Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection

Abstract

BackgroundLeishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA.MethodsLAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification.ResultsThe use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 103 parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica.ConclusionsThe simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed.

Chagas Disease Diagnostic Applications

“…Serological tests, however show substantial variations in their sensitivity in different geographical areas (Verani et al, 2009), and display sub-optimal performances (Sanchez-Camargo et al, 2014). Unfortunately, and despite being widely used in different parasitic diseases (Mondal et al, 2016; Sriworarat et al, 2015), PCR-based methods applicable in PoC settings such as those based on simple colorimetric loop-mediated isothermal amplification or Recombinase Polymerase Amplification (RPA) techniques are not yet available for T. cruzi detection.…”

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection

“…Although, a few studies employing LAMP assay to diagnose Leishmania infection are known [26–32] however, these assays are limited in their utility because of the false positivity due to cross contamination [29], or prolonged reaction time [31] or the use specialized equipment [32]. Here, we have developed a LAMP assay based on kinetoplast minicircle DNA (kDNA) of L.donovani for reliable and rapid diagnosis of Leishmania infection in a closed tube manner using SYBR Green I for detection of the amplified product.…”

Analytical Performance of a Loop-Mediated Isothermal Amplification Assay for Leishmania DNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis

“…In 2000, Notomi and collaborators designed LAMP, which is characterized as an isothermic technique with a fast reaction time and high sensitivity that offers several alternatives for the visualization of results including turbidity, fluorescence, and/or color changes. [8][9][10][12][13][14] In 2016, Mondal et al 54 reported the use of a recombinant polymerase amplification assay for the detection of Leishmania donovani; this technique offers a shorter reaction time compared with LAMP. Despite advances in the development of new diagnostic techniques, studies often only report certain aspects relating to the sensitivity and specificity of these techniques.…”

“…10 Some studies have demonstrated the use of LAMP against extracts from macerated sandflies 8,11 and several alternatives exist for the visualization of the results including turbidity, fluorescence, and/or color changes. [8][9][10][12][13][14] To date, LAMP has been used for the diagnosis of bacterial, 15 fungal, 16,17 and viral 18,19 infections, as well as parasitic infections such as those caused by Trypanosoma brucei, 20 Plasmodium falciparum, 21 and Trypanosoma cruzi. 22 For the diagnosis of leishmaniasis, LAMP has been used with the implementation of primers targeting the small ribosomal subunit (18S rRNA gene), 10,11,23 kinetoplast DNA (kDNA) for the detection of Old World Leishmania species, [24][25][26] and the Internal Transcribed Spacer 1 (ITS-1).…”