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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 Dates: March 2017 - December 2022

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 the last quarter of 2021, Dr. Guizani and her team continued optimizing the lateral flow test they developed for Leishmania, working to hone its sensitivity and specificity to various strains. In November 2021, they hosted their Lebanese collaborator Prof. Nabil Haddad for two weeks to train him on molecular techniques they developed for DNA identification of Leishmania parasites in archived slides of samples from CL patients. They used various techniques to analyze the parasitic content of the slides, including PCR amplification and sequence analyses. Prof. Haddad brought with him DNA extracts from archived slides made from samples from Syrian CL patients (N=135) prior to the Syrian civil conflict. Their analysis showed some interesting geographic patterns of Leishmania eco-epidemiology in an area where little previous research has been done, but more work is needed to follow up on these insights. The findings could ultimately help to inform the Syrian Ministry of Health, form the baseline profile of transmitted parasites in the region, and facilitate the implementation of monitoring based on molecular evidence.

Also during the last quarter, the research team visited the Parasitology Department at Farhat Hached Hospital Sousse and met with three parasitologists who are responsible for samples and data collection for the PEER project, in addition to their responsibilities for CL routine diagnosis. Dr. Guizani and her colleagues presented updates on their recent progress and discussed options for technology transfer and implementation of the new tools they are developing. Another highlight of the quarter was the team’s submission of a new manuscript on Leishmania DNA diagnostics. A citation and link will be added to this page once the paper is published. In the coming months, the researchers will continue working on assay optimizations and drafting manuscripts on their results. When the current COVID surge subsides in Tunisia, they hope organize a workshop session about their new L. major detection tool, as well as content on ethics and clinical and laboratory good practices. A no-cost extension has been issued through December 2022.

Publications:

Bel Hadj Ali I, Chouaieb H, Saadi Ben Aoun Y, Harigua-Souiai E, Souguir H, Yaacoub A, Oussaïma El Dbouni, Zoubir Harrat, Maowia M. Mukhtar, Moncef Ben Said, Nabil Haddad, Akila Fathallah-Mili, Ikram Guizani. (2021) Dipeptidyl peptidase III as a DNA marker to investigate epidemiology and taxonomy of Old World Leishmania species. PLoS Negl Trop Dis 15(7): e0009530. https://doi.org/10.1371/journal.pntd.0009530

Daoui O., M. Ait Kbaich, I. Mhaidi, S. El Kacem, L. Hjiyej Andaloussi, K. Akarid, and M. Lemrani. (2020) The role of sampling by cotton swab in the molecular diagnosis of cutaneous leishmaniasis. Transbound Emerg Dis. October 22, 2020. doi: 10.1111/tbed.13886. Online ahead of print. PMID: 33094519


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