Date Published: February 17, 2019
Publisher: The American Society of Tropical Medicine and Hygiene
Author(s): Vera Kühne, Philippe Büscher.
Current diagnostic tests for visceral leishmaniasis (VL) are either not adapted for use in resource-poor settings or are insufficiently accurate in Eastern Africa. Only the direct agglutination test (DAT), based on whole Leishmania promastigotes, is highly reliable in all endemic regions, but its implementation is hampered by the need for a cold chain, minimal laboratory conditions, and long incubation times. Integrating the DAT antigen(s) in an immunochromatographic rapid diagnostic test (RDT) would overcome these disadvantages. Unfortunately, the identity of the DAT antigen(s) involved in the agglutination reaction is unknown. For this study, we reviewed all publications that might shed some light on this issue. We conclude that the DAT antigen is a mixture of Leishmania-specific epitopes of protein, carbohydrate, and lipid nature. To develop an accurate RDT for VL diagnosis in Eastern Africa, we suggest to complement the classical protein antigen discovery with approaches to identify carbohydrate and lipid epitopes.
With more than 100 years of research on the diagnosis of visceral leishmaniasis (VL), the direct agglutination test (DAT) remains the most reliable serodiagnostic test for the disease in the field.1
References for this review were identified through searches between January 2017 and July 2017 of PubMed and Google Scholar for articles published until July 2017, by use of the terms “DAT AND Leishmania,” “Direct Agglutination Test AND Leishmania,” “diagnosis AND Leishmania,” “membrane AND Leishmania,” “antigen AND Leishmania,” “Wasserman test,” “aldehyde test,” “formol-gel test,” “lipophosphoglycan (LPG),” “trypsin AND Leishmania,” “beta-mercaptoethanol AND Leishmania,” “carbohydrate AND antigen,” and “lipid AND antigen.” Other relevant articles were identified through searches in the authors’ and collaborators’ personal files. Articles resulting from these searches and relevant references cited in those articles were reviewed. Articles published in English, French, and German were included.
Historically, two tests challenge the need for Leishmania-specific antigens in the serodiagnosis of VL: the Wasserman test that makes use of bacterial extracts to detect VL with a sensitivity of 74% and a specificity of 75%17 and the aldehyde test, or “formol-gel” test, that suggests VL without making use of any particular antigen but that shows surprisingly high diagnostic accuracy; sensitivity of 98% and specificity of 98% in its first description by Napier,18 whereas in a more recent study in Nepal by Boelaert et al.,19 a sensitivity of 34% (27–41%) and specificity of 99% (96–100%) was detected. By adding formaldehyde to a patient’s serum, an opaque gel develops in the case of VL, whereas for non-VL cases, the serum remains clear and liquid. This solidification reaction with formaldehyde is postulated to be caused by the very high concentration of immunoglobulins in VL patients.18
The surface of a L. donovani promastigote cell is covered by a dense glycocalyx of 20–40-nm thickness (Figure 2).27 This glycocalyx is composed of LPG, glycosylphosphatidylinositol (GPI)-anchored glycoproteins, and glycoinositolphospholipids. Interestingly, amastigotes lack this prominent glycocalyx.27
From what precedes (as summarized in Table 1), it is clear that the identity of the Leishmania-specific antigens or epitopes that form the basis of the high diagnostic accuracy of the DAT remains enigmatic. Considering their abundance, it is likely that there are antigenic proteins on the surface of the promastigotes, but there is no clear evidence for or against protein epitopes to be involved in the DAT reaction. Most probably, parts of LPG and potentially other lipid components, as well as carbohydrate epitopes play the major role.