Pan and species-specific SHERLOCK assays for human and animal African trypanosome infections.

(A) Schematic of the target regions used in the SHERLOCK4AAT toolbox. T. congolense, T. theileri (multiplex), T. simiae, Pan- Trypanozoon and Pan-trypanosomatid SHERLOCK assays were designed using the 18S rRNA gene as a target; T. vivax SHERLOCK assay using the IFX gene and T. suis test using the gGAPDH gene. Gene specific accession number in brackets below the assay name. Details of the regions amplified are provided for each SHERLOCK assay (amplicon). RPA primers are represented by black arrows, the extension in the forward primer represents the T7 polymerase promoter sequence. The regions targeted by the crRNAs (CRISPR guides) are also indicated in the diagram. The schematic representation is to scale. (B) Evaluation of the specificity of single pan- trypanosomatid and pan-Trypanozoon SHERLOCK tests RNA from T. b. brucei (Tbb), T. b. gambiense (Tbg), T. b. rhodesiense (Tbr), T. evansi A (TevA) and B (TevB), T. equiperdum (Teq) T. congolense (Tcg), T. vivax (Tvx), T. theileri (Tth), Leishmania major (Lm), Plasmodium falciparum (Pfl) and HEK cells. The boxplot means are represented by single lines, and error bars correspond to minimum and maximum values. N=3 for the RPA amplifications and three Cas-13a detections for each RPA amplification, totalling 9 replicates for each assay. T. theileri was used in fewer replicates as an input due to the limited genetic material available. (C) Evaluation of the specificity of T. congolense, T. vivax, T. theileri, T. simiae and T. suis-specific SHERLOCK tests on RNA from Tbb, Tbg, Tcg, Tvx, Tth. Synthetic controls for T. simiae (Tsm) and T. suis (Tsu) were used to evaluate the species-specificity of the assays. N=1 RPA amplification and three Cas-13a detections totalling 3 replicates for each assay. Error bars represent standard deviations from the mean of the replicates. All inputs were used at a concentration of 5 ng/µL.

Evaluation of the sensitivity of SHERLOCK assays targeting trypanosome species causing AAT.

(A) Limit of detection (LoD) assays for the single pan- trypanosomatid, pan-Trypanozoon, T. vivax, T. congolense and T. theileri assays were performed using RNAs from bloodstream parasites diluted to obtain equivalent concentrations of parasites per mL, ranging from 107 to 0.1 parasites/mL. (B) LoD of T. simiae and T. suis assays were performed on consecutive dilutions of the synthetic controls, ranging from 1010 to 101 fM. N=24 for pan-trypanosomatid assay, N=12 for pan-Trypanozoon and T. theileri assays, N=6 for T. simiae assay and N=3 for T. congolense, T. vivax and T. suis assays. Error bars represent standard deviations from the mean of the replicates; open circle represents each replicate.

Comparison of the single and multiplex 18S pan-trypanosomatid SHERLOCK assays efficiency.

(A) Schematic of the single (one crRNA) and multiplex (two crRNAs) pan-trypanosomatid SHERLOCK assays designed using the 18S rRNA gene as a target. Gene specific accession number in brackets below the assay name. Details of the amplified gene regions are provided (amplicon). RPA primers are represented by black arrows; the forward primer extension represents the T7 polymerase promoter sequence. The regions targeted by the crRNAs are indicated in the diagram. (B) Posterior sensitivity estimates of the single (green) and multiplex (purple) pan-trypanosomatid SHERLOCK assays on the 224 pig blood samples from Côte d’Ivoire presented in Figure 4. Estimates were obtained with the Monte Carlo method (Gibbs sampling) proceeding in an iterative chain. (C) Results of pan- Trypanozoon SHERLOCK assay plotted against results of the single (left panel) and multiplex (right panel) 18S pan-trypanosomatid SHERLOCK assays on pig samples from Côte d’Ivoire. The coloured dashed lines represent the detection thresholds set for each of the tests: single pan-trypanosomatid (green), multiplex pan-trypanosomatid (purple), and pan-Trypanozoon (orange). Numbers and proportions (%) of double positive samples are indicated at the top right of each graph and only positive for pan- trypanosomatid in the bottom right part of each graph.

Diversity of blood trypanosomatids among free-range pigs from Côte d’Ivoire assessed by the SHERLOCK4AAT toolbox.

(A) Map of the Côte d’Ivoire, the region where the samples were collected is highlighted in purple. (B) Results of the SHERLOCK4AAT screening of 224 pig samples collected in Côte d’Ivoire. The coloured dashed lines represent the detection thresholds set for each assay. Each colour represents a specific test: multiplex pan-trypanosomatid (green), pan- Trypanozoon (orange), T. b. gambiense (yellow), T. congolense (light green), T. vivax (purple), T. theileri (red), T. simiae (blue) and T. suis (black). (C) Table summarizing the number of positive samples as well as the percentage (%) positive infections for each test, calculated on the total number of samples analysed. The total number of samples analysed were 219 for the multiplex pan-trypanosomatid assay, 173 for the T. vivax-specific assay, and 224 for all other SHERLOCK assays. (D) Venn diagram representing the crossover between the positive samples analysed with the three tests (multiplex pan-trypanosomatid (green), pan-Trypanozoon (orange) and T. congolense (purple)) giving the highest prevalence. The percentage of the total is shown in brackets below the number of samples constituting each fraction.

Diversity of blood trypanosomatids among farm pigs from Guinea assessed by the SHERLOCK4AAT toolbox.

(A) Map of Guinea showing the N’zérékoré prefecture where samples were collected in pink. (B) Results of the SHERLOCK4AAT screening of 200 pig samples collected in Guinea. The coloured dashed lines represent the detection thresholds set for each of the tests. Each colour represents a specific test: multiplex pan-trypanosomatid (green), pan-Trypanozoon (orange), T. b. gambiense (yellow), T. congolense (light green), T. vivax (purple), T. theileri (red), T. simiae (blue) and T. suis (black). (C) Table summarizing the number of positive samples as well as the percentage (%) positive infections for each test, calculated on the total number of samples analysed (n=200). (D) Venn diagram representing the crossover between the positive samples analysed with the three tests (multiplex pan- trypanosomatid (green), pan-Trypanozoon (orange) and T. congolense (purple)) giving the highest prevalence. The percentage of the total is shown in brackets below the number of samples constituting each fraction.

A: Representation of the proportions of identity between 18S rRNA (A) and GAPDH (B) genes from Microsporidia, Ciliophora, Haemosporida, Diplomonadida, Symbiontida, Diplonemea, Euglenida and Kinetoplastea, focusing on several species within last family.

(A) Alignments for 18S Pan-trypanosomatid SHERLOCK target region. The sequences used for the alignments are as follows: Microsporidia (Nosema adalia, KC412706.1), Ciliophora (Vorticella sp., DQ487201.2), Haemosporida (Plasmodium falciparum, XR_002966679.1), Diplomonadida (Giargia intestinalis, XR_005248679.1), Symbiontida (Calkinsia aureus, EU753419.1), Diplonemea (Diplonema ambulator, AY425009.1), Euglenida (Euglena sanguinea, JQ281806.1) and Kinetoplastea (Trypanosoma (T) brucei brucei, XR_002989632.1). (B) Alignments for 18S Pan-Trypanozoon SHERLOCK target region. The sequences used for the alignments are as follows: Human (H, pdb|5VYC|i), Plasmodium falciparum (Pfl, XR_002966679.1), Leishmania major (Lm, GQ332361.1), T. cruzi (Tcz, AJ009147.1), T. vivax (Tvx, EU477537.1), T. congolense (Tcg, AJ009146.1), T. theileri (Tth, AJ009163.1), T. simiae (Tsm, AJ404608.1), T. suis (Tsu, MK962702.1) and T. brucei brucei (Tbb, XR_002989632.1). Non-conserved bases between sequences are highlighted in bold.

Summary of BLAST analysis obtained against the 18S pan- trypanosomatid SHERLOCK amplicon and evaluation of the homology of the target region of each crRNA.

The percentage identity of the target amplicon of pan- trypanosomatid RPA was evaluated using different degrees of exclusion, to assess its percentage of identity within the different groups and especially in the Phylum Euglenozoa, excluding the target group Kinetoplastea. The number of sequences evaluated is shown, as well as the percentage of sequences where the target regions of the pan-trypanosomatid guides (crRNAs) are conserved.

Screening of different RPA pairs of primers and/or crRNAs guides for each SHERLOCK assay optimization.

In each evaluation a positive control (RNA from a target species, coloured bar) and a negative control (RNA from a non- target species, grey bar) were used. RPA pairs of primers are named A, B or C, while crRNAs were name from 1 to 10. The time point used is indicated. # indicates the combination of RPA primers and crRNA selected for the rest of the analysis. All inputs were used at a concentration of 5 ng/µL.

Alignments of the 18S T. congolense SHERLOCK target region.

The sequences used for the alignments are from T. simiae, T. godfreyi, T. sapaensis, T. vivax, T. theileri and T. thomasbancrofti. Non-conserved bases between sequences are highlighted in grey. The percentage identity of each sequence was analysed against the RPA amplicon in the T. congolense-specific SHERLOCK assay.

Comparison of the limit of detection of the single and multiplex 18S pan-trypanosomatid SHERLOCK assays.

Single and multiplex 18S pan- trypanosomatid SHERLOCK assays on dilutions of T. b. brucei RNA and on RNA extracted from HEK human cells as negative control. N=9 for each dilution. Error bars represent standard deviations from the mean of the replicates.

Background cross-reactivity of the 18S pan-trypanosomatid (A) and 18S pan-Trypanozoon (B) SHERLOCK assays using genetic material extracted from pig blood as input.

Means are represented by single lines. Quantities of porcine and T. b. brucei genetic material extracts included in each trial are as follows: “-“ none, “+” 1 ng/µL, “++” 2 ng/µL and “+++” 3 ng/µL.

Threshold determination for all new SHERLOCK4AAT assays.

(A) Compilation of positive and negative results obtained using multiplex pan- trypanosomatid and pan-Trypanozoon SHERLOCK tests and ROC curves obtained for each analysis. The red dotted line marks the selected threshold for each test. (B) Compilation of positive and negative results obtained using the species-specific SHERLOCK assays and ROC curve obtained for each analysis. The red dotted line marks the selected threshold for each test. (C) Summary of the analytical features of each SHERLOCK assay. Analytical sensitivity (95 % of CI), specificity (95 % of CI) and fold change cut-off.

Posterior sensitivity estimates of three SHERLOCK assays (multiplex pan-trypanosomatid, pan-Trypanozoon and T. congolense-specific SHERLOCK assays, red curve) to the three parasitological tests (buffy coat examination, mAECT and /or isolation in mice, orange curve) performed in parallel on the 224 pig blood samples from Côte d’Ivoire.

Estimates were obtained with the Monte Carlo method (Gibbs sampling) proceeding in an iterative chain.