Volume 102, Issue 4
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



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; = 0.5890). Following a similar reconstitution procedure as for the 7-year expired antigen, using significantly lower promastigote concentration (1.4 × 10/mL) than in the non-expired (9.0 × 10/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 ( = 21.00; = 0.0868) provided the required safety for the test procedure similar to the widely applied LQ-DAT.


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  • Received : 08 Oct 2019
  • Accepted : 02 Jan 2020
  • Published online : 10 Feb 2020
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