Validation of an improved reference freeze-dried direct agglutination test for detecting leishmaniasis in the canine...

Introduction. Proper identification and management of post-kala-azar dermal leishmaniasis (PKDL) and canine leishmaniasis (CanL) cases are among the prerequisites to the effective control of visceral leishmaniasis worldwide. Unlike PKDL, CanL still awaits effective improvement because of its cryptic nature, absence of Leishmania parasites in lesions or lymph nodes and not complete sensitivity of some diagnostic tools in use. Because of the need for certain skills and equipment, both the liquid direct agglutination test and freeze-dried direct agglutination test (FD-DAT) versions are, in comparison with the indirect immunofluorescence antibody test (IFAT) or enzyme-linked immunosorbent assay (ELISA), practical and feasible diagnostic alternatives. Aim. Validate the performance of an improved FD-DAT to suit routine and large-scale applications in CanL endemic areas. Methodology. Introducing citrate-saline formaldehyde (CSF) as an anti-clumping agent to replace normal saline for antigen reconstitution and drastically, however, eligibly lower the concentration of promastigotes (1.4×10⁷) in comparison with the original FD-DAT reference (>5×10⁷ ml^-1). To ensure optimal safety, β-mercaptoethanol was replaced by urea or SDS as a serum-reducing agent. Results. By improving the procedure for reconstitution of FD-DAT antigen with CSF, a 150% reduction in the test application cost was achieved. Expired test batches (±4 years earlier) were successfully revitalized to full validity. As compared to the 48 h shelf-life time for the original, an FD-DAT batch reconstituted here with CSF maintained stability for ±12 months. Conclusions. The highly concordant results with IFAT and ELISA (one-way ANOVA test, P=0.142, homogeneity of variances P=0.009) as routine CanL diagnostics further motivate the application of the improved FD-DAT for the detection of the disease in endemic areas.