The fully automated Sysmex XN-31 hematology analyzer can detect bloodstream form Trypanosoma brucei.

BACKGROUND: For fully automated detection and quantification of Plasmodium parasites, Sysmex developed the XN-31 hemocytometer. This study investigated whether the XN-31 can also detect and quantify bloodstream form trypanosomes (trypomastigotes). METHODS: Axenic cultures of Trypanosoma brucei brucei were used to prepare two dilution series of trypomastigotes in the whole blood of a healthy donor, which were subsequently examined by the XN-31 as well as by microscopic examination of thin and thick blood films. Trypomastigote intactness during the procedures was evaluated by microscopy. RESULTS: The XN-31 hemocytometer detected trypomastigotes with a detection limit of 26 trypomastigotes/µL. Scattergram patterns of Trypanosoma and Plasmodium parasites were clearly distinct, but current interpretation settings do not allow the identification of trypomastigotes yet, and therefore, need future refinement. CONCLUSION: Proof of concept was provided for an automated fluorescent flow cytometry method that can detect and quantify Plasmodium spp., as well as Trypanosoma brucei trypomastigotes.

Modelling gambiense human African trypanosomiasis infection in villages of the Democratic Republic of Congo using Kolmogorov forward equations.

Stochastic methods for modelling disease dynamics enable the direct computation of the probability of elimination of transmission. For the low-prevalence disease of human African trypanosomiasis (gHAT), we develop a new mechanistic model for gHAT infection that determines the full probability distribution of the gHAT infection using Kolmogorov forward equations. The methodology allows the analytical investigation of the probabilities of gHAT elimination in the spatially connected villages of different prevalence health zones of the Democratic Republic of Congo, and captures the uncertainty using exact methods. Our method provides a more realistic approach to scaling the probability of elimination of infection between single villages and much larger regions, and provides results comparable to established models without the requirement of detailed infection structure. The novel flexibility allows the interventions in the model to be implemented specific to each village, and this introduces the framework to consider the possible future strategies of test-and-treat or direct treatment of individuals living in villages where cases have been found, using a new drug.

A Wandering Missionary's Burden: Persistent Fever and Progressive Somnolence in a Returning Traveler.

Human African trypanosomiasis incidence has declined, but diagnosis remains difficult, especially in nonendemic areas. Our patient presented with fever, progressive lethargy, and weight loss for 5 months and had previously traveled to Ghana and Cameroon but had not been to areas with recently reported African trypanosomiasis. Extensive workup was negative, except for lymphocytic pleocytosis in cerebrospinal fluid; ultimately, a bone marrow aspiration revealed necrotizing granulomatous inflammation with 2 trypanosomes discovered on the aspirate smear, consistent with Trypanosoma brucei. The patient was treated with combination nifurtimox and eflornithine with full recovery.

Cost-effectiveness modelling to optimise active screening strategy for gambiense human African trypanosomiasis in endemic areas of the Democratic Republic of Congo.

BACKGROUND: Gambiense human African trypanosomiasis (gHAT) has been brought under control recently with village-based active screening playing a major role in case reduction. In the approach to elimination, we investigate how to optimise active screening in villages in the Democratic Republic of Congo, such that the expenses of screening programmes can be efficiently allocated whilst continuing to avert morbidity and mortality. METHODS: We implement a cost-effectiveness analysis using a stochastic gHAT infection model for a range of active screening strategies and, in conjunction with a cost model, we calculate the net monetary benefit (NMB) of each strategy. We focus on the high-endemicity health zone of Kwamouth in the Democratic Republic of Congo. RESULTS: High-coverage active screening strategies, occurring approximately annually, attain the highest NMB. For realistic screening at 55% coverage, annual screening is cost-effective at very low willingness-to-pay thresholds (20.4 per disability adjusted life year (DALY) averted), only marginally higher than biennial screening (14.6 per DALY averted). We find that, for strategies stopping after 1, 2 or 3 years of zero case reporting, the expected cost-benefits are very similar. CONCLUSIONS: We highlight the current recommended strategy-annual screening with three years of zero case reporting before stopping active screening-is likely cost-effective, in addition to providing valuable information on whether transmission has been interrupted.

Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes.

Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.

Environmental neurology in the tropics.

We address the impact of the tropical environment on the human nervous system using the multifaceted approach characteristic of environmental neurology. First, environmental factors are examined according to their nature (physical, chemical and biological) and in relation to human activity and behavior. Some factors are specific to the tropics (climate and infections), while others are non-specific (chemicals, human communities and their way of life). Second, we examine the major role of human adaptation to the success of Homo sapiens, with emphasis on the linkage between thermoregulation and sleep-wake regulation. Third, we examine the performance of environmental neurology as a clinical discipline in tropical climates, with focus on the diagnostic and therapeutic challenges posed by human African trypanosomiasis. Finally, the prevention, early detection and monitoring of environmental neurological diseases is examined, as well as links with political and economic factors. In conclusion, practitioners of environmental neurology seek a global, multidisciplinary and holistic approach to understanding, preventing and treating neurological disorders within their purview. Environmental neurology integrates an expanded One Health concept by linking health and wellness to the interaction of plants, animals, humans and the ecosystem. Recent epidemics and the current COVID-19 pandemic exemplify the need for worldwide action to protect human health and biodiversity.

Basic Biology of Trypanosoma brucei with Reference to the Development of Chemotherapies.

Trypanosoma brucei are protozoan parasites that cause the lethal human disease African sleeping sickness and the economically devastating disease of cattle, Nagana. African sleeping sickness, also known as Human African Trypanosomiasis (HAT), threatens 65 million people and animal trypanosomiasis makes large areas of farmland unusable. There is no vaccine and licensed therapies against the most severe, late-stage disease are toxic, impractical and ineffective. Trypanosomes are transmitted by tsetse flies, and HAT is therefore predominantly confined to the tsetse fly belt in sub-Saharan Africa. They are exclusively extracellular and they differentiate between at least seven developmental forms that are highly adapted to host and vector niches. In the mammalian (human) host they inhabit the blood, cerebrospinal fluid (late-stage disease), skin, and adipose fat. In the tsetse fly vector they travel from the tsetse midgut to the salivary glands via the ectoperitrophic space and proventriculus. Trypanosomes are evolutionarily divergent compared with most branches of eukaryotic life. Perhaps most famous for their extraordinary mechanisms of monoallelic gene expression and antigenic variation, they have also been investigated because much of their biology is either highly unconventional or extreme. Moreover, in addition to their importance as pathogens, many researchers have been attracted to the field because trypanosomes have some of the most advanced molecular genetic tools and database resources of any model system. The following will cover just some aspects of trypanosome biology and how its divergent biochemistry has been leveraged to develop drugs to treat African sleeping sickness. This is by no means intended to be a comprehensive survey of trypanosome features. Rather, I hope to present trypanosomes as one of the most fascinating and tractable systems to do discovery biology.

Predicting 19 F†NMR Chemical Shifts: A Combined Computational and Experimental Study of a Trypanosomal Oxidoreductase-Inhibitor Complex.

The absence of fluorine from most biomolecules renders it an excellent probe for NMR spectroscopy to monitor inhibitor-protein interactions. However, predicting the binding mode of a fluorinated ligand from a chemical shift (or vice versa) has been challenging due to the high electron density of the fluorine atom. Nonetheless, reliable 19 F chemical-shift predictions to deduce ligand-binding modes hold great potential for in silico drug design. Herein, we present a systematic QM/MM study to predict the 19 F NMR chemical shifts of a covalently bound fluorinated inhibitor to the essential oxidoreductase tryparedoxin (Tpx) from African trypanosomes, the causative agent of African sleeping sickness. We include many protein-inhibitor conformations as well as monomeric and dimeric inhibitor-protein complexes, thus rendering it the largest computational study on chemical shifts of 19 F nuclei in a biological context to date. Our predicted shifts agree well with those obtained experimentally and pave the way for future work in this area.

Phosphoproteomic analysis of mammalian infective Trypanosoma brucei subjected to heat shock suggests atypical mechanisms for thermotolerance.

The symptoms of African sleeping sickness, caused by the parasite Trypanosoma brucei, can include periods of fever as high as 41 °C which triggers a heat shock response in the parasite. To capture events involved in sensing and responding to heat shock in the mammalian infective form we have conducted a SILAC-based quantitative proteomic and phosphoproteomic analysis of T. brucei cells treated at 41 °C for 1h. Our analysis identified 193 heat shock responsive phosphorylation sites with an average of 5-fold change in abundance, but only 20 heat shock responsive proteins with average of 1.5-fold change. These data indicate that protein abundance does not rapidly respond (≤1 h) to heat shock, and that the changes observed in phosphorylation site abundance are larger and more widespread. The heat shock responsive phosphorylation sites showed enrichment of RNA binding proteins with putative roles in heat shock response included P-body / stress granules and the eukaryotic translation initiation 4F complex. The ZC3H11-MKT1 complex, which stabilises mRNAs of thermotolerance proteins, appears to represent a key signal integration node in the heat shock response. SIGNIFICANCE: We report the first quantitative study of changes in protein and phosphorylation site abundance in response to heat shock in the clinically relevant form of the human parasite Trypanosoma brucei. The identification of heat shock responsive phosphorylation sites on proteins with putative roles in thermotolerance including the ZC3H11-MKT1 complex provides evidence of the role dynamic phosphorylation of RNA binding proteins in co-ordinating heat shock. Temperature changes in the host are a major physiological challenge to parasites and factors conferring tolerance to heat shock constitute overlooked virulence factors. A better understanding of these virulence factors will pave the way for the development of novel drug therapies which selectively target T. brucei.

From Colonial Research Spirit to Global Commitment: Bayer and African Sleeping Sickness in the Mirror of History.

In the early 20th century, a series of epidemics across equatorial Africa brought African sleeping sickness (human African trypanosomiasis, HAT) to the attention of the European colonial administrations. This disease presented an exciting challenge for microbiologists across Europe to study the disease, discover the pathogen and search for an effective treatment. In 1923, the first "remedy for tropical diseases"-Suramin-manufactured by Bayer AG came onto the market under the brand name "Germanin." The development and life cycle of this product-which today is still the medicine of choice for Trypanosoma brucei (T.b), hodesiense infections-reflect medical progress as well as the successes and failures in fighting the disease in the context of historic political changes over the last 100 years.

Macromolecular Targets of Antiparasitic Germacranolide Sesquiterpenoids: An In Silico Investigation.

BACKGROUND: The parasitic protozoal infections leishmaniasis, human African trypanosomiasis, and Chagas disease are neglected tropical diseases that pose serious health risks for much of the world's population. Current treatment options suffer from limitations, but plantderived natural products may provide economically advantageous therapeutic alternatives. Several germacranolide sesquiterpenoids have shown promising antiparasitic activities, but the mechanisms of activity have not been clearly established. OBJECTIVE: The objective is to use in silico screening of known antiparasitic germacranolides against recognized protozoal protein targets in order to provide insight into the molecular mechanisms of activity of these natural products. METHODS: Conformational analyses of the germacranolides were carried out using density functional theory, followed by molecular docking. A total of 88 Leishmania protein structures, 86 T. brucei protein structures, and 50 T. cruzi protein structures were screened against 27 antiparasitic germacranolides. RESULTS: The in-silico screening has revealed which of the protein targets of Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are preferred by the sesquiterpenoid ligands.

Michel Jouvet and "exotic" sleep.

Michel Jouvet directed my medical thesis on paradoxical sleep in cats obtained in 1969, and my research on sleep in extreme environments (Antarctica, Arctic winter cold, physical exercise), which was the subject of my Ph.D. dissertation in 1984. As a military MD and scientist, I was posted in "exotic" (far away) places (Antarctica, Canada, Niger) and participated in several remote field trials (Canada, Côte d'Ivoire, Congo, Angola). Michel Jouvet supervised my research activity, allowing me the use of his laboratory facilities. He co-authored the work on sleep in Antarctica in 1987. In 1988, he was invited to Niamey (Niger) to preside on the international jury of medical doctorate dissertations. He then examined one of my patients with narcolepsy-like sleep attacks, suspect of sleeping sickness. Jouvet also co-authored our work on nitric oxide in the rat model of sleeping sickness. His scientific curiosity led him to study REM sleep eye movements in Bassari people, an isolated ethnic group in Senegal. With Monique Gessain, he co-authored a book on the Bassari oneiric activity. He was convinced that research in electricity-free villages was capital for understanding past mankind story. The present contribution recognizes the tremendous work capacity and scientific curiosity of Michel Jouvet.

Shedding light on lipid metabolism in Kinetoplastida: A phylogenetic analysis of phospholipase D protein homologs.

Unicellular flagellates that make up the class Kinetoplastida include multiple parasites responsible for public health concerns, including Trypanosoma brucei and T. cruzi (agents of African sleeping sickness and Chagas disease, respectively), and various Leishmania species, which cause leishmaniasis. These diseases are generally difficult to eradicate, with treatments often having lethal side effects and/or being effective only during the acute phase of the diseases, when most patients are still asymptomatic. Phospholipid signaling and metabolism are important in the different life stages of Trypanosoma, including playing a role in transitions between stages and in immune system evasion, thus, making the responsible enzymes into potential therapeutic targets. However, relatively little is understood about how the pathways function in these pathogens. Thus, in this study we examined evolutionary history of proteins from one such signaling pathway, namely phospholipase D (PLD) homologs. PLD is an enzyme responsible for synthesizing phosphatidic acid (PA) from membrane phospholipids. PA is not only utilized for phospholipid synthesis, but is also involved in many other signaling pathways, including biotic and abiotic stress response. 37 different representative Kinetoplastida genomes were used for an exhaustive search to identify putative PLD homologs. The genome of Bodo saltans was the only one of surveyed Kinetoplastida genomes that encoded a protein that clustered with plant PLDs. The representatives from other Kinetoplastida species clustered together in two different clades, thought to be homologous to the PLD superfamily, but with shared sequence similarity with cardiolipin synthases (CLS), and phosphatidylserine synthases (PSS). The protein structure predictions showed that most Kinetoplastida sequences resemble CLS and PSS, with the exception of 5 sequences from Bodo saltans that shared significant structural similarities with the PLD sequences, suggesting the loss of PLD-like sequences during the evolution of parasitism in kinetoplastids. On the other hand, diacylglycerol kinase (DGK) homologs were identified for all species examined in this study, indicating that DGK could be the only pathway for the synthesis of PA involved in lipid signaling in these organisms due to genome streamlining during transition to parasitic lifestyle. Our findings offer insights for development of potential therapeutic and/or intervention approaches, particularly those focused on using PA, PLD and/or DGK related pathways, against trypanosomiasis, leishmaniasis, and Chagas disease.

Alpha-Difluoromethylornithine, an Irreversible Inhibitor of Polyamine Biosynthesis, as a Therapeutic Strategy against Hyperproliferative and Infectious Diseases.

The fluorinated ornithine analog α-difluoromethylornithine (DFMO, eflornithine, ornidyl) is an irreversible suicide inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme of polyamine biosynthesis. The ubiquitous and essential polyamines have many functions, but are primarily important for rapidly proliferating cells. Thus, ODC is potentially a drug target for any disease state where rapid growth is a key process leading to pathology. The compound was originally discovered as an anticancer drug, but its effectiveness was disappointing. However, DFMO was successfully developed to treat African sleeping sickness and is currently one of few clinically used drugs to combat this neglected tropical disease. The other Food and Drug Administration (FDA) approved application for DFMO is as an active ingredient in the hair removal cream Vaniqa. In recent years, renewed interest in DFMO for hyperproliferative diseases has led to increased research and promising preclinical and clinical trials. This review explores the use of DFMO for the treatment of African sleeping sickness and hirsutism, as well as its potential as a chemopreventive and chemotherapeutic agent against colorectal cancer and neuroblastoma.

Human African Trypanosomiasis in Emigrant Returning to China from Gabon, 2017.

Human African trypanosomiasis is endemic to parts of sub-Saharan Africa and should be considered in the differential diagnosis of patients who have visited or lived in Africa. We report a 2017 case of stage 2 Trypanosoma brucei gambiense disease in an emigrant who returned to China from Gabon.

Ornithine Decarboxylase Inhibition: A Strategy to Combat Various Diseases.

Ornithine decarboxylase is the first enzyme in the polyamine biosynthetic pathway. It is the rate-limiting enzyme which is included during the change of ornithine to putrescine which is the first polyamine. Polyamines (putrescine, spermidine, spermine) are natural and synthetic compounds which contain two or more amino group. Polyamines are highly implicated in cellular functions such as cell growth & multiplication, DNA stabilization, gene transcription and translation, ion-channel activity, etc. Elevated levels of polyamines are found in highly proliferating tumor cells. Hence inhibition of this enzyme was found useful in cancer. α-DL-difluoromethylornithine(DFMO) (Eflornithine) an enzyme- activated irreversible inhibitor was the first of this type. However, its use as an anticancer agent did not continue for long due to various reasons. Polyamines have also been found to play an important role in other infectious microorganisms. Eflornithine is successfully used in diseases such as African sleeping sickness and is being researched against a number of tropical diseases. It is widely used against hirsutism in women. Various other product (putrescine) based analogs and transition state or PLP (cofactor) based analogs are being synthesized against diseases such as Leishmaniasis, Malaria and others discussed in the article.

Indole and Benzimidazole Bichalcophenes: Synthesis, DNA Binding and Antiparasitic Activity.

A novel series of indole and benzimidazole bichalcophene diamidine derivatives were prepared to study their antimicrobial activity against the tropical parasites causing African sleeping sickness and malaria. The dicyanoindoles needed to synthesize the target diamidines were obtained through Stille coupling reactions while the bis-cyanobenzimidazoles intermediates were made via condensation/cyclization reactions of different aldehydes with 4-cyano-1,2-diaminobenzene. Different amidine synthesis methodologies namely, lithium bis-trimethylsilylamide (LiN[Si(CH3)3]2) and Pinner methods were used to prepare the diamidines. Both types (indole and benzimidazole) derivatives of the new diamidines bind strongly with the DNA minor groove and generally show excellent in vitro antitrypanosomal activity. The diamidino-indole derivatives also showed excellent in vitro antimalarial activity while their benzimidazole counterparts were generally less active. Compound 7c was highly active in vivo and cured all mice infected with Trypanosoma brucei rhodesiense, a model that mimics the acute stage of African sleeping sickness, at a low dose of 4 × 5 mg/kg i.p. and hence 7c is more potent in vivo than pentamidine.

Cell Cycle Inhibition To Treat Sleeping Sickness.

African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens.IMPORTANCE The development of drugs to treat infections with eukaryotic pathogens is challenging because many key virulence factors have closely related homologues in humans. Drug toxicity greatly limits these development efforts. For pathogens that replicate at a high rate, especially in the blood, an alternative approach is to target the cell cycle directly, much as is done to treat some hematologic malignancies. The results presented here indicate that targeting the cell cycle via inhibition of ribonucleotide reductase is effective at killing trypanosomes and prolonging the survival of infected animals.

Novel 1,2-dihydroquinazolin-2-ones: Design, synthesis, and biological evaluation against Trypanosoma brucei.

In 2014, a published report of the high-throughput screen of>42,000 kinase inhibitors from GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary compound synthesis and SAR studies, we have confirmed the previously reported activity of 1 in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1, while also showing good in vitro metabolic stability. The SAR developed on 24 provides new directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery.