Abstract
BackgroundLeishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application.MethodsWe have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL).ResultsThe assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse.ConclusionsThe study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.
Highlights
Leishmaniasis is a spectrum of diseases with great relevance to public health
Cloning and sequence analysis of kinetoplast minicircle DNA (kDNA) minicircle polymerase chain reaction (PCR)-amplification of kDNA from total DNA isolated from L. donovani, L. major and L. tropica, using F3 and B3 primers yielded the product of 200 bp size of variable intensity (Fig. 2a)
Determination of kDNA copy number in Leishmania spp. using Quantitative Real-time PCR (Q-PCR) For evaluation of differences in copy number of kDNA minicircle in different Leishmania spp, a standard curve was established with serially diluted known number of kDNA plasmid copies (103 to 108) from L. donovani
Summary
Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. Leishmaniasis, a group of diseases caused by protozoan parasite Leishmania is transmitted to humans by the bite of infected phlebotomine female sandflies. In India and Bangladesh, PKDL is reported in 5-15% of patients treated for VL, usually after an interval of a few months to several years [2, 3], in Sudan 50-60% of VL cases develop PKDL within a few weeks of treatment [4]. The need to identify persons affected with PKDL and treat them as a part of VL control programs have been emphasized since PKDL patients are considered as an important reservoir for the parasite during inter-epidemic periods of VL [4]. In India, CL is primarily endemic in the western Thar region of Rajasthan, in Bikaner, where L. tropica is the major causative agent [5]
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