Evaluation of the TbgI 2 and TbgI 17 Tandem Repeat Antigens as Potential Antigens for the Diagnosis of Trypanosoma brucei rhodesiense

Human African trypanosomiasis (HAT) affects up to half a million people every year in sub-Saharan Africa. Interruption of transmission of the disease by early diagnosis and treatment is core to the control and eventual elimination of HAT. The routine diagnostic method for HAT is light microscopy of blood samples. The present study sought to evaluate the potential of TbgI 2 and TbgI 17 tandem repeat antigens as candidates for the diagnosis of Trypanosoma brucei rhodesiense. The expressed proteins were purified and the antigenic reactivity evaluation was done using multiplex assay using sera obtained from HAT patients. Receiver operating characteristic analysis showed that recombinant antigen, TbgI 2 had high sensitivity for sera from patients infected with T. b. rhodesiense with the area under the curve being 0.577 and a sensitivity of 0.641 and specificity 0.650. The results suggest that TbgI 2 is a potential biomarker for T. b. rhodesiense HAT serodiagnostic tests.


Introduction
Human African trypanosomiasis (HAT) is a vector born disease caused by flagellated protozoa of the genus Trypanosoma which is transmitted to humans through the bite of a tsetse fly [1] [2]. The disease exists in two forms: a chronic form of HAT caused by Trypanosoma brucei gambiense (Tbg), predominantly found in the Central and West Africa and the acute form of HAT caused by Trypanosoma brucei rhodesiense (Tbr) which is present in East and Southern Africa. The reservoir host for Tbg is the human while Tbr is a zoonosis [3]. The symptoms are exhibited in two stages: the early haemolymphatic stage where the trypanosomes are in blood, lymph and other tissues which is characterized by non-specific features such as chancre, fever, headache, lymphadenopathy and the late encephalitic stage where the trypanosomes cross the blood brain barrier and invade the central nervous system (CNS) causing neuropsychiatric disorders and sleep disorders which leads ultimately to death if untreated [4] [5] [6] [7]. this to be achieved, there is a need for robust diagnostic tools for both Tbg and Tbr [3] [6] [8].
Diagnosis of HAT is not done routinely even in known endemic areas. Parasite detection by microscopy is the first line of diagnosis. Other methods also used include lymph node examination, mini anion exchange centrifugation technique (mAECT), capillary tube centrifugation (CTC), molecular methods such as polymerase chain reaction (PCR), and nucleic acid sequence-based amplification (NASBA). Serological methods such as card agglutination trypanosomiasis test (CATT), rapid diagnostic tests (RDTs), enzyme-linked immunosorbent assay (ELISA) is also used. CATT is widely used for active serological screening of Tbg. The reported sensitivity of the CATT on filter paper ranges from 91% to 92.7% while specificity varies from 93.7% to 100% [9] [10] [11] [12]. Nonetheless, CATT has challenges of under diagnosis since it relies on the variable surface glycoproteins (VSG) LiTat 1.3 which are often switched off by the parasite to evade the host immune system by the antigenic variation process [2] [13] [14]. Furthermore, it presents a high incidence of false positives, which makes it particularly problematic in regions where the prevalence is low. There is still no equivalent test to the CATT widely available for the screening of Tbr HAT; hence, the screening for Tbr infection relies on clinical features due to a lack of serological tests. This can lead to the underestimation of its burden due to similarity of symptoms with other infections [8] [15]. Thus, there is a need for robust diagnostic markers, which can capture low transmission accurately and V. Irumva et al. Open Journal of Clinical Diagnostics effectively. It is known that Trypanosoma brucei subspecies possess genes encoding proteins with large tandem repeat (TR) domains as do other trypanosomatid and that they are potent B cell antigens. Two of these antigens I 2 and I 17 have been characterized at the molecular level and have been shown to allow a sensitive and specific detection of infections with different species of trypanosomatids, and then could be diagnostically useful [16] [17]. Therefore, in this study, we evaluated the specificity and sensitivity of two invariable TR intracellular antigens, which were recombinantly produced, namely TbgI 2 and TbgI 17 against Rhodesiense, and Gambiense HAT sera with a focus on Tbr infections on a bead-based platform.

Antigens Expression and Purification
The antigens in pET52b expression vector were transformed in ECOS-BL21 The PVDF membrane was washed five times, 10 min each in 100 ml at room temperature. The blot was then incubated in chemiluminescence (ECL) (Lot#9659209, GE Healthcare UK Limited) for five minutes at room temperature. For CBB, the gel was stained in Coommassie Brilliant Blue (40% Methanol, 10% Acetic Acid, 0.25% (w/v) CBB-R250) for protein purity visualization and gently shaken for two hours, prior to the gel destaining overnight in destaining solution containing 10% Methanol, 7.5% Acetic Acid in water.
The image was captured in LAS500 while the protein concentrations of the purified antigens were determined using the Pierce BCA protein assay kit (Thermo Scientific, USA).

Coupling Confirmation
To determine the coupling effectiveness, a coupling efficiency assay was per-

Statistical Analysis
Box Plots were generated to describe samples' variability in antigens reactivity.
To assess the antigens reactivity, sensitivity and specificity, the area under the curve (AUC) was generated using the Receiver Operating Characteristics (ROC) to illustrate the diagnostic ability and the accuracy of the TbgI 2 and TbgI 17 recombinant antigens. Box plots and ROC were obtained using XLSTAT 2019 (Addinsoft).

Human Samples
Antigen reactivity was evaluated using 119 human sera provided by the WHO Specimen Biobank. The samples consisted of 20 negative sera and 40 positive sera for Tbg; 20 negative sera and 39 positive sera for Tbr.

Ethics
Ethical approval was obtained from the Jomo Kenyatta University of Agriculture
The density was measured for TbgI 2 and TbgI 17 as shown in Table 1.
Western Blot was performed and its analysis confirmed that the TbgI 2 and TbgI 17 recombinant antigens were effectively produced while the Coomassie Brilliant Blue analysis showed that all the two recombinant antigens were of good purity (Figure 1 & Figure 2).
Following protein expression and purification confirmation, protein quantification was performed by Pierce BCA (Bincinchoninic Acid) Protein Assay kit (Thermo scientific) as shown in Table 2.

Coupling Efficiency
Subsequently, the recombinant proteins were coupled to Magplex magnetic beads. Figure 3 illustrates the average of the different median fluorescence intensity (MFI) values generated during coupling confirmation.

Discussion
In this study, we report the evaluation of the potential of invariable cytoskeleton flagellar antigens, TbgI 2 and TbgI 17 , in the diagnosis of Tbr.
Following antigens coupling, the diagnosis capacity of the TbgI 2 and TbgI 17 antigens was evaluated in a multiplex platform. In this study, the multiplex platform allowed to evaluate the TbgI 2 and TbgI 17 antigens simultaneously using a small amount of HAT sera (two microlitres) within a short time. These observations are in agreement with previous studies, where several antigens were evaluated against several infectious diseases using the simultaneous bead-based technology and highlighted several advantages of this technology in antigen evaluation such as rapidity, reproducibility, simultaneous detection and less amount of samples aligned with cost-effectiveness [18] [19] [20].
The data generated suggested that Gambiense HAT negative control sera highly reacted with the TbgI 2 and TbgI 17 TR recombinant antigens, which demonstrate a low specificity of the two antigens against Tbg infections.
For Tbr, some control sera also reacted. This non-specific reaction to Tbr could be explained by several other reasons: 1) a non-specific activation of B lymphocytes leading to nonspecific binding, which has been already reported to occur during trypanosomal infections [21]; 2) cross reactivity or 3) might indi- proteins were used to detect antibodies in sera from mice infected with Gambiense HAT but did not highly detect the antibodies [22].
Our results showed that TbgI 2 exhibited a better sensitivity and a considerable specificity while TbgI 17 despite a good specificity, exhibited a low sensitivity for sera from Tbr patients. Therefore, TbgI 2 could be more diagnostically useful in Tbr infections than TbgI 17 . This is in contrast with what was previously described by another study where TbgI 2 and TbgI 17 were both considered to allow early sensitive and specific detection with various Trypanosomatids species [16].
Finding biomarkers for Rhodesiense HAT has been challenging. Our data gives light for further research on TbgI 2 invariable tandem repeat intracellular antigen and its potential application in Rhodesiense HAT diagnosis. These findings are in agreement with previous studies where the results indicated that the African trypanosomes present certain invariant molecules which could be immunogenic [23].
Evaluating the 2 antigens in another assay should be considered for comparison purposes. Also, the evaluation of these antigens with a larger number of samples, with the different stages of the disease represented, should be considered in order to evaluate whether the antigens can be used to diagnose HAT staging. Finally, cross reactivity of these antigens with other parasitic diseases such as malaria and filariasis which has been already reported to occur in the CATT screening test should be further investigated [24].

Conclusions
Using a multiplex format, we were able to assess the potential immunoreactivity of two repetitive proteins from the flagellar cytoskeleton of African Trypanosomes, recombinantly produced, namely TbgI 2 and TbgI 17 against Tbr infections.
The data showed that TbgI 2 was better than TbgI 17 and could be considered as the first attempt to develop a reasonable diagnostic tool for East African Trypanosomiasis, which lacks a serological test.
Improvement of this study should be done since it was limited by the number of stage 1 Rhodesiense HAT sera and the low reactivity of the antigens to human sera.