Development of a Reverse Transcriptase Loop-Mediated Isothermal Amplification (LAMP) Assay for the Sensitive Detection of Leishmania Parasites in Clinical Samples

Abstract

Abstract. Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved region of the 18S ribosomal RNA (rRNA) gene; amplification was visualized by the pre-amplification addition of fluorescent detection reagent (FDR) and a simple UV lamp. By using a reverse-transcriptase step, the system detected infections between 10 and 100 parasites per mL. The assay was tested on a range of nucleic acid extracts from Leishmania species, visceral leishmaniasis (VL) patients from Sudan, and cutaneous leishmaniasis (CL) patients from Suriname. The sensitivity of RT-LAMP from the blood of VL patients was 83% ( N = 30) compared with microscopy of bone-marrow and lymph-node aspirates; for CL patients the observed sensitivity was 98% ( N = 43). The potential to use LAMP as a diagnostic tool for leishmaniasis is discussed.

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

“…This new detection method could be helpful for high-throughput DNA detection in basic research and pointof-care testing. Several studies have used LAMP assays for detecting various pathogens, but mostly using a real-time turbidimeter (Parida et al, 2007;Yoneyama et al, 2007;Toriniwa et al, 2006;Thekisoe et al, 2010), a real-time PCR system (Cai et al, 2008;Ohtsuki et al, 2008) or 14 fluorescent detection reagents (FDR) (Eiken) (Adams et al, 2010), which require a UV illuminator for LAMP product discrimination. The use of expensive and specialized equipment reduces the versatility of LAMP and greatly limits the wide use of this technique, especially in developing countries.…”

Species Typing in Dermal Leishmaniasis

“…Unlike PCR, it does not require a thermocycler, and the amplified product is visualized in the reaction tube and seen by the naked eye, not requiring gels or fluorescence detection. Even though the technique has been used primarily in first-line diagnostics for parasite detection (159,160), an L. donovani-specific assay based on kDNA minicircles was developed (161,162). It was tested on only a few type strains for some species, and hence it currently has limited validity.…”

Health-seeking behaviour, diagnostics and transmission dynamics in the control of visceral leishmaniasis in the Indian subcontinent

“…The only commercially available product, KAtex, has challenges in utility and suboptimal sensitivity, although we would argue that low sensitivity should not necessarily be considered a barrier to implementation. Simplified molecular diagnostic tools that can be adapted for field situations are under development, including loop-mediated isothermal amplification 20 . These tests, along with standard polymerase chain reaction, are able to detect circulating DNA in the blood of individuals who are actively infected.…”