Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of “resistance” related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined.

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Drug resistance and treatment failure in leishmaniasis: A 21st century challenge

The WHO recognizes human African trypanosomiasis, Chagas disease and the leishmaniases as neglected tropical diseases. These diseases are caused by parasitic trypanosomatids and range in severity from mild and self-curing to near invariably fatal. Public health advances have substantially decreased the effect of these diseases in recent decades but alone will not eliminate them. In this Review, we discuss why new drugs against trypanosomatids are required, approaches that are under investigation to develop new drugs and why the drug discovery pipeline remains essentially unfilled. In addition, we consider the important challenges to drug discovery strategies and the new technologies that can address them. The combination of new drugs, new technologies and public health initiatives is essential for the management, and hopefully eventual elimination, of trypanosomatid diseases from the human population.

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Anti-trypanosomatid drug discovery: an ongoing challenge and a continuing need

In the recent past the standard treatment of kala-azar involved the use of pentavalent antimonials Sb(V). Because of progressive rise in treatment failure to Sb(V) was limited its use in the treatment program in the Indian subcontinent. Until now the mechanism of action of Sb(V) is not very clear. Recent studies indicated that both parasite and hosts contribute to the antimony efflux mechanism. Interestingly, antimonials show strong immunostimulatory abilities as evident from the upregulation of transplantation antigens and enhanced T cell stimulating ability of normal antigen presenting cells when treated with Sb(V) in vitro. Recently, it has been shown that some of the peroxovanadium compounds have Sb(V)-resistance modifying ability in experimental infection with Sb(V) resistant Leishmania donovani isolates in murine model. Thus, vanadium compounds may be used in combination with Sb(V) in the treatment of Sb(V) resistance cases of kala-azar.

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Use of antimony in the treatment of leishmaniasis: current status and future directions.

Background: Leishmania and other intracellular pathogens have evolved strategies that support invasion and persistence within host target cells. In some cases the underlying mechanisms involve the export of virulence factors into the host cell cytosol. Previous work from our laboratory identified one such candidate leishmania effector, namely elongation factor-1α, to be present in conditioned medium of infectious leishmania as well as within macrophage cytosol after infection. To investigate secretion of potential effectors more broadly, we used quantitative mass spectrometry to analyze the protein content of conditioned medium collected from cultures of stationary-phase promastigotes of Leishmania donovani, an agent of visceral leishmaniasis. Results: Analysis of leishmania conditioned medium resulted in the identification of 151 proteins apparently secreted by L. donovani. Ratios reflecting the relative amounts of each leishmania protein secreted, as compared to that remaining cell associated, revealed a hierarchy of protein secretion, with some proteins secreted to a greater extent than others. Comparison with an in silico approach defining proteins potentially exported along the classic eukaryotic secretion pathway suggested that few leishmania proteins are targeted for export using a classic eukaryotic amino-terminal secretion signal peptide. Unexpectedly, a large majority of known eukaryotic exosomal proteins was detected in leishmania conditioned medium, suggesting a vesicle-based secretion system. Conclusion: This analysis shows that protein secretion by L. donovani is a heterogeneous process that is unlikely to be determined by a classical amino-terminal secretion signal. As an alternative, L. donovani appears to use multiple nonclassical secretion pathways, including the release of exosomelike microvesicles.

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Proteomic analysis of the secretome of Leishmania donovani

Leishmaniasis is a parasitic disease caused by hemoflagellate, Leishmania spp. The parasite is transmitted by the bite of an infected female phlebotomine sandfly. The disease is prevalent throughout the world and in at least 88 countries. Nearly 25 compounds are reported to have anti-leishmanial effects but not all are in use. The pentavalent antimony compounds have remained mainstay for nearly 75 years. Pentavalent antimony is a prodrug that is reduced by glutathione to active trivalent species catalyzed by thiol-dependent-reductase. However, emergence of resistance led to the use of other compounds -amphotericin B, pentamidine, paromomycin, allopurinol etc. Amphotericin B, an antifungal macrolide polyene is characterized by the hydrophilic polyhydroxyl and hydrophobic polyene faces on it long axis. Presently, it is the only drug with highest cure rate. It acts on membrane sterols resulting in parasite cell lysis. Its lipid formulations have been developed to minimize side effects. Other anti-fungals like ketoconazole, fluconazole and terbinafine are found less effective. Recently, anticancer alkylphosphocholines have been found most effective oral compounds. These act as membrane synthetic ether-lipid analogues, and consist of alkyl chains in the lipid portions. Most promising of these are miltefosine (hexadecylphosphocholine), edelfosine (ET-18-OCH3) and ilmofosine (BM 41.440). However, the recent focus has been on identifying newer therapeutic targets in the parasite such as DNA topoisomerases. The present review describes the current understanding of different drugs against leishmaniasis, their chemistry, mode of action and the mechanism of resistance in the parasite. Future perspectives in the area of new anti-leishmanial drug targets are also enumerated. However, due to the vastness of the topic main emphasis is given on visceral leishmaniasis.

Chemotherapy of leishmaniasis: past, present and future.

Leishmaniasis is a vector-borne disease caused by different species of protozoan parasites of the genus Leishmania. It is a major health problem yet neglected tropical diseases, with approximately 350 million people worldwide at risk and more than 1.5 million infections occurring each year. Leishmaniasis has different clinical manifestations, including visceral (VL or kala-azar), cutaneous (CL), mucocutaneous (MCL), diffuse cutaneous (DCL) and post kala-azar dermal leishmaniasis (PKDL). Currently, the only mean to treat and control leishmaniasis is by rational medications and vector control. However, the number of available drugs is limited and even these are either exorbitantly priced, have toxic side effects or prove ineffective due to the emergence of resistant strains. On the other hand, the vector control methods are not so efficient. Therefore, there is an urgent need for developing a safe, effective, and affordable vaccine for the prevention of leishmaniasis. Although in recent years a large body of researchers has concentrated their efforts on this issue, yet only three vaccine candidates have gone for clinical trial, until date. These are: (i) killed vaccine in Brazil for human immunotherapy; (ii) live attenuated vaccine for humans in Uzbekistan; and (iii) second-generation vaccine for dog prophylaxis in Brazil. Nevertheless, there are at least half a dozen vaccine candidates in the pipeline. One can expect that, in the near future, the understanding of the whole genome of Leishmania spp. will expand the vaccine discovery and strategies that may provide novel vaccines. The present review focuses on the development and the status of various vaccines and potential vaccine candidates against leishmaniasis.

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Possibilities and challenges for developing a successful vaccine for leishmaniasis.

Miltefosine unresponsive and relapse cases of visceral leishmaniasis (VL) are increasingly being reported. However, there has been no laboratory confirmed reports of miltefosine resistance in VL. Here, we report two laboratory confirmed cases of VL from India. Two patients with VL were referred to us with suspected VL. The first patient was a native of the VL endemic state of Bihar, but residing in Delhi, a VL non-endemic area. He was treated with broad-spectrum antibiotics and antipyretics but was unresponsive to treatment. The second patient was from Jharkhand state in eastern India (adjoining Bihar), another endemic state for VL. He was refractory to anti-leishmanial treatment, which included administration of miltefosine. Following investigation, both patients were serologically positive for VL, and blood buffy coat from both patients grew Leishmania donovani. The isolates derived from both cases were characterized for their drug susceptibility, genetically characterised, and SNPs typed for LdMT and LdROS gene expression. Both patients were successfully treated with amphotericin B. The in vitro drug susceptibility assays carried out on both isolates showed good IC50 values to amphotericin B (0.1 ± 0.0004 μg/ml and 0.07 ± 0.0019 μg/ml). One isolate was refractory to SbIII with an IC50 of > 200 μM while the second isolate was sensitive to SbIII with an IC50 of 36.70 ± 3.2 μM. However, in both the isolates, IC50 against miltefosine was more than 10-fold higher (> 100 μM) than the standard strain DD8 (6.8 ± 0.1181 μM). Furthermore, genetic analyses demonstrated single nucleotide polymorphisms (SNPs) (354Tyr↔Phe and 1078Phe↔Tyr) in the LdMT gene of the parasites. Here, we document two laboratory confirmed cases of miltefosine resistant VL from India. Our finding highlights the urgent need to establish control measures to prevent the spread of these strains. We also propose that LdMT gene mutation analysis could be used as a molecular marker of miltefosine resistance in L. donovani.

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Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India.

Miltefosine resistance in Leishmania donovani involves suppression of oxidative stress-induced programmed cell death

Visceral leishmaniasis (VL) is a disease that has both zoonotic and anthroponotic etiologies. In India, VL is endemic, considered to be anthroponotic, and caused by Leishmania donovani. Anthroponotic diseases are maintained by transmission from human to human and to a lesser extent from human to animals. Serum samples from 1,220 animals from 7 human VL endemic districts of Bihar, India, were tested for antibodies to a recombinant kinetoplast antigen (rK39 antigen) present in amastigotes of visceralizing Leishmania species, i.e., L. donovani complex. Additionally, PCR was used to examine samples positive by rK39 antigen serology. Antibodies to rK39 indicative of VL were detected in 33 of 1,220 animals. Thirty-one of 867 goats (Capra hircus), 1 of 161 cattle (Bos indicus), and 1 of 54 wild rats (Rattus sp.) were positive by rK39 serology. None of 106 chickens (Gallus domesticus), 26 sheep (Ovis aries), 3 water buffaloes (Bubalus bubalus), or 3 dogs (Canis familiaris) was positive by rK39 serology...

https://bioone.org/journals/Journal-of-Parasitology/volume-99/issue-1/GE-3085.1/Animal-Reservoirs-of-Visceral-Leishmaniasis-in-India/10.1645/GE-3085.1.pdf

Jyotsna Mishra

Papers

Chemotherapy of leishmaniasis: past, present and future.

Possibilities and challenges for developing a successful vaccine for leishmaniasis.

Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India.

Miltefosine resistance in Leishmania donovani involves suppression of oxidative stress-induced programmed cell death

Animal Reservoirs of Visceral Leishmaniasis in India