A CRISPR-based diagnostic tool to survey drug resistance in human African trypanosomiasis.

The World Health Organization aims to eliminate human African trypanosomiasis caused by Trypanosoma brucei gambiense (gHAT) by 2030. With the decline of reported cases, maintaining active surveillance is essential, including for the potential emergence of drug-resistant parasites. We have developed new highly specific diagnostic tools, using the Cas13a-based Specific High-Sensitivity Reporter Enzymatic UnLOCKing (SHERLOCK) technology, for the detection of drug-resistant genotypes that (i) are already circulating, such as the AQP2/3(814) chimera providing resistance to pentamidine and melarsoprol or (ii) could emerge, such as the TbCPSF3 (N232H) mutation, associated with acoziborole resistance under laboratory conditions. The AQP2/3(814) SHERLOCK assay detected RNA from both cultured parasites and field strains isolated from gHAT patients who relapsed following melarsoprol or pentamidine treatment. The CPSF3(SNV) SHERLOCK assay discriminated between wild-type CPSF3 RNA and CPSF3 bearing a single A-C mutation that confers resistance to acoziborole in vitro. These SHERLOCK assays are amenable for use as a high-throughput screening method to monitor for drug-resistant-associated mutations in Trypanosoma brucei, providing a new molecular tool for epidemiological surveillance during the gHAT elimination phase.