Browsing by Author "Jean-Claude Dujardin"

Now showing 1 - 20 of 24

A Novel Bioimpedance-Based Detection of Miltefosine Susceptibility Among Clinical Leishmania donovani Isolates of the Indian Subcontinent Exhibiting Resistance to Multiple Drugs
(Frontiers Media S.A., 2021) Souradeepa Ghosh; Souvik Biswas; Sandip Mukherjee; Arijit Pal; Aaditya Saxena; Shyam Sundar; Jean-Claude Dujardin; Soumen Das; Syamal Roy; Rupkatha Mukhopadhyay; Budhaditya Mukherjee
The extent of susceptibility towards miltefosine (Mil), amphotericin B (AmpB), and paromomycin (Paro) was measured among 19 clinical isolates of Leishmania donovani (LD). Thirteen of these clinical isolates were reported to exhibit low susceptibility towards sodium stibogluconate (SSG-R), while six of them were highly susceptible (SSG-S). The degree of clearance of amastigotes (EC50) for these predefined SSG-R- and SSG-S-infected macrophages was determined against Mil, AmpB, and Paro. Two out of the 13 SSG-R isolates (BHU575 and BHU814) showed low susceptibility towards all three drugs studied, while the rest of the 11 SSG-R isolates showed varying degrees of susceptibility either towards none or only towards individual drugs. Interestingly, all the SSG-S isolates showed high susceptibility towards Mil/AmpB/Paro. The total intracellular non-protein thiol content of the LD promastigotes, which have been previously reported to be positively co-related with EC50 towards SSG, was found to be independent from the degree of susceptibility towards Mil/AmpB/Paro. Impedance spectra analysis, which quantifies membrane resistance, revealed lower impedimetric values for all those isolates exhibiting low efficacy to Mil (Mil-R). Our analysis points out that while non-protein thiol content can be an attribute of SSG-R, lower impedimetric values can be linked with lower Mil susceptibility, although neither of these parameters seems to get influenced by the degree of susceptibility towards AmpB/Paro. Finally, a correlation analysis with established biological methods suggests that impedance spectral analysis can be used for the accurate determination of lower Mil susceptibility among LD isolates, which is further validated in the LD-infected in vivo hamster model. Copyright © 2021 Ghosh, Biswas, Mukherjee, Pal, Saxena, Sundar, Dujardin, Das, Roy, Mukhopadhyay and Mukherjee.
Antimony-resistant but not antimony-sensitive Leishmania donovani up-regulates host IL-10 to overexpress multidrug-resistant protein 1
(2013) Budhaditya Mukherjee; Rupkatha Mukhopadhyay; Bijoylaxmi Bannerjee; Sayan Chowdhury; Sandip Mukherjee; Kshudiram Naskar; Uday Sankar Allam; Dipshikha Chakravortty; Shyam Sundar; Jean-Claude Dujardin; Syamal Roy
The molecular mechanism of antimony-resistant Leishmania donovani (Sb RLD)-driven up-regulation of IL-10 and multidrug-resistant protein 1 (MDR1) in infected macrophages (Mφs) has been investigated. This study showed that both promastigote and amastigote forms of SbRLD, but not the antimony-sensitive form of LD, express a unique glycan with N-acetylgalactosamine as a terminal sugar. Removal of it either by enzyme treatment or by knocking down the relevant enzyme, galactosyltransferase in SbRLD (KD SbRLD), compromises the ability to induce the above effects. Infection of Mφs with KD SbRLD enhanced the sensitivity toward antimonials compared with infection with SbRLD, and infection of BALB/c mice with KD SbRLD caused significantly less organ parasite burden compared with infection induced by SbRLD. The innate immune receptor, Toll-like receptor 2/6 heterodimer, is exploited by SbRLD to activate ERK and nuclear translocation of NF-κB involving p50/c-Rel leading to IL-10 induction, whereas MDR1 up-regulation is mediated by PI3K/Akt and the JNK pathway. Interestingly both recombinant IL-10 and SbRLD up-regulate MDR1 in Mφ with different time kinetics, where phosphorylation of PI3K was noted at 12 h and 48 h, respectively, but Mφs derived from IL-10-/- mice are unable to show MDR1 up-regulation on infection with SbRLD. Thus, it is very likely that an IL-10 surge is a prerequisite for MDR1 up-regulation. The transcription factor important for IL-10-driven MDR1 up-regulation is c-Fos/c-Jun and not NF-κB, as evident from studies with pharmacological inhibitors and promoter mapping with deletion constructs.
Antimony-resistant Leishmania donovani Exploits MIR-466i to deactivate host MyD88 for regulating IL-10/IL-12 levels during early hours of infection
(American Association of Immunologists, 2015) Budhaditya Mukherjee; Joydeep Paul; Sandip Mukherjee; Rupkatha Mukhopadhyay; Shantanabha Das; Kshudiram Naskar; Shyam Sundar; Jean-Claude Dujardin; Bhaskar Saha; Syamal Roy
Infection with antimony-resistant Leishmania donovani (SbRLD) induces aggressive pathology in the mammalian hosts as compared with ones with antimony-sensitive L. donovani (SbSLD) infection. SbRLD, but not SbSLD, interacts with TLR2/TLR6 to induce IL-10 by exploiting p50/c-Rel subunits of NF-κB in infected macrophages (Mfs). Most of the TLRs exploit the universal adaptor protein MyD88 to activate NF-κB. We now show that infection of Mfs from MyD882/2 mice with SbRLD gave rise to significantly higher intracellular parasite number coupled with elevated IL-10/IL-12 ratio in the culture supernatant as compared with infection in wild type (WT) Mfs. These attributes were not seen with SbSLD in similar experiments. Further, SbRLD infection upregulated miR-466i, which binds with 39-untranslated region, leading to the downregulation of MyD88. Infection of MyD882/2 Mf or IL-12-/- Mf with SbRLD induced IL-10 surge at 4 h, whereas the same in WT Mf started from 12 h. Thus, absence of IL-12 in MyD882/2 mice favored early binding of NF-κB subunits to the IL-10 promoter, resulting in IL-10 surge. Infection of MyD882/2 mice with SbRLD showed significantly higher organ parasites coupled with ill-defined and immature hepatic granulomas, whereas in WT mice there were less organ parasites and the granulomas were well defined. From the survival kinetics it was observed that SbRLD-infected MyD882/2 mice died by 60 d postinfection, whereas the WT mice continued to survive. Our results demonstrate that SbRLD has evolved a unique strategy to evade host antileishmanial immune repertoire by manipulating host MyD88 to its advantage. © 2015 by The American Association of Immunologists, Inc.
Efficacy of miltefosine in the treatment of visceral leishmaniasis in India after a decade of use
(2012) Shyam Sundar; Anup Singh; Madhukar Rai; Vijay K. Prajapati; Avinash K. Singh; Bart Ostyn; Marleen Boelaert; Jean-Claude Dujardin; Jaya Chakravarty
Background. Miltefosine 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 III trial in the 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. Methods. An open-label, noncomparative study was performed in which 567 patients received oral miltefosine (50 mg for patients weighing <25 kg, 100 mg in divided doses for those weighing =25 kg, and 2.5 mg per kg for those aged <12 years, daily for 28 days) in a directly observed manner. Patients were followed up for 6 months to see the response to therapy. Results. At the end of treatment the initial cure rate was 97.5% (intention to treat), and 6 months after the end of treatment the final cure rate was 90.3%. The overall death rate was 0.9% (5 of 567), and 2 deaths were related to drug toxicity. Gastrointestinal intolerance was frequent (64.5%). The drug was interrupted in 9 patients (1.5%) because of drug-associated adverse events. Conclusions. As compared to the phase III trial that 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. © The Author 2012.
Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent
(eLife Sciences Publications Ltd, 2016) Hideo Imamura; Tim Downing; Frederik van den Broeck; Mandy J. Sanders; Suman Rijal; Shyam Sundar; An Mannaert; Manu Vanaerschot; Maya Berg; Géraldine de Muylder; Franck Dumetz; Bart Cuypers; Ilse Maes; Malgorzata Domagalska; Saskia Decuypere; Keshav Rai; Surendra Uranw; Narayan Raj Bhattarai; Basudha Khanal; Vijay Kumar Prajapati; Smriti Sharma; Olivia Stark; Gabriele Schönian; Harry P. de Koning; Luca Settimo; Benoit Vanhollebeke; Syamal Roy; Bart Ostyn; Marleen Boelaert; Louis Maes; Matthew Berriman; Jean-Claude Dujardin; James A. Cotton
Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector- borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment. © Imamura et al.
Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: Outcome dependent on in vitro selection protocol
(2012) Sarah Hendrickx; Raquel Andrea Inocêncio da Luz; Vasundhra Bhandari; Kristel Kuypers; Craig D. Shaw; Julien Lonchamp; Poonam Salotra; Katharine Carter; Shyam Sundar; Suman Rijal; Jean-Claude Dujardin; Paul Cos; Louis Maes
Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 μM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 μM inclusion level that did not affect normal growth. Comparison of the IC50 values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC50 = 199 μM) compared to the parent strain (IC50 = 45 μM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field. © 2012 Hendrickx et al.
Failure of miltefosine treatment for visceral leishmaniasis in children and men in South-East Asia
(Public Library of Science, 2014) Bart Ostyn; Epco Hasker; Thomas P. C. Dorlo; Suman Rijal; Shyam Sundar; Jean-Claude Dujardin; Marleen Boelaert
Background: High frequency of relapse in miltefosine-treated visceral leishmaniasis (VL) patients in India and Nepal followed up for twelve months. Objective: To identify epidemiological and clinical risk factors for relapse of VL in patients recently treated with standard dosing of miltefosine in India and Nepal. Design: Prospective observational study in three Primary Health Centers and one reference center in Muzaffarpur district, Bihar, India; and two zonal hospitals and a university hospital in South-east Nepal; records of all consenting patients diagnosed with VL and treated with miltefosine according to the current treatment guidelines of the Kala azar elimination program between 2009 and 2011. Results: We compared the clinical records of 78 cases of relapse with those of 775 patients who had no record of subsequent relapse. Relapse was 2 times more common amongst male patients (IRR 2.14, 95% CI 1.27-3.61), and 2 to 3 times more frequent in the age groups below 15 compared to the over 25 year olds (age 10 to 14: IRR 2.53; 95% CI 1.37-4.65 and Age 2 to 9: IRR 3.19; 95% CI 1.77-5.77). History of earlier VL episodes, or specific clinical features at time of diagnosis such as duration of symptoms or spleen size were no predictors of relapse. Conclusions: Young age and male gender were associated with increased risk of VL relapse after miltefosine, suggesting that the mechanism of relapse is mainly host-related i.e. immunological factors and/or drug exposure (pharmacokinetics). The observed decrease in efficacy of miltefosine may be explained by the inclusion of younger patients compared to the earlier clinical trials, rather than by a decreased susceptibility of the parasite to miltefosine. Our findings highlight the importance of proper clinical trials in children, including pharmacokinetics, to determine the safety, efficacy, drug exposure and therapeutic response of new drugs in this age group. © 2014 Ostyn et al.
Four layer multi-omics reveals molecular responses to aneuploidy in Leishmania
(Public Library of Science, 2022) Bart Cuypers; Pieter Meysman; Ionas Erb; Wout Bittremieux; Dirk Valkenborg; Geert Baggerman; Inge Mertens; Shyam Sundar; Basudha Khanal; Cedric Notredame; Jean-Claude Dujardin; Malgorzata A. Domagalska; Kris Laukens
Aneuploidy causes system-wide disruptions in the stochiometric balances of transcripts, proteins, and metabolites, often resulting in detrimental effects for the organism. The protozoan parasite Leishmania has an unusually high tolerance for aneuploidy, but the molecular and functional consequences for the pathogen remain poorly understood. Here, we addressed this question in vitro and present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. Our analyses unambiguously establish that aneuploidy in Leishmania proportionally impacts the average transcript- and protein abundance levels of affected chromosomes, ultimately correlating with the degree of metabolic differences between closely related aneuploid strains. This proportionality was present in both proliferative and non-proliferative in vitro promastigotes. However, as in other Eukaryotes, we observed attenuation of dosage effects for protein complex subunits and in addition, non-cytoplasmic proteins. Differentially expressed transcripts and proteins between aneuploid Leishmania strains also originated from non-aneuploid chromosomes. At protein level, these were enriched for proteins involved in protein metabolism, such as chaperones and chaperonins, peptidases, and heat-shock proteins. In conclusion, our results further support the view that aneuploidy in Leishmania can be adaptive. Additionally, we believe that the high karyotype diversity in vitro and absence of classical transcriptional regulation make Leishmania an attractive model to study processes of protein homeostasis in the context of aneuploidy and beyond. © 2022 Cuypers 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.
Genetic markers for SSG resistance in leishmania donovani and SSG treatment failure in visceral leishmaniasis patients of the Indian subcontinent
(2012) Manu Vanaerschot; Saskia Decuypere; Tim Downing; Hideo Imamura; Olivia Stark; Simonne De Doncker; Syamal Roy; Bart Ostyn; Louis Maes; Basudha Khanal; Marleen Boelaert; Gabriele Schönian; Matthew Berriman; François Chappuis; Jean-Claude Dujardin; Shyam Sundar; Suman Rijal
The current standard to assess pentavalent antimonial (SSG) susceptibility of Leishmania is a laborious in vitro assay of which the result has little clinical value because SSG-resistant parasites are also found in SSG-cured patients. Candidate genetic markers for clinically relevant SSG-resistant parasites identified by full genome sequencing were here validated on a larger set of clinical strains. We show that 3 genomic locations suffice to specifically detect the SSG-resistant parasites found only in patients experiencing SSG treatment failure. This finding allows the development of rapid assays to monitor the emergence and spread of clinically relevant SSG-resistant Leishmania parasites. © The Author 2012.
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 Schönian
The 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.
Imipramine exploits histone deacetylase 11 to increase the IL-12/IL-10 ratio in macrophages infected with antimony-resistant Leishmania donovani and clears organ parasites in experimental infection
(American Association of Immunologists, 2014) Sandip Mukherjee; Budhaditya Mukherjee; Rupkatha Mukhopadhyay; Kshudiram Naskar; Shyam Sundar; Jean-Claude Dujardin; Syamal Roy
The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kalaazar patients infected with antimony-resistant Leishmania donovani (SbRLD). Previously we showed that MDR-1 upregulation in SbRLD infection is IL-10-dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from SbRLD-infected macrophages (SbRLD-Mφs) and favors accumulation of surrogates of antimonials. It inhibits IL-10-driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from SbRLDMfs. It abrogates SbRLD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in SbRLD-Mφs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in SbRLD-Mφs. Furthermore, exogenous rIL-12 inhibits intracellular SbRLD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in SbRLD-Mφs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ SbRLD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on SbRLD clearance from infected hosts. Copyright © 2014 by The American Association of Immunologists, Inc.
In vitro susceptibility of leishmania donovani to miltefosine in Indian visceral leishmaniasis
(2013) Vijay Kumar Prajapati; Smriti Sharma; Madhukar Rai; Bart Ostyn; Poonam Salotra; Manu Vanaerschot; Jean-Claude Dujardin; Shyam Sundar
Promastigote miltefosine (MIL) susceptibility was performed on Leishmania donovani isolates from Indian patients with visceral leishmaniasis treated with MIL. Isolates that were obtained before the onset of MIL treatment, after completion of treatment (29th day), or at the time of treatment failure, were screened using in vitro promastigote assay. The MIL susceptibility of the pre-treatment isolates (N = 24, mean IC50 ± SEM= 3.74 ± 0.38 mM) was significantly higher than that of the post-treatment group (N = 26, mean IC50 ± SEM = 6.15 ± 0.52 mM; P = 0.0006) but was similar in the cured patients (N = 22, mean IC50 ± SEM= 5.58 ± 0.56 mM) and those who failed treatment (N = 28, mean IC50 ± SEM= 4.53 ± 0.47 mM). The pre/post-treatment results thus showed a 2-fold difference, whereas isolated from cured versus failed patients showed a similar susceptibility, suggesting that this higher tolerance is not responsible forMIL-treatment failure. Our work highlights the need for careful monitoring of MIL susceptibility for implementation in national VL elimination programs. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.
Model-Based Investigations of Different Vector-Related Intervention Strategies to Eliminate Visceral Leishmaniasis on the Indian Subcontinent
(Public Library of Science, 2014) Anette Stauch; Hans-Peter Duerr; Albert Picado; Bart Ostyn; Shyam Sundar; Suman Rijal; Marleen Boelaert; Jean-Claude Dujardin; Martin Eichner
The elimination of infectious diseases requires reducing transmission below a certain threshold. The Visceral Leishmaniasis (VL) Elimination Initiative in Southeast Asia aims to reduce the annual VL incidence rate below 1 case per 10,000 inhabitants in endemic areas by 2015 via a combination of case management and vector control. Using a previously developed VL transmission model, we investigated transmission thresholds dependent on measures reducing the sand fly density either by killing sand flies (e.g., indoor residual spraying and long-lasting insecticidal nets) or by destroying breeding sites (e.g., environmental management).Model simulations suggest that elimination of VL is possible if the sand fly density can be reduced by 67% through killing sand flies, or if the number of breeding sites can be reduced by more than 79% through measures of environmental management.These results were compared to data from two recent cluster randomised controlled trials conducted in India, Nepal and Bangladesh showing a 72% reduction in sand fly density after indoor residual spraying, a 44% and 25% reduction through the use of long-lasting insecticidal nets and a 42% reduction after environmental management.Based on model predictions, we identified the parameters within the transmission cycle of VL that predominantly determine the prospects of intervention success. We suggest further research to refine model-based predictions into the elimination of VL. © 2014 Stauch et al.
Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India
(2013) Pankaj Srivastava; Kamlesh Gidwani; Albert Picado; Gert Van der Auwera; Puja Tiwary; Bart Ostyn; Jean-Claude Dujardin; Marleen Boelaert; Shyam Sundar
Objectives: Recent epidemiological reports indicate that asymptomatic human infections with Leishmania donovani, the causative agent of visceral leishmaniasis or Kala-azar (KA), occur frequently in India. We explored markers of infection. Methods: Blood samples were collected from 286 healthy subjects from 16 villages in the Muzaffarpur district of Bihar. These individuals were classified into three groups: (i) persons with no history of KA and living in a house where no KA cases were previously reported, (ii) persons with no history of KA but living in a house where KA cases were diagnosed at the time of sampling or in the past, and (iii) successfully treated KA patients. Each sample was tested using a Leishmania-specific PCR to detect Leishmania DNA, and two serological tests to demonstrate anti-Leishmania antibodies: the Direct Agglutination Test and rK39 ELISA. Results: PCR positivity was similar among the three groups (20-25%). In contrast, among treated patients, the percentage of serologically positive individuals was roughly five times that of healthy individuals with no KA history, as measured with either test. Living in a house where KA had been reported did not affect seropositivity. Conclusion: A significant proportion of asymptomatic infections of Leishmania exist in endemic regions. Using a combination of molecular and serological tests increases the capacity to detect infections at different stages. Further work is required to understand the kinetics of the markers. © 2013 Blackwell Publishing Ltd.
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 Schönian
In 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.
Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
(Public Library of Science, 2019) Bruno Cesar Bremer Hinckel; Tegwen Marlais; Stephanie Airs; Tapan Bhattacharyya; Hideo Imamura; Jean-Claude Dujardin; Sayda El-Safi; Om Prakash Singh; Shyam Sundar; Andrew Keith Falconar; Bjorn Andersson; Sergey Litvinov; Michael A. Miles; Pascal Mertens
Background The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a crude lysate antigen (CLA) as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs. Methodology Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera. Results Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60—97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive. Conclusion We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL. © 2019 Bremer Hinckel et al.
Research priorities for neglected infectious diseases in Latin America and the Caribbean Region
(2010) Jean-Claude Dujardin; Socrates Herrera; Virgilio do Rosario; Jorge Arevalo; Marleen Boelaert; Hernan J. Carrasco; Rodrigo Correa-Oliveira; Lineth Garcia; Eduardo Gotuzzo; Theresa W. Gyorkos; Alexis M. Kalergis; Gustavo Kouri; Vicente Larraga; Pascal Lutumba; Maria Angeles Macias Garcia; Pablo C. Manrique-Saide; Farrokh Modabber; Alberto Nieto; Gerd Pluschke; Carlos Robello; Antonieta Rojas de Arias; Martin Rumbo; Jose Ignacio Santos Preciado; Shyam Sundar; Jaime Torres; Faustino Torrico; Patrick van der Stuyft; Kathleen Victoir; Ole F. Olesen
[No abstract available]
Retrospective Quarterly Cohort Monitoring for patients with Visceral Leishmaniasis in the Indian subcontinent: Outcomes of a pilot project
(2013) Bart Ostyn; Paritosh Malaviya; Epco Hasker; Surendra Uranw; Rudra P. Singh; Suman Rijal; Shyam Sundar; Jean-Claude Dujardin; Marleen Boelaert
Objective: To evaluate a new tool for the monitoring of Visceral Leishmaniasis (VL) treatment outcomes in primary healthcare (PHC) settings, adapted from the standardised Retrospective Quarterly Cohort Monitoring done in tuberculosis control. Methods: We developed standard case definitions for early and late VL treatment outcomes, a single register allowing for one-line entry per patient as registration tool, and quarterly reporting formats for the clinical outcomes. We pilot-tested these tools in three Indian Primary Health Centres and two Nepalese district hospitals, as well as in a charity VL treatment centre and a university hospital. Results: Data collection for early treatment outcome was easily implemented but information on late treatment outcome was hard to obtain. Effectiveness of Miltefosine under routine care conditions was about 87% at end of treatment, and 76% at 6 months post-treatment related to the high number of patients lost to follow up at the latter end point. Conclusion: A retrospective cohort monitoring methodology is conceptually a good framework for monitoring clinical outcomes for chronic conditions as VL. The monitoring of early outcomes of VL treatment is perfectly feasible in Primary Care settings. The completeness of information on late outcomes can be improved by a number of strategies that remain to be field tested. Generally, clinical outcome monitoring should be strengthened in the VL control programmes. © 2013 John Wiley & Sons Ltd.
Simple colorimetric trypanothione reductase-based assay for high-throughput screening of drugs against leishmania intracellular amastigotes
(2014) Erika Van Den Bogaart; Gerard J. Schoone; Paul England; Dorien Faber; Kristina M. Orrling; Jean-Claude Dujardin; Shyam Sundar; Henk D.F.H. Schallig; Emily R. Adams
Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assays. © 2014, American Society for Microbiology.
Treatment of Visceral Leishmaniasis: Model-Based Analyses on the Spread of Antimony-Resistant L. donovani in Bihar, India
(2012) Anette Stauch; Hans-Peter Duerr; Jean-Claude Dujardin; Manu Vanaerschot; Shyam Sundar; Martin Eichner
Background: Pentavalent antimonials have been the mainstay of antileishmanial therapy for decades, but increasing failure rates under antimonial treatment have challenged further use of these drugs in the Indian subcontinent. Experimental evidence has suggested that parasites which are resistant against antimonials have superior survival skills than sensitive ones even in the absence of antimonial treatment. Methods and Findings: We use simulation studies based on a mathematical L. donovani transmission model to identify parameters which can explain why treatment failure rates under antimonial treatment increased up to 65% in Bihar between 1980 and 1997. Model analyses suggest that resistance to treatment alone cannot explain the observed treatment failure rates. We explore two hypotheses referring to an increased fitness of antimony-resistant parasites: the additional fitness is (i) disease-related, by causing more clinical cases (higher pathogenicity) or more severe disease (higher virulence), or (ii) is transmission-related, by increasing the transmissibility from sand flies to humans or vice versa. Conclusions: Both hypotheses can potentially explain the Bihar observations. However, increased transmissibility as an explanation appears more plausible because it can occur in the background of asymptomatically transmitted infection whereas disease-related factors would most probably be observable. Irrespective of the cause of fitness, parasites with a higher fitness will finally replace sensitive parasites, even if antimonials are replaced by another drug. © 2012 Stauch et al.