Development of multiplex serological assay for the detection of human African trypanosomiasis

Abstract

Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance. We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance.

Rapid diagnosis of parasitic diseases: current scenario and future needs

“…However, there are still some challenges in this area. In the future, therefore, we will specifically need to: [140] Develop new, specific biomarkers: a) which will provide serodiagnosis of trichinellosis during the early stage of disease (3e4 weeks between Trichinella infection and specific antibody positivity) [213], b) for Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense infections considering the fact that these parasites express variable surface antigens which significantly limits diagnostic usefulness of serological examination [214], c) for Toxoplasma gondii infection suitable to divide recent from past infections and replace a panel of tests (Toxoplasma gondii serologic profile (TSP)) which is still in use for this purpose [215], d) which will allow serological differentiation between Toxocara canis and Toxocara cati (or Toxocara malaysiensis) infections [216]. Develop scalable tests, particularly for use in field situations (e.g.…”

Development of a microsphere-based immunoassay for the serological diagnosis of equine trypanosomosis

“…The relatively low fluorescence intensity obtained for these last five antigens could be explained either by a weak coupling of the antigens to the fluorescent microspheres or by the conformation of the coated antigens that made the HisTag inaccessible to the anti-HisTag antibodies. Discrepancies between the coating measurement with anti-HisTag antibodies and the efficacy of coupling antigens in diagnostic tests have also been described by others 18 , so we decided to perform serum reactivity assays for the eight antigens. The results obtained confirmed that coupling controls using anti-HisTag antibodies cannot accurately predict the reactivity of the antigen during these experiments since high fluorescence intensities were obtained for antigens with low fluorescence intensities using anti-HisTag antibodies (Figs.…”

“…Antigens enolase, PFR1 and PFR2 were also excluded due to low fluorescence intensities for positive sera (lack of sensitivity). The exclusion of seven out of the eight antigens originally selected in this study was unexpected, as several of these antigens (such as Rotat1.2 20 and ISG65 18 ) have already been used by other teams developing diagnostic tools for trypanosomosis. The reasons for the lack of efficacy remain unidentified.…”

“…Technically, indirect immunoassays using xMAP® technology may be used to detect antibodies through capture antigens coated on microspheres similar to a microsphere-based ELISA. The potential of this strategy has been demonstrated in particular by Nzou, et al 18 who developed an immunoassay for human African trypanosomiasis screening.…”

Evaluation of Antigens for Development of a Serological Test for Human African Trypanosomiasis

“…It is possible that other antigens that were either not included or had not been identified at that time could perform equally well or better than assessed in the present study. For example, the flagellar calcium-binding protein TB-17 [20,60], the Gene Related to Expression Site Associated Gene (GRESAG) 4, and transferrin receptor subunits ESAG 6 and 7 have also been suggested as potential candidates [20]. Since the most reactive antigens include the native VSG LiTat 1.3 and VSG LiTat 1.5 antigens, which are costly and difficult to produce, one approach might be to generate recombinant versions of these proteins.…”