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Among the parasitic diseases, amoebic liver abscess (ALA) ranks second to malaria in terms of mortality. Due to the poor sensitivity of conventional diagnostic methods, there is a need for the development of effective and rapid diagnostic methods for ALA. Thus, the purpose of this work was to develop a real‐time loop‐mediated isothermal amplification (RT‐LAMP) assay specific to Entamoeba histolytica. Further, we compared the performance of real‐time LAMP with conventional and real‐time PCR (RT‐PCR) targeting 18S small subunit ribosomal RNA (18S SSU rRNA) gene of E. histolytica in patients with ALA. A total of 126 liver samples were obtained for the study. Of these, 96 aspirated pus samples were obtained from patients suffering from an ALA (serology confirmed, anti‐amoebic immunoglobulin IgG positive), 19 aspirated pus samples from patients with pyogenic liver abscess (PLA, 16S RNA gene positive) and 11 autopsy liver tissues. The results showed that the DNA of E. histolytica was detected in 81 samples by conventional PCR, 93 by RT‐PCR and 95 by RT‐LAMP. The analytical sensitivity of the RT‐LAMP assay was much higher than the other two techniques. RT‐LAMP assay was able to amplify up to one copy of the targeted gene of E. histolytica while conventional PCR and RT‐PCR could amplify up to 103 and 102 copies of the targeted gene of E. histolytica, respectively. In conclusion, RT‐LAMP proved to be a sensitive, specific and rapid test which can be utilised as an effective tool for the diagnosis of ALA. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a vector-borne parasitic infection caused by the infective bite of female Phlebotomine sandflies. Treatment of leishmaniasis by conventional synthetic compounds is met by challenges pertaining to adverse effects which call for the discovery of newer anti-leishmanial molecules. This study was performed to evaluate the effect and modes of action of a sesquiterpene alcoholic molecule Farnesol on Leishmania major, the causative agent of Zoonotic CL. The cytotoxic effect of Farnesol against L.major promastigotes, amastigotes and macrophages was assessed by MTT test and counting. The IC50 on promastigotes by Farnesol on L.major was also evaluated by flow cytometry. In the findings, promastigotes were reduced at 167μM. The mean numbers of L.major amastigotes in macrophages were significantly decreased on exposure to Farnesol at 172μM. In addition, Farnesol induced significant apoptosis dose-dependent on L.major promastigotes. In silico protein-ligand_binding analyses indicated the effect of Farnesol in perturbation of the ergosterol synthesis pathway of Leishmania with attributes suggesting inhibition of Lanosterol-α-demethylase, the terminal enzyme of ergosterol synthesis machinery. Findings from flow cytometry reveal the role of Farnesol in apoptosis-induced killing in promastigotes. Farnesol was effective at very lower concentrations when compared to Paromomycin. Further studies are crucial to evaluate the therapeutic potential of Farnesol alone or in combination with other conventional drugs in animal models. [ABSTRACT FROM AUTHOR]

Background & objectives The emergence of drug resistance in leishmaniasis has remained a concern. Even new drugs have been found to be less effective within a few years of their use. Coupled with their related side effects and cost-effectiveness, this has prompted the search for alternative therapeutic options. In this study, the Computer Aided Drug Design (CADD) approach was used to repurpose already existing drugs against Leishmania major. The enzyme lanosterol 14-alpha demethylase (CYP51), in L. major , was chosen as the drug target since it is a key enzyme involved in synthesizing ergosterol, a crucial component of the cell membrane. Methods A library of 1615 FDA-approved drugs was virtually screened and docked with modeled CYP51 at its predicted binding site. The drugs with high scores and high affinity were subjected to Molecular Dynamics (MD) simulations for 100 ns. Finally, the compounds were tested in vitro using an MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay against the promastigotes of L. major. Results Computational screening of FDA-approved drugs identified posaconazole and isavuconazole as promising candidates, as both drugs target the CYP51 enzyme in fungi. Molecular dynamics (MD) simulations demonstrated that both drugs form stable complexes with the target enzyme. In vitro studies of posaconazole and isavuconazole against promastigotes of L. major demonstrated significant efficacy, with IC50 values of 2.062±0.89 µg/ml and 1.202±0.47 µg/ml, respectively. Interpretation & conclusions The study showed that the existing FDA-approved drugs posaconazole and isavuconazole can successfully be repurposed for treating L. major by targeting the CYP51 enzyme, demonstrating significant efficacy against promastigotes. [ABSTRACT FROM AUTHOR]