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
Graphical abstract
Colorimetric malachite green based FTA-LAMP assay for cutaneous leishmaniasis.
Introduction
Leishmaniasis is a wide spectrum of diseases caused by an intracellular protozoan parasite of the genus Leishmania, transmitted by the bite of an infected female sand fly. Leishmania infection can result in three main clinically distinct manifestations in the human host; cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL). A critical component towards an understanding of the epidemiology and proper control/treatment of leishmaniasis is early and accurate diagnosis.
Conventionally, leishmaniasis is diagnosed by microscopic examination of skin smear/biopsy samples or aspirates from lesions for CL and MCL, and splenic or bone marrow aspirates for VL (Reithinger and Dujardin, 2007, De Vries et al., 2015). Despite high specificity, these methods are insensitive, invasive, and also require technical expertise (Reithinger and Dujardin, 2007, De Vries et al., 2015). Molecular approaches such as polymerase chain reaction (PCR) assays have been employed in the diagnosis of leishmaniasis (Reithinger and Dujardin, 2007; De Ruiter et al., 2014; De Vries et al., 2015); however, the need for expensive specialized equipment, the long time to result and lack of field applicability have greatly hindered the integration of these techniques into the diagnostic algorithm in endemic areas.
Recently, a rapid and simplified molecular technique, the loop-mediated isothermal amplification (LAMP) has been shown to be an effective tool in detection of human pathogenic infectious agents (Notomi et al., 2000, Mori et al., 2012, Dhama et al., 2014). The technique has been applied in the detection of Leishmania using purified DNA extracted from patient’s materials (Takagi et al., 2009, Adams et al., 2010, Khan et al., 2012) or swab boiled samples from CL model mice (Direct Boil-LAMP method; Mikita et al., 2014). However, the efficiency of the reported Direct Boil-LAMP method as a rapid diagnostic tool for CL remains to be demonstrated with clinical samples. Furthermore, the Foundation for Innovative New Diagnostics (FIND) has also devoted its effort towards reducing the burden of visceral leishmaniasis through innovative LAMP technique (http://www.finddiagnostics.org/). Despite the progress made with LAMP in diagnosis of leishmaniasis, a simple and efficient procedure for field and clinic sample collection and storage without the need for liquid handling and refrigerant/cold storage, is necessary. Flinders Technology Associates (FTA) cards (Whatman) lyse spotted cells and pathogens, and protect the nucleic acids from oxidation, nucleases and UV damage at room temperature for long storage. Studies have shown the utility of FTA cards for nested PCR analyses in epidemiological studies of leishmaniasis (Kato et al., 2010, Kato et al., 2011). However, the potential usefulness of FTA cards as a direct sampling tool for diagnosis of leishmaniasis by LAMP assay has not yet been well-explored. Therefore, this study reports the establishment of a quick, one-step, and single-tube, sensitive colorimetric malachite green (MG) based LAMP in combination with an FTA card for the detection of Leishmania DNA from patients’ cutaneous lesion-materials.
Section snippets
Parasites and template preparation
A WHO reference strain of Leishmania (Leishmania) major (MHOM/SU/1973/5ASKH) was cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal calf serum (Cansera International, Etobicoke Ontario, Canada), 2 mM l-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin at 25 °C. Parasites were harvested in the log phase and suspended in phosphate–buffered saline (PBS), counted using a Neubauer counting chamber (Hirschmann, Germany), and then 106 to 1
Results and discussion
The LAMP assay could detect all the L. (L.) major (106 to 1 parasites) levels from 2.0-mm-diameter FTA cards templates. No amplification was detected in the negative controls. Additionally, the FTA-LAMP assay had high detection sensitivity down to a level of 0.01 parasites/μl (Fig. 1A), which agreed with our previous report using purified DNA as a template (Nzelu et al., 2014). All the positive reactions turned light blue while the negative reactions became colorless without adding any reagent
Acknowledgements
The authors thanks the patients for giving consent to be a part of the study and all medical doctors and health staff at the different centers in Peru for their active participation during sampling. This study was financially supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Grant nos. 25257501 and 23256002), and the Program for Leading Graduate Schools “Fostering Global Leaders in Veterinary Science for Contributing to One Health” (FO1), MEXT,
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