Table 1.
What is the DAT antigen?
| Hypothesis | Pro | Contra | Conclusion |
|---|---|---|---|
| DAT antigen is a non-Leishmania antigen | |||
| Nonspecific antigen on promastigote surface or medium contaminants (Wasserman test reinvented) | Wasserman test does not contain Leishmania antigens but has high diagnostic accuracy17 | – | DAT antigen is Leishmania-specific but nonspecific antigens might contribute to the reaction |
| Molecular mimicry between Leishmania antigens and human auto-antigens24,25 | – | – | |
| Latex beads coated with glutaraldehyde-cross-linked HSA*Glut agglutinate with VL sera20 | Trypanosoma cruzi epimastigotes prepared in the same way as DAT do not cross-react with VL sera26 | – | |
| HAS*Glut in ELISA is more specific for VL than crude promastigote extract20 | Promastigotes grown in albumin-free medium agglutinate with VL sera and even with a monoclonal antibody against HSA*Glut22 | – | |
| Monoclonal antibody against HSA*Glut reacts in DAT20 | – | – | |
| Nonspecific detection of hyperimmunoglobulinemia (aldehyde test reinvented) | Aldehyde test contains no antigen but has high diagnostic accuracy18 | – | – |
| VL patient serum generally contains high levels of nonspecific immunoglobulins21 | – | – | |
| VL patient sera often test false positive in non-VL ELISA51 | – | – | |
| DAT reacts with sera from patients with other diseases known to induce hyperimmunoglobulinemia23 | End-titer of VL sera in DAT is much higher than end-titer of sera from patients with other causes of hyperimmunglobulinemia23 | – | |
| DAT antigen is an LPG | |||
| LPG in ELISA has a high diagnostic accuracy to detect VL31 | LPG masks the antigen on the promastigote surface32 | Core-anchor fragment of LPG almost certainly DAT antigen | |
| Monoclonal antibody against LPG reacts in DAT20 | End-titers of VL sera are higher with LPG-minus mutant Leishmania promastigotes than with wild-type promastigotes32 | – | |
| LPG reactivity with VL sera in ELISA is completely inhibited by its core-anchor fragment32 | In immunofluorescence, VL sera react with LPG-minus mutant Leishmania promastigotes but not with wild-type promastigotes32 | – | |
| Fixation process during DAT preparation disrupts masking effect of LPG32 and potentially exposes the LPG core-anchor fragment | – | – | |
| DAT antigen is a carbohydrate or a glycoprotein | |||
| ME treatment of promastigotes increases ConA-binding sites and DAT sensitivity36,38 | ME treatment might release carbohydrates as well as other epitopes | Despite trypsin treatment (which removes carbohydrates in T. cruzi), carbohydrate involvement in DAT is not excluded, in contrary carbohydrates likely involved in DAT ag | |
| Trypsin treatment of Leishmania promastigotes does not remove the ConA-binding sites of DAT antigen34,36,37 | – | – | |
| VL sera react with many Leishmania glycoproteins in Western blot34,38,39 | – | – | |
| gp63 is the DAT antigen | Monoclonal antibodies against gp63 might not be against the epitope in DAT | Monoclonal antibodies against gp63, which has been proposed as DAT epitope,38 do not react in DAT 19 | – |
| DAT antigen is a lipid | |||
| Wasserman test uses lipids and has high diagnostic accuracy17 | – | Lipids are likely involved in the DAT reaction | |
| Lipase decreases sensitivity of DAT38 | Other molecules associated to lipids, e.g., embedded in the cell membrane, might be cleaved by lipase as well | – | |
| DAT is more sensitive than ELISA with hydrosoluble Leishmania antigens41,42 | May be due to differences in test format between ELISA and DAT | – | |
| Lipid fraction of Leishmania parasites is reactive with VL sera43 | Proteins and carbohydrates remaining in the lipid fraction might react as well | – | |
| DAT antigen is a protein | |||
| Proteins might be shielded from proteases, such as trypsin, by the glycocalyx, which in turn is removed during fixation of the Leishmania promastigotes | Proteases do not have an effect on DAT sensitivity38 | Protein involvement in DAT reaction is uncertain but not excluded | |
| DAT antigen is a mixture of antigens | |||
| Geographic diversity of Leishmania donovani antigens,44 yet high diagnostic accuracy of DAT irrespective of geographic distribution1 | – | DAT ag is most probably composed of a mixture of antigens | |
| Studies with monoclonal antibodies show that at least three different epitopes are involved in the DAT test: LPG, an HAS*Glut like epitope, and a yet unidentified epitope20 | – | – | |
| “Whole promastigote cells” confer higher sensitivity to DAT than any single antigen known so far42 | – | – | |
ConA = concanavalin A; DAT = direct agglutination test; ELISA = enzyme-linked immunosorbent assay; HSA*Glut = human serum albumin; LPG = lipophosphoglycan; ME = mercaptoethanol; VL = visceral leishmaniasis.