A rapid molecular diagnosis of cutaneous leishmaniasis by colorimetric malachite green-loop-mediated isothermal amplification (LAMP) combined with an FTA card as a direct sampling tool

Abstract

Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64 degrees C for 60 mm using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per mu l. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas. (C) 2015 Elsevier B.V. All rights reserved

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

“…Implementing a molecular technique such as qPCR for field work in endemic areas for leishmaniasis leads to having a special infrastructure as well as the acquisition of costly equipment and reagents (its cost is reported to be up to three times higher than that of conventional PCR) (Bock and Langer, 1993). This reflects the need to develop new technologies more sensitive but easy to acquire and to be implemented in this type of regions, such as LAMP (Notomi et al, 2000; Tomita et al, 2008; Nzelu et al, 2014, 2016; Abbasi et al, 2016) and nanoparticles (Andreadou et al, 2014). Although microscopy remains the gold standard for routine diagnosis, the high incidence of CL in different regions of South America highlights the need to rethink the implementation of specific strategies for the correct and timely diagnosis of this disease.…”

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

“…For this reason, several authors have combined the LAMP technique with FTA cards (Flinders Technology Associates) that protect the nucleic acids from oxidation, nucleases and UV damage at room temperature for long storage periods for the diagnosis of leishmaniasis. In this sense, Nzelu et al, 2016 have developed a one-step, single-tube, highly sensitive LAMP assay for rapid detection of Leishmania DNA from tissue spotted on an FTA card, which has incorporated malachite green for the final visualisation step. The validation of this system was carried out on tissue samples obtained by aspirating or scraping the lesions of CL patients from Peru.…”

“…The validation of this system was carried out on tissue samples obtained by aspirating or scraping the lesions of CL patients from Peru. The developed system was fast (30 min), sensitive (detection levels as low as 0.01 parasites/µl) and specific (without cross-reactivity with the genomic DNA of Trypanosoma cruzi, Trypanosoma brucei gambiense, human and dog) [91].…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…The study showed that LAMP was efficient to detect 10 1 copies of DNA. They concluded that the newly developed LAMP was helpful when leishmania parasite density is very low, especially during the early infection stage (Nzelu et al 2014(Nzelu et al , 2016. There was a number of other reported significant practices of LAMP including Trichomonas viganalis (Reyes et al 2014), Necator americanus (Mugambi et al 2015) and Strongyloides stercoralis (Watts et al 2014), and it was shown that LAMP is at least 1000 times more sensitive than the conventional PCR.…”

“…Other dyes such as malachite green dye (Sriworarat et al 2015;Lucchi et al 2016;Nzelu et al 2016), Picogreen dye (Batra et al 2015), Gelred dye (Wozniakowski et al 2013;Wassermann et al 2014), SYTO fluorescent dye (Liu et al 2013;Watts et al 2014;Mwendwa et al 2017) Zhou et al, 2014). The reaction between free calcein and magnesium ions intensified the fluorescence of LAMP product (ii.)…”