A SHERLOCK toolbox for the eco-epidemiological surveillance of animal African trypanosomosis reveals a similar parasite diversity in domestic pigs in two ancient sleeping sickness foci in Western Africa

Reviewer #1 (Public review):

Summary:

This study addresses a critical gap in veterinary diagnostics by developing a CRISPR-based diagnostic toolbox (SHERLOCK4AAT) for detecting animal African trypanosomosis. It describes the development and field deployment of SHERLOCK4AAT, a CRISPR-Cas13-based diagnostic toolbox for the eco-epidemiological surveillance of animal African trypanosomosis (AAT) in West Africa.

The authors successfully created and validated species-specific assays for multiple trypanosomes, including T. congolense, T. vivax, T. theileri, T. simiae, and T. suis, alongside pan-trypanosomatid and pan-Trypanozoon assays. The field validation in pigs from Guinea and Côte d'Ivoire revealed high trypanosome prevalence (62.7%), frequent co-infections, and importantly identified T. b. gambiense in one animal at each site, suggesting pigs may serve as potential reservoirs for this human-infective parasite.

A major strength of the study lies in its methodological innovation. By adapting SHERLOCK to target both conserved and species-discriminating sequences, the authors achieved high sensitivity and specificity in detecting Trypanosoma species. Their use of dried blood spots, validated thresholds through ROC analyses, and statistical robustness (e.g., Bayesian latent class modeling) provides a strong foundation for their conclusions.

The results are significant: over 60% of pigs tested positive for at least one trypanosome species, with co-infections observed frequently and T. b. gambiense detected in pigs at both sites. These findings have direct implications for the role of animal reservoirs in human disease transmission and underscore the value of pigs as sentinel hosts in gHAT elimination efforts.

The limitations are well acknowledged, particularly the suboptimal sensitivity of the T. vivax assay and the reliance on synthetic controls for T. suis and T. simiae. However, these limitations do not undermine the overall conclusions, and the paper provides a clear roadmap for further assay refinement and implementation.

This study offers a timely, impactful, and well-substantiated contribution to the field. The SHERLOCK4AAT toolbox holds promise for improving AAT diagnostics in resource-limited settings and advancing One Health surveillance frameworks.

Strengths:

(1) The adaptation of SHERLOCK technology for AAT represents a significant technical advancement, offering higher sensitivity than traditional parasitological methods and the ability to detect multiple species simultaneously.

(2) Rigorously performed with validation using appropriate controls, ROC curve analyses, and Bayesian latent class modelling, establishing clear analytical sensitivity and specificity for most assays.

(3) Testing 424 pig samples across two countries provides robust evidence of the tool's utility and reveals important epidemiological insights about trypanosome diversity and prevalence.

(4) The identification of T. b. gambiense in pigs at both sites has significant implications for HAT elimination strategies and highlights the need for integrated One Health approaches.

(5) The use of dried blood spots and RNA detection for active infections makes the approach practical for field surveillance in resource-limited settings.

Weaknesses:

(1) The manuscript would benefit from more detailed discussion of practical considerations such as cost, equipment requirements, and training needs for implementing SHERLOCK in endemic areas and rural settings which would improve applicability.

(2) Limited discussion of pig selection criteria: More justification for choosing pigs as sentinel animals and discussion of potential limitations of this approach would strengthen the manuscript.

(3) More details on why certain genes were targeted would strengthen the methods.

(4) Table formatting could be improved for readability.

(5) Some figures are complex and would benefit from additional explanations in the legends.

Reviewer #2 (Public review):

Summary:

The manuscript is important due to the significance of the findings. The strength of evidence is convincing.

Strengths:

(1) Using a Novel SHERLOCK4AAT toolkit for diagnosis.

(2) Identification of various sub-species of Trypanosomes.

(3) Differentiating the animal subspecies from the human one.

Weaknesses:

(1) The title is too long, and the use of definite articles should be reduced in the title.

(2) The route of blood sample collection in the animals should be well defined and explained.

Reviewer #3 (Public review):

Summary:

The study adapts CRISPR-based detection toolkit (SHERLOCK assay) using conserved and species-specific targets for the detection of some members of the Trypanosomatidae family of veterinary importance and species-specific assays to differentiate between the six most common animal trypanosome species responsible for AAT (SHERLOCK4AAT). The assays were able to discriminate between Trypanozoon (T. b. brucei, T. evansi, and T. equiperdum), T. congolense (Savanah, Forest Kilifi, and Dzanga sangha), T. vivax, T. theileri, T. simiae, and T. suis. The design of both broad and species-specific assays was based primarily on sequences of the 18S rRNA, GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), and invariant flagellum antigen (IFX) genes for species identification. Most importantly, the authors showed varying limits of detection for the different SHERLOCK assays, which is somewhat comparable to PCR-derived molecular techniques currently used for detecting animal trypanosomes, even though some of these methodologies have used other primers that target genes such as ITS1 and 7SL sRNA.

The data presented in the study are particularly useful and of significant interest for the diagnosis of AAT in affected areas.

Strengths:

The assays convincingly allow for the analysis and detection of most trypanosomes in AAT.

Weaknesses:

Inability for the assay to distinguish T. b. brucei, T. evansi, and T. equiperdum using the 18S rRNA gene, as well as the IFX gene, not achieving the sensitivity requirements for detection of T. vivax. Both T. brucei brucei and T. vivax are the most predominant infective species in animals (in addition to T. congolense), therefore, a reliable assay should be able to convincingly detect these to allow for proper use of the diagnostic assay.