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

Introduction: Proper identification and management of reservoir post-kala-azar dermal leishmaniasis (PKDL) and canine visceral leishmaniasis (CVL) cases are prerequisites to the effective control of visceral leishmaniasis (VL) worldwide. Unlike PKDL, CVL still awaits effective improvement because of its cryptic nature, absence of Leishmania parasite in lesions or lymph nodes, and insensitivity of tools in current use. Because of the need for certain skills and equipment, both the liquid direct agglutination test (LQ-DAT) and freeze-dried direct agglutination test (FD-DAT) versions, present in comparison with the indirect immunofluorescence (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 CVL endemic areas. Methodology Introducing of citrate-saline formaldehyde (CSF) as an anti-clumping agent to replace normal saline for antigen reconstitution and drastically however eligibly lowering the concentration of promastigotes (1.4X107) in comparison with the original reference FD-DAT (> 5X107/ml), To ensure optimal safety, β-mercaptoethanol (β-ME) was replaced by urea or sodium dodecyl sulphate (SDS) as a serum reducing agent. Results Through 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 48-hour shelf-life time for the original, an FD-DAT batch re-constituted here with CSF maintained validity 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 CVL diagnostics further motivate the application of the improved FD-DAT for detection of the disease in endemic areas.