdistrict. Despite the relatively less optimal conditions encountered, 5854 inhabitants from 7 villages appendant to Trishal were assessed for VL. The demographic distribution for sero-positivity obtained at the rural setting was comparable to that found by DAT as executed at the central laboratory (IEDC&R, Dhaka) on 9619 inhabitants from the same upazila. The overall sero-prevalence rate was 4.4% compared to 3.7% obtained in the population assessed at the central laboratory. In either study, similar VL prevalence rates of 2.1% were obtained in the male populations. Irrespective of sex, younger population (< 20 years) in both studies appeared to have higher VL incidence rate (2.3% and 2.6%) than others of 21- > or = 90 years (1.4% and 1.8%). Local production of DAT antigen employing an authochtonus L. donovani isolate was attempted at the central laboratory (IEDC&R) in Dhaka. By comparison with the reference antigen, titres obtained in all 33 VL sera tested were equally higher (1:6400- > or =: 51200) than in 35 out of 38 negative controls (< or = 1:400-1:1600). A comparable level of reactivity was also obtained in 53 VL and 52 negative control sera using a well characterized L. donovani strain (MHOM/IN/80/D88) from India. However, unlike the reference strain, titres obtained in 7 endemic controls were significantly higher with the authochtonous and homologous antigen (1:3200 - 1:6400) than with the reference (1:100 - 1:1600). The results signify the advantage of employing indigenous L. donovani isolates to further improve DAT sensitivity for detection of early and sub-clinical VL.

Positive response to sodium antimony gluconate administration in visceral leishmaniasis seropositive patients.

In a prospective study conducted in Mymensingh district of Bangladesh, 1, 273 patients were assessed for the presence of visceral leishmaniasis (VL). Sodium antimony gluconate (SAG) was successfully administered to 715 patients with parasitologically confirmed infection. In the remaining 558, although there was clinical indication of VL, Leishmania donovani parasites could not be demonstrated. Administration of SAG in this group was on the grounds of the prevailing symptoms, exclusion of malaria and a positive direct agglutination test (DAT). Significant improvements in the clinical and hematological parameters were observed in 547 (98%) of the unconfirmed VL cases. On the basis of the parasitological findings or positive response to specific anti-Leishmania chemotherapy, the sensitivity and specificity of the DAT were 99.6% and 97.7% respectively. The results supported the reliability of DAT for diagnosis of VL at levels below that of parasitological detection.