Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patients
IntroductionCutaneous leishmaniases (CL), a wide range of cutaneous diseases caused by diverse species of Leishmania genus parasites, are among the most neglected infectious diseases. While they are non-fatal, CL are highly morbid with disfiguring lesions, which could be chronic, leaving lifelong un...
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2025-01-01
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| author | Insaf Bel Hadj Ali Yusr Saadi-Ben Aoun Imen Khammeri Hejer Souguir Emna Harigua-Souiai Hamed Chouaieb Ahmed S. Chakroun Meryem Lemrani Aicha Kallel Kalthoum Kallel Nabil Haddad Oussaima El Dbouni Rhea N. Coler Rhea N. Coler Rhea N. Coler Steven G. Reed Akila Fathallah-Mili Akila Fathallah-Mili Ikram Guizani |
| author_facet | Insaf Bel Hadj Ali Yusr Saadi-Ben Aoun Imen Khammeri Hejer Souguir Emna Harigua-Souiai Hamed Chouaieb Ahmed S. Chakroun Meryem Lemrani Aicha Kallel Kalthoum Kallel Nabil Haddad Oussaima El Dbouni Rhea N. Coler Rhea N. Coler Rhea N. Coler Steven G. Reed Akila Fathallah-Mili Akila Fathallah-Mili Ikram Guizani |
| author_sort | Insaf Bel Hadj Ali |
| collection | DOAJ |
| description | IntroductionCutaneous leishmaniases (CL), a wide range of cutaneous diseases caused by diverse species of Leishmania genus parasites, are among the most neglected infectious diseases. While they are non-fatal, CL are highly morbid with disfiguring lesions, which could be chronic, leaving lifelong unsightly scars; they are combined with psychological distress and social stigma. The efficiency of treatment highly depends on the infecting Leishmania species. Diagnosis is mainly based on microscopic direct examination (DE) of Giemsa-stained smears needing experienced microscopists. It can be laborious and time-consuming when the parasite load is low. DE is poorly sensitive and does not identify Leishmania species. So far, only DNA assays accurately identify the species. Despite their wide use for generic detection, PCR methods also require equipment and additional steps to identify causal Leishmania species. L. major is hyperendemic in many countries in Africa, the Middle East, and Asia, where other species co-occur with different endemicity levels according to the situations. This complicates disease management and treatment, particularly as distribution and epidemiology of leishmaniases remain poorly understood. Here, we aimed for a simple and rapid molecular diagnostic test to detect and identify L. major, a predominant CL causal species, which could be prone to become a control tool at the point of care, in endemic areas, using isothermal recombinase DNA amplification (recombinase polymerase amplification, RPA, or recombinase aided amplification, RAA) coupled to detection by the lateral flow (LF) chromatography on a PCRD cassette.MethodsTo develop an L. major species-specific RPA-LF assay, computational analysis of 70 Leishmania DNA targets, identified through bibliography and database searches, selected five targets. We designed and tested 7 primer pairs/probe sets to specifically amplify L. major DNAs. First, the primers were tested for species specificity and sensitivity using basic RPA chemistry. Then, to develop RPA-coupled LF detection, we shifted to the nfo chemistry.ResultsThis way, we retained one set for further investigation, which confirmed it is L. major species-specific. Tested on 86 human cutaneous samples, this selected set was able to detect 100% of L. major infections in confirmed CL patients. We did not observe any cross-reactivity with lesions due to L. infantum or L. tropica. |
| format | Article |
| id | doaj-art-8f1f991b922145a3ad2461c603a1c45a |
| institution | Kabale University |
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| language | English |
| publishDate | 2025-01-01 |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-8f1f991b922145a3ad2461c603a1c45a2025-01-27T06:40:08ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-01-011510.3389/fmicb.2024.15146841514684Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patientsInsaf Bel Hadj Ali0Yusr Saadi-Ben Aoun1Imen Khammeri2Hejer Souguir3Emna Harigua-Souiai4Hamed Chouaieb5Ahmed S. Chakroun6Meryem Lemrani7Aicha Kallel8Kalthoum Kallel9Nabil Haddad10Oussaima El Dbouni11Rhea N. Coler12Rhea N. Coler13Rhea N. Coler14Steven G. Reed15Akila Fathallah-Mili16Akila Fathallah-Mili17Ikram Guizani18Laboratory of Molecular Epidemiology and Experimental Pathology-LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, TunisiaLaboratory of Molecular Epidemiology and Experimental Pathology-LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, TunisiaParasitology Department, UH Farhat Hached, Faculty of Medicine of Sousse, University of Sousse, Sousse, TunisiaLaboratory of Molecular Epidemiology and Experimental Pathology-LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, TunisiaLaboratory of Molecular Epidemiology and Experimental Pathology-LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, TunisiaParasitology Department, UH Farhat Hached, Faculty of Medicine of Sousse, University of Sousse, Sousse, TunisiaLaboratory of Molecular Epidemiology and Experimental Pathology-LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, TunisiaInstitut Pasteur du Maroc, Casablanca, MoroccoParasitology Department, UH La Rabta, Faculty of Medicine of Tunis, Tunis, TunisiaParasitology Department, UH La Rabta, Faculty of Medicine of Tunis, Tunis, TunisiaFaculty of Public Health, Lebanese University, Beirut, LebanonRafic Hariri Hospital, Beirut, LebanonCenter for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA, United StatesDepartment of Pediatrics, University of Washington School of Medicine, Seattle, WA, United StatesDepartment of Global Health, University of Washington, Seattle, WA, United States0HDT Bio, Seattle, WA, United StatesLaboratory of Molecular Epidemiology and Experimental Pathology-LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, TunisiaParasitology Department, UH Farhat Hached, Faculty of Medicine of Sousse, University of Sousse, Sousse, TunisiaLaboratory of Molecular Epidemiology and Experimental Pathology-LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, TunisiaIntroductionCutaneous leishmaniases (CL), a wide range of cutaneous diseases caused by diverse species of Leishmania genus parasites, are among the most neglected infectious diseases. While they are non-fatal, CL are highly morbid with disfiguring lesions, which could be chronic, leaving lifelong unsightly scars; they are combined with psychological distress and social stigma. The efficiency of treatment highly depends on the infecting Leishmania species. Diagnosis is mainly based on microscopic direct examination (DE) of Giemsa-stained smears needing experienced microscopists. It can be laborious and time-consuming when the parasite load is low. DE is poorly sensitive and does not identify Leishmania species. So far, only DNA assays accurately identify the species. Despite their wide use for generic detection, PCR methods also require equipment and additional steps to identify causal Leishmania species. L. major is hyperendemic in many countries in Africa, the Middle East, and Asia, where other species co-occur with different endemicity levels according to the situations. This complicates disease management and treatment, particularly as distribution and epidemiology of leishmaniases remain poorly understood. Here, we aimed for a simple and rapid molecular diagnostic test to detect and identify L. major, a predominant CL causal species, which could be prone to become a control tool at the point of care, in endemic areas, using isothermal recombinase DNA amplification (recombinase polymerase amplification, RPA, or recombinase aided amplification, RAA) coupled to detection by the lateral flow (LF) chromatography on a PCRD cassette.MethodsTo develop an L. major species-specific RPA-LF assay, computational analysis of 70 Leishmania DNA targets, identified through bibliography and database searches, selected five targets. We designed and tested 7 primer pairs/probe sets to specifically amplify L. major DNAs. First, the primers were tested for species specificity and sensitivity using basic RPA chemistry. Then, to develop RPA-coupled LF detection, we shifted to the nfo chemistry.ResultsThis way, we retained one set for further investigation, which confirmed it is L. major species-specific. Tested on 86 human cutaneous samples, this selected set was able to detect 100% of L. major infections in confirmed CL patients. We did not observe any cross-reactivity with lesions due to L. infantum or L. tropica.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1514684/fullcutaneous leishmaniasesmolecular diagnosisRPA/RAALeishmania majorlateral flow chromatographypoint of care diagnosis |
| spellingShingle | Insaf Bel Hadj Ali Yusr Saadi-Ben Aoun Imen Khammeri Hejer Souguir Emna Harigua-Souiai Hamed Chouaieb Ahmed S. Chakroun Meryem Lemrani Aicha Kallel Kalthoum Kallel Nabil Haddad Oussaima El Dbouni Rhea N. Coler Rhea N. Coler Rhea N. Coler Steven G. Reed Akila Fathallah-Mili Akila Fathallah-Mili Ikram Guizani Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patients Frontiers in Microbiology cutaneous leishmaniases molecular diagnosis RPA/RAA Leishmania major lateral flow chromatography point of care diagnosis |
| title | Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patients |
| title_full | Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patients |
| title_fullStr | Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patients |
| title_full_unstemmed | Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patients |
| title_short | Recombinase-based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of Leishmania major in cutaneous leishmaniasis patients |
| title_sort | recombinase based amplification coupled with lateral flow chromatography for the specific and sensitive detection and identification of leishmania major in cutaneous leishmaniasis patients |
| topic | cutaneous leishmaniases molecular diagnosis RPA/RAA Leishmania major lateral flow chromatography point of care diagnosis |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1514684/full |
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