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

Abstract

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%.

Comparison of Nested-PCR and Loop-Mediated Isothermal Amplification for Cutaneous Leishmaniasis Diagnosis

“…Adams et al (34) conducted a study to evaluate LAMP for cutaneous leishmaniasis and visceral leishmaniasis diagnosis. To this end, lesion swab samples were obtained from 105 patients suspected to cutaneous leishmaniasis and blood samples were collected from 50 suspected visceral leishmaniasis patients.…”

Applying Loop-mediated Isothermal Amplification (LAMP) in the Diagnosis of Malaria, Leishmaniasis and Trypanosomiasis as Point-of-Care Tests (POCTs)

“…Target genes, 18S rDNA and kDNA, were chosen based on multiple strands and conservation across multiple strains and species of Leishmania across geographic areas, with the aim of being able to diagnose both VL and CL. Bst DNA polymerase is used for amplification, and the dry reagents include a calibration to allow for the visual detection of the amplified products without opening the reaction tubes [95].…”

“…The optimization of the assay was performed using samples of suspected CL patients from southwest Colombia (obtaining a sensitivity of 95% and a specificity of 86%). In the case of VL, 50 blood samples from Ethiopian patients were used, and the technique showed a sensitivity of 92.3% and a specificity of 100% on whole blood [95].…”

Introductory Chapter: Leishmaniasis: An Emerging Clinical Syndrome

“…Nonetheless, amastigote detection in bone marrow aspirates and positive rK39 immunochromatographic test should be further validated by nested PCR [60]. Recently, a loop-mediated isothermal amplification (LAMP) assay based on 18S rDNA and the conserved region of minicircle kDNA has been implicated with high sensitivity for visceral as well as CL diagnostics [61]. Further, Leishmania urine antigen has been explored as a probable biomarker for predicting treatment failure and relapse in VL/HIV-coinfected patients [62].…”