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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 - December 2021

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: 

During the second quarter of 2021, Dr. Guizani and her team focused on the set up of the multiplex species specific and sensitive RPA basic assays using a selection of Leishmania DNA markers. They have faced serious procurement issues due to the inability of vendors to supply necessary reagents and test kits, but they are adapting as best they can to continue using alternate supplies. The researchers recently identified a Chinese manufacturer of isothermal amplification kits called Recombinase Amplification Assays (RAA) based on the same technology of the recombinase polymerase amplification (RPA) kits they had planned to use. As soon as they receive the equivalent of the RPA kits, they will work to assess and adjust the reaction on the retained specific simplex tests. They will then optimize the sensitivity of both RPA reactions and lateral flow detection assays and assess their specificities and sensitivities on a larger set of samples collected in the course of this PEER study. In the coming months, they will also work on optimizing the two selected multiplex RPA basic assays to improve their sensitivities. Optimizations will include reaction time and temperature, primer concentrations and ratios, and Mg2+ concentration. The best one in term of analytical limit of detection will be evaluated on a selection of Leishmania samples in order to prove the principle of their use for the diagnosis of cutaneous leishmaniasis.

The PI and her colleagues had a manuscript on “Dipeptidyl peptidase III as a DNA marker to investigate epidemiology and taxonomy of Old World Leishmania species,” accepted in July 2021 for publication by PLOS Neglected Tropical Diseases. They also have one additional manuscript recently submitted and two more in preparation.

Dr. Guizani also reports some significant potential applications of her project results. The Leishmania major specific RPA-LF test developed as part of this project will be used to screen patients in the Hospital Farhat Hached Parasitology Department at Sousse, where the majority of Leishmania infections appear to be caused by L. major. In addition to a multiplex PCR developed in their lab, an algorithm will be elaborated and evaluated by the Clinical Investigation Center of the Institut Pasteur de Tunis (IPT) involving this assay and another one (multiplex PCR coupled to lateral flow detection for Leishmania species identification). Plans also call for implementing Lesionia, a platform developed during this project as a tool for routine data and patient management. Workshops and training sessions will be organized in order to ensure the transfer of the developed tools to the PEER team’s partners in Hospital Farhat Hached as a first step, after which the platform will be disseminated to other national and international partners. The PI reports that she has applied for support from the Ministry of Higher Education and Research under the auspices of the IPT Clinical Investigation Center.


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.

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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