Abstract
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
Highlights
Leishmania (Kinetoplastida, Trypanosomatidae) is a genus of parasitic protozoa transmitted by sand flies to a variety of mammal hosts including humans
Details on the SureSelect bait design are described in S1 Text and in S1A Fig. SuSL-seq was optimized on artificial mixtures (Leishmania DNA diluted in mammalian DNA in different ratios: see details and metrics in S1 Text and S1B and S1C Fig and S2 Fig): a percentage of Leishmania DNA of 0.006% was found to be the lowest limit for suitable analysis of genome diversity
SuSL-seq was applied to 63 clinical samples with Leishmania DNA percentage of at least 0.006%, 59 bone marrow and 4 splenic aspirate samples collected from visceral leishmaniasis (VL) patients in Nepal in the same region where we previously studied genome diversity of L. donovani isolates (S3 Fig)
Summary
Leishmania (Kinetoplastida, Trypanosomatidae) is a genus of parasitic protozoa transmitted by sand flies to a variety of mammal hosts including humans. Leishmania cause a broad spectrum of clinical presentations, the most severe being visceral leishmaniasis (VL) which annually affects up to 90,000 new individuals [1] and has a 10% mortality rate [2]. Like the one running in the Indian subcontinent (ISC) [3] could be challenged by several factors, including drug resistance [4,5] and emergence of new parasite variants [6,7]. Close surveillance is required to avoid new epidemics, including molecular tracking of the etiological agent. We previously resolved the hitherto cryptic population structure of Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), in the Indian subcontinent ISC [8]. We described an unprecedented level of aneuploidy and found genetic variants likely to underpin reduced efficacy of antimonial drugs in that region
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