Pushkar Shivam

Bio: Pushkar Shivam is an academic researcher from Rajendra Memorial Research Institute of Medical Sciences. The author has contributed to research in topics: Leishmania donovani & Antigen. The author has an hindex of 6, co-authored 15 publications receiving 174 citations. Previous affiliations of Pushkar Shivam include Indian Council of Medical Research.

DDT-based indoor residual spraying suboptimal for visceral leishmaniasis elimination in India.

Abstract: Indoor residual spraying (IRS) is used to control visceral leishmaniasis (VL) in India, but it is poorly quality assured. Quality assurance was performed in eight VL endemic districts in Bihar State, India, in 2014. Residual dichlorodiphenyltrichloroethane (DDT) was sampled from walls using Bostik tape discs, and DDT concentrations [grams of active ingredient per square meter (g ai/m(2))] were determined using HPLC. Pre-IRS surveys were performed in three districts, and post-IRS surveys were performed in eight districts. A 20% threshold above and below the target spray of 1.0 g ai/m(2) was defined as "in range." The entomological assessments were made in four districts in IRS and non-IRS villages. Vector densities were measured: pre-IRS and 1 and 3 mo post-IRS. Insecticide susceptibility to 4% DDT and 0.05% deltamethrin WHO-impregnated papers was determined with wild-caught sand flies. The majority (329 of 360, 91.3%) of pre-IRS samples had residual DDT concentrations of <0.1 g ai/m(2). The mean residual concentration of DDT post-IRS was 0.37 g ai/m(2); 84.9% of walls were undersprayed, 7.4% were sprayed in range, and 7.6% were oversprayed. The abundance of sand flies in IRS and non-IRS villages was significantly different at 1 mo post-IRS only. Sand flies were highly resistant to DDT but susceptible to deltamethrin. The Stockholm Convention, ratified by India in 2006, calls for the complete phasing out of DDT as soon as practical, with limited use in the interim where no viable IRS alternatives exist. Given the poor quality of the DDT-based IRS, ready availability of pyrethroids, and susceptibility profile of Indian sand flies, the continued use of DDT in this IRS program is questionable.

Synthesis, characterization, and mechanistic studies of a gold nanoparticle-amphotericin B covalent conjugate with enhanced antileishmanial efficacy and reduced cytotoxicity.

Abstract: Background Amphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity. Methods Citrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet-visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB. Results Infrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory Th1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01). Conclusion GNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.

Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis.

Abstract: The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the Leishmania antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of L. donovani proteins in circulating antigens were further validated using anti-Ld actin and anti-α tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit.

Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance

Abstract: Several Glutathione S-transferases (GSTs) enzymes, in insects, have previously been implicated in resistance developed against DDT and other insecticides. The GST enzyme particularly sigma class have important physiological role in detoxification of lipid peroxidation by-products in insects. Phlebotomus argentipes has been intensely exposed to DDT over years due to Indoor Residual Spray (IRS) programme for Kala-azar elimination in Bihar, India. However, in P. argentipes, role of GSTs in DDT resistance have not been elucidated. Here, sigma class GST of P. argentipes (Parg-GSTσ) was successfully cloned, expressed and purified by affinity chromatography. The recombinant Parg-GSTσ was found to be highly active towards cumene hydroperoxide and 4-HNE having specific activity 92.47 & 203.92 µM/min/mg of protein, respectively and exhibited low activity towards universal substrate CDNB i.e., 8.75 µM/min/mg of protein. RT-PCR and immunoblot analysis showed at least 2 and 1.8 fold overexpression of Parg-GSTσ in the single exposed and non exposed DDT resistant P. argentipes as compared to susceptible, implicating Parg-GSTσ also involved in DDT resistance probably by imparting enhanced stress tolerance. The DDT, H2O2 and temperature induction assays demonstrated stress-dependent induction of Parg-GSTσ expression indicating its important role in oxidative stress redressal.

Infection and immunity

Abstract: Mutation of the Maturase Lipoprotein Attenuates the Virulence of Streptococcus equi to a Greater Extent than Does Loss of General Lipoprotein Lipidation Andrea Hamilton,†‡ Carl Robinson,† Iain C. Sutcliffe, Josh Slater, Duncan J. Maskell, Nick Davis-Poynter, Ken Smith, Andrew Waller, and Dean J. Harrington* University of Sunderland, SR1 3SD Tyne and Wear, United Kingdom; Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, United Kingdom; Northumbria University, Newcastle upon Tyne NE1 8ST, United Kingdom; Royal Veterinary College, Hertfordshire AL9 7TA, United Kingdom; Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom; and University of Bradford, West Yorkshire BD7 1DP, United Kingdom

Global trends in the production and use of DDT for control of malaria and other vector-borne diseases

Abstract: DDT was among the initial persistent organic pollutants listed under the Stockholm Convention and continues to be used for control of malaria and other vector-borne diseases in accordance with its provisions on acceptable purposes. Trends in the production and use of DDT were evaluated over the period 2001–2014. Available data on global production of DDT showed a 32% decline over the reporting period, from 5144 to 3491 metric tons of active ingredient p.a. Similarly, global use of DDT, for control of malaria and leishmaniasis, showed a 30% decline over the period 2001–2014, from 5388 metric tons p.a. to 3772 metric tons p.a. India has been by far the largest producer and user of DDT. In some countries, DDT is used in response to the development of resistance in malaria vectors against pyrethroid and carbamate insecticides. Some other countries have stopped using DDT, in compliance to the Convention, or in response to DDT resistance in malaria vectors. Progress has been made in establishing or amending national legal measures on DDT, with the majority of countries reportedly having measures in place that prohibit, or restrict, the production, import, export and use of DDT. Limitations in achieving the objectives of the Stockholm Convention with regard to DDT include major shortcomings in periodic reporting by Parties to the Stockholm Convention, and deficiencies in reporting to the DDT Register. Global production and global use of DDT have shown a modest decline since the adoption of the Stockholm Convention.

Elimination of visceral leishmaniasis on the Indian subcontinent

Abstract: Summary Visceral leishmaniasis is a serious public health problem on the Indian subcontinent, causing high morbidity and mortality. The governments in the region launched a visceral leishmaniasis elimination initiative in 2005. We review knowledge gaps and research priorities. Key challenges include low coverage of health services for those most at risk, drug resistance, the absence of a vaccine, and the complex biology of the sandfly–human host transmission cycle. Vector control is an essential component, but innovation in this field is insufficient. Substantial progress has been made in the area of diagnostic, therapeutic, and vaccine development, but there are still many hurdles to overcome. For visceral leishmaniasis elimination to become a reality, effective deployment of these existing and new tools is essential. A strong commitment at community level is imperative, and appropriate diagnostic and treatment services as well as effective epidemiological surveillance need to be ensured.

Recent advances and new strategies on leishmaniasis treatment

Abstract: Leishmaniasis is one of the most important tropical neglected diseases according to the World Health Organization. Even after more than a century, we still have few drugs for the disease therapy and their great toxicity and side effects put in check the treatment control program around the world. Moreover, the emergence of strains resistant to conventional drugs, co-infections such as HIV/Leishmania spp., the small therapeutic arsenal (pentavalent antimonials, amphotericin B and formulations, and miltefosine), and the low investment for the discovery/development of new drugs force researchers and world health agencies to seek new strategies to combat and control this important neglected disease. In this context, the aim of this review is to summarize new advances and new strategies used on leishmaniasis therapy addressing alternative and innovative treatment paths such as physical and local/topical therapies, combination or multi-drug uses, immunomodulation, drug repurposing, and the nanotechnology-based drug delivery systems.Key points• The treatment of leishmaniasis is a challenge for global health agencies.• Toxicity, side effects, reduced therapeutic arsenal, and drug resistance are the main problems.• New strategies and recent advances on leishmaniasis treatment are urgent.• Immunomodulators, nanotechnology, and drug repurposing are the future of leishmaniasis treatment.