User and environment friendly direct agglutination test for the sero-diagnosis of visceral leishmaniasis: exclusion of formaldehyde and β-mercaptoethanol in test execution

Purpose. Based on world-wide evaluation, the direct agglutination test (DAT) is now generally acknowledged as one of the leading diagnostics for visceral leishmaniasis (VL). To enhance more routine and mass application, but simultaneously ensure safety to both user and environment, further improvements need to be introduced. Methodology. In the current format, a two-sixfold titre decrease was observed due to using formaldehyde as an antigen preservative in DAT. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3% (wt/vol) for conserving promastigote status after beta-mercaptoethanol (beta-ME) treatment and repeating exposure of the parasite to the fixative after Coomassie Brilliant Blue staining. Results. Microbial contamination was not observed in any of the antigen aliquots preserved in 0.05% (wt/vol) sodium dichloroisocyanurate (chlorine) instead of formaldehyde for 6 months or longer. By excluding formaldehyde, restoring the normal antibody level, prior to treatment of sera with b-ME only minimally influenced the test outcome. A comparable successful reduction in non-specific agglutination, as with b-ME, was achieved by incorporating urea (0.3% wt/vol) in the improved DAT procedure (P=0.646; T=23.0). As with the current procedure, the improved equivalent (formaldehyde and b-ME free) showed good reliability for VL detection (VL - Fr=52.39, W=0.70, P<0.001; and non-VL -Fr=65.97, W=0.83, P<0.001). A much lower cut-off (titre 1 : 400 versus 1 : 3200) for VL diagnosis can be adopted if urea is integrated in the improved procedure. Conclusions. By introducing the modifications mentioned, we think we have succeeded to a reasonable degree in increasing the DAT potential for VL control.