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

Abstract

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.

Use of glycerol as an alternative to freeze‐drying for long‐term preservation of antigen for the direct agglutination test

“…The use of a single L. donovani isolate (1‐s) for production of the freeze‐dried antigen constitutes a major limitation for applicability in VL endemic areas where L. infantum and L. chagasi are involved. Previous studies have shown that sensitivity for VL diagnosis can significantly be increased in DAT by incorporating the indigenous or homologous L. donovani subspecies (Harith et al. 1995).…”

“…Promastigotes were harvested 7 days post‐inoculation of the basic LIT mass culture. Antigen processing was according to the improved procedures described by Harith et al. (1995).…”

“…The high ambient temperatures and the lack of reliable cold chain facilities in most of the VL endemic areas have limited application of the test to central and district laboratories. Several improvements were introduced to DAT antigen processing in order to maintain stability under rural conditions (Harith et al. 1989,1995).…”

Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan

“…Cross-reactions with other pathogens may also occur . Several improvements in antigen quality and delivery have been made (Harith et al 1995;Meredith et al 1995). Although the DAT was adapted for ®eld use (Meredith et al 1995;Zijlstra et al 1997), there is a need for a simple, rapid and accurate test with good sensitivity and speci®city that can be used in the ®eld and for which no speci®c expertise is required.…”

Experimental Evidence on the Nature of the Antigen in the Direct Agglutination Test for Visceral Leishmaniasis

“…18 Interestingly, β-ME treatment of promastigotes increases concanavalin A (ConA)-binding sites and also increases the sensitivity of the DAT. 19 Based on these observations, we hypothesized that carbohydrates are likely to be part of the DAT Ag.…”