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

Abstract

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.

Figure 1.

(A) Sensitivity of LAMP assays for the detection of Leishmania DNA using serial dilutions of Leishmania tropica. After incubation at 67°C for 50 minutes, LAMP reactions were inspected by agarose gel electrophoresis of LAMP products. Lanes 1–6 show the typical ladder-shaped pattern of a positive reaction. (B) Specificity of Leishmania major LAMP assay tested on different species: lane 1: 1-kb ladder, lane 2: L. major (L3171), lane 3: Leishmania infantum (MHOM/TN/1980/IPT1), lane 4: L. tropica (AM), lane 5: Leishmania donovani (PKDL306), and lane 6: negative control.

Figure 2.

Diagram describing the workflow of samples from cutaneous leishmaniasis suspects in the study.