Human leishmaniasis is a severe health problem in many countries around the world. Hence, a cheap, reliable, and accurate diagnostic test is required to fight this disease. Perhaps the direct agglutination test (DAT) meets these criteria, but antigen elaboration involves many difficulties. We have developed a new antigen elaboration method, the EasyDAT method, that avoids the problems associated with the DAT. In this study, we compared the traditional DAT antigen method with our EasyDAT antigen method by using canine sera. The sensitivities (100%) and specificities (98.7%) were the same for both methods; we therefore concluded that the EasyDAT Leishmania antigen method simplifies serologic diagnosis, making this method easier and cheaper to use.Human visceral leishmaniasis, caused by a protozoan of the genus Leishmania, is a zoonotic disease whose main reservoirs are dogs. Visceral leishmaniasis is a potentially fatal disease that affects an estimated 500,000 people each year (9). Even though leishmaniasis is a serious public health problem in underdeveloped areas of the world, prevalence is quite low in the Mediterranean basin. Early diagnosis is the preferable way to fight this disease, which has been targeted by the World Health Organization.The main objective of this research was to develop a new protocol for antigen elaboration that avoids all the inconveniences of the traditional direct agglutination test (DAT) (3, 4) without compromising either the sensitivity or specificity. For EasyDAT antigen elaboration ( Fig. 1), we cultured Leishmania infantum (MHOM/FR/78/LEM-75) at 26.4°C in half-liter flasks containing RPMI 1640 with L-glutamine and NaHCO 3 (Sigma) and 5 mM HEPES, 100 IU of penicillin per ml, 100 g of streptomycin per ml, and 10% heat-inactivated fetal bovine serum, until the majority of the organisms had developed into the elongated promastigote forms. Promastigotes usually appear 3 to 5 days after culturing is begun, although this time varies between strains. The concentration of the culture was determined by counting the promastigotes in a Neubauer chamber and standardizing the concentration at 10 9 promastigotes/ml; 0.2 g of trypsin (at a 1:250 dilution with ␥-irradiated porcine pancreas; Panreac) was added to the culture, which was maintained at 37°C for 45 min. After this time, the culture was placed in a frozen water bath to stop trypsinization. Then 130 l of formalin (37 to 38% p/p ethanol stabilized; Panreac) was added. The culture was stirred gently for 1 h to fix the promastigotes properly. For harvesting, the culture was centrifuged (in 50-ml Falcon tubes at 2,000 ϫ g for 10 min) two times in order to concentrate the promastigotes and one time with citrate saline solution to remove excess formalin. Finally, the pellet was dissolved in 25 ml of citrate saline solution. To stain the promastigotes, we used Coomassie brilliant blue (R-250; Merck) diluted to 0.5% (wt/vol); we added 25 ml of this solution to the fixed promastigotes, which produced a final color concentration of 0.25%. After ...
