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

Autor: Mandy Sanders, Manu Vanaerschot, Suman Rijal, Keshav Rai, Ilse Maes, Erika D’Haenens, Narayan Raj Bhattarai, Jean-Claude Dujardin, Frederik Van den Broeck, Malgorzata A. Domagalska, James Cotton, Matthew Berriman, Hideo Imamura
Přispěvatelé: Rogers, Matthew Brian, Clinical sciences, Medical Genetics, Department of Bio-engineering Sciences
Jazyk: angličtina
Rok vydání: 2019
Předmět:
0301 basic medicine
Leishmania Donovani
Life Cycles
Heredity
Physiology
RC955-962
Protozoology
Parasite load
Genome
0302 clinical medicine
Bone Marrow
Immune Physiology
Arctic medicine. Tropical medicine
INFECTION
Medicine and Health Sciences
Genome Sequencing
Copy-number variation
Child
Protozoans
Leishmania
Genetics
0303 health sciences
NEPAL
Eukaryota
Genomics
3. Good health
Genetic Mapping
Infectious Diseases
Child
Preschool
Leishmaniasis
Visceral
Protozoan Life Cycles
Public aspects of medicine
RA1-1270
Life Sciences & Biomedicine
INFANTUM
Research Article
Adolescent
Genotype
Immunology
030231 tropical medicine
Leishmania donovani
Variant Genotypes
Biology
Research and Analysis Methods
Microbiology
DNA sequencing
Specimen Handling
Molecular Genetics
03 medical and health sciences
Nepal
Tropical Medicine
medicine
Humans
Molecular Biology Techniques
Sequencing Techniques
Molecular Biology
030304 developmental biology
Whole genome sequencing
Science & Technology
Whole Genome Sequencing
Intracellular parasite
Promastigotes
Organisms
Public Health
Environmental and Occupational Health
Biology and Life Sciences
Computational Biology
Infant
AMPLIFICATION
DNA
Protozoan
Genome Analysis
Genomic Libraries
medicine.disease
biology.organism_classification
Parasitic Protozoans
030104 developmental biology
Visceral leishmaniasis
Immune System
Parasitology
Human medicine
Developmental Biology
Zdroj: PLoS Neglected Tropical Diseases, Vol 13, Iss 12, p e0007900 (2019)
PLoS Neglected Tropical Diseases
PLoS neglected tropical diseases
ISSN: 1935-2727
DOI: 10.1101/676163
Popis: 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
Author summary Visceral leishmaniasis (VL) is caused by parasitic protozoa of the Leishmania donovani complex and is lethal in the absence of treatment. Whole Genome Sequencing (WGS) of L. donovani clinical isolates revealed hitherto cryptic population structure in the Indian Sub-Continent and provided insights into the epidemiology and potential mechanisms of drug resistance. However, several biases are likely introduced during the culture step. We report here the development of a method that allows determination of parasite genomes directly in clinical samples, and validate it on bone marrow and splenic aspirates of VL patients in Nepal. Our study sheds a new light on the biology of Leishmania in the human host: we found that intracellular parasites of the patients had very low levels of aneuploidy, in sharp contrast to the situation in cultivated isolates. Moreover, the observed differences in genomes between intracellular amastigotes of the patient and the derived cultured parasites suggests polyclonality of infections, with different clones dominating in clinical samples and in culture, likely due to fitness differences. We believe this method is most suitable for clinical studies and for molecular tracking in the context of elimination programs.
Databáze: OpenAIRE
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