Deep kinetoplast genome analyses result in a novel molecular assay for detecting <i>Trypanosoma brucei gambiense</i> -specific minicircles

ABSTRACT 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. Here, we aim at improving molecular diagnosis of Tbg 1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of Trypanosoma brucei 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 Tbg 1-specific, of which three were shared by the 18 studied Tbg 1 strains. We then developed a novel multiplex quantitative PCR assay ( g -qPCR3) targeting one Tbg 1-specific minicircle and three Tbg 1-specific or Trypanozoon-specific markers. Molecular analyses revealed that the minicircle-based assay is applicable on animals and is as specific as the TgsGP -based assay, the current golden standard for molecular detection of Tbg 1. The median copy number of the targeted minicircle was equal to eight, suggesting that our minicircle-based assay may be used for the sensitive detection of Tbg 1 parasites. Finally, 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 Tbg 1 populations. These results demonstrated that our minicircle-based assay is a promising new tool for reliable and sensitive detection of Tbg 1 infections in humans and animals.