SHERLOCK4HAT: a CRISPR-based tool kit for diagnosis of Human African Trypanosomiasis

Abstract

Elimination of Human African Trypanosomiasis (HAT) requires highly specific and sensitive tools for both diagnostic at point of care and epidemiological surveys. We have adapted SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the detection of trypanosome nucleic acids. Our SHERLOCK4HAT diagnostic tool kit, using 7SLRNA, TgSGP and SRA targets, distinguishes between Trypanosoma brucei (T. b.) brucei, T. b. gambiense (g) and T. b. rhodesiense (r) without cross-reactivity and with sensitivity between 0.01 and 0.1 parasite/uL. SHERLOCK4HAT can accurately detect a trypanosome infection in cryo-banked patient buffy coats, with 85.1% sensitivity and 98.4% specificity for gHAT, and 100% sensitivity and 94.1% specificity for rHAT. Our SHERLOCK4HAT diagnostic showed 85.6% correlation with a reference standard qPCR in gHAT patients, 96.2% correlation in rHAT patients, discriminates between r/gHAT with 100% accuracy and is compatible with lateral flow assay readout for use at the point of care.

Performance of clinical signs and symptoms, rapid and laboratory diagnostic tests for diagnosis of human African trypanosomiasis by passive screening in Guinea: a non-interventional, prospective cross-sectional study

“…For example, inhibition ELISA could replace trypanolysis [36], increasing the feasibility of implementation of DBS testing incountry and reducing DBS storage time. New nucleic acid detection tests including SHERLOCK [42] or (RT)-qPCRs [39] should be evaluated prospectively, including optimized specimen collection methods.…”

Trypanosome spliced leader RNA for diagnosis of acoziborole treatment outcome in gambiense human African trypanosomiasis: A longitudinal follow-up study

“…Other topics for future research include the reason of persistence of SL-RNA in some patients up to 11 days after acoziborole treatment, confirmation of the sensitivity of SL-RNA in blood for diagnosis of relapses, and importantly, evaluation of more sensitive targets for RNA detection allowing earlier detection of relapse in blood, including but not limited to 7SL-derived small RNA or SHERLOCK. 35 , 36 …”

Isothermal Nucleic Acid Amplification to Detect Infection Caused by Parasites of the Trypanosomatidae Family: A Literature Review and Opinion on the Laboratory to Field Applicability

“…These methodologies present many advantages since they increase sensitivity by the fluorescent read-out and specificity conferred by the guide crDNA or crRNA. For example, CRISPR-Cas 13 (SHERLOCK) [ 109 ] or CRISPR-Cas12 (HOLMES: a one-Hour Low-cost Multipurpose highly Efficient System) [ 110 ] were tested with success on CL and HAT clinical samples [ 111 , 112 ]. These methodologies detect Nucleic acid but require an initial step for amplifying the genetic material (RNA or DNA) by isothermal or classical PCR methodologies.…”

“…The CRISPR-Cas-based nucleic acid detection methodologies can also improve sensitivity and specificity but with limitations for their field deployment (see Chapter 4.4). It has been tested with RPA to detect Trypanosomes responsible for HAT [ 112 ].…”