Dataset Information

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

ABSTRACT:

Background

To achieve elimination of Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense (gHAT), the development of highly sensitive diagnostics is needed. We have developed a CRISPR based diagnostic for HAT using SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) that is readily adaptable to a field-based setting.

Methods

We adapted SHERLOCK for the detection of T. brucei species. We targeted 7SLRNA, TgSGP and SRA genes and tested SHERLOCK against RNA from blood, buffy coat, dried blood spots (DBS), and clinical samples.

Findings

The pan-Trypanozoon 7SLRNA and T. b. gambiense-specific TgSGP SHERLOCK assays had a sensitivity of 0.1 parasite/μL and a limit of detection 100 molecules/μL. T. b. rhodesiense-specific SRA had a sensitivity of 0.1 parasite/μL and a limit of detection of 10 molecules/μL. TgSGP SHERLOCK and SRA SHERLOCK detected 100% of the field isolated strains. Using clinical specimens from the WHO HAT cryobank, the 7SLRNA SHERLOCK detected trypanosomes in gHAT samples with 56.1%, 95% CI [46.25-65.53] sensitivity and 98.4%, 95% CI [91.41-99.92] specificity, and rHAT samples with 100%, 95% CI [83.18-100] sensitivity and 94.1%, 95% CI [80.91-98.95] specificity. The species-specific TgSGP and SRA SHERLOCK discriminated between the gambiense/rhodesiense HAT infections with 100% accuracy.

Interpretation

The 7SLRNA, TgSGP and SRA SHERLOCK discriminate between gHAT and rHAT infections, and could be used for epidemiological surveillance and diagnosis of HAT in the field after further technical development.

Funding

Institut Pasteur (PTR-175 SHERLOCK4HAT), French Government's Investissement d'Avenir program Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (LabEx IBEID), and Agence Nationale pour la Recherche (ANR-PRC 2021 SherPa).

SUBMITTER: Sima N

PROVIDER: S-EPMC9626900 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Sima Núria N Dujeancourt-Henry Annick A Perlaza Blanca Liliana BL Ungeheuer Marie-Noelle MN Rotureau Brice B Glover Lucy L

EBioMedicine 20221027

<h4>Background</h4>To achieve elimination of Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense (gHAT), the development of highly sensitive diagnostics is needed. We have developed a CRISPR based diagnostic for HAT using SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) that is readily adaptable to a field-based setting.<h4>Methods</h4>We adapted SHERLOCK for the detection of T. brucei species. We targeted 7SLRNA, TgSGP and SRA genes and tested SHERLOCK ag ...[more]

PMID: 36374773

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Project description:Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).

Project description:Human African trypanosomiasis (HAT) or sleeping sickness leads to a complex neuropsychiatric syndrome with characteristic sleep alterations. Current division into a first, hemolymphatic stage and second, meningoencephalitic stage is primarily based on the detection of white blood cells and/or trypanosomes in the cerebrospinal fluid. The validity of this criterion is, however, debated, and novel laboratory biomarkers are under study. Objective clinical HAT evaluation and monitoring is therefore needed. Polysomnography has effectively documented sleep-wake disturbances during HAT, but could be difficult to apply as routine technology in field work. The non-invasive, cost-effective technique of actigraphy has been widely validated as a tool for the ambulatory evaluation of sleep disturbances. In this pilot study, actigraphy was applied to the clinical assessment of HAT patients.Actigraphy was recorded in patients infected by Trypanosoma brucei gambiense, and age- and sex-matched control subjects. Simultaneous nocturnal polysomnography was also performed in the patients. Nine patients, including one child, were analyzed at admission and two of them also during specific treatment. Parameters, analyzed with user-friendly software, included sleep time evaluated from rest-activity signals, rest-activity rhythm waveform and characteristics. The findings showed sleep-wake alterations of various degrees of severity, which in some patients did not parallel white blood cell counts in the cerebrospinal fluid. Actigraphic recording also showed improvement of the analyzed parameters after treatment initiation. Nocturnal polysomnography showed alterations of sleep time closely corresponding to those derived from actigraphy.The data indicate that actigraphy can be an interesting tool for HAT evaluation, providing valuable clinical information through simple technology, well suited also for long-term follow-up. Actigraphy could therefore objectively contribute to the clinical assessment of HAT patients. This method could be incorporated into a clinical scoring system adapted to HAT to be used in the evaluation of novel treatments and laboratory biomarkers.

Dataset Information

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

Background

Methods

Findings

Interpretation

Funding

Publications

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

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