Deep kinetoplast genome analyses result in a novel molecular assay for detecting trypanosoma brucei gambiense-specific minicircles

The World Health Organization targeted Trypanosoma brucei gambiense (Tbg) human African trypanosomiasis for elimination of transmission by 2030. Sensitive molecular markers that specifically detect Tbg type 1 (Tbg1) parasites will be important tools to assist in reaching this goal. We aim at improving molecular diagnosis of Tbg1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of these parasites. Using Next-Generation Sequencing of total cellular DNA extracts, we assembled and annotated the kinetoplast genome and investigated minicircle sequence diversity in 38 animal- and human-infective trypanosome strains. Computational analyses recognized a total of 241 Minicircle Sequence Classes as Tbg1-specific, of which three were shared by the 18 studied Tbg1 strains. We developed a minicircle-based assay that is applicable on animals and as specific as the TgsGP-based assay, the current golden standard for molecular detection of Tbg1. The median copy number of the targeted minicircle was equal to eight, suggesting our minicircle-based assay may be used for the sensitive detection of Tbg1 parasites. Annotation of the targeted minicircle sequence indicated that it encodes genes essential for the survival of the parasite and will thus likely be preserved in natural Tbg1 populations, the latter ensuring the reliability of our novel diagnostic assay

Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense

Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT

Box blots of the percent positivity (PP) obtained in ELISA with respectively native LiTat 1.3 (2 µg/ml), rLiTat 1.3_{H-SUMO-24-372-Strep} (4 µg/ml), native LiTat 1.5 (2 µg/ml), rLiTat 1.5_{H-SUMO-33-426-Strep} (4 µg/ml) and mixtures of both native (1+1 µg/ml) and recombinant (2+2 µg/ml) antigens tested with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.

<p>Box blots of the percent positivity (PP) obtained in ELISA with respectively native LiTat 1.3 (2 µg/ml), rLiTat 1.3_{H-SUMO-24-372-Strep} (4 µg/ml), native LiTat 1.5 (2 µg/ml), rLiTat 1.5_{H-SUMO-33-426-Strep} (4 µg/ml) and mixtures of both native (1+1 µg/ml) and recombinant (2+2 µg/ml) antigens tested with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.</p

Median percent positivity (PP) with 25^th and 75^th percentile obtained in ELISA with the different antigens and sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.

<p>Median percent positivity (PP) with 25^th and 75^th percentile obtained in ELISA with the different antigens and sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.</p

Percent positivity cut-off (max. Youden index) and sensitivity and specificity with 95% confidence intervals of the different antigens tested in ELISA with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.

<p><b>*</b> significant difference between recombinant and native.</p

Expression of rLiTat 1. 3_{H-SUMO-24-372-Strep} by <i>Pichia pastoris</i>.

<p>A. Western blot with anti-His tag antibody; B: Western blot with anti-Strep tag antibody; C: Coomassie stained 12% SDS-PAGE gel; Protein markers (M): ColorPlus Prestained Protein Marker (NEB; A and B) and LMW-SDS protein marker (GE Healthcare; C); lane 1: supernatant of transfected <i>Pichia pastoris</i> M5 after 25 h induction; lane 2: supernatant of transfected <i>Pichia pastoris</i> M5 after 44 h induction; lane 3: Ni-NTA purified recombinant LiTat 1.3; lane 4: 20× concentrated flow-through of Ni-NTA purified supernatant.</p

Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained with native LiTat 1.3 (2 µg/ml), rLiTat 1.3_{H-SUMO-24-372-Strep} (4 µg/ml), native LiTat 1.5 (2 µg/ml), rLiTat 1.5_{H-SUMO-33-426-Strep} (4 µg/ml) and mixtures of both native (1+1 µg/ml) and recombinant (2+2 µg/ml) antigens tested with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.

<p>Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained with native LiTat 1.3 (2 µg/ml), rLiTat 1.3_{H-SUMO-24-372-Strep} (4 µg/ml), native LiTat 1.5 (2 µg/ml), rLiTat 1.5_{H-SUMO-33-426-Strep} (4 µg/ml) and mixtures of both native (1+1 µg/ml) and recombinant (2+2 µg/ml) antigens tested with sera from 88 <i>T.b. gambiense</i> HAT patients and 74 non-HAT controls.</p

Expression of rLiTat 1. 5_{H-SUMO-33-426-Strep} by <i>Pichia pastoris</i>.

<p>A. Western blot with anti-His tag antibody; B: Western blot with anti-Strep tag antibody; C: Coomassie stained 12% SDS-PAGE gel; Protein markers (M): ColorPlus Prestained Protein Marker (NEB; A and B) and LMW-SDS protein marker (GE Healthcare; C); lane 1: supernatant of transfected <i>Pichia pastoris</i> M5 after 25 h induction; lane 2: supernatant of transfected <i>Pichia pastoris</i> M5 after 44 h induction; lane 3: Ni-NTA purified recombinant LiTat 1.5; lane 4: 20× concentrated flow-through of Ni-NTA purified supernatant.</p