Browsing by Author "Toolika Singh"

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A correlative study of splenic parasite score and peripheral blood parasite load estimation by quantitative PCR in visceral leishmaniasis
(American Society for Microbiology, 2015) Medhavi Sudarshan; Toolika Singh; Jaya Chakravarty; Shyam Sundar
Parasitological diagnosis of visceral leishmaniasis (VL) by splenic smear is highly sensitive, but it is associated with the risk of severe hemorrhage. In this study, the diagnosis of VL using quantitative PCR (qPCR) in peripheral blood was evaluated in 100 patients with VL. Blood parasitemia ranged from 5 to 93,688 leishmania parasite genomes/ml of blood and positively correlated with splenic score (P<0.0001; r2=0.58). Therefore, quantification of parasite genomes by qPCR can replace invasive procedures for diagnostic and prognostic evaluations. © 2015, American Society for Microbiology.
Cyanobacterial genome editing toolboxes: recent advancement and future projections for basic and synthetic biology researches
(Elsevier, 2020) Sandeep Kumar Singh; Alok Kumar Shrivastava; Ajay Kumar; Vipin Kumar Singh; Deepanker Yadav; Arpan Modi; Wenjing Wang; Avinash Singh; Toolika Singh; Viji Sitther; Prashant Kumar Singh
Cyanobacteria are ancient photosynthetic prokaryotes and serve as a model organism for studies such as photosynthesis and earth elements cycling. Cyanobacteria also termed microbial cell factories, because of their ability to utilize carbon dioxide, as well as sunlight absorption in one way, act as primary producers of the aquatic ecosystem while on the other way the diazotrophic forms fix atmospheric nitrogen in paddy fields. Moreover, the successful invention of several fuels as well as fine chemicals from cyanobacteria is indicative of the advancement in synthetic cyanobacteriology in one hand while on the other knocking the door of the augmented application shortly. Unfortunately, the limited availability of genetic manipulation tools for either at single-gene level or pathway to the whole genome in cyanobacteria compared to other organisms handicaps the fundamental biology researches as well as further application and advancement in synthetic cyanobacteriology. However, the role of genetic tools in tuning gene expression, genome-wide manipulations, and carbon flux redirections is available in cyanobacteria. Furthermore, recently a noteworthy headway evolving to familiarizing novel and efficient genetic manipulations tools such as riboswitches, promoters, ribosome-binding site engineering, small RNA regulatory tools, genome-scale modeling strategies, and clustered regularly interspaced short palindromic repeats-associated nuclease has revolutionized the cyanobacteriology. The present chapter disapprovingly recapitulates the recent advancement on the applications and development as well as technical limitations also for the future projections of the toolboxes for genetic manipulation of cyanobacterial genes/genomes. Besides this, the chapter also briefly discusses the toolkits feasible for large-scale cultivation of cyanobacteria. © 2020 Elsevier Inc. All rights reserved.
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 Sundar
Background: 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/.
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. Blackwell
HLA-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.
Genetic heterogeneity in clinical isolates of Leishmania donovani from India
(2011) Pankaj Srivastava; Toolika Singh; Shyam Sundar
Genetic diversity within 45 Indian Leishmania donovani isolates was analyzed using seven genetic markers. While kinetoplast DNA (kDNA) analysis revealed 15 genotypes, 8 genotypes were obtained by analysis of other markers. In contrast to earlier reports, our data suggest that significant genetic polymorphisms exist in L. donovani strains in Bihar, India. Our results confirm the presence of 2 zymodemes in India. Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Precision medicine: Tailoring therapeutics through advanced modeling strategies
(Elsevier, 2025) Sakshi Singh; Payal Singh; Toolika Singh
Precision medicine is an advanced approach towards developing new therapies to properly understand the cause of disease for providing better treatment and prevention. Each affected individual can be treated better through data-based decision-making approach. Mapping clinical history of the patient to take appropriate action regarding selection of drug, dosage, administration time, and other actions involving statistical research for treatment and care is possible due to personalized medication. It is the transition from treating disease based on symptoms and diagnosis to a method targeting disease prior to its occurrence, providing better cure if it cannot be avoided. Precision medicine provides improved patient outcomes through artificial intelligence, optimized use of tools and medicines, omics, social and environmental factors along with population and preventive medicine. Patient's genotypic and phenotypic data is maintained through biomarkers, laboratory, and radiological testings. This chapter will explain the importance of precision medicine and provide different strategies to deliver it in practice. © 2026 Elsevier Inc. All rights reserved.
Quantitative PCR in Epidemiology for Early Detection of Visceral Leishmaniasis Cases in India
(Public Library of Science, 2014) Medhavi Sudarshan; Toolika Singh; Abhishek Kumar Singh; Ankita Chourasia; Bhawana Singh; Mary E. Wilson; Jaya Chakravarty; Shyam Sundar
Studies employing serological, DTH or conventional PCR techniques suggest a vast proportion of Leishmania infected individuals living in regions endemic for Visceral Leishmaniasis (VL) remain asymptomatic. This study was designed to assess whether quantitative PCR (qPCR) can be used for detection of asymptomatic or early Leishmania donovani infection and as a predictor of progression to symptomatic disease.The study included 1469 healthy individuals living in endemic region (EHC) including both serology-positive and -negative subjects. TaqMan based qPCR assay was done on peripheral blood of each subject using kDNA specific primers and probes.A large proportion of EHC 511/1469 (34.78%) showed qPCR positivity and 56 (3.81% of 1469 subjects) had more than 1 calculated parasite genome/ml of blood. However, the number of individuals with parasite load above 5 genomes/ml was only 20 (1.36% of 1469). There was poor agreement between serological testing and qPCR (k = 0.1303), and 42.89% and 31.83% EHC were qPCR positive in seropositive and seronegative groups, respectively. Ten subjects had developed to symptomatic VL after 12 month of their follow up examination, of which eight were initially positive according to qPCR and among these, five had high parasite load.Thus, qPCR can help us to detect significant early parasitaemia, thereby assisting us in recognition of potential progressors to clinical disease. This test could facilitate early intervention, decreased morbidity and mortality, and possibly interruption of disease transmission. © 2014.
Suppression of host PTEN gene expression for Leishmania donovani survival in Indian visceral leishmaniasis
(Elsevier Masson SAS, 2016) Medhavi Sudarshan; Toolika Singh; Bhawana Singh; Jaya Chakravarty; Shyam Sundar
Lipid phosphatase, PTEN is amongst the host gene actively involved in determining disease susceptibility. Expression of pten and other genes in vicinity egr1 & 4e-bp1 were evaluated in splenic tissue before and after treatment in visceral leishmaniasis patients. Lower expression of egr1 in correlation with pten suppressed 4e-bp1 gene in active cases. The higher levels of pten mRNA expression post treatment confirmed its role in effective clearance of Leishmania. Therefore, it is hypothesized that lower mRNA expression of pten is due to suppression of egr1 activates PI3K signaling bestowing host the ability to cope up infection and continue its normal metabolic machinery. © 2016 Institut Pasteur
Transcriptional blood signatures for active and amphotericin B treated visceral leishmaniasis in India
(Public Library of Science, 2019) Michaela Fakiola; Om Prakash Singh; Genevieve Syn; Toolika Singh; Bhawana Singh; Jaya Chakravarty; Shyam Sundar; Jenefer M. Blackwell
Amphotericin B provides improved therapy for visceral leishmaniasis (VL) caused by Leishmania donovani, with single dose liposomal-encapsulated Ambisome providing the best cure rates. The VL elimination program aims to reduce the incidence rate in the Indian subcontinent to <1/10,000 population/year. Ability to predict which asymptomatic individuals (e.g. anti-leishmanial IgG and/or Leishmania-specific modified Quantiferon positive) will progress to clinical VL would help in monitoring disease outbreaks. Here we examined whole blood transcriptional profiles associated with asymptomatic infection, active disease, and in treated cases. Two independent microarray experiments were performed, with analysis focussed primarily on differentially expressed genes (DEGs) concordant across both experiments. No DEGs were identified for IgG or Quantiferon positive asymptomatic groups compared to negative healthy endemic controls. We therefore concentrated on comparing concordant DEGs from active cases with all healthy controls, and in examining differences in the transcriptome following different regimens of drug treatment. In these comparisons 6 major themes emerged: (i) expression of genes and enrichment of gene sets associated with erythrocyte function in active cases; (ii) strong evidence for enrichment of gene sets involved in cell cycle in comparing active cases with healthy controls; (iii) identification of IFNG encoding interferon-γ as the major hub gene in concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; (iv) enrichment for interleukin signalling (IL-1/3/4/6/7/8) and a prominent role for CXCL10/9/11 and chemokine signalling pathways in comparing active cases with treated cases; (v) the novel identification of Aryl Hydrocarbon Receptor signalling as a significant canonical pathway when comparing active cases with healthy controls or with treated cases; and (vi) global expression profiling support for more effective cure at day 30 post-treatment with a single dose of liposomal encapsulated amphotericin B compared to multi-dose non-liposomal amphotericin B treatment over 30 days. (296 words; 300 words allowed). © 2019 Fakiola et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.