Antibodies directed against L-tryptophan epitope (WE - W for tryptophan, E for epitope), a constant epitope borne by variant surface glycoproteins (VSG), have been detected in sera of all 152 Human African Trypanosomosis (HAT) patients from Angola. The WE is present in VSG hydrophobic regions of the C terminal domains. In the assay, L-tryptophan was linked to bovine serum albumin (BSA) with glutaraldehyde to synthesize W-G-BSA conjugate which was used in an enzyme-linked immunosorbent assay (ELISA) to detect the antibodies. A significant difference was found between HAT patients and controls confirming previous results obtained with a lower number of patients in Congo. A diagnostic test based on this synthetic epitope, especially in combination with other tests, might improve the HAT diagnostic test in field conditions.Key words: Tryptophan, enzyme-linked immunosorbent assay (ELISA), human African trypanosomosis, serological diagnostic

Chuchana, Paul

Courtois, Pierrette

Daulouède, Sylvie

Holzmuller, Philippe

Lemesre, Jean-loup

Nzoumbou-Boko, Romaric

Semballa, Silla

Truc, Philippe

Vincendeau, Philippe

English

AJOL - African Journals Online

 Vol. 15(36), pp. 1986-1990, 7 September, 2016 DOI: 10.5897/AJB2016.15575 Article Number: 273864A60390 ISSN 1684-5315 Copyright © 2016 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB African Journal of Biotechnology      Full Length Research Paper  Confirmation of antibodies against L-tryptophan-like epitope in human African trypanosomosis serological diagnostic  Silla Semballa1,2,3, Romaric Nzoumbou-Boko1,2,3 , Philippe Holzmuller4 , Paul Chuchana5 , Jean-loup Lemesre6, Philippe Vincendeau1,2, Pierrette Courtois1, Sylvie Daulouède1 and Philippe Truc7,8*  1Université de Bordeaux, IRD, UMR 177 «INTERTRYP», 33076 Bordeaux France. 2CHU de Bordeaux, Laboratoire de Parasitologie, 33076 Bordeaux France. 3Laboratoire des Sciences Biologiques et Agronomiques pour le Développement (LASBAD) Université de Bangui, Bangui, République Centrafricaine. 4CIRAD UMR Contrôle des maladies exotiques et émergentes (CMAEE), TA-A15/G, Campus International de Baillarguet, 34398, Montpellier Cedex, France. 5IRMB, Unité INSERM U1183, Hôpital Saint Eloi, 34295 Montpellier France. 6Institut de Recherche pour le Développement IRD, UMR 177 « INTERTRYP», Centre IRD de Montpellier, 34000 Montpellier Cedex, France. 7Institut de Recherche pour le Développement IRD, UMR 177 «INTERTRYP», Campus International de Baillarguet, 34398 Montpellier Cedex, France. 8Laboratoire de recherche sur les trypanosomoses, Un        e de  ec e c e        -CIRAD, Campus International de Baillarguet, 34398 Montpellier Cedex, France.  Received 20 July, 2016; Accepted 25 August, 2016  Antibodies directed against L-tryptophan epitope (WE - W for tryptophan, E for epitope), a constant epitope borne by variant surface glycoproteins (VSG), have been detected in sera of all 152 Human African Trypanosomosis (HAT) patients from Angola. The WE is present in VSG hydrophobic regions of the C terminal domains. In the assay, L-tryptophan was linked to bovine serum albumin (BSA) with glutaraldehyde to synthesize W-G-BSA conjugate which was used in an enzyme-linked immunosorbent assay (ELISA) to detect the antibodies. A significant difference was found between HAT patients and controls confirming previous results obtained with a lower number of patients in Congo. A diagnostic test based on this synthetic epitope, especially in combination with other tests, might improve the HAT diagnostic test in field conditions.   Key words: Tryptophan, enzyme-linked immunosorbent assay (ELISA), human African trypanosomosis, serological diagnostic.   INTRODUCTION  Trypanosoma brucei gambiense (T. b. gambiense) and T. b. rhodesiense, the causative  agents  of  Human  African Trypanosomosis (HAT) or sleeping sickness, are tsetse fly-transmitted  protozoan  parasites.  Diagnosis   of  HAT         remains a challenge for disease control as HAT is characterized by a non-specific clinical presentation and its definitive diagnosis requires trypanosome detection by microscopy (WHO, 2013). Additionally, the detection of this parasite is difficult because of the low parasite concentration in circulating blood, despite the use of concentration methods such as the mini-anion-exchange centrifugation technique (mAECT) (Lumsden et al., 1979). Moreover, molecular diagnostics based on parasite DNA or RNA detection are more sensitive but are not adapted to field conditions.   Serological diagnostic test of HAT, caused by T. b. gambiense, had been performed in the field during medical surveys since 1978 using the Card Agglutination Test with stained Trypanosomes (CATT) (Magnus et al., 1978). CATT/T. b. gambiense is still the reference test in the field conditions although, it requires refrigeration and an electric rotator, with drawbacks of false positive and negative results (Magnus et al., 1978). Trypanosomes produce a set of variant surface glycoproteins (VSG). Thus, new diagnostic approaches are based using native surface glycoproteins although the variable nature of the VSG is not satisfactory for a serological diagnostic test (WHO, 2013). Following an initiative of Foundation for Innovative New Diagnostics (FIND), a rapid diagnostic test is now available (Standart Diagnostic SD Bioline HAT, Sternberg et al., 2014). However, this latter is very useful for passive mode detection but not for mass screening survey (Büscher et al., 2014). The search of new biomarker candidates will permit an improvement to HAT diagnostic test based on CATT (Bonnet et al., 2015). Proteomic investigations are identifying potential immunodiagnostic parasite protein antigens and several biomarkers that are in preliminary evaluation or design for new diagnostic tests (Holzmuller et al., 2013). It has also been discussed how molecular diagnostics may contribute to the elimination of HAT (Büscher and Deborggraeve, 2015). A tryptophan-like epitope (WE) was found in VSGs from the T. brucei group (Semballa et al., 2007). The tryptophan residue is one of the conserved flanking residues of cysteine residues in their hydrophobic regions in the C terminal domain. Antibodies directed to WE were previously found in the sera of HAT patients detected during medical survey (active mode detection), but they were not detected in HIV infection, malaria, Chagas and Pa k nson’s d seases (Okomo-Assoumou et al., 1995). This latter investigation was performed in the Bouenza focus in the Republic of Congo. No other study has been done in a different HAT focus.   In the present study, anti-WE detection in sera of HAT patients  from   Angola   detected   by   passive   mode  at  Semballa et al.          1987    the Viana hospital compared to negative controls, in order to confirm the potential value of this immune marker in another endemic country, use in setting up a new test for serological HAT mass screening.   METHODS  Patients  Informed consent was obtained from all individual participants which were included in the study. Patients were diagnosed by passive mode detection in Viana hospital in Angola (Truc et al., 2012). Only CATT positive patients with trypanosomes that were detected in one body fluid (blood, lymph juice, cerebrospinal fluid) were selected. Stage determination of the disease was done by searching trypanosomes and white blood cells count in CSF: 13 in stage 1 (early period) and 139 in stage 2 (neurological period). A clinical examination allows detection of neurological signs. Sera from CATT negative subjects living in the same endemic area were used as controls. These controls were uninfected volunteer persons. Serum aliquots were kept at -80°C until use.   ELISA based on L-tryptophan conjugates  Enzyme-linked immunosorbent assay (ELISA) was performed as previously described (Okomo-Assoumou et al., 1995). Briefly, W and BSA (bovine serum albumin) were dissolved in 1.5 M acetate buffer, pH 8 before the addition of glutaraldehyde. After dialysis, the conjugate (W-G-BSA) or control (G-BSA) was added into polystyrene well plate. The well plate was filled with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-Tween), 10% glycerol and BSA (5 g/L). The well plate was incubated for 1 hr at 37°C to saturate it. The plate was rinsed twice with PBS-Tween. The well plate was then filled with 200 µL of diluted (2,000-fold) serum plus PBS-Tween containing BSA (5 g/L) and 10% glycerol. After washings, horseradish peroxidase-conjugated goat antibodies were added to human immunoglobulins. Thereafter, substrate solution and stop solution were successively added. The absorbance in the well plate was measured at 492 nm. The specific immunologic binding of sera was obtained by subtracting blank values read on well plates coated with BSA-G from experimental absorbance values.    Statistical method  Results are expressed in OD value for each individual. Comparison between the patients and the control groups was made using the nonparametric Wilcoxon–Mann–Whitney test with median and quintiles. The difference was considered significant when p<0.05. This comparison was illustrated by a graph performed with Graph Pad V6.0.   RESULTS AND DISCUSSION  Anti-WE antibodies were detected in serum of 152 patients (Table 1) and in 10 controls (Table 2). The limited  *Corresponding author. E-mail: philippe.truc@ird.fr. Tel.: +33 467593919. Fax: +33 467593893.  Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 1988          Afr. J. Biotechnol.    Table 1. Values of anti-WE antibodies in optic density and clinical stage for each patient.  Patient Code Stage OD Units (anti-WE) Patient Code Stage OD Units (anti-WE) V31 1 0.546 V117 2 0.750 V34 1 0.475 V119 2 0.725 V36 1 0.536 V122 2 3.808 V38 1 0.442 V124 2 0.546 V40 1 0.291 V125 2 1.346 V42 1 0.447 V128 2 0.722 V91 1 0.345 V129 2 0.635 V198 1 0.404 V130 2 0.451 V201 1 0.225 V133 2 0.716 V203 1 0.320 V134 2 0.676 V215 1 0.259 V135 2 0.602 V217 1 0.382 V137 2 0.593 V131 1 0.267 V138 2 0.604 V32 2 0.500 V139 2 0.505 V63 2 0.514 V141 2 0.592 V120 2 0.371 V142 2 0.779 V231 2 0.540 V143 2 0.780 V02 2 0.311 V144 2 0.736 V03 2 1.317 V145 2 0.634 V05 2 1.848 V146 2 0.592 V07 2 0.345 V147 2 1.288 V10 2 1.251 V150 2 0.707 V13 2 0.628 V151 2 0.852 V15 2 0.772 V152 2 0.509 V16 2 0.914 V153 2 0.724 V17 2 0.730 V156 2 0.777 V18 2 0.911 V157 2 0.557 V19 2 1.107 V159 2 0.691 V23 2 0.792 V160 2 0.504 V24 2 0.693 V162 2 0.622 V25 2 0.426 V164 2 0.871 V26 2 0.598 V165 2 0.602 V27 2 0.733 V166 2 0.512 V35 2 0.554 V167 2 1.234 V37 2 0.683 V168 2 0.730 V43 2 0.783 V170 2 0.760 V44 2 0.784 V171 2 0.715 V46 2 1.036 V172 2 0.850 V47 2 0.775 V173 2 0.530 V48 2 0.687 V174 2 0.517 V49 2 0.588 V176 2 0.642 V52 2 0.655 V177 2 0.736 V53 2 0.603 V178 2 0.546 V54 2 0.736 V179 2 0.794 V57 2 0.856 V180 2 0.577 V61 2 0.915 V182 2 0.607 V64 2 0.728 V183 2 1.055 V65 2 0.664 V187 2 0.573 V66 2 0.582 V188 2 0.719 V67 2 0.652 V189 2 0.746 V69 2 0.659 V191 2 0.700 Semballa et al.          1989    Table 1. Contd.  V70 2 0.846 V192 2 0.744 V75 2 0.561 V194 2 0.677 V80 2 0.684 V195 2 0.624 V82 2 1.389 V196 2 0.514 V83 2 0.685 V199 2 1.034 V85 2 0.643 V200 2 0.490 V86 2 0.594 V202 2 0.325 V87 2 1.015 V206 2 0.835 V88 2 0.558 V207 2 0.559 V89 2 0.604 V208 2 0.741 V90 2 0.898 V209 2 0.571 V92 2 0.841 V210 2 0.589 V93 2 0.843 V212 2 0.518 V94 2 0.781 V214 2 0.489 V96 2 0.364 V216 2 0.649 V98 2 1.637 V221 2 0.498 V100 2 0.996 V223 2 0.733 V104 2 0.802 V226 2 0.385 V105 2 1.099 V228 2 0.655 V106 2 0.944 V232 2 0.880 V107 2 0.700 V235 2 0.555 V109 2 0.758 V238 2 0.439 V110 2 3.090 V239 2 0.571 V113 2 0.796 V240 2 0.438 V114 2 0.719 V241 2 0.518    Table 2. Values of anti-WE antibodies in optic density afor each negative control individual.  Control Code OD Units (anti-WE) AG 0.109 AR 0.108 EB 0.096 MR 0.114 NF 0.089 BA 0.111 BH 0.192 MF 0.121 OE 0.117 OC 0.110    number of the control group was due to the difficulty in ob a n ng   e people’s consent at the Viana Hospital. In a survey carried out in Congo, including 22 controls and 76 patients (52 and 24 in stages 1 and 2 respectively), a very low immunological signal was detected in controls. The comparison between patients (median= 0.6622, n=152) and controls values is highly significant P <0.0001 (Figure 1). This confirms the use of anti-WE as a potential biomarker in HAT serological diagnostic test. Further investigations are required to compare the present results with the post-cure HAT persons and other parasitic diseases. Furthermore, among 139 stage 2 patients, 86 presented neurological disorders. There is no correlation between OD values and presence of neurological disorders P=0.58. Thus, the anti-WE value is not correlated to patient clinical state. Rapid tests are now available and are very promising but have been employed mainly for passive mode detection (Büscher et al., 2014). However, their observed specificity was relatively low. A larger study on their use in field condition in combination and in comparison with immune trypanolysis, the reference test, is under progress.  L-Tryptophan epitope conjugate production by peptide synthesis is now cheaper and easily compared to CATT antigen production, requiring T. b. gambiense infection in laboratory rodents, or production of recombinant proteins to develop diagnostic kits. Moreover, WE conjugate is thermo-stable and can be developed in a rapid test format. For instance, as most anti-WE antibodies are of IgM isotype, an agglutination assay is feasible. Reliably and easy to use, diagnostic tools are necessary to eliminate HAT as a public health problem in the year 2020 and zero transmission in the year 2030.  1990          Afr. J. Biotechnol.      Figure 1. Level of anti-WE antibodies, as assayed by ELISA, in the HAT patients and control groups. Black lines indicates median and interquartiles (Q25 and Q75).    Conflict of Interests  The authors have not declared any conflict of interests.    ACKNOWLEDGMENTS  The authors are grateful to the staff of Instituto de Combate e Controlo das Tripanossimiases, Ministry of Health, Angola.   REFERENCES  Bonnet J, Boudot C, Courtioux B (2015). Overview of the Diagnostic Methods Used in the Field for Human African Trypanosomiasis: What Could Change in the Next Years? Biomed. Res. Int. Volume 2015, Article ID 583262, 10 pages.  Büscher P, Deborggraeve S (2015). How can molecular diagnostics contribute to the elimination of human African trypanosomiasis? Expert Rev. Mol. Diagn. 15(5):607-615.  Büscher P, Mertens P, Leclipteux T, Gilleman Q, Jacquet D, Mumba-Ngoyi D, Pyana PP, Boelaert M, Lejon V (2014). Sensitivity and specificity of HAT Sero-K-SeT, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by Trypanosoma brucei gambiense: a case-control study. Lancet Glob. Health 2(6):e359-363. Holzmuller P, Grébaut P, Semballa S, Gonzatti MI, Geiger A (2013). Proteomics: a new way to improve human African trypanosomiasis diagnosis? Expert Rev. Proteomics 10(3):289-301.    Lumsden WH, Kimber CD, Evans DA, Doig SJ (1979). Trypanosoma brucei: Miniature anion-exchange/centrifugation technique for detection of low parasitaemias: Adaptation for field use. Trans. R. Soc. Trop. Med. Hyg. 73(3):312-317. Magnus E, Vervoort T, Van Meirvenne N (1978). A card-agglutination test with stained trypanosomes (CATT) for the serological diagnosis of T. b. gambiense trypanosomiasis. Ann. Soc. Belge Méd. Trop. 58:169-176. Okomo-Assoumou MC, Geffard M, Daulouede S, Chaugier C, Lemesre JL, Vincendeau P (1995). Circulating antibodies directed against tryptophan-like epitopes in sera of patients with human African trypanosomiasis. Am. J. Trop. Med. Hyg. 52(5):461-467.  Semballa S, Okomo-Assoumou MC, Holzmuller P, Büscher P, Magez S, Lemesre JL, Daulouede S, Courtois P, Geffard M, Vincendeau P (2007). Identification of a tryptophan-like epitope borne by the variable surface glycoprotein (VSG) of African trypanosomes. Exp. Parasitol. 115:173-180. S e nbe g J , G e l nsk   , B  le  S, Fe guson  AJ, Ndung’u J  (2014). Evaluation of the Diagnostic accuracy of prototype Rapid Tests for Human African Trypanosomiasis. PLoS Negl. Trop. Dis. 8(12):e3373.  Truc P, Lando A, Penchenier L, Vatunga G, Josenando T (2012). Human African trypanosomiasis in Angola: clinical observations, treatment, and use of PCR for stage determination of early stage of the disease. Trans. R. Soc. Trop. Med. Hyg. 106(1):10-14.                               WHO Control and surveillance of human African trypanosomiasis (2013). WHO Technical Report Series 984 p.  

Confirmation of antibodies against L-tryptophan-like epitope in human African trypanosomosis serological diagnostic

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Confirmation of antibodies against L-tryptophan-like epitope in human African trypanosomosis serological diagnostic

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