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 pronas e (400 mu 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 pre pared in this may performed satisfactorily when compared with the perf ormance of the initial trypsinated antigen. Even higher was the sensit ivity 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 va riants processed with pronase, lipase, pancreatin, or trypsin. By comb ining the use of indigenous Leishmania donovani subspecies from Sudan, Bangladesh, or Morocco and incorporating 2-ME instead of trypsin in t he 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 m onths, as evidenced by the absence of autoagglutination and the reprod ucibility of DAT readings with standard sera. The specificity of DAT w as 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. Tit ers ranging from 1:200 to 1:12,800 in the sera of patients and laborat ory animals infected with various Trypanosoma species were significant ly reduced (less than or equal to 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 posi tive or negative test in DAT were 100%. Sera from patients who formerl y had VL and who had been treated 6 to 36 months earlier remained reac tive (greater than or equal to 1:51,200) against 2-ME-processed antige n, despite the incorporation of urea into the DAT.