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PARTNERSHIPS FOR ENHANCED ENGAGEMENT IN RESEARCH (PEER)
Cycle 5 (2016 Deadline)


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


PI: Ikram Guizani (iguizani@yahoo.com), Institut Pasteur de Tunis
U.S. Partner: Steven Reed, Infectious Disease Research Institute

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: 

As of December 2017, Dr. Guizani and her team refined their study protocol in order to prioritize targets and establish a decision tree to facilitate the selection and prioritization of the primers designed. Targets were selected using past literature research, as well as public and local databases. Sequences were retrieved from databases and were complemented by a sequence survey for designing adequate and species specific primers. Then the team will proceed with kinetoplast data generation and continuation of screening genome resources to identify additional targets. The researchers are implementing a cost effective approach for first design primers using basic RPA with detection on agarose gels. Sample collection for patients with cutaneous leishmaniasis (CL) is ongoing at various hospital sites in Tunisia, Morocco, and Lebanon. During this reporting period, they collected 28 samples in Tunisia, 14 samples in Lebanon, and 55 samples in Morocco. The high number of samples in Morocco was due to a recent outbreak of CL in the province of Quarzazate/Zagora. The characterization of etiological agents is ongoing.


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