Molecular Preadaptation to Antimony Resistance in Leishmania donovani on the Indian Subcontinent.

Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (SbIII) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline SbIII susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to SbIII resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to SbIII resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of SbIII The main driver of this preadaptation was shown to be MRPA, a gene involved in SbIII sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.IMPORTANCE 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.

In vivo characterization of two additional Leishmania donovani strains using the murine and hamster model.

Leishmania donovani is a protozoan parasite causing the neglected tropical disease visceral leishmaniasis. One difficulty to study the immunopathology upon L. donovani infection is the limited adaptability of the strains to experimental mammalian hosts. Our knowledge about L. donovani infections relies on a restricted number of East African strains (LV9, 1S). Isolated from patients in the 1960s, these strains were described extensively in mice and Syrian hamsters and have consequently become 'reference' laboratory strains. L. donovani strains from the Indian continent display distinct clinical features compared to East African strains. Some reports describing the in vivo immunopathology of strains from the Indian continent exist. This study comprises a comprehensive immunopathological characterization upon infection with two additional strains, the Ethiopian L. donovani L82 strain and the Nepalese L. donovani BPK282 strain in both Syrian hamsters and C57BL/6 mice. Parameters that include parasitaemia levels, weight loss, hepatosplenomegaly and alterations in cellular composition of the spleen and liver, showed that the L82 strain generated an overall more virulent infection compared to the BPK282 strain. Altogether, both L. donovani strains are suitable and interesting for subsequent in vivo investigation of visceral leishmaniasis in the Syrian hamster and the C57BL/6 mouse model.

Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification.

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the New and Old World, using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Current PCR presents limitations in terms of sensitivity, which hampers its use for analyzing clinical and biological samples, and specificity, which makes it inappropriate to discriminate between Leishmania and other trypanosomatids. The aim of the study was to improve the sensitivity and specificity of a previously reported hsp70 PCR using alternative PCR primers and RFLPs. Following in silico analysis of available sequences, three new PCR primer sets and restriction digest schemes were tested on a globally representative panel of 114 Leishmania strains, various other infectious agents, and clinical samples. The largest new PCR fragment retained the discriminatory power from RFLP, while two smaller fragments discriminated less species. The detection limit of the new PCRs was between 0.05 and 0.5 parasite genomes, they amplified clinical samples more efficiently, and were Leishmania specific. We succeeded in significantly improving the specificity and sensitivity of the PCRs for hsp70 Leishmania species typing. The improved PCR-RFLP assays can impact diagnosis, treatment, and epidemiological studies of leishmaniasis in any setting worldwide.

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.

Universal PCR assays for the differential detection of all Old World Leishmania species.

For the epidemiological monitoring and clinical case management of leishmaniasis, determination of the causative Leishmania species gains importance. Current assays for the Old World often suffer from drawbacks in terms of validation on a geographically representative sample set and the ability to recognize all species complexes. We want to contribute to standardized species typing for Old World leishmaniasis. We determined the ribosomal DNA internal transcribed spacer 1 sequence of 24 strains or isolates, and validated four species-specific polymerase chain reactions (PCRs) amplifying this target. They discriminate L. aethiopica, L. tropica, L. major, and the L. donovani complex, use the same cycling conditions, and include an internal amplification control. Our PCRs amplify 0.1 pg of Leishmania DNA, while being 100% specific for species identification on an extensive panel of geographically representative strains and isolates. Similar results were obtained in an endemic reference laboratory in Kenya. Species could also be identified in clinical specimens. The presented PCRs require only agarose gel detection, and have several other advantages over many existing assays. We outline potential problems, suggest concrete solutions for transferring the technique to other settings, and deliver the proof-of-principle for analyzing clinical samples.

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.

Simple data-reduction method for high-resolution LC-MS data in metabolomics.

BACKGROUND: Metabolomics LC-MS experiments yield large numbers of peaks, few of which can be identified by database matching. Many of the remaining peaks correspond to derivatives of identified peaks (e.g., isotope peaks, adducts, fragments and multiply charged molecules). In this article, we present a data-reduction approach that automatically identifies these derivative peaks. RESULTS: Using data-driven clustering based on chromatographic peak shape correlation and intensity patterns across biological replicates, derivative peaks can be reliably identified. Using a test data set obtained from Leishmania donovani extracts, we achieved a 60% reduction of the number of peaks. After quality control filtering, almost 80% of the peaks could putatively be identified by database matching. CONCLUSION: Automated peak filtering substantially speeds up the data-interpretation process.

Evolutionary history of Trypanosoma cruzi according to antigen genes.

Trypanosoma cruzi, the agent of Chagas disease is associated with a very high clinical and epidemiological pleomorphism. This might be better understood through studies on the evolutionary history of the parasite. We explored here the value of antigen genes for the understanding of the evolution within T. cruzi. We selected 11 genes and 12 loci associated with different functions and considered to be involved in host-parasite interaction (cell adhesion, infection, molecular mimicry). The polymorphism of the respective genes in a sample representative of the diversity of T. cruzi was screened by PCR-RFLP and evolutionary relationships were inferred by phenetic analysis. Our results support the classification of T. cruzi in 2 major lineages and 6 discrete typing units (DTUs). The topology of the PCR-RFLP tree was the one that better fitted with the epidemiological features of the different DTUs: (i) lineage I, being encountered in sylvatic as well as domestic transmission cycles, (ii) IIa/c being associated with a sylvatic transmission cycle and (iii) IIb/d/e being associated with a domestic transmission cycle. Our study also supported the hypothesis that the evolutionary history of T. cruzi has been shaped by a series of hybridization events in the framework of a predominant clonal evolution pattern.

Gene expression profiling of Leishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation.

Gene expression profiling is increasingly used in the field of infectious diseases for characterization of host, pathogen and the nature of their interaction. The purpose of this study was to develop a robust, standardized method for comparative expression profiling and molecular characterization of Leishmania donovani clinical isolates. The limitations and possibilities associated with expression profiling in intracellular amastigotes and promastigotes were assessed through a series of comparative experiments in which technical and biological parameters were scrutinized. On a technical level, our results show that it is essential to use parasite harvesting procedures that involve minimal disturbance of the parasite's environment in order to 'freeze' gene expression levels instantly; this is particularly a delicate task for intracellular amastigotes and for specific 'sensory' genes. On the biological level, we demonstrate that gene expression levels fluctuate during in vitro development of both intracellular amastigotes and promastigotes. We chose to use expression-curves rather than single, specific, time-point measurements to capture this biological variation. Intracellular amastigote protocols need further refinement, but we describe a first generation tool for high-throughput comparative molecular characterization of patients' isolates, based on the changing expression profiles of promastigotes during in vitro differentiation.

Evaluation of an in vitro and in vivo model for experimental infection with Leishmania (Viannia) braziliensis and L. (V.) peruviana.

Leishmania (Viannia) braziliensis and L. (V.) peruviana are two parasite species characterized by a very different pathogenicity in humans despite a high genetic similarity. We hypothesized previously that L. (V.) peruviana would descend from L. (V.) braziliensis and would have acquired its 'peruviana' character during the southward colonization and adaptation of the transmission cycle in the Peruvian Andes. In order to have a first appreciation of the differences in virulence between both species, we evaluated an in vitro and in vivo model for experimental infection. A procedure was adapted to enrich culture forms in infective stages and the purified metacyclics were used to infect macrophage cell lines and golden hamsters. The models were tested with 2 representative strains of L. (V.) braziliensis from cutaneous and mucosal origin respectively and 2 representative strains of L. (V.) peruviana from Northern and Southern Peru respectively. Our models were reproducible and sensitive enough to detect phenotypic differences among strains. We showed in vitro as well as in vivo that the L. (V.) braziliensis was more infective than L. (V.) peruviana. Furthermore, we found that in vitro infectivity patterns of the 4 strains analysed, were in agreement with the geographical structuring of parasite populations demonstrated in our previous studies. Further work is needed to confirm our results with more strains of different geographical origin and their specific clinical outcome. However, our data open new perspectives for understanding the process of speciation in Leishmania and its implications in terms of pathogenicity.

Putative markers of infective life stages in Leishmania (Viannia) braziliensis.

Gene expression is known to vary significantly during the Leishmania life-cycle. Its monitoring might allow identification of molecular changes associated with the infective stages (metacyclics and amastigotes) and contribute to the understanding of the complex host-parasite relationships. So far, very few studies have been done on Leishmania (Viannia) braziliensis, one of the most pathogenic species. Such studies require, first of all, reference molecular markers. In the present work, we applied differential display analysis (DD analysis) in order to identify transcripts that might be (i) candidate markers of metacyclics and intracellular amastigotes of L. (V.) braziliensis or (ii) potential controls, i.e. constitutively expressed. In total, 48 DNA fragments gave reliable sequencing data, 29 of them being potential markers of infective stages and 12 potential controls. Eight sequences could be identified with reported genes. Validation of the results of DD analysis was done for 4 genes (2 differentially expressed and 2 controls) by quantitative real-time PCR. The infective insect stage-specific protein (meta 1) was more expressed in metacyclic-enriched preparations. The oligopeptidase b showed a higher expression in amastigotes. Two genes, glucose-6-phosphate dehydrogenase and a serine/threonine protein kinase, were found to be similarly expressed in the different biological samples.

Clonal propagation and the fast generation of karyotype diversity: An in vitro Leishmania model.

In the present work we studied the karyotype stability during long-term in vitro maintenance in 3 cloned strains of Leishmania (Viannia) peruviana, Leishmania (Viannia) braziliensis and a hybrid between both species. Only the L. (V.) peruviana strain showed an unstable karyotype, even after subcloning. Four chromosomes were studied in detail, each of them characterized by homologous chromosomes of different size (heteromorphy). Variations in chromosome patterns during in vitro maintenance were rapid and discrete, involving loss of heteromorphy or appearance of additional chromosome size variants. The resulting pattern was not the same according to experimental conditions (subinoculation rate or incubation temperature), and interestingly, this was associated with differences in growth behaviour of the respective parasites. No change in total ploidy of the cells was observed by flow cytometry. We discuss several mechanisms that might account for this variation of chromosome patterns, but we favour the occurrence of aneuploidy, caused by aberrant chromosome segregation during mitosis. Our results provide insight into the generation of karyotype diversity in natural conditions and highlight the relativity of the clone concept in parasitology.

Generic sodium stibogluconate is as safe and effective as branded meglumine antimoniate, for the treatment of tegumentary leishmaniasis in Isiboro Secure Park, Bolivia.

Human cutaneous leishmaniasis (CL) and mucous leishmaniasis (ML) are highly endemic in Isiboro Secure Park, which lies in the Bolivian department of Cochabamba--an area where branded meglumine antimoniate (Glucantime) is expensive and poorly distributed. The safety and efficacy of generic sodium stibogluconate (SSG), from Albert David Ltd, was therefore explored, in CL and ML cases from the park, who were treated with 20 mg/kg.day for 20 and 30 days, respectively. A questionnaire recording adverse effects was completed by a physician in each treatment centre. Efficacy of treatment was assessed at the end of treatment and at follow-ups 1 month and 3, 6 and 12 months later. Overall, 146 patients completed treatment with SSG in 2003-2004. No fatalities or severe adverse effects were reported but mild to moderate adverse effects were noted in 41 (28%) of the patients. The incidence of adverse effects was significantly higher among the cases of ML than among the cases of CL. Of the 86 patients with CL who completed 6 months of follow-up, 81 (94.2%) were considered to have been clinically cured; a comparable cohort of 69 CL cases who had been treated with Glucantime in 2001-2002 showed a similar frequency of clinical cure (90%). Generic SSG was shown to be safe and efficacious for the treatment of tegumentary leishmaniasis in Bolivia. Being several times cheaper than Glucantime, it could contribute to improving the access of CL and ML patients to treatment, not only in Bolivia but also in other countries of Latin America.