Publications by authors named "Jean-Claude Dujardin"

190 Publications

Application of CRISPR/Cas9-Based Reverse Genetics in : Conserved Roles for HSP100 and HSP23.

Genes (Basel) 2020 09 30;11(10). Epub 2020 Sep 30.

Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany.

The protozoan parasite () ) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of through reverse genetics analyses has so far lagged behind in comparison with Old World spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in that was previously developed for Old World and New World species. As proof of principle, we demonstrate the targeted replacement of a transgene () and two single-copy genes ( and ). We obtained homozygous Cas9-free - and -null mutants in that matched the phenotypes reported previously for the respective null mutants. The function of is indeed conserved throughout the Trypanosomatida as null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of by CRISPR-Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.
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http://dx.doi.org/10.3390/genes11101159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601497PMC
September 2020

Viruses of protozoan parasites and viral therapy: Is the time now right?

Virol J 2020 09 29;17(1):142. Epub 2020 Sep 29.

Unit of Foodborne and Neglected Parasitic Diseases, European Union Reference Laboratory for Parasites, Department of Infectious Diseases, Istituto Superiore Di Sanità, viale Regina Elena 299, 00186, Rome, Italy.

Infections caused by protozoan parasites burden the world with huge costs in terms of human and animal health. Most parasitic diseases caused by protozoans are neglected, particularly those associated with poverty and tropical countries, but the paucity of drug treatments and vaccines combined with increasing problems of drug resistance are becoming major concerns for their control and eradication. In this climate, the discovery/repurposing of new drugs and increasing effort in vaccine development should be supplemented with an exploration of new alternative/synergic treatment strategies. Viruses, either native or engineered, have been employed successfully as highly effective and selective therapeutic approaches to treat cancer (oncolytic viruses) and antibiotic-resistant bacterial diseases (phage therapy). Increasing evidence is accumulating that many protozoan, but also helminth, parasites harbour a range of different classes of viruses that are mostly absent from humans. Although some of these viruses appear to have no effect on their parasite hosts, others either have a clear direct negative impact on the parasite or may, in fact, contribute to the virulence of parasites for humans. This review will focus mainly on the viruses identified in protozoan parasites that are of medical importance. Inspired and informed by the experience gained from the application of oncolytic virus- and phage-therapy, rationally-driven strategies to employ these viruses successfully against parasitic diseases will be presented and discussed in the light of the current knowledge of the virus biology and the complex interplay between the viruses, the parasite hosts and the human host. We also highlight knowledge gaps that should be addressed to advance the potential of virotherapy against parasitic diseases.
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http://dx.doi.org/10.1186/s12985-020-01410-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522927PMC
September 2020

Ecological divergence and hybridization of Neotropical parasites.

Proc Natl Acad Sci U S A 2020 10 21;117(40):25159-25168. Epub 2020 Sep 21.

Department of Biomedical Sciences, Institute of Tropical Medicine, 2000 Antwerp, Belgium;

The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles-but not maxicircles-show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.
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http://dx.doi.org/10.1073/pnas.1920136117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547230PMC
October 2020

Evaluation of whole genome amplification and bioinformatic methods for the characterization of Leishmania genomes at a single cell level.

Sci Rep 2020 09 14;10(1):15043. Epub 2020 Sep 14.

Institute of Tropical Medicine Antwerp, Molecular Parasitology Unit, Antwerp, Belgium.

Here, we report a pilot study paving the way for further single cell genomics studies in Leishmania. First, the performances of two commercially available kits for Whole Genome Amplification (WGA), PicoPLEX and RepliG were compared on small amounts of Leishmania donovani DNA, testing their ability to preserve specific genetic variations, including aneuploidy levels and SNPs. We show here that the choice of WGA method should be determined by the planned downstream genetic analysis, PicoPLEX and RepliG performing better for aneuploidy and SNP calling, respectively. This comparison allowed us to evaluate and optimize corresponding bio-informatic methods. As PicoPLEX was shown to be the preferred method for studying single cell aneuploidy, this method was applied in a second step, on single cells of L. braziliensis, which were sorted by fluorescence activated cell sorting (FACS). Even sequencing depth was achieved in 28 single cells, allowing accurate somy estimation. A dominant karyotype with three aneuploid chromosomes was observed in 25 cells, while two different minor karyotypes were observed in the other cells. Our method thus allowed the detection of aneuploidy mosaicism, and provides a solid basis which can be further refined to concur with higher-throughput single cell genomic methods.
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http://dx.doi.org/10.1038/s41598-020-71882-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7490275PMC
September 2020

The Absence of C-5 DNA Methylation in Allows DNA Enrichment from Complex Samples.

Microorganisms 2020 Aug 18;8(8). Epub 2020 Aug 18.

Molecular Parasitology, Institute of Tropical Medicine, 2000 Antwerp, Belgium.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in , the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the genome contains a C-5 DNA methyltransferase () from the subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of promastigote and amastigote DNA with human DNA, resulting in average human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing clinical samples.
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http://dx.doi.org/10.3390/microorganisms8081252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463849PMC
August 2020

A Case-Control Study on the Association Between Intestinal Helminth Infections and Treatment Failure in Patients With Cutaneous Leishmaniasis.

Open Forum Infect Dis 2020 May 12;7(5):ofaa155. Epub 2020 May 12.

Instituto de Medicina Tropical "Alexander von Humboldt," Universidad Peruana Cayetano Heredia, Lima, Peru.

Background: Endemic regions of cutaneous leishmaniasis (CL) and intestinal helminthiasis overlap. CL treatment with systemic pentavalent antimonial drugs (Sb) fails in 10%-30% of patients. The study objective was to assess the etiological role of intestinal helminthiasis in CL treatment failure.

Methods: An unmatched case-control study was done in 4 CL treatment sites in Peru in 2012-2015. Cases were CL patients with Sb treatment failure; controls were CL patients with Sb treatment success. Patients with a parasitologically confirmed CL diagnosis who had received supervised Sb treatment and could be classified as cases or controls were eligible. The main exposure variables were intestinal helminthiasis and strongyloidiasis, diagnosed through direct examination, rapid sedimentation, Baermann, Kato-Katz, or agar culture of stool samples. Additional exposure variables were other infections (HIV, human T-lymphotropic virus 1, tuberculosis, hepatitis B, intestinal protozoa) and noninfectious conditions (diabetes, renal insufficiency, and immunosuppressive medication). Age, gender, CL history, probable exposure place, and species were treated as potential confounders in multiple logistic regression.

Results: There were 94 case and 122 control subjects. Overall, infectious and noninfectious comorbidities were frequent both among cases (64%) and controls (71%). The adjusted odds ratio (OR) for the association between any intestinal helminth infection and CL treatment failure was 0.65 (95% confidence interval [CI], 0.30-1.38), and the adjusted OR for the association between strongyloidiasis and CL treatment failure was 0.34 (95% CI, 0.11-0.92).

Conclusions: In the Peruvian setting, high Sb treatment failure rates are not explained by intestinal helminthiasis. On the contrary, strongyloidiasis had a protective effect against treatment failure.
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http://dx.doi.org/10.1093/ofid/ofaa155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252286PMC
May 2020

Global genome diversity of the complex.

Elife 2020 03 25;9. Epub 2020 Mar 25.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

Protozoan parasites of the complex - and - cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the complex.
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http://dx.doi.org/10.7554/eLife.51243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105377PMC
March 2020

Next-Generation Molecular Surveillance of TriTryp Diseases.

Trends Parasitol 2020 04 17;36(4):356-367. Epub 2020 Feb 17.

Molecular Parasitology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.

Elimination programs targeting TriTryp diseases (Leishmaniasis, Chagas' disease, human African trypanosomiasis) significantly reduced the number of cases. Continued surveillance is crucial to sustain this progress, but parasite molecular surveillance by genotyping is currently lacking. We explain here which epidemiological questions of public health and clinical relevance could be answered by means of molecular surveillance. Whole-genome sequencing (WGS) for molecular surveillance will be an important added value, where we advocate that preference should be given to direct sequencing of the parasite's genome in host tissues instead of analysis of cultivated isolates. The main challenges here, and recent technological advances, are discussed. We conclude with a series of recommendations for implementing whole-genome sequencing for molecular surveillance.
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http://dx.doi.org/10.1016/j.pt.2020.01.008DOI Listing
April 2020

Visceral Leishmaniasis, Northern Somalia, 2013-2019.

Emerg Infect Dis 2020 01;26(1):153-154

We identified visceral leishmaniasis caused by Leishmania donovani in a previously unknown focus in northern Somalia. Clinical and epidemiologic characteristics of 118 cases during 2013-2019 in Bosaso, the region's commercial capital, have raised suspicion of visceral leishmaniasis endemicity status there.
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http://dx.doi.org/10.3201/eid2601.181851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924893PMC
January 2020

Tracking of quiescence in Leishmania by quantifying the expression of GFP in the ribosomal DNA locus.

Sci Rep 2019 12 12;9(1):18951. Epub 2019 Dec 12.

Laboratorios de Investigación y Desarrollo de la Facultad de Ciencias y Filosofía & Instituto de Medicina Tropical "Alexander von Humboldt", Universidad Peruana Cayetano Heredia, Lima, Peru.

Under stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently discovered in Leishmania. We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.
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http://dx.doi.org/10.1038/s41598-019-55486-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908629PMC
December 2019

Genomes of Leishmania parasites directly sequenced from patients with visceral leishmaniasis in the Indian subcontinent.

PLoS Negl Trop Dis 2019 12 12;13(12):e0007900. Epub 2019 Dec 12.

Institute of Tropical Medicine Antwerp, Molecular Parasitology Unit, Antwerp, Belgium.

Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in clinical samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples.
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http://dx.doi.org/10.1371/journal.pntd.0007900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932831PMC
December 2019

ISC1, a new Leishmania donovani population emerging in the Indian sub-continent: Vector competence of Phlebotomus argentipes.

Infect Genet Evol 2019 12 17;76:104073. Epub 2019 Oct 17.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic. Electronic address:

Visceral leishmaniasis (VL), the most severe form of the disease, is caused by Leishmania donovani in the Indian sub-continent (ISC). Whole genome sequencing studies revealed that two parasite populations exist in the ISC: a main population named the Core Group (CG) found mostly in the lowlands, and a new, genetically different subpopulation called ISC1. Parasites belonging to the CG were shown to be responsible for the recent epidemics, while the ISC1 variant was originally identified in hilly districts of Nepal and was later on increasingly found in the lowlands. Importantly, the ISC1 and CG isolates differ in their drug susceptibility and virulence signatures, suggesting that ISC1 constitutes an emerging and functionally different variant of L. donovani. In present study we aimed to address the potential of ISC1 transmission by the natural vector of L. donovani in the lowlands, Phlebotomus argentipes. By experimental infection of sand flies with parasites of the different genotypes, we demonstrate that ISC1 and CG strains are developing similarly in P. argentipes, suggesting that P. argentipes is a fully competent vector for ISC1 parasites. Integration of previous and current findings shows thus that ISC1 is a new and different variant of L. donovani, fully adapted to spread in the ISC through the main vector. This information is directly useful for managers of the elimination program. Furthermore, integration of our successive studies (genotyping, phenotyping and vector competence) demonstrates the relevance of molecular surveillance and should be of interest for scientists working on vector borne diseases and control managers.
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http://dx.doi.org/10.1016/j.meegid.2019.104073DOI Listing
December 2019

Major changes in chromosomal somy, gene expression and gene dosage driven by Sb in Leishmania braziliensis and Leishmania panamensis.

Sci Rep 2019 07 1;9(1):9485. Epub 2019 Jul 1.

Grupo de Investigaciones Microbiológicas-UR (GIMUR), Programa de Biología, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia.

Leishmania braziliensis and Leishmania panamensis are two species clinically and epidemiologically important, among others because of their relative resistance to first-line drugs (antimonials). The precise mechanism underlying the ability of these species to survive antimony treatment remains unknown. Therefore, elucidating the pathways mediating drug resistance is essential. We herein experimentally selected resistance to trivalent antimony (Sb) in the reference strains of L. braziliensis (MHOM/BR75/M2904) and L. panamensis (MHOM/COL/81L13) and compared whole genome and transcriptome alterations in the culture promastigote stage. The results allowed us to identify differences in somy, copy number variations in some genes related to antimony resistance and large-scale copy number variations (deletions and duplications) in chromosomes with no somy changes. We found mainly in L. braziliensis, a direct relation between the chromosomal/local copy number variation and the gene expression. We identified differentially expressed genes in the resistant lines that are involved in antimony resistance, virulence, and vital biological processes in parasites. The results of this study may be useful for characterizing the genetic mechanisms of these Leishmania species under antimonial pressure, and for clarifying why the parasites are resistant to first-line drug treatments.
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http://dx.doi.org/10.1038/s41598-019-45538-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603004PMC
July 2019

Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis.

PLoS Negl Trop Dis 2019 05 6;13(5):e0007353. Epub 2019 May 6.

Coris BioConcept, Gembloux, Belgium.

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.
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http://dx.doi.org/10.1371/journal.pntd.0007353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522066PMC
May 2019

A Guide to Next Generation Sequence Analysis of Leishmania Genomes.

Methods Mol Biol 2019 ;1971:69-94

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Next generation sequencing (NGS) technology transformed Leishmania genome studies and became an indispensable tool for Leishmania researchers. Recent Leishmania genomics analyses facilitated the discovery of various genetic diversities including single nucleotide polymorphisms (SNPs), copy number variations (CNVs), somy variations, and structural variations in detail and provided valuable insights into the complexity of the genome and gene regulation. Many aspects of Leishmania NGS analyses are similar to those of related pathogens like trypanosomes. However, the analyses of Leishmania genomes face a unique challenge because of the presence of frequent aneuploidy. This makes characterization and interpretation of read depth and somy a key part of Leishmania NGS analyses because read depth affects the accuracy of detection of all genetic variations. However, there are no general guidelines on how to explore and interpret the impact of aneuploidy, and this has made it difficult for biologists and bioinformaticians, especially for beginners, to perform their own analyses and interpret results across different analyses. In this guide we discuss a wide range of topics essential for Leishmania NGS analyses, ranging from how to set up a computational environment for genome analyses, to how to characterize genetic variations among Leishmania samples, and we will particularly focus on chromosomal copy number variation and its impact on genome analyses.
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http://dx.doi.org/10.1007/978-1-4939-9210-2_3DOI Listing
August 2019

Genomic Analysis of Colombian Leishmania panamensis strains with different level of virulence.

Sci Rep 2018 11 26;8(1):17336. Epub 2018 Nov 26.

Grupo Biología y Control de Enfermedades Infecciosas, Universidad de Antioquia, Medellín, Colombia.

The establishment of Leishmania infection in mammalian hosts and the subsequent manifestation of clinical symptoms require internalization into macrophages, immune evasion and parasite survival and replication. Although many of the genes involved in these processes have been described, the genetic and genomic variability associated to differences in virulence is largely unknown. Here we present the genomic variation of four Leishmania (Viannia) panamensis strains exhibiting different levels of virulence in BALB/c mice and its application to predict novel genes related to virulence. De novo DNA sequencing and assembly of the most virulent strain allowed comparative genomics analysis with sequenced L. (Viannia) panamensis and L. (Viannia) braziliensis strains, and showed important variations at intra and interspecific levels. Moreover, the mutation detection and a CNV search revealed both base and structural genomic variation within the species. Interestingly, we found differences in the copy number and protein diversity of some genes previously related to virulence. Several machine-learning approaches were applied to combine previous knowledge with features derived from genomic variation and predict a curated set of 66 novel genes related to virulence. These genes can be prioritized for validation experiments and could potentially become promising drug and immune targets for the development of novel prophylactic and therapeutic interventions.
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http://dx.doi.org/10.1038/s41598-018-35778-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255768PMC
November 2018

Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

mBio 2018 11 6;9(6). Epub 2018 Nov 6.

Unité de Parasitologiemoléculaire et Signalisation, Institut Pasteur, Paris, France

Protozoan parasites of the genus adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast growth. Together our data draw a complex picture of genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. Protozoan parasites of the genus cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the , , and complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.
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http://dx.doi.org/10.1128/mBio.01399-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222132PMC
November 2018

Mitonuclear genomics challenges the theory of clonality in Trypanosoma congolense: Reply to Tibayrenc and Ayala.

Mol Ecol 2018 09 24;27(17):3425-3431. Epub 2018 Aug 24.

Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

We recently published the first genomic diversity study of Trypanosoma congolense, a major aetiological agent of Animal African Trypanosomiasis. We demonstrated striking levels of SNP and indel diversity in the Eastern province of Zambia as a consequence of hybridization between divergent trypanosome lineages. We concluded that these and earlier findings in T. congolense challenge the predominant clonal evolution (PCE) model. In a recent comment, Tibayrenc and Ayala claim that there are many features in T. congolense supporting their theory of clonality. While we can follow the reasoning of the authors, we also identify major limitations in their theory and interpretations that resulted in incorrect conclusions. First, we argue that each T. congolense subgroup should be analysed independently as they may represent different (sub)species rather than "near-clades". Second, the authors neglect major findings of two robust population genetic studies on Savannah T. congolense that provide clear evidence of frequent recombination. Third, we reveal additional events of introgressive hybridization in T. congolense by analysing the maxicircle coding region using next-generation sequencing analyses. At last, we pinpoint two important misinterpretations by the authors and show that there are no spatially and temporally widespread clones in T. congolense. We stand by our earlier conclusions that the clonal framework is unlikely to accurately model the population structure of T. congolense. Other theoretical frameworks such as Maynard Smith's epidemic model may better represent the complex ancestry seen in T. congolense, where clones delimited in space and time arise against a background of recombination.
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http://dx.doi.org/10.1111/mec.14809DOI Listing
September 2018

Importance of secondary screening with clinical isolates for anti-leishmania drug discovery.

Sci Rep 2018 08 6;8(1):11765. Epub 2018 Aug 6.

Diseases of the developing world, GlaxoSmithKline, Tres cantos, Spain.

The growing drug resistance (DR) raises major concerns for the control of visceral leishmaniasis (VL), a neglected disease lethal in 95 percent of the cases if left untreated. Resistance has rendered antimonials (SSG) obsolete in the Indian Sub-Continent (ISC) and the first miltefosine-resistant Leishmania donovani were isolated. New chemotherapeutic options are needed and novel compounds are being identified by high-throughput screening (HTS). HTS is generally performed with old laboratory strains such as LdBOB and we aimed here to validate the activity of selected compounds against recent clinical isolates. In this academic/industrial collaboration, 130 compounds from the GSK "Leishbox" were screened against one SSG-sensitive and one SSG-resistant strain of L. donovani recently isolated from ISC patients, using an intracellular assay of L. donovani-infected THP1-derived macrophages. We showed that only 45% of the compounds were active in both clinical isolates and LdBOB. There were also different compound efficiencies linked to the SSG susceptibility background of the strains. In addition, our results suggested that the differential susceptibility profiles were chemical series-dependent. In conclusion, we demonstrate the potential value of including clinical isolates (as well as resistant strains) in the HTS progression cascade.
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http://dx.doi.org/10.1038/s41598-018-30040-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078976PMC
August 2018

Accuracy of a Rapid Diagnostic Test Based on Antigen Detection for the Diagnosis of Cutaneous Leishmaniasis in Patients with Suggestive Skin Lesions in Morocco.

Am J Trop Med Hyg 2018 09 5;99(3):716-722. Epub 2018 Jul 5.

Department of Public Health, Institute of Tropical Medicine, Antwerp, Belgium.

In rural areas in Morocco, diagnosing cutaneous leishmaniasis (CL) can be challenging. We evaluated the accuracy of a rapid diagnostic test (RDT) based on antigen detection, CL Detect Rapid Test (Inbios International Inc., Seattle, WA), in this setting. We consecutively recruited patients with new skin ulcers in nine primary health centers. We took a dental broach sample for the RDT and two other tissue samples by scraping the border and center of the lesion with a scalpel and smearing it on a slide. We duplicated each smear by pressing a clean slide against it and processed the slides by microscopy, polymerase chain reaction (PCR) internal transcribed spacer 1, and kDNA minicircle PCR. In a subgroup with positive PCR, the species was identified using PCR-restriction fragment length polymorphism and PCR-sequencing of genes. A participant with positive microscopy and/or PCR was considered a confirmed CL case. We computed sensitivity (Se) and specificity (Sp) of the RDT compared with this reference standard (ClinicalTrials.gov registration: NCT02979002). Between December 2016 and July 2017, we included 219 patients, 50% of them were under 18 years old. Rapid diagnostic test Se was 68% [95% confidence interval (CI): 61-74], Sp 94% [95% CI: 91-97], positive predictive value 95% [95% CI: 92-98], and negative predictive value 64% [95% CI: 58-70]. Despite its low Se, this novel RDT is a useful addition to clinical management of CL in Morocco, especially in isolated localities. Rapid diagnostic test-positive lesions can be treated as CL; but when RDT negative, microscopy should be done in a second step. The Se of the RDT can probably be optimized by improving the sampling procedure.
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http://dx.doi.org/10.4269/ajtmh.18-0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169188PMC
September 2018

Genomic and transcriptomic alterations in Leishmania donovani lines experimentally resistant to antileishmanial drugs.

Int J Parasitol Drugs Drug Resist 2018 08 13;8(2):246-264. Epub 2018 Apr 13.

Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain. Electronic address:

Leishmaniasis is a serious medical issue in many countries around the World, but it remains largely neglected in terms of research investment for developing new control and treatment measures. No vaccines exist for human use, and the chemotherapeutic agents currently used are scanty. Furthermore, for some drugs, resistance and treatment failure are increasing to alarming levels. The aim of this work was to identify genomic and trancriptomic alterations associated with experimental resistance against the common drugs used against VL: trivalent antimony (Sb, S line), amphotericin B (AmB, A line), miltefosine (MIL, M line) and paromomycin (PMM, P line). A total of 1006 differentially expressed transcripts were identified in the S line, 379 in the A line, 146 in the M line, and 129 in the P line. Also, changes in ploidy of chromosomes and amplification/deletion of particular regions were observed in the resistant lines regarding the parental one. A series of genes were identified as possible drivers of the resistance phenotype and were validated in both promastigotes and amastigotes from Leishmania donovani, Leishmania infantum and Leishmania major species. Remarkably, a deletion of the gene LinJ.36.2510 (coding for 24-sterol methyltransferase, SMT) was found to be associated with AmB-resistance in the A line. In the P line, a dramatic overexpression of the transcripts LinJ.27.T1940 and LinJ.27.T1950 that results from a massive amplification of the collinear genes was suggested as one of the mechanisms of PMM resistance. This conclusion was reinforced after transfection experiments in which significant PMM-resistance was generated in WT parasites over-expressing either gene LinJ.27.1940 (coding for a D-lactate dehydrogenase-like protein, D-LDH) or gene LinJ.27.1950 (coding for an aminotransferase of branched-chain amino acids, BCAT). This work allowed to identify new drivers, like SMT, the deletion of which being associated with resistance to AmB, and the tandem D-LDH-BCAT, the amplification of which being related to PMM resistance.
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http://dx.doi.org/10.1016/j.ijpddr.2018.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039315PMC
August 2018

Integrated genomic and metabolomic profiling of ISC1, an emerging Leishmania donovani population in the Indian subcontinent.

Infect Genet Evol 2018 08 19;62:170-178. Epub 2018 Apr 19.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address:

Leishmania donovani is the responsible agent for visceral leishmaniasis (VL) in the Indian subcontinent (ISC). The disease is lethal without treatment and causes 0.2 to 0.4 million cases each year. Recently, reports of VL in Nepalese hilly districts have increased as well as VL cases caused by L. donovani from the ISC1 genetic group, a new and emerging genotype. In this study, we perform for the first time an integrated, untargeted genomics and metabolomics approach to characterize ISC1, in comparison with the Core Group (CG), main population that drove the most recent outbreak of VL in the ISC. We show that the ISC1 population is very different from the CG, both at genome and metabolome levels. The genomic differences include SNPs, CNV and small indels in genes coding for known virulence factors, immunogens and surface proteins. Both genomic and metabolic approaches highlighted dissimilarities related to membrane lipids, the nucleotide salvage pathway and the urea cycle in ISC1 versus CG. Many of these pathways and molecules are important for the interaction with the host/extracellular environment. Altogether, our data predict major functional differences in ISC1 versus CG parasites, including virulence. Therefore, particular attention is required to monitor the fate of this emerging ISC1 population in the ISC, especially in a post-VL elimination context.
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http://dx.doi.org/10.1016/j.meegid.2018.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261844PMC
August 2018

Tegumentary leishmaniasis and coinfections other than HIV.

PLoS Negl Trop Dis 2018 03 1;12(3):e0006125. Epub 2018 Mar 1.

Department of Public Health, Institute of Tropical Medicine Antwerp, Antwerp, Belgium.

Background: Tegumentary leishmaniasis (TL) is a disease of skin and/or mucosal tissues caused by Leishmania parasites. TL patients may concurrently carry other pathogens, which may influence the clinical outcome of TL.

Methodology And Principal Findings: This review focuses on the frequency of TL coinfections in human populations, interactions between Leishmania and other pathogens in animal models and human subjects, and implications of TL coinfections for clinical practice. For the purpose of this review, TL is defined as all forms of cutaneous (localised, disseminated, or diffuse) and mucocutaneous leishmaniasis. Human immunodeficiency virus (HIV) coinfection, superinfection with skin bacteria, and skin manifestations of visceral leishmaniasis are not included. We searched MEDLINE and other databases and included 73 records: 21 experimental studies in animals and 52 studies about human subjects (mainly cross-sectional and case studies). Several reports describe the frequency of Trypanosoma cruzi coinfection in TL patients in Argentina (about 41%) and the frequency of helminthiasis in TL patients in Brazil (15% to 88%). Different hypotheses have been explored about mechanisms of interaction between different microorganisms, but no clear answers emerge. Such interactions may involve innate immunity coupled with regulatory networks that affect quality and quantity of acquired immune responses. Diagnostic problems may occur when concurrent infections cause similar lesions (e.g., TL and leprosy), when different pathogens are present in the same lesions (e.g., Leishmania and Sporothrix schenckii), or when similarities between phylogenetically close pathogens affect accuracy of diagnostic tests (e.g., serology for leishmaniasis and Chagas disease). Some coinfections (e.g., helminthiasis) appear to reduce the effectiveness of antileishmanial treatment, and drug combinations may cause cumulative adverse effects.

Conclusions And Significance: In patients with TL, coinfection is frequent, it can lead to diagnostic errors and delays, and it can influence the effectiveness and safety of treatment. More research is needed to unravel how coinfections interfere with the pathogenesis of TL.
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http://dx.doi.org/10.1371/journal.pntd.0006125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832191PMC
March 2018

The blackfly vectors and transmission of Onchocerca volvulus in Mahenge, south eastern Tanzania.

Acta Trop 2018 May 2;181:50-59. Epub 2018 Feb 2.

National Institute for Medical Research, Tukuyu Research Centre, Tukuyu, Tanzania.

The Mahenge Mountains onchocerciasis focus in south eastern Tanzania was historically one of the most heavily infected areas in the country. The vectors of Onchocerca volvulus are mainly Simulium damnosum complex blackflies, but a species of the Simulium neavei group may also contribute to transmission in some areas. The only detailed studies of parasite transmission in Mahenge were conducted in the late 1960s. The taxonomy of the S. damnosum complex has since been revised and onchocerciasis control through annual community directed treatment with ivermectin (CDTI) commenced in 1997. This study aimed to provide a cytogenetic and molecular update of the S. damnosum complex cytoforms present in Mahenge, and to evaluate the current status of O. volvulus transmission by blackflies following 19 years of annual CDTI. Rivers were surveyed to identify sites of S. damnosum s.l. breeding among the eastern slopes of the mountains, and human landing collections of adult female blackflies were made close to breeding sites. Identification of S. damnosum complex cytoforms was by cytotaxonomy of late-instar larvae and ITS1 amplicon size polymorphisms of larvae and adults. Adult blackflies were pool screened for O. volvulus infection using a triplex real-time PCR. The cytoforms 'Nkusi', Simulium kilibanum and 'Turiani' were found breeding in perennial rivers. 'Nkusi' and S. kilibanum were collected on human bait at 7/7 catch sites and possessed ITS1 profiles most closely resembling the molecular forms 'Nkusi J' and S. kilibanum 'T'. Whereas 'Turiani' was present in rivers, it was not collected on human bait and appears to be zoophilic. Simulium nyasalandicum was collected in low numbers on human bait at 3/7 catch sites. In total, 12,452 S. damnosum s.l. were pool screened and O. volvulus infection was detected in 97/104 pools of bodies and 51/104 pools of heads. The estimated percentage of S. damnosum s.l. carrying infective L3 stage parasites was 0.57% (95% CI 0.43%-0.74%). Onchocerca volvulus transmission by S. damnosum s.l. is continuing in the Mahenge Mountains after 19 years of annual CDTI. Infection rates appear similar to those reported in the 1960s, but a more detailed study is required to fully understand the epidemiological significance of the ongoing transmission. These results provide further evidence that annual CDTI may be insufficient to eliminate the parasite in formerly hyperendemic foci.
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http://dx.doi.org/10.1016/j.actatropica.2018.01.009DOI Listing
May 2018

Drug resistance and treatment failure in leishmaniasis: A 21st century challenge.

PLoS Negl Trop Dis 2017 Dec 14;11(12):e0006052. Epub 2017 Dec 14.

Research Center in Infectious Diseases, CHU de Quebec Research Center and Department of Microbiology-Infectious Disease and Immunology, University Laval, Quebec, Canada.

Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of "resistance" related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined.
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http://dx.doi.org/10.1371/journal.pntd.0006052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730103PMC
December 2017

Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani.

Nat Ecol Evol 2017 Dec 6;1(12):1961-1969. Epub 2017 Nov 6.

Institut Pasteur, INSERM U1201, Unité de Parasitologie Moléculaire et Signalisation, 75015, Paris, France.

The parasite Leishmania  donovani causes a fatal disease termed visceral leishmaniasis. The process through which the parasite adapts to environmental change remains largely unknown. Here we show that aneuploidy is integral for parasite adaptation and that karyotypic fluctuations allow for selection of beneficial haplotypes, which impact transcriptomic output and correlate with phenotypic variations in proliferation and infectivity. To avoid loss of diversity following karyotype and haplotype selection, L. donovani utilizes two mechanisms: polyclonal selection of beneficial haplotypes to create coexisting subpopulations that preserve the original diversity, and generation of new diversity as aneuploidy-prone chromosomes tolerate higher mutation rates. Our results reveal high aneuploidy turnover and haplotype selection as a unique evolutionary adaptation mechanism that L. donovani uses to preserve genetic diversity under strong selection. This unexplored process may function in other human diseases, including fungal infection and cancer, and stimulate innovative treatment options.
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http://dx.doi.org/10.1038/s41559-017-0361-xDOI Listing
December 2017

Transcriptome profiling identifies genes/pathways associated with experimental resistance to paromomycin in Leishmania donovani.

Int J Parasitol Drugs Drug Resist 2017 12 10;7(3):370-377. Epub 2017 Oct 10.

ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, India. Electronic address:

Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL) in the Indian subcontinent. Paromomycin (PMM), an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug. In this study, PMM resistant lines were selected up to 100 μM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed 6 fold decreases in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R) and the corresponding PMM sensitive (PMM-S) parasites revealed modulated expression of 500 genes (1.5 fold cut off) in PMM-R parasites. Selected genes were validated for their modulated expression by quantitative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant lines which included a) reduced oxidative phosphorylation; b) increased glycosomal succinate fermentation and substrate level phosphorylation; c) dependency on lipids and amino acids for energy generation; d) reduced DNA synthesis and increased DNA damage repair and e) decreased protein synthesis and degradation. Interestingly, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, antagonists of Ca channel that are also modulators of ABC transporters. Moreover, infection of macrophages by PMM-R parasites led to modulated nitric oxide (NO) levels while reactive oxygen species (ROS) level remained unaltered. The present study highlights the putative mechanisms of PMM resistance in Leishmania.
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http://dx.doi.org/10.1016/j.ijpddr.2017.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645162PMC
December 2017
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