Abstract
.The direct agglutination test (DAT) for visceral leishmaniasis (VL) is the serodiagnostic test for VL that has the most robust sensitivity and specificity in the field across all endemic regions. It is based on trypsin-treated and formaldehyde-fixed whole promastigote cells from Leishmania donovani. The exact identity and nature of the epitopes on the DAT antigen that cause agglutination with VL patients’ sera are currently unknown. In this study, we performed antigen-inhibition studies which revealed that lipophosphoglycan (LPG) and the DAT antigen share epitopes. Antibody inhibition with a monoclonal antibody directed against the phosphoglycan repeat epitope of LPG showed that this is not the epitope that reacts with human sera. Oxidation of carbohydrates by sodium metaperiodate did not alter the reactivity of human sera with the DAT antigen and LPG. This indicates that carbohydrates do not play a role in the reaction of the DAT antigen with antibodies in serum from VL patients, and that they also are not involved in the reaction of LPG with the same serum. We conclude that the noncarbohydrate moiety of LPG, that is, the core–anchor fragment, and potentially other noncarbohydrate epitopes on the surface of the DAT antigen are responsible for its agglutination with antibodies from VL patients. As LPG plays a role in the DAT reaction, this could facilitate the following: 1) incorporation of LPG, preferably the synthetic version of the core–anchor fragment, into an immunochromatographic test format that is more adapted as a point-of-care test (short incubation, little training, and equipment needed) than DAT and 2) enhancing the quality control for the production of the DAT antigen.
Highlights
Visceral leishmaniasis (VL) is a fatal disease that is caused mainly by protozoan parasites of the species Leishmania donovani and Leishmania infantum
In the ELISA, CA7AE reacted at an average ODc450nm of 1.09 (±0.55) (Supplemental Figure 3A), and the end-titer of CA7AE in direct agglutination test (DAT) was 1:400
The inhibition experiments with DAT Ag coated in ELISA microtiter plates revealed that preincubation with neither the monoclonal CA7AE nor with VL patient sera leads to a decreased reactivity of, respectively, VL patient sera or CA7AE, suggesting that human sera and CA7AE do not react with the same epitope of LPG
Summary
Visceral leishmaniasis (VL) is a fatal disease that is caused mainly by protozoan parasites of the species Leishmania donovani and Leishmania infantum. Its diagnostic sensitivity and specificity are high across all geographic regions endemic for VL with 94.8% (95% CI: 92.7–96.4%) sensitivity and 97.1% (95% CI: 93.9–98.7%) specificity.[1] The antigen in the DAT consists of whole promastigote cells from a Sudanese L. donovani isolate (MHOM/--/SD/1-S) that have been trypsin treated, formaldehyde fixed and stained with Coomassie brilliant blue (DAT Ag).[2] This test is performed by incubating the serum, diluted in a buffer containing beta-mercaptoethanol (β-ME) and albumin, with the antigen in V-bottom microtiter plates for 18 hours.[1,3]. The need for small laboratory equipment, well-trained personnel, and long incubation times makes the DAT less field applicable than the rapid diagnostic immunochromatographic test (ICT). Production of the promastigotes is subject to considerable batch-to-batch variation and is difficult to quality control
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