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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 - January 2023
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, aimed 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 designed and selected species-specific primers and probes for sensitive amplification of DNA in single-target reactions. Upon screening, the most relevant RPA-LF tests were 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 was carried out in the laboratory on clinical samples from selected sites in the MENA region with appropriate institutional review board approval. The newly developed test was then 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 also helped to 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.
Final Summary of Project Activities:
The main aim of this study was to address needs in cutaneous leishmaniases (CL) diagnosis in the Old World (OW) for point of care (POC) diagnostic tests that are low cost, rapid (<1h), reliable, simple, sensitive, and species-specific. The researchers worked to develop lateral flow tests for concomitant detection and identification of the most prevalent Leishmania (L.) species in the OW: L. major (L.m), L. tropica (L.t) and L. infantum (L.i; expected cross-reaction with L. donovani, L.d). They also aimed at building capacity and networks for translation and POC test implementation in health structures.
The team adopted Recombinase Polymerase Amplification (RPA) with the detection of amplicons by lateral flow chromatography (LF). First, they developed the study protocol and procedures and had them reviewed by the Ethical Review Boards of the Institut Pasteur de Tunis, Institut Pasteur du Maroc, and Rafik Hariri Hospital, Lebanon, to ensure ethical standards and good practices across the consortium. Policies, procedures, and relevant training were put in place regarding research ethics, good lab practices, and data management and confidentiality. The sample and data collection were done at the study sites in Tunisia, Lebanon, and Morocco following a harmonized data collection sheet. For efficient data collection and management, the team developed a web-based application, Lesionia (www.lesionia.pasteur.tn), which provides all registered users with real-time data entry, retrieval, updating, and analysis. Then, they developed mLesionia, a mobile version that offers in addition an "on-the-go" advantage over Leisionia. It was provided to healthcare facilities and agencies for validation during the project closing workshops.
The team collected a total of 298 well-documented cutaneous samples from consented patients referred for routine diagnosis to parasitology departments in Tunisia (213 ), or treatment in Lebanon (14), or during field visits in endemic foci in Morocco (71). The DNA was then extracted from the samples and analyzed to identify markers for the various L. species and strains, after which primers and probes for each were designed. The researchers next focused on developing Multiplex RPA assays for simultaneous detection and identification of L. species. They set multiplex basic RPA tests by screening 11 primer combinations. For the LF detection, due to the timeline, high costs of development, and unavailability of multiple line antibody-based devices, they opted for customized multiline dipsticks for DNA-DNA detection. Two out of the 11 tested combinations were kept and used in a Taguchi experimental design adopted to define best reaction factors, which are now being studied.
Although the PEER project has ended, Dr. Guizani and her team will continue working on various aspects of the research. The available version of mLesionia will be tested for its ergonomic potential and stability by selected end users and will be subject to continuous enhancement to better meet their expectations. The developed tests will be applied to larger number of cutaneous samples, and samples with ambiguous results will be tested using other molecular tests to properly assign species to the corresponding samples. A Material Transfer Agreement will be signed to collect additional L. infantum samples from Morocco for use in further evaluations of the L. infantum-specific test. The team will continue working on the set-up of the multiplex RPA/LF assays and testing the reproducibility of the selected multiplex test on well characterized Leishmania DNA. The end goal, of course, is to produce a well-proven clinical diagnostic tool that can be put into practice in the healthcare system in Tunisia and beyond. In addition to the multiplex PCR developed in Dr. Guizani’s lab, an algorithm will be elaborated and evaluated at the Clinical Investigation Center of the Institut Pasteur de Tunis. Additional workshops and training sessions will be organized to ensure the transfer of the developed tools to the team’s partners at Hospital Farhat of Sousse, Tunisia, as a first step, after which it will be extended to other national and international partners. The researchers are finalizing research articles and a policy paper on the results from their project, and they plan to submit patent applications for the primers/probe sets developed as tools for Leishmania species diagnosis.
Following the research-to-action plan developing with guidance provided by USAID’s Research Technical Assistance Center, Dr. Guizani and her colleagues have taken the initiative to gather and lead an African consortium dedicated to capacity building in research, training, and evidence-based policy to control leishmaniases. The consortium involves Algeria, Ethiopia, Mali, Morocco, Nigeria, Sudan, Tunisia, and Spain, with collaborators in Morocco, Tunisia, and the United States. Funding support for the consortium is being provided by a grant from the Science for Africa Foundation. Through this program, Dr. Guizani and her colleagues will be able to develop studies related to POC diagnostics evaluation, implementation involving control programs, cost-effectiveness analysis, and policy briefings. They will also promote the use of DNA tools to develop risk predictive and distribution maps and to support vector and reservoir control.
Beyond its research results, the project was also valuable for the scope of the opportunities it provided to its majority-female team for training, career progress, and networking. The project included hands-on training workshops on the cutting-edge DNA methods used in the project, on the Leisionia and mLeisionia platforms, and on the RPA LF tests developed. Thanks to their participation in the project, the younger researchers involved were encouraged and mentored in progressing in their careers and taking up new leadership roles as they engaged in networks and innovative projects and collaborations.
Publications:
Bel Hadj Ali, I.; Saadi-Ben Aoun, Y.; Hammami, Z.; Rhouma, O.; Chakroun, A.S.; Guizani, I. 2023. Handheld ultra-fast duplex polymerase chain reaction assays and lateral flow
detection and identification of Leishmania parasites for cutaneous Leishmaniases diagnosis. Pathogens 12: 1292. https://doi.org/10.3390/pathogens12111292
Daoui O, Bennaid H, Kbaich MA, Mhaidi I, Aderdour N, Rhinane H, Bouhout S, Akarid K, Lemrani M. 2022. Environmental, climatic, and parasite molecular factors impacting the incidence of cutaneous Leishmaniasis due to Leishmania tropica in three Moroccan foci. Microorganisms 2022, 10, 1712. https://doi.org/10.3390/microorganisms10091712
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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