Manu Vanaerschot

Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance

Visceral leishmaniasis is a potentially fatal disease endemic to large parts of Asia and Africa, primarily caused by the protozoan parasite Leishmania donovani. Here, we report a high-quality reference genome sequence for a strain of L. donovani from Nepal, and use this sequence to study variation in a set of 16 related clinical lines, isolated from visceral leishmaniasis patients from the same region, which also differ in their response to in vitro drug susceptibility. We show that whole-genome sequence data reveals genetic structure within these lines not shown by multilocus typing, and suggests that drug resistance has emerged multiple times in this closely related set of lines. Sequence comparisons with other Leishmania species and analysis of single-nucleotide diversity within our sample showed evidence of selection acting in a range of surface- and transport-related genes, including genes associated with drug resistance. Against a background of relative genetic homogeneity, we found extensive variation in chromosome copy number between our lines. Other forms of structural variation were significantly associated with drug resistance, notably including gene dosage and the copy number of an experimentally verified circular episome present in all lines and described here for the first time. This study provides a basis for more powerful molecular profiling of visceral leishmaniasis, providing additional power to track the drug resistance and epidemiology of an important human pathogen.

Increasing Failure of Miltefosine in the Treatment of Kala-azar in Nepal and the Potential Role of Parasite Drug Resistance, Reinfection, or Noncompliance

BACKGROUND Miltefosine (MIL), the only oral drug for visceral leishmaniasis (VL), is currently the first-line therapy in the VL elimination program of the Indian subcontinent. Given the paucity of anti-VL drugs and the looming threat of resistance, there is an obvious need for close monitoring of clinical efficacy of MIL. METHODS In a cohort study of 120 VL patients treated with MIL in Nepal, we monitored the clinical outcomes up to 12 months after completion of therapy and explored the potential role of drug compliance, parasite drug resistance, and reinfection. RESULTS The initial cure rate was 95.8% (95% confidence interval [CI], 92.2-99.4) and the relapse rate at 6 and 12 months was 10.8% (95% CI, 5.2-16.4) and 20.0% (95% CI, 12.8-27.2) , respectively. No significant clinical risk factors of relapse apart from age <12 years were found. Parasite fingerprints of pretreatment and relapse bone marrow isolates within 8 patients were similar, suggesting that clinical relapses were not due to reinfection with a new strain. The mean promastigote MIL susceptibility (50% inhibitory concentration) of isolates from definite cures was similar to that of relapses. Although more tolerant strains were observed, parasite resistance, as currently measured, is thus not likely involved in MIL treatment failure. Moreover, MIL blood levels at the end of treatment were similar in cured and relapsed patients. CONCLUSIONS Relapse in one-fifth of the MIL-treated patients observed in our study is an alarming signal for the VL elimination campaign, urging for further review and cohort monitoring.

Molecular Mechanisms of Drug Resistance in Natural Leishmania Populations Vary with Genetic Background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogens genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability.

Linking In Vitro and In Vivo Survival of Clinical Leishmania donovani Strains

Background Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these ‘hostile’ environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent. Methodology/Principal Findings We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain. Conclusions/Significance The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis.

Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent

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. DOI: http://dx.doi.org/10.7554/eLife.12613.001

Evaluation of normalization methods to pave the way towards large-scale LC-MS-based metabolomics profiling experiments.

Combining liquid chromatography-mass spectrometry (LC-MS)-based metabolomics experiments that were collected over a long period of time remains problematic due to systematic variability between LC-MS measurements. Until now, most normalization methods for LC-MS data are model-driven, based on internal standards or intermediate quality control runs, where an external model is extrapolated to the dataset of interest. In the first part of this article, we evaluate several existing data-driven normalization approaches on LC-MS metabolomics experiments, which do not require the use of internal standards. According to variability measures, each normalization method performs relatively well, showing that the use of any normalization method will greatly improve data-analysis originating from multiple experimental runs. In the second part, we apply cyclic-Loess normalization to a Leishmania sample. This normalization method allows the removal of systematic variability between two measurement blocks over time and maintains the differential metabolites. In conclusion, normalization allows for pooling datasets from different measurement blocks over time and increases the statistical power of the analysis, hence paving the way to increase the scale of LC-MS metabolomics experiments. From our investigation, we recommend data-driven normalization methods over model-driven normalization methods, if only a few internal standards were used. Moreover, data-driven normalization methods are the best option to normalize datasets from untargeted LC-MS experiments.

Genome-wide SNP and microsatellite variation illuminate population-level epidemiology in the **Leishmania donovani** species complex

Highlights ► Microsatellite typing of Leishmania donovani complex isolates discriminates intercontinental groups. ► Genome-wide SNP profiling reveals diversity in a homogeneous population. ► Identification of a novel divergent lineage within a small geographic region. ► SNP-typing of samples resistant and sensitive to treatment drugs.

Metabolic adaptations of Leishmania donovani in relation to differentiation, drug resistance, and drug pressure

Antimonial (sodium stibogluconate, SSG) resistance and differentiation have been shown to be closely linked in Leishmania donovani, with SSG‐resistant strains showing an increased capacity to generate infectious (metacyclic) forms. This is the first untargeted LC‐MS metabolomics study which integrated both phenomena in one experimental design and provided insights into metabolic differences between three clinical L. donovani strains with a similar genetic background but different SSG‐susceptibilities. We performed this analysis at different stages during promastigote growth and in the absence or presence of drug pressure. When comparing SSG‐resistant and SSG‐sensitive strains, a number of metabolic changes appeared to be constitutively present in all growth stages, pointing towards a clear link with SSG‐resistance, whereas most metabolic changes were only detected in the stationary stage. These changes reflect the close intertwinement between SSG‐resistance and an increased metacyclogenesis in resistant parasites. The metabolic changes suggest that SSG‐resistant parasites have (i) an increased capacity for protection against oxidative stress; (ii) a higher fluidity of the plasma membrane; and (iii) a metabolic survival kit to better endure infection. These changes were even more pronounced in a resistant strain kept under SbIII drug pressure.

Detection of Leptomonas sp. parasites in clinical isolates of Kala-azar patients from India

We report here nine unusual cases of Kala-azar, of which parasites were isolated and found by 18S rRNA gene sequencing to be most similar to Leptomonas species. One of these isolates was used to inoculate Balb/c mice; organs were collected and directly submitted to a genus-specific rDNA-ITS1 PCR analysis: this revealed the presence of both Leptomonas sp. and Leishmania donovani. Therefore, we conclude that there was a mixed infection of Leptomonas sp. and L. donovani in this isolate. We consider that mixed infection may be present in the patients themselves, Leptomonas persisting in them because of the immuno-suppression associated with Kala-azar.

Increased metacyclogenesis of antimony-resistant Leishmania donovani clinical lines

Mathematical models predict that the future of epidemics of drug-resistant pathogens depends in part on the competitive fitness of drug-resistant strains. Considering metacyclogenesis (differentiation process essential for infectivity) as a major contributor to the fitness of Leishmania donovani, we tested its relationship with pentavalent antimony (SbV) resistance in clinical lines. Different methods for the assessment of metacyclogenesis were cross-validated: gene expression profiling (META1 and SHERP), morphometry (microscopy and FACS), in vitro infectivity to macrophages and resistance to complement lysis. This was done on a model constituted by 2 pairs of reference strains cloned from a SbV-resistant and -sensitive isolate. We selected the most adequate parameter and extended the analysis of metacyclogenesis diversity to a sample of 20 clinical lines with different in vitro susceptibility to the drug. The capacity of metacyclogenesis, as measured by the complement lysis test, was shown to be significantly higher in SbV-resistant clinical lines of L. donovani than in SbV-sensitive lines. Together with other lines of evidence, it is concluded that L. donovani constitutes a unique example and model of drug-resistant pathogens with traits of increased fitness. These findings raise a fundamental question about the potential risks of selecting more virulent pathogens through massive chemotherapeutic interventions.