The “antibiotic resistance crisis” is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.

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

Pathogenic variants in the RBM10 gene cause a rare X‐linked disorder described as TARP (Talipes equinovarus, Atrial septal defect, Robin sequence, and Persistent left vena cava superior) syndrome. We report two novel patients with truncating RBM10 variants in view of the literature, presenting a total of 26 patients from 15 unrelated families. Our results illustrate the highly pleiotropic nature of RBM10 pathogenic variants, beyond the classic TARP syndrome features. Major clinical characteristics include severe developmental delay, failure to thrive, brain malformations, neurological symptoms, respiratory issues, and facial dysmorphism. Minor features are growth retardation, cardiac, gastrointestinal, limb, and skeletal abnormalities. Additional recurrent features include genital and renal abnormalities as well as hearing and visual impairment. Thus, RBM10 loss of function variants typically cause an intellectual disability and congenital malformation syndrome that requires assessment of multiple organ systems at diagnosis and for which provided clinical features might simplify diagnostic assessment. Furthermore, evidence for an RBM10‐related genotype–phenotype correlation is emerging, which can be important for prognosis.

The CAMTA1‐associated phenotype was initially defined in patients with intragenic deletions and duplications who showed nonprogressive congenital ataxia, with or without intellectual disability. Here, we describe 10 individuals with CAMTA1 variants: nine previously unreported (likely) pathogenic variants comprising one missense, four frameshift and four nonsense variants, and one missense variant of unknown significance. Six patients were diagnosed following whole exome sequencing and four individuals with exome‐based targeted panel analysis. Most of them present with developmental delay, manifesting in speech and motor delay. Other frequent findings are hypotonia, cognitive impairment, cerebellar dysfunction, oculomotor abnormalities, and behavioral problems. Feeding problems occur more frequently than previously observed. In addition, we present a systematic review of 19 previously published individuals with causal variants, including copy number, truncating, and missense variants. We note a tendency of more severe cognitive impairment and recurrent dysmorphic features in individuals with a copy number variant. Pathogenic variants are predominantly observed in and near the N‐ and C‐ terminal functional domains. Clinical heterogeneity is observed, but 3′‐terminal variants seem to associate with less pronounced cerebellar dysfunction.

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 patient 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 summaryVisceral 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.