BackgroundMiltefosine (MIL) is an oral antileishmanial drug used for treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Recent reports indicate a significant decline in its efficacy with a high rate of relapse in VL as well as post kala-azar dermal leishmaniasis (PKDL). We investigated the parasitic factors apparently involved in miltefosine unresponsiveness in clinical isolates of Leishmania donovani.MethodologyL. donovani isolated from patients of VL and PKDL at pretreatment stage (LdPreTx, n = 9), patients that relapsed after MIL treatment (LdRelapse, n = 7) and parasites made experimentally resistant to MIL (LdM30) were included in this study. MIL uptake was estimated using liquid chromatography coupled mass spectrometry. Reactive oxygen species and intracellular thiol content were measured fluorometrically. Q-PCR was used to assess the differential expression of genes associated with MIL resistance.ResultsLdRelapse parasites exhibited higher IC50 both at promastigote level (7.92 ± 1.30 μM) and at intracellular amastigote level (11.35 ± 6.48 μM) when compared with LdPreTx parasites (3.27 ± 1.52 μM) and (3.85 ± 3.11 μM), respectively. The percent infectivity (72 hrs post infection) of LdRelapse parasites was significantly higher (80.71 ± 5.67%, P<0.001) in comparison to LdPreTx (60.44 ± 2.80%). MIL accumulation was significantly lower in LdRelapse parasites (1.7 fold, P<0.001) and in LdM30 parasites (2.4 fold, P<0.001) when compared with LdPreTx parasites. MIL induced ROS levels were significantly lower (p<0.05) in macrophages infected with LdRelapse while intracellular thiol content were significantly higher in LdRelapse compared to LdPreTx, indicating a better tolerance for oxidative stress in LdRelapse isolates. Genes associated with oxidative stress, metabolic processes and transporters showed modulated expression in LdRelapse and LdM30 parasites in comparison with LdPreTx parasites.ConclusionThe present study highlights the parasitic factors and pathways responsible for miltefosine unresponsiveness in VL and PKDL.

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.

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Leishmania donovani is the causative agent of anthroponotic visceral leishmaniasis in the Indian subcontinent. Oral miltefosine therapy has recently replaced antimonials in endemic areas. However, the drug is at risk of emergence of resistance due to unrestricted use, and, already, there are indications towards decline in treatment efficacy. Hence, understanding the mechanism of miltefosine resistance in the parasite is crucial. We employed genomic microarray analysis to compare the gene expression patterns of miltefosine-resistant and miltefosine-sensitive L. donovani. Three hundred eleven genes, representing ∼3.9% of the total Leishmania genome, belonging to various functional categories including metabolic pathways, transporters, and cellular components, were differentially expressed in miltefosine-resistant parasite. Results in the present study highlighted the probable mechanisms by which the parasite sustains miltefosine pressure including (1) compromised DNA replication/repair mechanism, (2) reduced protein synthesis and degradation, (3) altered energy utilization via increased lipid degradation, (4) increased ABC 1-mediated drug efflux, and (5) increased antioxidant defense mechanism via elevated trypanothione metabolism. The study provided the comprehensive insight into the underlying mechanism of miltefosine resistance in L. donovani that may be useful to design strategies to increase lifespan of this important oral antileishmanial drug.

The main focus of this study is to conduct a systematic literature review to integrate lean, agile, resilient, green and sustainable (LARGS) paradigms in the supply chain (SC) domain. To achieve this aim, several research questions were designed: First, how to locate LARGS research in context of SC domain? For this, it is important to understand which types of research articles should be selected for the study? Further, where such studies were conducted (geographical location)? Second, what is the focus of research in LARGS paradigm in SCs? For this, it is important to study, which types of industries or sectors have been targeted in literature? In addition, which tools and techniques have been used mostly? Third, what are the current trends in the relationships of LARGS paradigms, among themselves, and with SC performance measures? Fourth, what are the emerging issues, unexplored areas in this field, based on these what could be future research avenues in this subject domain have been proposed? A total of 160 relevant articles published during 1999–2019 were used for analysis. Based on analysis, findings are summarised, and main research issues and possible future research directions in LARGS paradigms in SCs are highlighted.

SummaryProtein modification by ubiquitin (Ub) and Ub-like molecules (Ubls) is a diverse biological process that regulates the activity of the target proteins. Systematic studies of Ubls in trypanosomatids like Leishmania, the causative organism of potentially fatal visceral leishmaniasis, would yield a better understanding of the disease pathogenesis and identify novel therapeutic targets. The present study is the first to characterize Leishmania donovani-specific Ub-related modifier-1 (LdUrm1) and the associated conjugation pathway. Based on homology modeling, LdUrm1 was found to possess a β-grasp fold and a C-terminal di-glycine motif unique to Ub/Ubls, essential for its conjugation to the target proteins. We identified LdUba4 as the E1 enzyme for LdUrm1 and demonstrated its energydependent enzymatic activity. LdUrm1 was immunolocalized anteriorly near the flagellar reservoir, while LdUba4 was cytoplasmic, both in promastigotes and axenic amastigotes. Expression of nonconjugatable LdUrm1 in L. donovani resulted in depleted parasite growth suggesting its role in the pathogenesis. By mass spectrometry, we identified Rab5, a known mediator of early endosome regulated hemoglobin endocytosis in Leishmania, as a target of LdUrm1. Our data suggest that LdUrm1 conjugation pathway may have a role in early endosome-mediated heme uptake in Leishmania that may be explored as a drug target.

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Leprosy and post–kala-azar dermal leishmaniasis (PKDL) are co-endemic neglected tropical diseases often misdiagnosed because of close resemblance in their clinical manifestations. The test that aids in differential diagnosis of leprosy and PKDL would be useful in endemic areas. Here, we report development of a multiplex loop-mediated isothermal amplification (m-LAMP) assay for differential detection of Mycobacterium leprae and Leishmania donovani using a real-time fluorometer. The m-LAMP assay was rapid with a mean amplification time of 15 minutes, and analytical sensitivity of 1 fg for L. donovani and 100 fg for M. leprae. The distinct mean Tm values for M. leprae and L. donovani allowed differentiation of the two organisms in the m-LAMP assay. Diagnostic sensitivity of the assay was evaluated by using confirmed cases of leprosy (n = 40) and PKDL (n = 40) (tissue and slit aspirate samples). All the leprosy and PKDL samples used in this study were positive by organism-specific QPCR and loop-mediated isothermal amplification assays. The diagnostic sensitivity of the m-LAMP assay was 100% (95% CI: 91.2–100.0%) for detecting PKDL and 95% for leprosy (95% CI: 83.1–99.4%). Our m-LAMP assay was successfully used to detect both M. leprae and L. donovani in a patient coinfected with leprosy and macular PKDL. The m-LAMP assay is rapid, accurate, and applicable for differential diagnosis of leprosy versus PKDL, especially in endemic areas.