Shyam Sundar

Publications of Shyam Sundar

• Efficacy of Miltefosine in the Treatment of Visceral Leishmaniasis after a Decade of use in India.

  Authors: Shyam Sundar, Anup Singh, Madhukar Rai, Vijay K Prajapati, Avinash K Singh, Bart Ostyn, Marleen Boelaert, Jean-Claude Dujardin, Jaya Chakravarty

  Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 05/2012;

  BackgroundMiltefosine is the only oral drug available for treatment of Indian Visceral Leishmaniasis (VL) which was shown to have an efficacy of 94% in a phase 3 trial in Indian subcontinent. ItsBackgroundMiltefosine is the only oral drug available for treatment of Indian Visceral Leishmaniasis (VL) which was shown to have an efficacy of 94% in a phase 3 trial in Indian subcontinent. Its unrestricted use has raised concern about its continued effectiveness. This study evaluates the efficacy and safety of miltefosine for the treatment of VL after a decade of use in India.MethodsIt was an open-label non-comparative study, in which 567 patients received oral Miltefosine (50 mg for those weighing <25 kg, or 100 mg in divided doses for those ≥25 kg, and 2.5 mg per kg for children < 12years, daily for 28 days) in a directly observed manner. Patients were followed up for six months to see the response to therapy.ResultAt the end of treatment the initial cure rate was 97.5% (intention to treat) and at six month the final cure rate was 90.3%. Overall death rate was 0.9% (5/567) and two deaths were drug-toxicity related. Gastrointestinal intolerance was frequent (64.5%). The drug was interrupted in 9 (1.5%) patients due to the adverse events of the drug.ConclusionAs compared to the phase 3 trial which led to registration of the drug a decade ago, there is a substantial increase in the failure rate of oral miltefosine for treatment of VL in India.

• A reassessment of immune correlates in human visceral leishmaniasis as defined by cytokine release in whole blood.

  Authors: Om Prakash Singh, Kamlesh Gidwani, Rajiv Kumar, Stephen L Jones, Marleen Boelaert, David Sacks, Shyam Sundar

  Clinical and vaccine immunology : CVI. 04/2012;

  Depressed cell mediated immunity in human visceral leishmaniasis (VL), revealed as the inability of peripheral blood mononuclear cells (PBMCs) to respond to Leishmania antigen, remains a hallmark ofDepressed cell mediated immunity in human visceral leishmaniasis (VL), revealed as the inability of peripheral blood mononuclear cells (PBMCs) to respond to Leishmania antigen, remains a hallmark of and is thought to underlie the progressive nature of this disease. We have recently reported the ability of a whole blood, IFN-γ release assay to detect sub-clinical infections amongst healthy individuals living in the kala-azar endemic zone in Bihar, India, and the surprising result that patients with active VL also secreted significant levels of antigen-specific IFN-γ in this assay. We were interested to extend these findings to a larger cohort of subjects, and to employ the whole blood assay to detect additional cytokines that might better correlate with the disease status of infected individuals. We evaluated IFN-γ, TNF-α and IL-10 release in 35 patients with active VL, 54 cured VL, 27 patients with other diseases, 52 Non-Endemic Healthy Controls (NEHC), and 147 Endemic Healthy Controls (EHC). The cellular response of the EHCs was correlated with their serological antibody titers against L. donovani and Phlebotomus argentipes saliva. The whole blood cells from the majority of both active (80%) and cured (85%) VL patients, as well as 24% of EHCs with presumed sub-clinical infections, produced significantly elevated levels of IFN-γ. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the active VL patients also produced IL-10, which in conjunction with IFN-γ better reflects the immune responses that distinguish active cases from cured or sub-clinically infected, immune individuals.

• Genetic and functional evaluation of the role of DLL1 in susceptibility to visceral leishmaniasis in India.

  Authors: Sanjana Mehrotra, Michaela Fakiola, Anshuman Mishra, Medhavi Sudharshan, Puja Tiwary, Deepa Selvi Rani, Kumarasamy Thangaraj, Madhukar Rai, Shyam Sundar, Jenefer M Blackwell

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 04/2012;

  Chromosome 6q26-27 is linked to susceptibility to visceral leishmaniasis (VL) in Brazil and Sudan. DLL1 encoding the Delta-like 1 ligand for Notch 3 was implicated as the etiological gene. DLL1Chromosome 6q26-27 is linked to susceptibility to visceral leishmaniasis (VL) in Brazil and Sudan. DLL1 encoding the Delta-like 1 ligand for Notch 3 was implicated as the etiological gene. DLL1 belongs to the family of Notch ligands known to selectively drive antigen-specific CD4 T helper 1 cell responses, which are important in protective immune response in leishmaniasis. Here we provide further genetic and functional evidence that supports a role for DLL1 in a well-powered population-based study centred in the largest global focus of VL in India. Twenty-one single nucleotide polymorphisms (SNPs) at PHF10/C6orf70/DLL1/FAM120B/PSMB1/TBP were genotyped in 941 cases and 992 controls. Logistic regression analysis under an additive model showed association between VL and variants at DLL1 and FAM120B, with top associations (rs9460106, OR=1.17, 95%CI 1.01-1.35, P=0.033; rs2103816, OR=1.16, 95%CI 1.01-1.34, P=0.039) robust to analysis using caste as a covariate to take account of population substructure. Haplotype analysis taking population substructure into account identified a common 2-SNP risk haplotype (frequency 0.43; P=0.028) at FAM120B, while the most significant protective haplotype (frequency 0.18; P=0.007) was a 5-SNP haplotype across the interval 5' of both DLL1 (negative strand) and FAM120B (positive strand) and extending to intron 4 of DLL1. Quantitative RT/PCR was used to compare expression of 6q27 genes in paired pre- and post-treatment splenic aspirates from VL patients (N=19). DLL1 was the only gene to show differential expression that was higher (P<0.0001) in pre- compared to post-treatment samples, suggesting that regulation of gene expression was important in disease pathogenesis. This well-powered genetic and functional study in an Indian population provides evidence supporting DLL1 as the etiological gene contributing to susceptibility to VL at Chromosome 6q27, confirming the potential for polymorphism at DLL1 to act as a genetic risk factor across the epidemiological divides of geography and parasite species.

• rK39 Antigen for the Diagnosis of Visceral Leishmaniasis by Using Human Saliva.

  Authors: Manisha Vaish, Om Prakash Singh, Jaya Chakravarty, Shyam Sundar

  The American journal of tropical medicine and hygiene. 04/2012; 86(4):598-600.

  Abstract. The rK39 rapid immunochromatographic test (ICT) is now being widely used in the diagnosis of visceral leishmaniasis (VL) using serum. We evaluated the presence of anti-rK-39 antibody inAbstract. The rK39 rapid immunochromatographic test (ICT) is now being widely used in the diagnosis of visceral leishmaniasis (VL) using serum. We evaluated the presence of anti-rK-39 antibody in human saliva being noninvasive to replace the invasive procedures of diagnosis. Enzyme-linked immunosorbent assay (ELISA) and ICT assays were performed in 300 subjects: 114-confirmed VL patients, 95 and 47 healthy controls from endemic and nonendemic regions, respectively, and 44 subjects with different diseases. Sensitivity in saliva was 83.3% by ELISA and 82.5% by ICT, compared with 100% for both ICT and ELISA in serum. Specificity in saliva was 100%, 90.5%, and 88.6% with ELISA, and 91.48%, 91.57%, and 84.06% using ICT, in nonendemic, endemic, and different diseases, respectively. In serum, specificity was 97%, 88.5%, and 89% by ELISA and 100%, 94.7%, and 95.5% by ICT in nonendemic, endemic, and different diseases, respectively. Saliva is not suitable for diagnosis of VL because of low sensitivity.

• Identification and Characterization of a Novel Leishmania donovani Antigen for Serodiagnosis of Visceral Leishmaniasis.

  Authors: Subodh Kumar, Dinesh Kumar, Jaya Chakravarty, Madhukar Rai, Shyam Sundar

  The American journal of tropical medicine and hygiene. 04/2012; 86(4):601-5.

  Abstract. Despite several drawbacks, rK39-based rapid immunochromatographic test is widely used for the diagnosis of visceral leishmaniasis (VL) in the Indian subcontinent. There is an urgent need toAbstract. Despite several drawbacks, rK39-based rapid immunochromatographic test is widely used for the diagnosis of visceral leishmaniasis (VL) in the Indian subcontinent. There is an urgent need to develop a better antigen. In this study we separated crude soluble antigens of Leishmania donovani by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hybridized with pool sera from pre- and post-treated VL patients, 6 months follow-up, endemic healthy (EHC), and nonendemic healthy controls (NEHC) by Western blotting. The sensitivity of enzyme-linked immunosorbent assay with identified protein was 95% (confidence interval [CI] = 89.6-98.01%), whereas the specificity for EHC, NEHC, and different disease groups were 96.3% (CI = 89.8-98.6%), 100% (CI = 95.8-100%), and 97.4% (CI = 91.02-99.3%), respectively. This specific antigen was subjected to two-dimensional gel electrophoresis and after tryptic digestion, antigen was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Further analysis showed that it is a member of the heat shock protein family of 70 kDa, designated as BHUP1, and has great potential in the diagnosis of VL.

• Identification and Characterization of a Novel Leishmania donovani Antigen for Serodiagnosis of Visceral Leishmaniasis

  Authors: Subodh Kumar, Dinesh Kumar, Jaya Chakravarty, Madhukar Rai, Shyam Sundar

  The American journal of tropical medicine and hygiene. 04/2012; 4(86-86(4)):601–605.

  Despite several drawbacks, rK39-based rapid immunochromatographic test is widely used for the diagnosis of visceral leishmaniasis (VL) in the Indian subcontinent. There is an urgent need to develop aDespite several drawbacks, rK39-based rapid immunochromatographic test is widely used for the diagnosis of visceral leishmaniasis (VL) in the Indian subcontinent. There is an urgent need to develop a better antigen. In this study we separated crude soluble antigens of Leishmania donovani by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hybridized with pool sera from pre- and post-treated VL patients, 6 months follow-up, endemic healthy (EHC), and nonendemic healthy controls (NEHC) by Western blotting. The sensitivity of enzyme-linked immunosorbent assay with identified protein was 95% (confidence interval [CI] = 89.6–98.01%), whereas the specificity for EHC, NEHC, and different disease groups were 96.3%(CI = 89.8–98.6%), 100% (CI = 95.8–100%), and 97.4%(CI = 91.02–99.3%), respectively. This specific antigen was subjected to two-dimensional gel electrophoresis and after tryptic digestion, antigen was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Further analysis showed that it is a member of the heat shock protein family of 70 kDa, designated as BHUP1, and has great potential in the diagnosis of VL.

• A novel recombinant Leishmania donovani p45, a partial coding region of methionine aminopeptidase, generates protective immunity by inducing a Th1 stimulatory response against experimental visceral leishmaniasis.

  Authors: Reema Gupta, Pramod K Kushawaha, Chandra Dev Pati Tripathi, Shyam Sundar, Anuradha Dube

  International journal for parasitology. 03/2012;

  The development of a vaccine against visceral leishmaniasis (VL) conferring long-lasting immunity remains a challenge. Identification and proteomic characterization of parasite proteins led to theThe development of a vaccine against visceral leishmaniasis (VL) conferring long-lasting immunity remains a challenge. Identification and proteomic characterization of parasite proteins led to the detection of p45, a member of the methionine aminopeptidase family. To our knowledge the present study is the first known report that describes the molecular and immunological characterization of p45. Recombinant Leishmania donovani p45 (rLdp45) induced cellular responses in cured hamsters and generated Th1-type cytokines from peripheral blood mononuclear cells of cured/endemic VL patients. Immunization with rLdp45 exerted considerable prophylactic efficacy (?85%) supported by an increase in mRNA expression of iNOS, IFN-?, TNF-? and IL-12 and decrease in TGF-? and IL-4, indicating its potential as a vaccine candidate against VL.

• Prevalence of Sand Flies and Leishmania donovani Infection in a Natural Population of Female Phlebotomus argentipes in Bihar State, India.

  Authors: Puja Tiwary, Dinesh Kumar, Rudra Pratap Singh, Madhukar Rai, Shyam Sundar

  Vector borne and zoonotic diseases (Larchmont, N.Y.). 01/2012;

  Abstract Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand flyAbstract Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90-17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.

• Evaluation of Leishmania donovani Protein Disulfide Isomerase as a Potential Immunogenic Protein/Vaccine Candidate against Visceral Leishmaniasis.

  Authors: Pramod Kumar Kushawaha, Reema Gupta, Chandra Dev Pati Tripathi, Shyam Sundar, Anuradha Dube

  PloS one. 01/2012; 7(4):e35670.

  In Leishmania species, Protein disulfide isomerase (PDI) - a redox chaperone, is reported to be involved in its virulence and survival. This protein has also been identified, through proteomics, as aIn Leishmania species, Protein disulfide isomerase (PDI) - a redox chaperone, is reported to be involved in its virulence and survival. This protein has also been identified, through proteomics, as a Th1 stimulatory protein in the soluble lysate of a clinical isolate of Leishmania donovani (LdPDI). In the present study, the molecular characterization of LdPDI was carried out and the immunogenicity of recombinant LdPDI (rLdPDI) was assessed by lymphocyte proliferation assay (LTT), nitric oxide (NO) production, estimation of Th1 cytokines (IFN-γ and IL-12) as well as IL-10 in PBMCs of cured/endemic/infected Leishmania patients and cured L. donovani infected hamsters. A significantly higher proliferative response against rLdPDI as well as elevated levels of IFN-γ and IL-12 were observed. The level of IL-10 was found to be highly down regulated in response to rLdPDI. A significant increase in the level of NO production in stimulated hamster macrophages as well as IgG2 antibody and a low level of IgG1 in cured patient's serum was observed. Higher level of IgG2 antibody indicated its Th1 stimulatory potential. The efficacy of pcDNA-LdPDI construct was further evaluated for its prophylactic potential. Vaccination with this construct conferred remarkably good prophylactic efficacy (∼90%) and generated a robust cellular immune response with significant increases in the levels of iNOS transcript as well as TNF-α, IFN-γ and IL-12 cytokines. This was further supported by the high level of IgG2 antibody in vaccinated animals. The in vitro as well as in vivo results thus indicate that LdPDI may be exploited as a potential vaccine candidate against visceral Leishmaniasis (VL).

• Leishmania donovani: immunostimulatory cellular responses of membrane and soluble protein fractions of splenic amastigotes in cured patient and hamsters.

  Authors: Shraddha Kumari, Pragya Misra, Rati Tandon, Mukesh Samant, Shyam Sundar, Anuradha Dube

  PloS one. 01/2012; 7(1):e30746.

  Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal ifVisceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI) and is usually fatal if not treated properly. An estimated 350 million people worldwide are at risk of acquiring infection with Leishmania parasites with approximately 500,000 cases of VL being reported each year. In the absence of an efficient and cost-effective antileishmanial drug, development of an appropriate long-lasting vaccine against VL is the need of the day. In VL, the development of a CMI, capable of mounting Th1-type of immune responses, play an important role as it correlate with recovery from and resistance to disease. Resolution of infection results in lifelong immunity against the disease which indicates towards the feasibility of a vaccine against the disease. Most of the vaccination studies in Leishmaniasis have been focused on promastigote--an infective stage of parasite with less exploration of pathogenic amastigote form, due to the cumbersome process of its purified isolation. In the present study, we have isolated and purified splenic amastigotes of L. donovani, following the traditional protocol with slight modification. These were fractionated into five membranous and soluble subfractions each i.e MAF1-5 and SAF1-5 and were subjected for evaluation of their ability to induce cellular responses. Out of five sub-fractions from each of membrane and soluble, only four viz. MAF2, MAF3, SAF2 and SAF3 were observed to stimulate remarkable lymphoproliferative, IFN-γ, IL-12 responses and Nitric Oxide production, in Leishmania-infected cured/exposed patients and hamsters. Results suggest the presence of Th-1 type immunostimulatory molecules in these sub-fractions which may further be exploited for developing a successful subunit vaccine from the less explored pathogenic stage against VL.

• Genetic and functional evaluation of the role of CXCR1 and CXCR2 in susceptibility to visceral leishmaniasis in north-east India.

  Authors: Sanjana Mehrotra, Michaela Fakiola, Joyce Oommen, Sarra E Jamieson, Anshuman Mishra, Medhavi Sudarshan, Puja Tiwary, Deepa Selvi Rani, Kumarasamy Thangaraj, Madhukar Rai, Shyam Sundar, Jenefer M Blackwell

  BMC medical genetics. 12/2011; 12:162.

  IL8RA and IL8RB, encoded by CXCR1 and CXCR2, are receptors for interleukin (IL)-8 and other CXC chemokines involved in chemotaxis and activation of polymorphonuclear neutrophils (PMN). Variants atIL8RA and IL8RB, encoded by CXCR1 and CXCR2, are receptors for interleukin (IL)-8 and other CXC chemokines involved in chemotaxis and activation of polymorphonuclear neutrophils (PMN). Variants at CXCR1 and CXCR2 have been associated with susceptibility to cutaneous and mucocutaneous leishmaniasis in Brazil. Here we investigate the role of CXCR1/CXCR2 in visceral leishmaniasis (VL) in India. Three single nucleotide polymorphisms (SNPs) (rs4674259, rs2234671, rs3138060) that tag linkage disequilibrium blocks across CXCR1/CXCR2 were genotyped in primary family-based (313 cases; 176 nuclear families; 836 individuals) and replication (941 cases; 992 controls) samples. Family- and population-based analyses were performed to look for association between CXCR1/CXCR2 variants and VL. Quantitative RT/PCR was used to compare CXCR1/CXCR2 expression in mRNA from paired splenic aspirates taken before and after treatment from 19 VL patients. Family-based analysis using FBAT showed association between VL and SNPs CXCR1_rs2234671 (Z-score = 2.935, P = 0.003) and CXCR1_rs3138060 (Z-score = 2.22, P = 0.026), but not with CXCR2_rs4674259. Logistic regression analysis of the case-control data under an additive model of inheritance showed association between VL and SNPs CXCR2_rs4674259 (OR = 1.15, 95%CI = 1.01-1.31, P = 0.027) and CXCR1_rs3138060 (OR = 1.25, 95%CI = 1.02-1.53, P = 0.028), but not with CXCR1_rs2234671. The 3-locus haplotype T_G_C across these SNPs was shown to be the risk haplotype in both family- (TRANSMIT; P = 0.014) and population- (OR = 1.16, P = 0.028) samples (combined P = 0.002). CXCR2, but not CXCR1, expression was down regulated in pre-treatment compared to post-treatment splenic aspirates (P = 0.021). This well-powered primary and replication genetic study, together with functional analysis of gene expression, implicate CXCR2 in determining outcome of VL in India.

• An oral formulation of amphotericin B attached to functionalized carbon nanotubes is an effective treatment for experimental visceral leishmaniasis.

  Authors: Vijay Kumar Prajapati, Kalpna Awasthi, Thakur Prasad Yadav, Madhukar Rai, Onkar Nath Srivastava, Shyam Sundar

  The Journal of infectious diseases. 12/2011; 205(2):333-6.

  Amphotericin B (AmB), is a highly effective antileishmanial agent used as first-line treatment in different formulations in visceral leishmaniasis endemic areas of Bihar, India. However, parenteralAmphotericin B (AmB), is a highly effective antileishmanial agent used as first-line treatment in different formulations in visceral leishmaniasis endemic areas of Bihar, India. However, parenteral infusion, prolonged hospitalization, and toxicity are major hurdles. Our previous work demonstrated the efficacy and stability of functionalized carbon nanotubes as a delivery mechanism for AmB. In this study, using the hamster model, we have shown that this novel formulation of AmB can be administered orally, resulting in 99% inhibition of parasite growth following a 5-day course at 15 mg/kg body weight.

• Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance.

  Authors: Tim Downing, Hideo Imamura, Saskia Decuypere, Taane G Clark, Graham H Coombs, James A Cotton, James D Hilley, Simonne de Doncker, Ilse Maes, Jeremy C Mottram, Mike A Quail, Suman Rijal, Mandy Sanders, Gabriele Schönian, Olivia Stark, Shyam Sundar, Manu Vanaerschot, Christiane Hertz-Fowler, Jean-Claude Dujardin, Matthew Berriman

  Genome research. 12/2011; 21(12):2143-56.

  Visceral leishmaniasis is a potentially fatal disease endemic to large parts of Asia and Africa, primarily caused by the protozoan parasite Leishmania donovani. Here, we report a high-qualityVisceral leishmaniasis is a potentially fatal disease endemic to large parts of Asia and Africa, primarily caused by the protozoan parasite Leishmania donovani. Here, we report a high-quality reference genome sequence for a strain of L. donovani from Nepal, and use this sequence to study variation in a set of 16 related clinical lines, isolated from visceral leishmaniasis patients from the same region, which also differ in their response to in vitro drug susceptibility. We show that whole-genome sequence data reveals genetic structure within these lines not shown by multilocus typing, and suggests that drug resistance has emerged multiple times in this closely related set of lines. Sequence comparisons with other Leishmania species and analysis of single-nucleotide diversity within our sample showed evidence of selection acting in a range of surface- and transport-related genes, including genes associated with drug resistance. Against a background of relative genetic homogeneity, we found extensive variation in chromosome copy number between our lines. Other forms of structural variation were significantly associated with drug resistance, notably including gene dosage and the copy number of an experimentally verified circular episome present in all lines and described here for the first time. This study provides a basis for more powerful molecular profiling of visceral leishmaniasis, providing additional power to track the drug resistance and epidemiology of an important human pathogen.

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• Residual activity and integrity of PermaNet(®) 2.0 after 24 months of household use in a community randomised trial of long lasting insecticidal nets against visceral leishmaniasis in India and Nepal.

  Authors: Albert Picado, Shri Prakash Singh, Veerle Vanlerberghe, Surendra Uranw, Bart Ostyn, Harparkash Kaur, Murari Lal Das, Shyam Sundar, Suman Rijal, Patrick Tungu, Marleen Boelaert, Mark Rowland

  Transactions of the Royal Society of Tropical Medicine and Hygiene. 12/2011; 106(3):150-9.

  The World Health Organization (WHO) recommends several brands of long lasting insecticidal net (LN) for protection against insect vectors but also advises national programmes to monitor and evaluateThe World Health Organization (WHO) recommends several brands of long lasting insecticidal net (LN) for protection against insect vectors but also advises national programmes to monitor and evaluate performance under local conditions to help them select the most suitable LN for their setting. During the course of a community randomised trial of LNs against visceral leishmaniasis in northern India and Nepal, opportunity arose to assess the efficacy of PermaNet 2.0 (Vestergaard-Frandsen, Denmark) after two years of use against sandfly vectors. Between 63% (India) and 78% (Nepal) of LNs became holed over the course of two years, deltamethrin residues fell from 55mg/m(2) to an average of 11.6mg/m(2) (India) and 27.9mg/m(2) (Nepal), but on the basis of bioassay criteria all LNs tested still met the WHO Pesticide Evaluation Scheme standard for LN effectiveness. Nets had on average only been washed 2.5 times (India) and 0.6 times (Nepal) by householders over the course of two years. The loss of insecticide was attributed to factors which had little or nothing to do with washing, such as handling, friction and torsion during daily use. Under conditions pertaining in this region of south Asia, and for two years at least, this brand of net continues to meet the criteria established by WHO for LNs.

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• Genome-wide SNP and microsatellite variation illuminate population-level epidemiology in the Leishmania donovani species complex.

  Authors: Tim Downing, Olivia Stark, Manu Vanaerschot, Hideo Imamura, Mandy Sanders, Saskia Decuypere, Simonne de Doncker, Ilse Maes, Suman Rijal, Shyam Sundar, Jean-Claude Dujardin, Matthew Berriman, Gabriele Schönian

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 11/2011; 12(1):149-59.

  The species of the Leishmania donovani species complex cause visceral leishmaniasis, a debilitating infectious disease transmitted by sandflies. Understanding molecular changes associated withThe species of the Leishmania donovani species complex cause visceral leishmaniasis, a debilitating infectious disease transmitted by sandflies. Understanding molecular changes associated with population structure in these parasites can help unravel their epidemiology and spread in humans. In this study, we used a panel of standard microsatellite loci and genome-wide SNPs to investigate population-level diversity in L. donovani strains recently isolated from a small geographic area spanning India, Bihar and Nepal, and compared their variation to that found in diverse strains of the L. donovani complex isolates from Europe, Africa and Asia. Microsatellites and SNPs could clearly resolve the phylogenetic relationships of the strains between continents, and microsatellite phylogenies indicated that certain older Indian strains were closely related to African strains. In the context of the anti-malaria spraying campaigns in the 1960s, this was consistent with a pattern of episodic population size contractions and clonal expansions in these parasites that was supported by population history simulations. In sharp contrast to the low resolution provided by microsatellites, SNPs retained a much more fine-scale resolution of population-level variability to the extent that they identified four different lineages from the same region one of which was more closely related to African and European strains than to Indian or Nepalese ones. Joining results of in vitro testing the antimonial drug sensitivity with the phylogenetic signals from the SNP data highlighted protein-level mutations revealing a distinct drug-resistant group of Nepalese and Indian L. donovani. This study demonstrates the power of genomic data for exploring parasite population structure. Furthermore, markers defining different genetic groups have been discovered that could potentially be applied to investigate drug resistance in clinical Leishmania strains.

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• Elongation factor-2, a Th1 stimulatory protein of Leishmania donovani, generates strong IFN-γ and IL-12 response in cured Leishmania-infected patients/hamsters and protects hamsters against Leishmania challenge.

  Authors: Pramod K Kushawaha, Reema Gupta, Shyam Sundar, Amogh A Sahasrabuddhe, Anuradha Dube

  Journal of immunology (Baltimore, Md. : 1950). 11/2011; 187(12):6417-27.

  In visceral leishmaniasis, Th1 types of immune responses correlate with recovery from and resistance to disease, and resolution of infection results in lifelong immunity against the disease.In visceral leishmaniasis, Th1 types of immune responses correlate with recovery from and resistance to disease, and resolution of infection results in lifelong immunity against the disease. Leishmanial Ags that elicit proliferative and cytokine responses in PBMCs from cured/exposed/Leishmania patients have been characterized through proteomic approaches, and elongation factor-2 is identified as one of the potent immunostimulatory proteins. In this study, we report the cloning and expression of Leishmania donovani elongation factor-2 protein (LelF-2) and its immunogenicity in PBMCs of cured/exposed Leishmania-infected patients and hamsters (Mesocricetus auratus). Leishmania-infected cured/exposed patients and hamsters exhibited significantly higher proliferative responses to recombinant Lelf-2 (rLelF-2) than those with L. donovani-infected hosts. The soluble L. donovani Ag stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLelF-2 stimulated the production of IFN-γ, IL-12, and TNF-α but not IL-4 or IL-10. Further, rLelF-2 downregulated LPS-induced IL-10 as well as soluble L. donovani Ag-induced IL-4 production by Leishmania patient PBMCs. The immunogenicity of rLelF-2 was also checked in hamsters in which rLelF-2 generates strong IL-12- and IFN-γ-mediated Th1 immune response. This was further supported by a remarkable increase in IgG2 Ab level. We further demonstrated that rLelF-2 was able to provide considerable protection (∼65%) to hamsters against L. donovani challenge. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFN-γ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-β. Hence, it is inferred that rLelF-2 elicits a Th1 type of immune response exclusively and confers considerable protection against experimental visceral leishmaniasis.

• Downregulation of mitogen-activated protein kinase 1 of Leishmania donovani field isolates is associated with antimony resistance.

  Authors: Ashutosh, Mansi Garg, Shyam Sundar, Robert Duncan, Hira L Nakhasi, Neena Goyal

  Antimicrobial agents and chemotherapy. 11/2011; 56(1):518-25.

  Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating inEmergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan Leishmania donovani (LdMAPK1) that was consistently downregulated in antimony-resistant field isolates. The expression level of the gene was validated by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed at the protein level in resistant isolates. Primary structure analysis of LdMAPK1 revealed the presence of all of the characteristic features of MAPK1. When expressed in Escherichia coli, the recombinant enzyme showed kinase activity with myelin basic protein as the substrate and was inhibited by staurosporine. Interestingly, overexpression of this gene in a drug-sensitive laboratory strain and a resistant field isolate resulted in increased the sensitivity of the transfectants to potassium antimony tartrate, suggesting that it has a role in antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may be in part correlated with antimony drug resistance in Indian VL isolates.

• Visceral leishmaniasis in the Indian subcontinent: modelling epidemiology and control.

  Authors: Anette Stauch, Ram Rup Sarkar, Albert Picado, Bart Ostyn, Shyam Sundar, Suman Rijal, Marleen Boelaert, Jean-Claude Dujardin, Hans-Peter Duerr

  PLoS neglected tropical diseases. 11/2011; 5(11):e1405.

  In the Indian subcontinent, about 200 million people are at risk of developing visceral leishmaniasis (VL). In 2005, the governments of India, Nepal and Bangladesh started the first regional VLIn the Indian subcontinent, about 200 million people are at risk of developing visceral leishmaniasis (VL). In 2005, the governments of India, Nepal and Bangladesh started the first regional VL elimination program with the aim to reduce the annual incidence to less than 1 per 10,000 by 2015. A mathematical model was developed to support this elimination program with basic quantifications of transmission, disease and intervention parameters. This model was used to predict the effects of different intervention strategies. Parameters on the natural history of Leishmania infection were estimated based on a literature review and expert opinion or drawn from a community intervention trial (the KALANET project). The transmission dynamic of Leishmania donovani is rather slow, mainly due to its long incubation period and the potentially long persistence of parasites in infected humans. Cellular immunity as measured by the Leishmanin skin test (LST) lasts on average for roughly one year, and re-infection occurs in intervals of about two years, with variation not specified. The model suggests that transmission of L. donovani is predominantly maintained by asymptomatically infected hosts. Only patients with symptomatic disease were eligible for treatment; thus, in contrast to vector control, the treatment of cases had almost no effect on the overall intensity of transmission. Treatment of Kala-azar is necessary on the level of the individual patient but may have little effect on transmission of parasites. In contrast, vector control or exposure prophylaxis has the potential to efficiently reduce transmission of parasites. Based on these findings, control of VL should pay more attention to vector-related interventions. Cases of PKDL may appear after years and may initiate a new outbreak of disease; interventions should therefore be long enough, combined with an active case detection and include effective treatment.

• Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples.

  Authors: Jason L Weirather, Selma M B Jeronimo, Shalini Gautam, Shyam Sundar, Mitchell Kang, Melissa A Kurtz, Rashidul Haque, Albert Schriefer, Sinésio Talhari, Edgar M Carvalho, John E Donelson, Mary E Wilson

  Journal of clinical microbiology. 11/2011; 49(11):3892-904.

  The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCRThe Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.

• Incidence of symptomatic and asymptomatic Leishmania donovani infections in high-endemic foci in India and Nepal: a prospective study.

  Authors: Bart Ostyn, Kamlesh Gidwani, Basudha Khanal, Albert Picado, François Chappuis, Shri Prakash Singh, Suman Rijal, Shyam Sundar, Marleen Boelaert

  PLoS neglected tropical diseases. 10/2011; 5(10):e1284.

  Incidence of Leishmania donovani infection and Visceral Leishmaniasis (VL) was assessed in a prospective study in Indian and Nepalese high-endemic villages. DAT-seroconversion was used as marker ofIncidence of Leishmania donovani infection and Visceral Leishmaniasis (VL) was assessed in a prospective study in Indian and Nepalese high-endemic villages. DAT-seroconversion was used as marker of incident infection in 3 yearly surveys. The study population was followed up to month 30 to identify incident clinical cases. In a cohort of 9034 DAT-negative individuals with neither active signs nor history of VL at baseline, 42 VL cases and 375 asymptomatic seroconversions were recorded in the first year, giving an infection:disease ratio of 8.9 to 1. In the 18 months' follow-up, 7 extra cases of VL were observed in the seroconverters group (N=375), against 14 VL cases among the individuals who had not seroconverted in the first year (N=8570) (RR=11.5(4.5<RR<28.3)). Incident asymptomatic L. donovani infection in VL high-endemic foci in India and Nepal is nine times more frequent than incident VL disease. About 1 in 50 of these new but latent infections led to VL within the next 18 months.