Modifications in a Reference Freeze-Dried Direct Agglutination Test to Improve Visceral Leishmaniasis Detection

Abstract

Currently, a significantly lower temperature (35°C) than initially established (56°C) is indicated as the maximum temperature storage for the commercial reference visceral leishmaniasis (VL) freeze-dried direct agglutination test (FD-DAT). Despite an approximately 50% loss in the number of promastigotes in an FD-DAT batch that expired 7 years earlier, the promastigotes maintained a similar morphology to the equivalent valid batch implying most likely that auto-agglutination, rather than aging, is the main reason for expiry. The substitution of normal saline which was initially recommended for reconstitution, by citrate-saline/formaldehyde (CSF) as an anti-clumping/preservative agent resulted in restoration of validity comparable with that of the freeze-dried original or the liquid direct agglutination test (LQ-DAT) version (Friedman ANOVA test = 1.0588; P = 0.5890). Following a similar reconstitution procedure as for the 7-year expired antigen, using significantly lower promastigote concentration (1.4 × 10 7 /mL) than in the non-expired (9.0 × 10 7 /mL), good reliability for VL detection and stability at 4°C (> 12 months) were achieved. In comparison with the original version using normal saline ($32.0/vial), the cost-effectiveness of the FD-DAT was appreciably improved by the CSF incorporation and lowering of promastigote concentration per unit suspension medium ($12.8/vial). With diagnostic reliability comparable with the full-out titration used, FD-DAT procedure based on single sample dilution at the VL cutoff (1:3,200) permitted the use of significantly smaller antigen volumes (0.1 mL vs. > 1.5 mL), therefore contributing to a further reduction in the application cost. The successful replacement of β-mercaptoethanol (β-ME) by urea (T = 21.00; P = 0.0868) provided the required safety for the test procedure similar to the widely applied LQ-DAT.

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

“…Through successfull replacement of normal saline by formaldehyde/citrate saline (CSF) as an anticlumping agent for antigen reconstitution, and drastically but eligibly lowering promastigote concentration per unit antigen suspension medium, signi cant improvement in test feasibility was achieved (12). Larger volume (12 ml) of the an anti-clumping agent CFS could therefore be used, in comparison with the 5-ml normal saline in the original reference FD-DAT.…”

“…The original reference FD-DAT was reconstituted according to the manufacturer's instructions with 5 ml normal saline per vial. Both valid and expired FD-DAT batches were reconstituted with an anti-clumping solution (0.056 M sodium citrate and 0.15 M sodium chloride) supplemented with 1.2% w/v formaldehyde (CSF) instead of normal saline as previously reported to attain a least however eligible promastigote concentration of 1.4x10 7 /ml, instead of the 8.7-9.0x10 7 /ml originally reported (12). The valid or expired FD-DAT was stored at 4°C until required.…”

“…During the past decade signi cant progress was made that included the production of the LQ-DAT locally and introduction of essential improvements to the FD-DAT to ensure optimal safety for VL routine diagnosis in Sudan and elsewhere. Aside from pronounced reduction in test cost using the valid batches, FD-DAT batches that were expired seven years earlier were successfully revitalized contributing to a further lowering the test expenses (12) In this study we intended to assess performances of an improved valid or expired FD-DAT version in comparison with the original reference, a liquid test version (LQ-DAT), IFAT and an enzyme-linked immunosorbent assay (ELISA) version employing a recombinant antigen for the detection of CVL in an endemic dog population from Croatia.…”

Haematological malignancies as disorders negatively impacting specificities of the direct agglutination and rapid rK39 strip tests as reference diagnostics for visceral leishmanisis

“…Based on merits here and previously enumerated included high diagnostic reliability, antigen stability under adverse conditions and the required safety for test execution, the new SDS-DAT is expected to comply with most of the criteria needed for an appropriate and safe management of VL [13,15].…”