A simple and economical direct agglutination test for the detection of visceral leishmaniasis is described. Trypsin-treated, Coomassie Brilliant Blue-stained, formalin-preserved promastigotes were used as antigen in re-usable V-well microtitre plates. In 21 patients with recent kala-azar, titres of 1:51200 or higher were found. Cured kala-azar patients treated 4 to 14 months before testing, showed titres in the range of 1:3,200 to greater than 1:51,200. Healthy and diseased controls had titres below 1:1,600 with the exception of African trypanosomiasis patients who showed titres of 1:200 to 1:12,800, overlapping with the titres of cured kala-azar patients. Where trypanosomiasis is not a consideration, a titre of 1:1,600 could be considered indicative of visceral leishmaniasis, the sensitivity and specificity were then 100%. The test was applied to sera of 280 inhabitants of Baringo District, a known focus of visceral leishmaniasis in Kenya. When treated cases were included, the test showed a sensitivity of 100% and specificity of 99.3%. This test could be used in district hospitals and health centres in endemic areas as an aid in diagnosis of kala-azar and in the field for sero-epidemiological studies.

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 3rC. 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.

Assessment of the resilience of canine leishmaniasis to control or, more ambitiously, the effort needed to eradicate infection, requires an estimate of the basic case reproduction number (R0). This paper applies the theoretical results of Hasibeder, Dye & Carpenter (1992) to data from a cross-sectional survey on the Maltese island of Gozo in which dogs of known age, sex and occupation (pet, guard etc) were subjected to three different serological tests for the presence of specific antibody (IFAT, DAT and ELISA). Difficulties in interpreting these test results, and hence of determining the proportion of dogs infected, present the main obstacle to estimating R0: estimates are critically dependent on the choice of threshold separating seropositives from seronegatives. The data do, however, allow a robust comparative analysis of risk which shows that the force of infection experienced by working dogs is about three times higher than that of pet dogs, a degree of non-homogeneous contact which actually has little effect on estimates of R0. We suggest a cautious point estimate of R0 congruent to 11, and comment briefly on its significance for leishmaniasis control.

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°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.

Diagnostic tests for visceral leishmaniasis that are based on antigens of a single Leishmania strain can have low diagnostic performance in regions where heterologous parasites predominate. The aim of this study was to investigate and compare the performance of five serological tests, based on different Leishmania antigens, in three endemic countries for visceral leishmaniasis. A total number of 231 sera of symptomatic and asymptomatic cases and controls from three endemic regions of visceral leishmaniasis in East Sudan, North India and South France were evaluated by following serological tests: rKLO8- and rK39 ELISA, DAT (ITMA-DAT) and two rapid tests of rK39 (IT LEISH) and rKE16 (Signal-KA). Overall, rKLO8- and rK39 ELISA were most sensitive in immunocompetent patients from all endemic regions (96–100%) and the sensitivity was reduced to 81.8% in HIV co-infected patients from France. Sera of patients from India demonstrated significantly higher antibody responses to rKLO8 and rK39 compared with sera from Sudan (p<0.0001) and France (p<0.0037). Further, some Indian and Sudanese patients reacted better with rKLO8 than rK39. Sensitivity of DAT (ITMA-DAT) was high in Sudan (94%) and India (92.3%) but low in France being 88.5% and 54.5% for VL and VL/HIV patients, respectively. In contrast, rapid tests displayed high sensitivity only in patients from India (96.2%) but not Sudan (64–88%) and France (73.1–88.5% and 63.6–81.8% in VL and VL/HIV patients, respectively). While the sensitivity varied, all tests showed high specificity in Sudan (96.7–100%) and India (96.6%).Heterogeneity of Leishmania parasites which is common in many endemic regions complicates the diagnosis of visceral leishmaniasis. Therefore, tests based on homologous Leishmania antigens are required for particular endemic regions to detect cases which are difficult to be diagnosed with currently available tests.