Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection. Issue 1 (December 2015)

• Record Type: Journal Article
• Authors: Sriworarat, Chaichontat; Phumee, Atchara; Mungthin, Mathirut; Leelayoova, Saovanee; Siriyasatien, Padet
• Is Part Of: Parasites & vectors. Volume 8:Issue 1(2015)
• Page Start: 1
• Page End: 8
• Publication Date: 2015-12
• Subjects: Leishmania martiniquensis -- L. siamensis -- LAMP -- Malachite green -- Diagnosis; Parasitism -- Periodicals; Parasites -- Periodicals; Vector-pathogen relationships -- Periodicals; Animals as carriers of disease -- Periodicals; Insects as carriers of disease -- Periodicals; 616.96
• DOI: 10.1186/s13071-015-1202-x
• Languages: English
• ISSNs: 1756-3305

Abstract

Abstract Background Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genusLeishmania . Recently, an increasing number of autochthonous leishmaniasis cases caused byL. martiniquensis and the novel speciesL. 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 ofLeishmania DNA. Methods LAMP 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 ofL. 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. Results The use of MG resulted in an unambiguous differentiation of positive and negative controls. ForL. siamensis, the detection limit was 103 parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of activeLeishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multipleLeishmania species other thanL. siamensis andL. martiniquensis, includingL. aethiopica, L. braziliensis, L. donovani andL. tropica . Conclusions The simplicity and sensitivity of LAMP in detecting activeLeishmania 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.