Metabolomics to unveil and understand phenotypic diversity between pathogen populations

Visceral leishmaniasis is caused by a parasite called Leishmania donovani, which every year infects about half a million people and claims several thousand lives. Existing treatments are now becoming less effective due to the emergence of drug resistance. Improving our understanding of the mechanisms used by the parasite to adapt to drugs and achieve resistance is crucial for developing future treatment strategies. Unfortunately, the biological mechanism whereby Leishmania acquires drug resistance is poorly understood. Recent years have brought new technologies with the potential to increase greatly our understanding of drug resistance mechanisms. The latest mass spectrometry techniques allow the metabolome of parasites to be studied rapidly and in great detail. We have applied this approach to determine the metabolome of drug-sensitive and drug-resistant parasites isolated from patients with leishmaniasis. The data show that there are wholesale differences between the isolates and that the membrane composition has been drastically modified in drug-resistant parasites compared with drug-sensitive parasites. Our findings demonstrate that untargeted metabolomics has great potential to identify major metabolic differences between closely related parasite strains and thus should find many applications in distinguishing parasite phenotypes of clinical relevance

Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

Leishmania spp. are parasitic protozoa responsible for a spectrum of diseases known as leishmaniasis. There are few drugs available for the treatment of these diseases, and miltefosine is the first oral drug used in treatment of visceral leishmaniasis, a form of the disease that can be lethal if not treated. In this study, we seek to understand the mechanism of action and identify targets of the drug by generating promastigote mutants highly resistant to miltefosine. Two independent mutants were submitted to short read whole genome sequencing. Genome analysis of these mutants has permitted us to identify point mutations in three genes (P-type ATPase, pyridoxal kinase and α-adaptin like protein) that were also present in other independent miltefosine resistant mutants. Some of the new genes identified here could be useful as potential markers for miltefosine resistance in Leishmania. Moreover, our approach has permitted us to highlight that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. This may have practical applications while studying resistance

Metabolic Variation during Development in Culture of Leishmania donovani Promastigotes

The genome sequencing of several Leishmania species has provided immense amounts of data and allowed the prediction of the metabolic pathways potentially operating. Subsequent genetic and proteomic studies have identified stage-specific proteins and putative virulence factors but many aspects of the metabolic adaptations of Leishmania remain to be elucidated. In this study, we have used an untargeted metabolomics approach to analyze changes in the metabolite profile as promastigotes of L. donovani develop during in vitro cultures from logarithmic to stationary phase. The results show that the metabolomes of promastigotes on days 3–6 of culture differ significantly from each other, consistent with there being distinct developmental changes. Most notable were the structural changes in glycerophospholipids and increase in the abundance of sphingolipids and glycerolipids as cells progress from logarithmic to stationary phase

Drug Resistance in Eukaryotic Microorganisms

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies

The genomic substrate for adaptive radiation in African cichlid fish

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification

Etat des lieux de la lutte contre la tuberculose à Madagascar de 1996 à 2004

Objectif. Le programme de lutte contre la tuberculose à Madagascar est fonctionnel depuis 1991, et la présente étude consiste à décrire l'évolution des résultats de cette lutte. Méthodologie. Il s'agit d'une étude rétrospective des rapports envoyés par les centres de diagnostic et de traitement au service de lutte contre la tuberculose à Madagascar, entre 1996 et 2004. L'étude a été focalisée sur les nouveaux cas de tuberculose à frottis positif. Résultats. Durant la période étudiée, l'incidence annuelle des cas à frottis positifs a augmenté de 65 à 82 pour 100 000 habitants. La maladie affecte beaucoup plus la population active. Le taux de réussite du traitement est passé de 64,4à 70,8chez les cas à frottis positifs. Le taux d'abandon a diminué de 21à 16,5. Une discordance de données entre le nombre de cas dépistés et le nombre de cas traités est observée. Conclusion. Une amélioration lente des résultats de la lutte contre la tuberculose est observée. Ces résultats ne permettent pas néanmoins d'identifier les stratégies pour améliorer encore les performances du programme. Une analyse plus détaillée des données en fonction des contextes serait nécessaire

Facteurs contextuels de l’efficacité du contrôle de la tuberculose à Madagascar : étude de validité au niveau national

[Contextual factors regarding the effectiveness of tuberculosis control in Madagascar: a nationwide validity study] INTRODUCTION: This study assesses the nationwide applicability of results from a study in the tuberculosis (TB) diagnostic and treatment centers (DTCs) in a sample of six districts in Madagascar, which identified adaptations of national guidelines and local initiatives that might explain the effectiveness of individual DTCs in improving adherence to TB treatment and thus reducing treatment default. OBJECTIVE: To assess, at a national level, the importance of these adaptations/initiatives for TB treatment adherence. METHODS: This analytical cross-sectional study assessed the responses to a questionnaire based on the previously identified adaptations/initiatives, which was sent to the heads of all 205 DTCs in Madagascar. RESULTS: Decentralization of TB care decreased the rate of patient default. The private DTCs report better results than public DTCs. Adaptations/initiatives in relation to local contexts often lead to good results. The relation between some adaptations/initiatives and continued adherence sometimes varies with the local context of the DTC; the same initiatives can result in better adherence or in higher of treatment default rates, depending on the setting. CONCLUSION: These initiatives should be applied after adaptation to the context

Validation of a Simple Resazurin-Based Promastigote Assay for the Routine Monitoring of Miltefosine Susceptibility in Clinical Isolates of Leishmania Donovani

Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases (n017) against standard amastigote assay, in two different labs in India. The interstage MIL susceptibility correlated strongly (r00.70, p0 0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation (r00.72, p00.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions (n07, r00.78, p00.046) and MIL-induced parasites (r00.92, p00.0001; n03) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patient