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Development and Assessment of Leishmania major and Leishmania tropica Specific Loop-Mediated Isothermal Amplification Assays for the Diagnosis of Cutaneous Leishmaniasis in Tunisia

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  • 1 Laboratory of Medical Parasitology, Biotechnology and Biomolecules LR 11 IPT 06, Institut Pasteur de Tunis, Tunis, Tunisia;
  • | 2 Laboratory of Bioinformatics, Biomathematics and Biostatistics LR 16 IPT 09, Institut Pasteur de Tunis, Tunis, Tunisia;
  • | 3 Laboratory of Parasitology and Mycology, Institut Pasteur de Tunis, Tunis, Tunisia
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Cutaneous leishmaniasis (CL) remains one of the world’s most prevalent neglected diseases, particularly in developing countries. Identification of the involved Leishmania species is an important step in the diagnosis and case management process. In this study, we tested simple, rapid, and highly sensitive loop-mediated isothermal amplification (LAMP) assays for Leishmania DNA species-specific detection from cutaneous lesions. Two LAMP assays, targeting cysteine protease B (cpb) gene, were developed to detect and identify Leishmania major and Leishmania tropica species. Loop-mediated isothermal amplification specificity was examined using DNA samples from other Leishmania species and Trypanosoma species. No cross-reactions were detected. The developed LAMP assays exhibited sensitivity with a detection limit of 20 fg and 200 fg for L. major and L. tropica, respectively. Both tests were applied on clinical samples of CL suspected patients living in endemic Tunisian regions and compared with kinetoplast DNA quantitative PCR (qPCR), microscopic, and conventional cpb-based polymerase chain reaction (PCR) assays. Our LAMP tests were able to discriminate between L. major and L. tropica species and showed a sensitivity of 84% and a specificity of 100%. However, when compared with the performance of the diagnostic tests with latent class analysis (LCA), our LAMP assays show a sensitivity of 100%. These assays can be used as a first-line molecular test for early diagnosis and prompt management of CL cases in public health programs.

Author Notes

Address correspondence to Melek Chaouch, Laboratory of Medical Parasitology, Biotechnology and Biomolecules LR 11 IPT 06, Institut Pasteur de Tunis, Tunis 1002, Tunisia. E-mail: mcmelek@msn.com

Financial support: This study received financial support from the WHO-TDR Small Grants Scheme for implementation research on infectious diseases of poverty (WHO Reference 2015/525274-0).

Authors’ addresses: Melek Chaouch, Laboratory of Medical Parasitology, Biotechnology and Biomolecules LR 11 IPT 06, Institut Pasteur de Tunis, Tunis, Tunisia, and Laboratory of Bioinformatics, Biomathematics and Biostatistics LR 16 IPT 09, Institut Pasteur de Tunis, Tunis, Tunisia, E-mail: mcmelek@msn.com. Karim Aoun and Aida Bouratbine, Laboratory of Medical Parasitology, Biotechnology and Biomolecules LR 11 IPT 06, Institut Pasteur de Tunis, Tunis, Tunisia, and Laboratory of Parasitology and Mycology, Institut Pasteur de Tunis, Tunis, Tunisia, E-mails: karim.aoun@pasteur.rns.tn and aida.bouratbine@pasteur.rns.tn. Souad Ben Othman, Meriem Ben Abid, Ines Ben Sghaier, and Souha Ben Abderrazak, Laboratory of Medical Parasitology, Biotechnology and Biomolecules LR 11 IPT 06, Institut Pasteur de Tunis, Tunis, Tunisia, E-mails: souaad.bo@gmail.com, meriem_benabid@yahoo.fr, ines.bensghaier@gmail.com, and souha.benabderrazak@pasteur.rns.tn.

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