Saskia Decuypere

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.

Metabolomics to Unveil and Understand Phenotypic Diversity between Pathogen Populations

Leishmaniasis is a debilitating disease caused by the parasite Leishmania. There is extensive clinical polymorphism, including variable responsiveness to treatment. We study Leishmania donovani parasites isolated from visceral leishmaniasis patients in Nepal that responded differently to antimonial treatment due to differing intrinsic drug sensitivity of the parasites. Here, we present a proof-of-principle study in which we applied a metabolomics pipeline specifically developed for L. donovani to characterize the global metabolic differences between antimonial-sensitive and antimonial-resistant L. donovani isolates. Clones of drug-sensitive and drug-resistant parasite isolates from clinical samples were cultured in vitro and harvested for metabolomics analysis. The relative abundance of 340 metabolites was determined by ZIC-HILIC chromatography coupled to LTQ-Orbitrap mass spectrometry. Our measurements cover approximately 20% of the predicted core metabolome of Leishmania and additionally detected a large number of lipids. Drug-sensitive and drug-resistant parasites showed distinct metabolic profiles, and unsupervised clustering and principal component analysis clearly distinguished the two phenotypes. For 100 metabolites, the detected intensity differed more than three-fold between the 2 phenotypes. Many of these were in specific areas of lipid metabolism, suggesting that the membrane composition of the drug-resistant parasites is extensively modified. Untargeted metabolomics has been applied on clinical Leishmania isolates to uncover major metabolic differences between drug-sensitive and drug-resistant isolates. The identified major differences provide novel insights into the mechanisms involved in resistance to antimonial drugs, and facilitate investigations using targeted approaches to unravel the key changes mediating drug resistance.

American Tegumentary Leishmaniasis: Is Antimonial Treatment Outcome Related to Parasite Drug Susceptibility?

BACKGROUND Antimonials are the first drug of choice for the treatment of American tegumentary leishmaniasis (ATL); however, their efficacy is not predictable, and this may be linked to parasite drug resistance. We aimed to characterize the in vitro antimony susceptibility of clinical isolates of Peruvian patients with ATL who were treated with sodium stibogluconate and to correlate this in vitro phenotype with different treatment outcomes. METHODS Thirty-seven clinical isolates were obtained from patients with known disease and treatment histories. These isolates were typed, and the susceptibility of intracellular amastigotes to pentavalent (SbV) and trivalent (SbIII) antimonials was determined. RESULTS We observed 29 SbV-resistant isolates among 4 species of subgenus Viannia, most of which exhibited primary resistance; isolates resistant only to SbIII; and 3 combinations of in vitro phenotypes: (1) parasites sensitive to both drugs, (2) parasites resistant to both drugs, and (3) parasites resistant to SbV only (the majority of isolates fell into this category). There was no correlation between in vitro susceptibility to both antimonials and the clinical outcome of therapy. CONCLUSION Antimony insensitivity might occur in a stepwise fashion (first to SbV and then to SbIII). Our data question the definition of true parasite resistance to antimonials. Further studies of treatment efficacy should apply standardized protocols and definitions and should also consider host factors.

Characterisation of antimony-resistant Leishmania donovani isolates: Biochemical and biophysical studies and interaction with host cells

Recent clinical isolates of Leishmania donovani from the hyperendemic zone of Bihar were characterised in vitro in terms of their sensitivity towards sodium stibogluconate in a macrophage culture system. The resulting half maximal effective concentration (EC(50)) values were compared with those of known sensitive isolates. Fifteen of the isolates showed decreased sensitivity towards SSG with an average EC(50) of 25.7 ± 4.5 μg/ml pentavalent antimony (defined as antimony resistant), whereas nine showed considerable sensitivity with an average EC(50) of 4.6 ± 1.7 μg/ml (defined as antimony sensitive). Out of those nine, seven were recent clinical isolates and the remaining two were known sensitive isolates. Compared with the antimony sensitive, resistant isolates showed enhanced expression of thiol metabolising enzymes in varying degrees coupled with increased intracellular non-protein thiol content, decreased fluorescence anisotropy (inversely proportional with membrane fluidity) and over-expression of the terminal glycoconjugates (N-acetyl-d-galactosaminyl residue). Macrophages infected with resistant but not with sensitive showed up-regulation of the ATP Binding Cassette transporter multidrug resistance protein 1 and permeability glycoprotein, while the supernatant contained abundant IL-10. The above results reinforce the notion that antimony resistant parasites have undergone a number of biochemical and biophysical changes as part of their adaptation to ensure their survival in the host.

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.

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.

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.

Drug-resistant microorganisms with a higher fitness – can medicines boost pathogens?

Drug-resistant microorganisms (DRMs) are generally thought to suffer from a fitness cost associated with their drug-resistant trait, inflicting them a disadvantage when the drug pressure reduces. However, Leishmania resistant to pentavalent antimonies shows traits of a higher fitness compared to its sensitive counterparts. This is likely due the combination of an intracellular pathogen and a drug that targets the parasite’s general defense mechanisms while at the same time stimulating the host’s immune system, resulting in a DRM that is better adapted to withstand the host’s immune response. This review aims to highlight how this fitter DRM has emerged and how it might affect the control of leishmaniasis. However, this unprecedented example of fitter antimony-resistant Leishmania donovani is also of significance for the control of other microorganisms, warranting more caution when applying or designing drugs that attack their general defense mechanisms or interact with the host’s immune system.

Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony

INTRODUCTION Evaluation of Leishmania drug susceptibility depends on in vitro Sb(V) susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of Sb(V)-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as Sb(V)-resistant and -sensitive, in order to identify potential resistance markers. METHODS The differential expression of 13 genes involved in Sb(V) metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. RESULTS Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of Sb(V)-resistant compared to the group of Sb(V)-sensitive parasites (P<0.01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. DISCUSSION Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro Sb(V) resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro Sb(V) susceptibility assays, but interfering with the gene expression patterns.