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PublicationArticle Multilocus microsatellite typing (MLMT) reveals genetic homogeneity of Leishmania donovani strains in the Indian subcontinent(2009) Mohammad Zahangir Alam; Katrin Kuhls; Carola Schweynoch; Shyam Sundar; Suman Rijal; Abul Khair M. Shamsuzzaman; Balaraju Venkata Subba Raju; Poonam Salotra; Jean-Claude Dujardin; Gabriele SchönianIn this population genetic study of Leishmania donovani parasites in the Indian subcontinent, 132 isolates obtained from patients in Bangladesh, India, Nepal and Sri Lanka suffering from Kala-azar (100), post-Kala-azar dermal leishmaniasis (PKDL) (25) and cutaneous leishmaniasis (CL) (2), and from 5 patients whose clinical patterns were not defined, were analysed by using 15 hyper-variable microsatellite loci. Multilocus microsatellite typing (MLMT) data were analysed by using a Bayesian model-based clustering algorithm and constructing phylogenic tree based on genetic distances. In total, 125 strains from Bangladesh, Bihar (India) and Nepal formed a very homogeneous population regardless of geographical origin, clinical manifestation, and whether they presented in vitro or in vivo susceptibility to antimonial drugs. Identical multilocus microsatellite profiles were found for 108 strains, other strains differed in only one marker. Considerably different microsatellite profiles were identified for three Indian strains most closely related to L. donovani from Kenya, and for four strains from Indian and Sri Lankan CL cases. The circulation of a single homogeneous population of L. donovani in Bihar (India), Bangladesh and Nepal is, most probably, related to the epidemic spread of visceral leishmaniasis in this area. © 2008 Elsevier B.V. All rights reserved.PublicationArticle Genome-wide SNP and microsatellite variation illuminate population-level epidemiology in the Leishmania donovani species complex(2012) 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 SchönianThe 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. © 2011 Elsevier B.V.PublicationReview Nanodiagnostics in leishmaniasis: A new frontiers for early elimination(Wiley-Blackwell, 2021) Mallikarjuna Rao Gedda; Prasoon Madhukar; Ashish Shukla; Shyam Lal Mudavath; Onkar Nath Srivastava; Om Prakash Singh; Shyam SundarVisceral leishmaniasis (VL) is still a major public health concern in developing countries having the highest outbreak and mortality potential. While the treatment of VL has greatly improved in recent times, the current diagnostic tools are limited for use in the post-elimination setting. Although conventional serological methods of detection are rapid, they can only differentiate between active disease in strict combination with clinical criteria, and thus are not sufficient enough to diagnose relapse patients. Therefore, there is a dire need for a portable, authentic, and reliable assay that does not require large space, specialized instrument facilities, or highly trained laboratory personnel and can be carried out in primary health care settings. Advances in the nanodiagnostic approaches have led to the expansion of new frontiers in the concerned area. The nanosized particles are blessed with an ability to interact one-on-one with the biomolecules because of their unique optical and physicochemical properties and high surface area to volume ratio. Biomolecular detection systems based on nanoparticles (NPs) are cost-effective, rapid, nongel, non-PCR, and nonculture based that provide fast, one-step, and reliable results with acceptable sensitivity and specificity. In this review, we discuss different NPs that are being used for the identification of molecular markers and other biomarkers, such as toxins and antigens associated with leishmaniasis. The most promising diagnostic approaches have been included in the article, and the ability of biomolecular recognition, advantages, and disadvantages have been discussed in detail to showcase the enormous potential of nanodiagnostics in human and veterinary medicine. This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Diagnostic Tools > Biosensing. © 2020 Wiley Periodicals LLC.PublicationArticle Epitope-binding characteristics for risk versus protective DRB1 alleles for visceral leishmaniasis(American Association of Immunologists, 2018) Toolika Singh; Michaela Fakiola; Joyce Oommen; Akhil Pratap Singh; Abhishek K. Singh; Noel Smith; Jaya Chakravarty; Shyam Sundar; Jenefer M. BlackwellHLA-DRB1 is the major genetic risk factor for visceral leishmaniasis (VL). We used SNP2HLA to impute HLA-DRB1 alleles and SNPTEST to carry out association analyses in 889 human cases and 977 controls from India. NetMHCIIpan 2.1 was used to map epitopes and binding affinities across 49 Leishmania vaccine candidates, as well as across peptide epitopes captured from dendritic cells treated with crude Leishmania Ag and identified using mass spectrometry and alignment to amino acid sequences of a reference Leishmania genome. Cytokines were measured in peptide-stimulated whole blood from 26 cured VL cases and eight endemic healthy controls. HLA-DRB1*1501 and DRB1*1404/DRB1*1301 were the most significant protective and risk alleles, respectively, with specific residues at aa positions 11 and 13 unique to protective alleles. We observed greater peptide promiscuity in sequence motifs for 9-mer core epitopes predicted to bind to risk (*1404/*1301) compared with protective (*1501) DRB1 alleles. There was a higher frequency of basic amino acids in DRB1*1404/*1301-specific epitopes compared with hydrophobic and polar amino acids in DRB1*1501-specific epitopes at anchor residues pocket 4 and pocket 6, which interact with residues at DRB1 positions 11 and 13. Cured VL patients made variable, but robust, IFN-g, TNF, and IL-10 responses to 20-mer peptides based on captured epitopes, with peptides based on DRB1*1501-captured epitopes resulting in a higher proportion (odds ratio 2.23, 95% confidence interval 1.17–4.25, p = 0.017) of patients with IFN-g/IL-10 ratios > 2-fold compared with peptides based on DRB1*1301-captured epitopes. Our data provide insight into the molecular mechanisms underpinning the association of HLA-DRB1 alleles with risk versus protection in VL in humans. Copyright © 2018 by The American Association of Immunologists, Inc.PublicationArticle Comparative proteomics and glycoproteomics of plasma proteins in Indian visceral leishmaniasis(BioMed Central Ltd., 2014) Arup K. Bag; Sutapa Saha; Shyam Sundar; Bibhuti Saha; Abhijit Chakrabarti; Chitra MandalBackground: Visceral leishmaniasis (VL) is a deadly parasitic diseases caused by Leishmania donovani; it is a major health problem in many countries. A lack of proper understanding of the disease biology, poor diagnostic methods and increasing drug resistance are the main reasons for the growing burden of VL infection. Comparative plasma proteomics are a relatively useful technique that can be used to investigate disease-associated alterations that can help in understanding host responses against pathogens, and might be useful in disease management and diagnosis.Result: In this study, a comparative proteomics and glycoproteomics approach using 2DE and 2D-DIGE was employed between early diagnosed VL patients of all age groups and healthy endemic and non-endemic controls in order to aid the recognition of disease-associated alterations in host plasma. Comparative proteomics was performed by the depletion of seven highly abundant plasma proteins. Comparative glycoproteomics was performed by the depletion of albumin and IgG, followed by purification of plasma glycoproteins using a multi lectin affinity column. From these two approaches, 39 differentially expressed protein spots were identified and sequenced using MALDI-TOF/TOF mass spectrometry. This revealed ten distinct proteins that appeared in multiple spots, suggesting micro-heterogeneity. Among these proteins, alpha-1-antitrypsin, alpha-1-B glycoprotein and amyloid-A1 precursor were up-regulated, whereas vitamin-D binding protein, apolipoprotein-A-I and transthyretin were down-regulated in VL. Alterations in the levels of these proteins in VL-infected plasma were further confirmed by western blot and ELISA.Conclusions: These proteins may be involved in the survival of parasites, resisting neutrophil elastase, and in their multiplication in macrophages, potentially maintaining endogenous anti-inflammatory and immunosuppressive conditions. Consequently, the results of this study may help in understanding the host response against L.donovani, which could help in the discovery of new drugs and disease management. Finally, these alterations on protein levels might be beneficial in improving early diagnosis considering those as biomarkers in Indian VL. © 2014 Bag et al.; licensee BioMed Central Ltd.PublicationArticle Ocular manifestations and human immunodeficiency virus retinopathy in patients with acquired immunodeficiency syndrome in North India(2006) V. Satya Suresh Attili; Vijay P. Singh; A.V. Bhaskar Reddy; Datla V. Varma; Madhukar Rai; Kumar A. Gulati; Shyam SundarAim: To evaluate the spectrum of eye diseases in patients with human immunodeficiency virus, with special reference to human immunodeficiency virus retinopathy and its risk factors. Methods: All patient with human immunodeficiency virus attending the Infectious Disease Clinic at Sir Sunderlal Hospital, Institute of Medical Sciences, Banaras Hindu University, India, between January 2001 and December 2003 were enrolled. All patients underwent a thorough eye examination. Patients with acquired immunodeficiency syndrome retinopathy were analysed separately for predisposing factors. The risk factors analysed were positive C-reactive protein, low CD4 levels, associated central nervous system infections, and other eye diseases. Statistical analysis was done using Medcalc version 7.5. Results: Of 460 analysable patients, 88 patients (19%) had some eye manifestations, and 54 patients had human immunodeficiency virus retinopathy. Univariate analysis disclosed that immunosuppression (CD4 levels, <20/μL), positive C-reactive protein, associated eye lesions, and any central nervous system pathology were significant risk factors for the development of human immunodeficiency virus/acquired immunodeficiency syndrome retinopathy. Conclusions: Human immunodeficiency virus could be a risk factor for retinopathy as a significant number of patients did not have any other findings or risk factors for the development of retinopathy. However, it would be premature to draw definitive conclusions about the risk factors, as the number of patients analysed was small. © 2006 Scientific Communications International Limited.PublicationArticle Relationship between skin diseases and CD4 cell counts in a hospital-based cohort of HIV-infected adults in North India(2008) V. Satya Suresh Attili; V.P. Singh; Shyam Sundar; A.K. Gulati; D.V. Varma; M. RaiBackground: Dermatological manifestations are seen at every stage of HIV/AIDS, and are often the presenting features. These manifestations not only act as markers but also reflect the underlying immune status. Objectives: To examine the relationship between various skin diseases and CD4 cell counts in a hospital-based cohort of HIV-infected adults in and around Varanasi, North India. Patients and methods: All HIV patients attending the SS (Sir Sunderlal) hospital, Varanasi, between January, 2001 and December, 2003 were studied. The relationship between CD4 counts and various skin diseases was analysed. Results: Rate of cutaneous diseases was 223 episodes per 1,000 PYO. CD4 counts were significantly lower in individuals with some skin diseases (warts, psoriasis, etc.) as compared to healthy HIV persons and some other skin disorders (dermatophyte skin infections, scabies, etc.). In HIV patients, there is a significant fall in the CD4 levels from baseline, when the patient develops a skin disease and recovery of CD4 is also prompt as soon as the patient recovers from the skin disease. Conclusion: There is a strong negative association between CD4 counts and the incidence and severity of skin disease in the HIV/AIDS patients. Fluctuations in CD4 levels observed during skin disease require further studies to establish the underlying pathophysiology. It may not be advisable to rely on CD4 levels in HIV patients having active skin disease, as transient but reversible fall in CD4 counts are known during the period of active skin disease.PublicationArticle Leishmania Specific CD4 T Cells Release IFNγ That Limits Parasite Replication in Patients with Visceral Leishmaniasis(Public Library of Science, 2014) Rajiv Kumar; Neetu Singh; Shalini Gautam; Om Prakash Singh; Kamlesh Gidwani; Madhukar Rai; David Sacks; Shyam Sundar; Susanne NylénVisceral leishmaniasis (VL) is associated with increased circulating levels of multiple pro-inflammatory cytokines and chemokines, including IL-12, IFNγ, and TNFα, and elevated expression of IFNγ mRNA in lesional tissue such as the spleen and bone marrow. However, an immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) typically fail to respond to stimulation with leishmanial antigen. Unexpectedly, it was recently shown that Leishmania specific IFNγ, can readily be detected when a whole blood stimulation assay (WBA) is used. We sought to define the conditions that permit whole blood cells to respond to antigen stimulation, and clarify the biological role of the IFNγ found to be released by cells from VL patients. CD4+ T cells were found to be crucial for and the main source of the IFNγ production in Leishmania stimulated whole blood (WB) cultures. Complement, antibodies and red blood cells present in whole blood do not play a significant role in the IFNγ response. The IFNγ production was reduced by blockade of human leukocyte antigen (HLA)-DR, indicating that the response to leishmanial antigens observed in WB of active VL patients is a classical HLA- T cell receptor (TCR) driven reaction. Most importantly, blockade of IFNγ in ex-vivo splenic aspirate cultures demonstrated that despite the progressive nature of their disease, the endogenous IFNγ produced in patients with active VL serves to limit parasite growth. © 2014.PublicationArticle Single-dose liposomal amphotericin B in the treatment of visceral leishmaniasis in India: A multicenter study(2003) Shyam Sundar; T.K. Jha; C.P. Thakur; M. Mishra; V.P. Singh; R. BuffelsWidespread antimony resistance renders conventional amphotericin B the only option for the treatment of visceral leishmaniasis (VL) in North Bihar, India. Because of its excellent safety profile, a large dose (7.5 mg/ kg) of liposomal amphotericin B (L-AmB) was given to each of 203 patients with VL at 4 treatment centers, and the patients were discharged the next day. At initial clinical and parasitological follow-up, performed on day 30 after treatment, evidence of a cure was seen in 195 (96%) of 203 patients (95% CI, 92-98); 4 patients experienced treatment failure. Two patients were lost to follow-up, 2 died (one due to progressive disease and another, 5 months after treatment, due to an unrelated illness), and 12 experienced relapses during follow-up. Thus, 183 patients (90%; 95% CI, 85-94) had obtained final cure 6 months after treatment. Very few adverse events (fever with rigor, in 9.8% of patients) were seen. Single-dose L-AmB (7.5 mg/kg) treatment is safe and effective, and it may be used for the mass treatment of VL in India.PublicationArticle Determinants for progression from asymptomatic infection to symptomatic visceral leishmaniasis: A cohort study(Public Library of Science, 2018) Jaya Chakravarty; Epco Hasker; Sangeeta Kansal; Om Prakash Singh; Paritosh Malaviya; Abhishek Kumar Singh; Ankita Chourasia; Toolika Singh; Medhavi Sudarshan; Akhil Pratap Singh; Bhawana Singh; Rudra Pratap Singh; Bart Ostyn; Michaela Fakiola; Albert Picado; Joris Menten; Jenefer M. Blackwell; Mary E. Wilson; David Sacks; Marleen Boelaert; Shyam SundarBackground: Asymptomatic Leishmania donovani infections outnumber clinical presentations, however the predictors for development of active disease are not well known. We aimed to identify serological, immunological and genetic markers for progression from L. donovani infection to clinical Visceral Leishmaniasis (VL). Methods: We enrolled all residents >2 years of age in 27 VL endemic villages in Bihar (India). Blood samples collected on filter paper on two occasions 6–12 months apart, were tested for antibodies against L. donovani with rK39-ELISA and DAT. Sero converters, (negative for both tests in the first round but positive on either of the two during the second round) and controls (negative on both tests on both occasions) were followed for three years. At the start of follow-up venous blood was collected for the following tests: DAT, rK39- ELISA, Quantiferon assay, SNP/HLA genotyping and L.donovani specific quantitative PCR. Results: Among 1,606 subjects enrolled,17 (8/476 seroconverters and 9/1,130 controls) developed VL (OR 3.1; 95% CI 1.1–8.3). High DAT and rK39 ELISA antibody titers as well as positive qPCR were strongly and significantly associated with progression from seroconversion to VL with odds ratios of 19.1, 30.3 and 20.9 respectively. Most VL cases arose early (median 5 months) during follow-up. Conclusion: We confirmed the strong association between high DAT and/or rK39 titers and progression to disease among asymptomatic subjects and identified qPCR as an additional predictor. Low predictive values do not warrant prophylactic treatment but as most progressed to VL early during follow-up, careful oberservation of these subjects for at least 6 months is indicated. © 2018, Public Library of Science. All rights reserved. https://creativecommons.org/publicdomain/zero/1.0/.