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 × 10⁷/mL) than in the non-expired (9.0 × 10⁷/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.

Figure 1.

An improved procedure for execution of a reference freeze-dried direct agglutination test (FD-DAT) in the diagnosis of visceral leishmaniasis, by using the citrate-saline/formaldehyde (CSF) as an antigen diluent and anti-clumping/preservative agent instead of normal saline as originally instructed. Six FD-DAT antigen aliquots reconstituted with CSF volumes of 5, 7, 9, 11, 12, or 13 mL with corresponding promastigote concentrations of 9.0 × 10⁷, 6.5 × 10⁷, 4.7 × 10⁷, 2.3 × 10⁷, 1.4 × 10⁷, and 0.8 × 10⁷/mL were tested against standard VL positive (+) and VL negative (−) sera starting at 1:100 serum dilution up to 1: 6,400. Reading of the test results was similar for all six antigen aliquots by locating a circumscribed blue spot (end point) in the titration row that resembles the one in the control well containing sample diluent only (extreme left column); the serum dilution that preceded the end point is considered the titer of the serum sample. Titers ≥ 1:3,200 are indicative of VL. All six antigen aliquots showed similar test readings: titers ≥ 1:6,400 in the VL (+) and ≤ 1:100 in the non-VL (−). The antigen aliquot reconstituted with the highest CSF volume (13 mL) had the corresponding lowest promastigote concentration (0.8 × 10⁷) and showed invalid agglutination reaction. This figure appears in color at www.ajtmh.org.