To increase the potential for the wide-scale application of our direct agglutination test for visceral leishmaniasis, modifications in the components and procedures were introduced. Supplementation with 0.056 M citrate of the suspension medium stabilized the antigen for 9 weeks at 37 degrees C. To circumvent the need for cooling systems in the field, 0.2% (wt/vol) gelatin was added to the serum diluent instead of fetal bovine serum, with reliable results. Specificity and sensitivity were improved by the incorporation of 0.1 M 2-mercaptoethanol in samples with borderline titers. The test could be performed on samples of whole blood; thus the difficulties of preparation and storage of serum, plasma, or filter paper blood are avoided. For mass screening programs, a single serum dilution of 1:6,400 could be employed, contributing to a further reduction in test expenses. Sera from different geographical areas showed equal reactivities in this direct agglutination test despite the nonhomologous Leishmania donovani antigens used.

https://jcm.asm.org/content/jcm/26/7/1321.full.pdf

Improvement of a direct agglutination test for field studies of visceral leishmaniasis.

A direct agglutination test (DAT) for detection of visceral leishmaniasis in humans has been developed. In this study, it was evaluated for applicability to detection of infections in dogs, a reservoir species. The reliability of the test was improved by treating the test sera with 0.2 M 2-mercaptoethanol and incubating them at 37 degrees C. Sensitivity was 100% and specificity was 98.9% when the test was used on serum samples from 220 dogs, including 26 with parasitologically confirmed canine leishmaniasis, 12 with suspected but unconfirmed leishmaniasis, and 182 with other conditions. The DAT detected specific antibodies in 10 dogs with canine leishmaniasis diagnosed by case history, clinical signs of leishmaniasis, and seropositivity in an immunofluorescence test using either promastigotes or amastigotes, as well as in 2 dogs suspected of having leishmaniasis. The performance of an antigen prepared from a homologous isolate of Leishmania infantum in the DAT was compared with that of an antigen from a laboratory-adapted strain of L. donovani (sensu lato). The homologous antigen compared favorably with the standard antigen, and the results provided further evidence of the potential of the DAT for detection of Leishmania infection in the canine reservoir host. The results of this study, together with those of our previous studies in human visceral leishmaniasis, demonstrate that the DAT is highly suitable for wide-scale epidemiological and ecological field work. This technique could also facilitate diagnosis of leishmaniasis in dogs in veterinary health services.

Application of a direct agglutination test for detection of specific anti-Leishmania antibodies in the canine reservoir.

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.

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

Control of endemic visceral leishmaniasis (VL) in large communities requires a feasible epidemiological indicator capable of monitoring on-going transmission rather than mere exposure to the parasite. Following confirmation of the desired level of reliability for laboratory diagnosis of VL, the direct agglutination test (DAT) was employed to estimate VL sero-prevalence in the endemicupazilas (subdistricts) of Trishal and Shahjadpur within Mymensingh and Sirajganj districts of Bangladesh. DAT antigen production was duly increased to allow coverage of a study population of 17826 inhabitants, 9619 of whom resided in Trishal, 7328 in Shahjadpur and 879 in Teknaf (Cox's Bazar), a knownLeishmania-free district in Bangladesh. Despite large-scale production in batches of 1120–4000 ml (each sufficient for 1176–6400 screening doses), all DAT antigen batches performed as required in quality control tests for sensitivity, specificity and stability. It was convenient for both collection and testing to take the required samples of whole blood by finger prick. A cross-sectional survey revealed VL point prevalences of 4.40% in Trishal and 6.75% in Shahjadpur, compared with an extremely low rate of 0.34% in non-endemic Teknaf. In both endemicupazilas (Trishal and Shahjadpur) VL was more prevalent (2.56–4.5%) in persons up to 20 years of age than in those 21 years of age and older (1.84–2.25%). Of 918 subjects recorded as seropositives, 539 were VL-asymptomatic and 379 were VL-symptomatic with various degrees of suspicion. Diagnosis of VL was established in 125 symptomatic seropositives subjects, either by demonstrating the presence ofLeishmania amastigotes (29), or by positivie DAT results combined with presentation of typical VL signs (96). All diagnosed patients responded favourably to sodium antimony gluconate administration; re-testing and follow-up procedures were started on the remaining 793 seropositives. The DAT can be considered as an appropriate and feasible alternative to the leishmanin and aldehyde tests for VL survey in large endemic communities.

Prevalence of agglutinating anti-Leishmania antibodies in two multi-thousand bengoli communities

A prototype test kit being developed, by the World Health Organization (WHO), for the diagnosis of visceral leishmaniasis (VL) was evaluated in the Baringo district of Rift Valley province in Kenya. The screening of approximately 10 000 individuals for the signs of VL produced 305 suspected cases. These cases and 304 controls matched for sex and age (± 2 years) were then tested with the kit, which is based on a direct agglutination test (DAT). The evaluation was a three-stage process. The first stage, the field screening, involved screening filter-paper samples of dried blood from the suspects and controls at a DAT titre of 1:500. The second stage, the laboratory titration, involved screening of the same individuals by testing freshly eluted filter-paper samples at 1:500 to 1:2000 dilution. In the third stage, the full-scale titration, all samples that had been positive at 1:2000 were titrated at 1:500–1:512 000. All the suspects giving DAT titres of 1:2000 or higher were considered positive for VL. This ...

Evaluation of a standardized direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis in Kenya.

A microagglutination test using trypsin-treated and Coomassie blue-stained Trypanosoma cruzi epimastigote antigen was adapted for the diagnosis of Chagas' disease. When incorporated in the test, 2-mercaptoethanol treatment of chagasic sera had no influence on antibody titer. In contrast, titers in sera from patients with visceral leishmaniasis, African trypanosomiasis, and autoimmune disorders, subjected to similar treatment, showed remarkable decline. Accordingly, a lower cut-off point for Chagas' disease serological negativity could be taken resulting in a higher sensitivity (95.6%); the specificity was 94.7%. Similar specificities were obtained with Leishmania donovani chagasi and L. d. donovani antigens applied to homologous visceral leishmaniasis and heterologous Chagas' sera. Of 316 nonchagasic sera, only 3 with leptospirosis and 1 with leprosy showed seropositive titers prior to and after 2-mercaptoethanol treatment.

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Papers

Trypsin-treated and coomassie blue-stained epimastigote antigen in a microagglutination test for Chagas' disease.