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A novel pan- Leishmania loop-mediated isothermal amplification (LAMP) assay for the diagnosis of cutaneous and visceral leishmaniasis (CL and VL) that can be used in near-patient settings was developed. Primers were designed based on the 18S ribosomal DNA (rDNA) and the conserved region of minicircle kinetoplast DNA (kDNA), selected on the basis of high copy number. LAMP assays were evaluated for CL diagnosis in a prospective cohort trial of 105 patients in southwest Colombia. Lesion swab samples from CL suspects were collected and were tested using the LAMP assay, and the results were compared to those of a composite reference of microscopy and/or culture in order to calculate diagnostic accuracy. LAMP assays were tested on samples (including whole blood, peripheral blood mononuclear cells, and buffy coat) from 50 suspected VL patients from Ethiopia. Diagnostic accuracy was calculated against a reference standard of microscopy of splenic or bone marrow aspirates. To calculate analytical specificity, 100 clinical samples and isolates from fever-causing pathogens, including malaria parasites, arboviruses, and bacteria, were tested. We found that the LAMP assay had a sensitivity of 95% (95% confidence interval [CI], 87.2% to 98.5%) and a specificity of 86% (95% CI, 67.3% to 95.9%) for the diagnosis of CL. With VL suspects, the sensitivity of the LAMP assay was 92% (95% CI, 74.9% to 99.1%) and its specificity was 100% (95% CI, 85.8% to 100%) in whole blood. For CL, the LAMP assay is a sensitive tool for diagnosis and requires less equipment, time, and expertise than alternative CL diagnostics. For VL, the LAMP assay using a minimally invasive sample is more sensitive than the gold standard. Analytical specificity was 100%.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis