Ayan Kumar Ghosh

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

Significance Visceral leishmaniasis (VL), also known as kala-azar, is a major parasitic disease on the Indian subcontinent, with 85% of the disease incidence in India. As with malaria, indoor residual spraying (IRS) can be used to control and, ultimately, eliminate VL as a public health problem. However, in India, widespread resistance to dichlorodiphenyltrichloroethane (DDT), the insecticide used, combined with poor quality assurance (QA) of IRS and limited entomological surveillance, is hindering the VL elimination effort. Here, we present entomological abundance and insecticide resistance data and data arising from QA of IRS to explore these issues and establish an evidence base for improving the Indian VL elimination program. Unless these issues are addressed, the South Asian VL elimination effort will fail. 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/m2)] 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/m2 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/m2. The mean residual concentration of DDT post-IRS was 0.37 g ai/m2; 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.

Ascorbate Peroxidase, a Key Molecule Regulating Amphotericin B Resistance in Clinical Isolates of Leishmania donovani

ABSTRACT Amphotericin B (AmB), a polyene macrolide, is now a first-line treatment of visceral leishmaniasis cases refractory to antimonials in India. AmB relapse cases and the emergence of secondary resistance have now been reported. To understand the mechanism of AmB, differentially expressed genes in AmB resistance strains were identified by a DNA microarray and real-time reverse transcriptase PCR (RT-PCR) approach. Of the many genes functionally overexpressed in the presence of AmB, the ascorbate peroxidase gene from a resistant Leishmania donovani strain (LdAPx gene) was selected because the gene is present only in Leishmania, not in humans. Apoptosis-like cell death after exposure to AmB was investigated in a wild-type (WT) strain in which the LdAPx gene was overexpressed and in AmB-sensitive and -resistant strains. A higher percentage of apoptosis-like cell death after AmB treatment was noticed in the sensitive strain than in both the resistant isolate and the strain sensitive to LdAPx overexpression. This event is preceded by AmB-induced formation of reactive oxygen species and elevation of the cytosolic calcium level. Enhanced cytosolic calcium was found to be responsible for depolarization of the mitochondrial membrane potential and the release of cytochrome c (Cyt c) into the cytosol. The redox behavior of Cyt c showed that it has a role in the regulation of apoptosis-like cell death by activating metacaspase- and caspase-like proteins and causing concomitant nuclear alterations, as determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and DNA fragmentation in the resistant strain. The present study suggests that constitutive overexpression of LdAPx in the L. donovani AmB-resistant strain prevents cells from the deleterious effect of oxidative stress, i.e., mitochondrial dysfunction and cellular death induced by AmB.

Metabolic reconfiguration of the central glucose metabolism: a crucial strategy of Leishmania donovani for its survival during oxidative stress

Understanding the mechanism that allows the intracellular protozoan parasite Leishmania donovani (Ld) to respond to reactive oxygen species (ROS) is of increasing therapeutic importance because of the continuing resistance toward antileishmanial drugs and for determining the illusive survival strategy of these parasites. A shift in primary carbon metabolism is the fastest response to oxidative stress. A 14CO2 evolution study, expression of glucose transporters together with consumption assays, indicated a shift in metabolic flux of the parasites from glycolysis toward pentose phosphate pathway (PPP) when exposed to different oxidants in vitro/ex vivo. Changes in gene expression, protein levels, and enzyme activities all pointed to a metabolic reconfiguration of the central glucose metabolism in response to oxidants. Generation of glucose‐6‐phosphate dehydrogenase (G6PDH) (~5‐fold) and transaldolase (TAL) (~4.2‐fold) overexpressing Ld cells reaffirmed that lethal doses of ROS were counterbalanced by effective manipulation of NADPH:NADP+ ratio and stringent maintenance of reduced thiol content. The extent of protein carbonylation and accumulation of lipid peroxidized products were also found to be less in overexpressed cell lines. Interestingly, the LD50 of sodium antimony gluconate (SAG), amphotericin‐B (AmB), and miltefosine were significantly high toward overexpressing parasites. Consequently, this study illustrates that Ld strategizes a metabolic reconfiguration for replenishment of NADPH pool to encounter oxidative challenges.—Ghosh, A. K., Sardar, A. H., Mandal, A., Saini, S., Abhishek, K., Kumar, A., Purkait, B., Singh, R., Das, S., Mukhopadhyay, R., Roy, S., Das, P. Metabolic reconfiguration of the central glucose metabolism: a crucial strategy of Leishmania donovani for its survival during oxidative stress. FASEB J. 29, 2081‐2098 (2015). www.fasebj.org

Spinigerin induces apoptotic like cell death in a caspase independent manner in Leishmania donovani

Antimicrobial peptides (AMPs) are multifunctional components of the innate immune system. Chemotherapeutic agents used for treatment of visceral leishmaniasis (VL) are now threatened due to the emergence of acquired drug resistance and toxicity. AMPs are attractive alternative to conventional pharmaceuticals. In this study, first time we explored the antileishmanial activity of spinigerin originally derived from Pseudacanthotermes spiniger. Leishmania donovani promastigotes present apoptosis-like cell death upon exposure to spinigerin (IC50, 150 μM). The infection rate was reduced by 20% upon exposure to 150 μM spinigerin but no cytotoxicity on host macrophages was observed. Elevation of intracellular ROS level and down-regulation of two ROS detoxifying enzymes, ascorbate peroxidase (APx) and trypanothione reductase (TR) suggested essential role of ROS machinery during spinigerin mediated cell death. About 97% cell population was found to be Annexin-V positive; 44% cells being highly Annexin-V positive. Moreover, we observed morphological changes like cell rounding, nuclear condensation, oligonucleosomal DNA degradation and TUNEL positive cells without loss of membrane integrity upon spinigerin exposure, suggests apoptosis-like death. Interestingly, collapse in mitochondrial membrane potential and increased level of intracellular ROS and calcium were not associated with caspase like activity. Computational analysis suggests spiningerin interacts with trypanothione reductase and thus probably interferes its function to detoxify the toxic ROS level. Therefore, spinigerin induces apoptosis-like cell death in L. donovani in a caspase-independent manner. The study elucidates the antileishmanial property of spinigerin that may be considered for future chemotherapeutic option alone or adjunct with other drug regimens for improved treatment of visceral leishmaniasis.

Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis.

Diagnosis of visceral leishmaniasis in Bihar India: comparison of the rK39 rapid diagnostic test on whole blood versus serum.

Background Antibody-detecting rapid diagnostic tests (RDTs) against rK39 are available to aid in the diagnosis of visceral leishmaniasis (VL). Although these rK39 RDTs have been developed, validated and approved for use with serum, they are universally performed using whole blood. It was therefore necessary to determine whether this RDT is as sensitive on whole blood as on serum. Method and Principal Findings In this study we compared the rK39 RDT on serum and blood samples from 624 individuals with symptoms of VL attending the outpatient clinic at the Rajendra Memorial Research Institute of Medical Sciences, Patna, India. A total of 251 cases (40%) were both serum and blood-positive and 26 cases (4%) were identified as blood-negative and serum-positive. These 26 individuals in general had low titer antibodies against rK39 as determined by ELISA and follow-up on most of these individuals revealed none had persistent VL symptoms. The Cohen kappa index comparing blood and serum was 0.88 indicating excellent concordance. Conclusion Although the concordance was excellent, it is possible to miss rK39 positive individuals when using blood and the titer of anti-rK39 antibodies is low. We recommend that when an individual from an endemic area has obvious clinical symptoms of VL and the whole blood rK39 RDT is negative, that the test should be redone 2–3 weeks later if the symptoms persist.

Up-regulation of silent information regulator 2 (Sir2) is associated with amphotericin B resistance in clinical isolates of Leishmania donovani

OBJECTIVE Silent information regulator 2 (Sir2) is involved in parasite survival and apoptosis. Here, we aimed to explore the involvement of Sir2 in amphotericin B (AmB) resistance mechanism in Leishmania donovani. METHODS The expression levels of Sir2, MDR1 and NAD(+) biosynthetic pathway enzymes in AmB-resistant and -susceptible parasites were measured and total intracellular NAD(+)/NADH ratios were compared. Overexpression and knockout constructs of Sir2 were transfected in AmB-resistant and -susceptible parasites. Both resistant and susceptible parasites were inhibited with sirtinol for 4 h. The deacetylase activity of Sir2, the expression level of MDR1, the rate of AmB efflux, concentrations of reactive oxygen species (ROS) and levels of apoptosis were examined in WT, inhibited and transfected parasites, and the AmB susceptibility of the respective parasites was measured by determining the LD50 of AmB. RESULTS Levels of mRNA, protein and NAD(+)-dependent deacetylase activity of Sir2 were elevated in resistant versus susceptible parasites. Inhibition and/or deletion of Sir2 allele showed a decreased mRNA level of MDR1, lower drug efflux, increased ROS concentration, apoptosis-like phenomenon and decreased LD50 of AmB in resistant parasites. In contrast, Sir2 overexpression in susceptible parasites reversed drug susceptibility producing a resistant phenotype. This was associated with increased LD50 of AmB along with increased expression levels of MDR1, drug efflux and reduced concentrations of ROS, corresponding to decreased apoptosis of resistant to WT sensitive. CONCLUSIONS Sir2 plays a critical role in AmB resistance by regulating MDR1, ROS concentration and apoptosis-like phenomena and may be a new resistance marker for visceral leishmaniasis.

Characterization of groundwater composition in Punjab state with special emphasis on uranium content, speciation and mobility

Abstract Groundwaters (borewell and handpump) were sampled from two districts (Bathinda and Mansa) of Punjab state and analyzed for their major ionic concentrations and uranium isotope compositions in order to assess the possible origins of the waters and water–rock interactions that occurred in the deep aquifer system. The major ionic concentrations of waters were plotted on a Piper diagram and grouped into four dominant hydrochemical facies as (Na+K)-SO4+Cl type (69% – 73%), (Ca+Mg)-SO4+Cl type (6% – 21%), (Ca+Mg)–HCO3 type (4% – 6%) and (Na+K)-HCO3 type (2% – 19%). It was observed that mobility of uranium in groundwater was very much influenced by TDS (total dissolved solids). To investigate the various mechanisms for deriving the elevated uranium concentrations in groundwater, 234U/238U activity ratios (ARs) were calculated using the determined activity levels of 234U and 238U. The mean ARs was found to be near unity (i.e. secular equilibrium) in the study regions confirmed that uranium in groundwaters was mainly resulted from its host/parent rocks through weathering processes. The concentration of HCO3− in ground water showed one order of magnitude higher than the total dissolved SiO2 indicates that carbonate weathering was the dominant process due to major water–rock interaction. The uranium speciation in groundwaters was investigated by an equilibrium model calculation using MEDUSA (make equilibrium diagrams using sophisticated algorithms) under the influence of redox conditions and complexant concentration. At the observed range of pH values, the predominant redox speciation of uranium was observed as hydroxo-carbonato complexes of (UO2)2(CO3)(OH)3− and hydroxyl complexes of UO2(OH)3− which might be caused for increasing the solubility of uranium. Due to very low concentration of phosphate in groundwater, its effects on U(VI)-aqueous speciation was negligible.

Unmethylated CpG motifs in the L. donovani DNA regulate TLR9-dependent delay of programmed cell death in macrophages

Regulation of macrophage PCD plays an important role in pathogenesis of leishmaniasis. However, the precise involvement of any parasite molecule in this process remains uncertain. In the current study, in silico wide analysis demonstrated that genes in the Leishmania donovani genome are highly enriched for CpG motifs, with sequence frequency of 8.7%. Here, we show that unmethylated species‐specific CpG motifs in LdDNA significantly (P = 0.01) delay macrophage PCD by endosomal interaction with TLR9 via the adaptor protein MyD88. Importantly, LdDNA triggered high levels of luciferase activity (P = 0.001) under NF‐κB‐dependent transcription in HEK‐TLR9 cells. Furthermore, the activation of caspases in macrophages was inhibited (P = 0.001) in the presence of LdDNA. Notably, the delay of PCD was mediated by modulation of the antiapoptotic proteins, Mcl‐1 and Bfl‐1, and impairment of loss of Δψm in macrophages through the neutralization of oxidative and nitrosative stress. The inhibition of caspase activation and up‐regulation of Mcl‐1 by LdDNA were TLR9 dependent. Analysis of the targets of LdDNA identified an early activation of the TLR9‐dependent PI3K/Akt and SFK pathways, which were required for the observation of the antiapoptotic effects in macrophages. Moreover, we demonstrate that LdDNA modulates the TLR9–IκB‐α pathway by promoting the tyrosine phosphorylation of TLR9 and the TLR9‐mediated recruitment of Syk kinase. The results have identified a novel, TLR9‐dependent antiapoptotic function of LdDNA, which will provide new opportunities for discovering and evaluating molecular targets for drug and vaccine designing against VL.

Characterization of chaotic dynamics—I: dynamical invariants of sheared polymer solutions

Stress measurements as a function of time in aqueous polymer solutions show the presence of sustained dynamic features for identifiable steady shear regions. The analysis of post-transient time-series data, reveals the presence of low-dimensional chaotic dynamics. Interestingly, the results obtained from different types of polymer solutions, viz., thermosensitive polymers and biopolymers, show similar complex dynamical features and suggest that mechanisms responsible are common. The experimental data have been analyzed with a view to characterizing the strange attractor in terms of invariant measures such as the correlation, embedding, Lyapunov dimensions and entropy.