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.

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Abstract. A prerequisite for the control of visceral leishmaniasis (VL) is the accessibility to reference diagnostics. The high price of the freeze-dried direct agglutination test (FD-DAT) and the short shelf-life time of the rK39 strip test (rK39) have limited the application of these tests in Sudan. An original liquid DAT (LQ-DAT) with high reproducibility compared with the FD-DAT and rK39 has been routinely produced in our laboratory since 1999. In this study, a 3.4-year-old batch (of more than 90 test batches produced to date) was chosen to validate the diagnostic performance of this test against microscopy, FD-DAT, and rK39 in 96 VL and 42 non-VL serum samples. Relatively higher sensitivity (95/96, 99.0%) was recorded for the LQ-DAT than for the FD-DAT (92/96, 95.8%) and rK39 (76/96, 79.2%), probably because of the use of the endemic autochthonous Leishmania donovani isolate as the antigen. Experience with the LQ-DAT, its low cost of production, ease of providing this test, and diagnostic reliability compared with the FD-DAT suggest that widescale implementation of the LQ-DAT can contribute to sustainable VL control in Sudan.

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 g/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 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 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 reduced (<1:200) or were rendered negative at a dilution of 1:25. Regardless of the infections caused by Trypanosoma species, the sensitivity, specificity, and predictive value of a positive or negative test in DAT were 100%. Sera from patients who formerly had VL and who had been treated 6 to 36 months earlier remained reactive (>1:51,200) against 2-ME-processed antigen, despite the incorporation of urea into the DAT.

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BackgroundCanine leishmaniosis (CanL) is an endemic zoonosis in the southern regions of Europe. This paper reports the trend in CanL seroprevalence in the municipality of Évora (southern Portugal), where the disease is endemic, over a period of 20 years. The work comprises three different studies that were conducted in the years of 1990 (n = 3,614), 1999 (n = 3,563) and 2010 (n = 1,485 dogs). Blood samples were collected during the anti-rabies vaccination campaigns. Anti-Leishmania antibodies were detected with the direct agglutination test (DAT).FindingsThe total percentages of DAT seropositive dogs were 3.9% (in 1990), 9.4% (in 1999) and 5.6% (in 2010). The overall seroprevalence was significantly higher in 1999 compared to 1990, but in 2010 a significant decrease was found in comparison with 1999. However, compared to 1990 the overall seroprevalence was still significantly higher in 2010. From 1990 to 2010 seroprevalence has switched from significantly lower to higher in the rural areas. Relatively few dogs showed clinical signs of overt disease (0.8% to 2.0%) with lymphadenopathy, onychogryphosis and skin involvement as most frequently observed. Gender associated differences in seroprevalence were not found, and most commonly seropositive dogs were working or stray animals. The mean age of seropositive dogs was significantly higher than seronegative dogs in all three sampling rounds.ConclusionsA high proportion of dogs, which are apparently healthy, yet seropositive, may remain an important factor in limiting the outcome of zoonotic leishmaniosis control efforts.

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.