key: cord-0296603-ybrczwsi authors: Sima, N.; Dujeancourt-Henry, A.; Perlaza, B. L.; Ungeheuer, M.-N.; Rotureau, B.; Glover, L. title: SHERLOCK4HAT: a CRISPR-based tool kit for diagnosis of Human African Trypanosomiasis date: 2022-03-12 journal: nan DOI: 10.1101/2022.03.09.22271543 sha: be87593f8ec41084b48baaa67f7d830fc3aa2d4d doc_id: 296603 cord_uid: ybrczwsi 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. within the Trypanozoon taxa, with 99% identity shared between the T. brucei sp., but more distant 114 to the co-endemic species T. congolense and T. vivax with 86.31% and 79.77% identity 115 respectively (data file S1). TgSGP gene is specific to Group Table 1 ). Exquisite specificities were shown for 7SLRNA as a pan-Trypanozoon diagnostic target, 149 and TgSGP and SRA as species-specific diagnostic targets for T. b. gambiense and T. b. S4 ). This 164 analytical sensitivity is similar to that reported previously for other molecular diagnostics that are 165 subgenus specific (29-35) and 10 to 100-fold more sensitive to those reported for subspecies- 166 specific tests (21, 36, 37) . 167 168 SHERLOCK is amenable to readout by lateral flow assay (LFA) (12). Importantly, using a 169 polyethylene glycol (PEG)-based CRISPR-optimized buffer (provided by Milenia Biotec), we 170 were able to detect the 7SLRNA, TgSGP and SRA SHERLOCK targets with the same respective 171 sensitivities as with the fluorescent readout, but with a reduced background signal as compared to CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 12, 2022. ; https://doi.org/10.1101/2022.03.09.22271543 doi: medRxiv preprint evaluated the performance of one-tube reactions at temperatures from 37°C to 42°C, and found 178 that reactions at 37°C had higher signal with reduced sample-to-result time ( fig. S6A ). An 179 additional consideration for the development of a PoC diagnosis for use in low-income countries 180 is affordability. We therefore tested three RT enzymes from different manufacturers and selected 181 ProtoScript II (NEB) as the most cost-effective reaction with a cost of 2.5 € / reaction ( fig. S6B ). 182 Given that Cas13a has uridine-cleavage preference (12, 39), we compared RNase Alert with a 183 6U-FAM reporter. The signal intensity obtained with the 6U-FAM reporter was lower and was 184 more prone to spontaneous degradation, as seen with the non-template control reaction ( fig. S6C ). 185 Thus, we selected RNase Alert as a reporter for an optimized one-tube SHERLOCK diagnostic 186 and used 8 μL of input material ( fig. S6D ). With these improvements, the 7SLRNA one-tube 187 SHERLOCK reaction had similar sensitivity than the two-step reaction and detected 1 parasite/μL 188 in less than 1 h (Fig. 1, F and G) . rhodesiense strains tested negative for SRA ( Fig. 2 and There is a critical need for highly sensitive and specific molecular detection tools that can be use 219 in a high-throughput format in the context of gHAT post-elimination phase. Adapted strategies 220 will be adopted to monitor for potential residual transmission and these tests will be performed in 221 regional reference centres. Methods to capture individual samples, such as dried blood spots 222 (DBSs) would allow easy collection in the field and safe transport and storage back to a lab. 223 SHERLOCK can detect both DNA and RNA, therefore, working with total nucleic acid (TNA) 224 instead of RNA alone can increase the sensitivity. However, for use as a test of cure it is 225 important to work with RNA only, since trypanosome DNA has been detected in the host up to 2 226 years after cure (35). To optimize the 7SLRNA SHERLOCK for epidemiological surveys, we 227 compared three methods of TNA extraction from DBS using non-infected sheep blood spiked 228 with cultured T. brucei parasites spotted on Whatman 903 TM Cards. Our 7SLRNA SHERLOCK 229 was able to detect 100 parasites/μL using a RNeasy kit (Qiagen) and 10 parasites/μL with the 260 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 12, 2022. ; https://doi.org/10.1101/2022.03.09.22271543 doi: medRxiv preprint endemic regions, but negative for rHAT, as further negative controls. Additionally, we analyzed 262 14 buffy coat samples from un-infected donors from non-endemic regions. As a positive control 263 for TNA extraction to validate negative SHERLOCK results in clinical samples, i.e. to ensure that 264 no SHERLOCK inhibitors were remaining in the sample, we designed an additional SHERLOCK 265 assay that targeted the human RNase P gene and validated its performance using RNA from 266 cultured human cells and parasites ( fig. S9 ). All samples were maintained at -80°C from 267 collection until delivery by the WHO HAT specimen biobank, and all the samples tested here 268 were more than 10 years old and stored without any preservative (41) . Therefore, the likelihood of 269 TNA deterioration was high, especially for RNA. As an additional control, we ran a Tb177 bp CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 12, 2022. ; https://doi.org/10.1101/2022.03.09.22271543 doi: medRxiv preprint can distinguish between the three T. brucei subspecies using a pan-Trypanozoon, gambiense-303 specific, or rhodesiense-specific targets. Although our subspecies-specific targets use TgSGP and 304 SRA, which are related to VSG genes, we do not see cross reactivity. In fact, in spite of the degree 305 of DNA sequence homology shared between SRA and VSG variants, we saw no false positives, 306 confirming that the selected target meets the specificity requirements for rHAT diagnosis. 307 As an RNA based diagnostic, SHERLOCK4HAT is a highly sensitive detection method for on-308 going infections with a simple set up. We show that the analytical sensitivity of 309 SHERLOCK4HAT for the Trypanozoon target is 0.1 parasite/µL (100 parasites/mL), which is to a commercial LFA), or 2 h 30 min for a two-step reaction (at 2.8 €), and these costs would be 337 notably reduced with an in-house manufactured strip for LFA. 338 One limitation for SHERLOCK4HAT, as for any molecular diagnostic method, is the NA 339 extraction step. Several extraction methods coupled to a CRISPR-based diagnostic reaction have 340 been published (8, 47, 48) , but remained to be tested in the context of HAT diagnostics. For high-341 throughput surveillance using SHERLOCK4HAT, automated NA extraction systems can be 342 implemented with higher reproducibility, reduced hand-on time and no cross-contamination. 343 Manual extraction methods, although more time consuming, showed an increased analytical 344 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The bio-banked clinical samples used here to validate SHERLOCK4HAT did not allow proper 352 analysis of sensitivity for gHAT patients. 44% of confirmed gHAT patient samples were negative 353 with SHERLOCK, and 53% were negative using standard qPCR analysis. The discrepancy 354 between our results compared to the original in-field diagnostic is most probably due to the 355 deterioration of the NA in these samples, that were stored at -80°C for more than 10 years without 356 preservative (41) . Low parasitemia is typical in gHAT infections, thus any NA degradation could 384 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) To produce the crRNA's, DNA IVT templates and T7-3G oligonucleotide were purchased from 408 ThermoFisher. crRNAs were synthesized as described in (13) 613 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 12, 2022. ; https://doi.org/10.1101/2022.03.09.22271543 doi: medRxiv preprint Human African trypanosomiasis. 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