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Cycle 5 (2016 Deadline)

Diagnosis of cutaneous leishmaniasis: Development and evaluation of multiplex POC DNA assays

PI: Ikram Guizani (, Institut Pasteur de Tunis
U.S. Partner: Steven Reed, Infectious Disease Research Institute
Project Dates: March 2017 - February 2020

Project Overview:

In the Old World, 1 million cutaneous leishmaniasis (CL) cases are reported each year. Some 80% of these cases occur in the Middle East and North Africa (MENA) region, caused by the four Leishmania species: L. major, L. tropica, L. infantum, and L. donovani. The MENA region is also at an increased risk for disease emergence and epidemics. Parasite detection and identification is central to treatment, patient management, epidemiology, and control. Currently, diagnosis is done by direct examination of lesion smears, a technique lacking sensitivity that does not allow for parasite identification. Laborious PCR tests allow their identification in well-equipped settings.

This project team, which includes researchers from Tunisia, Morocco, and Lebanon, aims to deliver a novel, sensitive, specific, rapid, and low-cost point-of-care (POC) CL diagnosis test to detect and identify the four Leishmania parasites in the Old World, using multiplexed isothermal Recombinase Polymerase Amplification (RPA) of DNA, coupled to lateral flow chromatography (LF) for the detection of the DNA products. They will design and select species-specific primers and probes for sensitive amplification of DNA in single-target reactions. Upon screening, the most relevant RPA-LF tests will then be combined to amplify and detect multiple targets (multiplex RPA-LF) in a single assay, thus enabling simultaneous sensitive detection and identification of the four Leishmania species. Proof-of-principle evaluation of this test will be done in the laboratory on clinical samples from selected sites in the MENA region with appropriate institutional review board approval. The newly developed test will be compared to direct examination and a valid real-time PCR screening for parasite detection and to PCR-RFLP assay of ITS1 genes for identification. This study should strengthen capacity building and empower young researchers while tackling public health research and development priorities using novel technologies and networks for technology transfer, research translation, implementation, and commercialization.

Summary of Recent Activities: 

In this reporting period, the PI and her team used public and local databases, computational screening and bibliography search to identify 63 nuclear targets. Available sequences of each target were retrieved from the databases, aligned and annotated. They selected the most interesting ones (N=25) in term of species specificity for further investigations. A well studied real time PCR (Nicolas et al. 2002) was selected and optimized in the lab and tested in a set of reference and clinical samples. This generic assay amplifies DNA from the species L.m, L.i, L.d and L.t. It is now used as molecular standard technique for L. parasites detection within CL samples. are still collecting samples and data in the different countries. In Tunisia and Lebanon the sampling was undertaken among patients attending health facilities and seeking for CL diagnosis confirmation and/or treatment. However in Morocco samples were collected during field visits in endemic foci.

A first survey was carried in Iminatnoute, an endemic L.t focus located in southwestern Morocco. Thirty patients presenting suspicious CL lesions have been sampled by dermal scraping. The scraping was suspended into a sterile tube containing preservative buffer and stored at -20°C until DNA extraction.

Future plans will be focused on the set up of the simplex RPA-LF assays. They will work on the optimization of the sensitivity of both RPA reactions and lateral flow detection assays. The best simplex RPA-LF tests will be tested on a selection of CL samples and pre-evaluated for statistical performance. Selected assays will be used to set up the multiplex RPA assays. For multiplex LF detection, the researchers will set up a training in the United States with the collaboration of IDRI and InBios to establish a multi-test line dipstick (three test lines and a control line). The developed LF will be then transferred to be used for the set up of the multiplex RPA-LF assays.

The LESIONIA platform will be made accessible online to enable all project partners to enter their data from their study sites. Once collected, the data will be analyzed and valorized. They will continue the characterization of CL samples using standard techniques, including real-time PCR and PCR-RFLP ITS1. These techniques will be used as the gold standard for the detection and the identification of Leishmania parasites within CL samples. The CL samples will be then used to evaluate the performances of the developed tool. Lastly, they will work with the collaboration of the Research Technical Assistance Center to elaborate their research to action plan and the communication product to reach potential stakeholders and impact policies and donors.

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