Genome-Wide SNP Analysis Reveals Distinct Origins of Trypanosoma evansi and Trypanosoma equiperdum.

YesTrypanosomes cause a variety of diseases in man and domestic animals in Africa, Latin America, and Asia. In the Trypanozoon subgenus, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis, whereas Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum are responsible for nagana, surra, and dourine in domestic animals, respectively. The genetic relationships between T. evansi and T. equiperdum and other Trypanozoon species remain unclear because the majority of phylogenetic analyses has been based on only a few genes. In this study, we have conducted a phylogenetic analysis based on genome-wide SNP analysis comprising 56 genomes from the Trypanozoon subgenus. Our data reveal that T. equiperdum has emerged at least once in Eastern Africa and T. evansi at two independent occasions in Western Africa. The genomes within the T. equiperdum and T. evansi monophyletic clusters show extremely little variation, probably due to the clonal spread linked to the independence from tsetse flies for their transmission.Funding was received from the Research Foundation Flanders (FWO, grants 1501413N and 1101614N) and the European DG Health and Food Safety (SANTE). We thank the Center of Medical Genetics at the University of Antwerp for hosting the NGS facility

Murine Models for Trypanosoma brucei gambiense Disease Progression—From Silent to Chronic Infections and Early Brain Tropism

Trypanosoma brucei gambiense is responsible for more than 90% of reported cases of human African trypanosomosis (HAT). Infection can last for months or even years without major signs or symptoms of infection, but if left untreated, sleeping sickness is always fatal. In the present study, different T. b. gambiense field isolates from the cerebrospinal fluid of patients with HAT were adapted to growth in vitro. These isolates belong to the homogeneous Group 1 of T. b. gambiense, which is known to induce a chronic infection in humans. In spite of this, these isolates induced infections ranging from chronic to silent in mice, with variations in parasitaemia, mouse lifespan, their ability to invade the CNS and to elicit specific immune responses. In addition, during infection, an unexpected early tropism for the brain as well as the spleen and lungs was observed using bioluminescence analysis. The murine models presented in this work provide new insights into our understanding of HAT and allow further studies of parasite tropism during infection, which will be very useful for the treatment and the diagnosis of the disease

Bioluminescent Imaging of Trypanosoma brucei Shows Preferential Testis Dissemination Which May Hamper Drug Efficacy in Sleeping Sickness

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs

Isolation of Trypanosoma brucei gambiense from Cured and Relapsed Sleeping Sickness Patients and Adaptation to Laboratory Mice

Human African trypanosomiasis, or sleeping sickness, is still a major public health problem in central Africa. Melarsoprol is widely used for treatment of patients where the parasite has already reached the brain. In some regions in Angola, Sudan, Uganda and Democratic Republic of the Congo, up to half of the patients cannot be cured with melarsoprol. From previous investigations it is not yet clear what causes these high relapse rates. Therefore we aimed to establish a parasite collection isolated from cured as well as relapsed patients for downstream comparative drug sensitivity profiling. From 360 sleeping sickness patients, blood and cerebrospinal fluid (CSF) was collected before treatment and along the prescribed 24 months follow-up. Blood and CSF were inoculated in thicket rats (Grammomys surdaster), Natal multimammate mice (Mastomys natalensis) and immunodeficient laboratory mice (Mus musculus). Thus, we established a unique collection of Trypanosoma brucei gambiense type I parasites, isolated in the same disease focus and within a limited period, including 12 matched strains isolated from the same patient before treatment and after relapse. This collection is now available for genotypic and phenotypic characterisation to investigate the mechanism behind abnormally high treatment failure rates in Mbuji-Mayi, Democratic Republic of the Congo

Preclinical Assessment of the Treatment of Second-Stage African Trypanosomiasis with Cordycepin and Deoxycoformycin

There is an urgent need to substitute the highly toxic arsenic compounds still in use for treatment of the encephalitic stage of African trypanosomiasis, a disease caused by infection with Trypanosoma brucei. We exploited the inability of trypanosomes to engage in de novo purine synthesis as a therapeutic target. Cordycepin was selected from a trypanocidal screen of a 2200-compound library. When administered together with the adenosine deaminase inhibitor deoxycoformycin, cordycepin cured mice inoculated with the human pathogenic subspecies T. brucei rhodesiense or T. brucei gambiense even after parasites had penetrated into the brain. Successful treatment was achieved by intraperitoneal, oral or subcutaneous administration of the compounds. Treatment with the doublet also diminished infection-induced cerebral inflammation. Cordycepin induced programmed cell death of the parasites. Although parasites grown in vitro with low doses of cordycepin gradually developed resistance, the resistant parasites lost virulence and showed no cross-resistance to trypanocidal drugs in clinical use. Our data strongly support testing cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Trypanosoma brucei gambiense: HMI-9 medium containing methylcellulose and human serum supports the continuous axenic in vitro propagation of the bloodstream form

Trypanosoma brucei (T.b.) gambiense causes the chronic form of human African trypanosomiasis or sleeping sickness. One of the major problems with studying T.b. gambiense is the difficulty to isolate it from its original host and the difficult adaptation to in vivo and in vitro mass propagation. The objective of this study was to evaluate if an established method for axenic culture of pleomorphic bloodstream form T.b. brucei strains, based on methylcellulose containing HMI-9 medium, also facilitated the continuous in vitro propagation of other bloodstream form Trypanozoon strains, in particular of T.b. gambiense. Bloodstream form trypanosomes from one T.b. brucei, two T.b. rhodesiense, one T. evansi and seven T.b. gambiense strains were isolated from mouse blood and each was concurrently cultivated in liquid and methylcellulose-containing HMI-9 based medium, either with or without additional human serum supplementation, for over 10 consecutive sub passages. Although HMI-9 based medium supplemented with 1.1% (w/v) methylcellulose supported the continuous cultivation of all non-gambiense strains better than liquid media could, the in vitro cultivation of all gambiense strains was only achieved in HMI-9 based medium containing 1.1% (w/v) methylcellulose, 15% (v/v) fetal calf serum and 5% (v/v) heat-inactivated human serum

Watching in vivo dendritic cells in action in the brain

Dendritic cells (DCs) present self or no-self antigens to T cells, to modulate the immune response. DCs are widely studied in peripheral organs, but little is known about their function in the brain. We recently demonstrated (Laperchia et al., 2013) that DCs can be monitored in vivo by two-photon microscopy in thy1GFP-M transgenic mice, in which a subset of DCs is tagged by green fluorescent protein (GFP-DCs). Here we investigated by two-photon microscopy the migratory pattern of brain GDP-DCs both in basal condition and during the meningo-encephalitic stage of an experimental model of African trypanosomiasis, also known as sleeping sickness, induced by the infection with parasites Trypanosome brucei brucei. Trough a chronically implanted brain window in thy1GFP-M mice we found, in basal conditions, GFP-DCs floating in the cerebrospinal fluid or static at the pia mater/parenchyma interface. At an early stage of the meningoencephalitis, circulating GFP-DCs were in contact with the parasites, maybe representing an antigen capture process. Subsequently, DCs roll and crawl on the inflamed endothelium and were massively recruited from the blood stream to the brain parenchyma, where exhibited rapid and wide displacements. At a late stage, the number of motile GFP-DCs was significantly reduced and, interestingly, GFP-DCs were mainly arranged in static clusters that incorporate the parasite. Our results show for the first time the migratory pattern of DCs during invasion of the inflamed brain and suggest a role of brain DCs in the passage from brain immune-resistance to immune-tolerance during a parasitic infection

Dendritic cells in the healthy and inflamed brain: A two photon microscopy investigation.

Dendritic cells (DCs) are professional antigen-presenting cells, derived from a bone marrow progenitor, which modulate the balance between suppression and induction of the immune response. DCs have been widely characterized in peripheral organs, especially in lymph nodes where antigen presentation mostly occurs. DCs have also been identified in the meninges and choroid plexus, as well as within the brain parenchyma. Several key aspects of DCs function in the brain are, however, still unexplored. Transgenic mice expressing fluorescent proteins in cell subsets provide valuable tools for in vivo investigations by two-photon fluorescence (TPF) microscopy. We recently demonstrated that in thy1GFP-M mice, engineered for green fluorescent protein (GFP) expression in neurons, also DCs express GFP. This murine line is, therefore, suited for the visualization of brain DCs. We here analyzed with TPF microscopy GFP-tagged brain DCs in healthy thy1GFP-M mice and during infection with Trypanosoma brucei (Tb). This parasite is the etiological agent of human African trypanosomiasis or sleeping sickness, whose encephalitic stage is fatal if untreated, and in which pathogenetic mechanisms of the neuroimmune response remain to be clarified. Our in vivo observations showed, in normal conditions, GFP-DCs in the subarachnoid space and meninges, where these cells were mainly static and occasionally in a probing-like motion. A motile behavior of GFP-DCs was also observed in the upper cortical layers, supporting a role of immunosurveillance of DCs in the healthy brain. Striking changes of motility and quantity of GFP-DCs were observed in the brain of Tb-infected thyGFP-M mice. During the early meningoencephalitic stage, GFP-DCs invaded the parenchyma with rapid and wide displacements, and also occurred in static adhesion to or crawling on the inflamed endothelium. With disease progression, a drastic decrease in the number of GFP-DCs was observed at the brain surface, and GFP-DCs appeared arranged in static clusters of cells exhibiting numerous processes, likely to increase the cell membrane surface on which antigens are exposed. Preliminary in vivo observations of thy1GFP-M mice infected with transgenic fluorescent Tb have shown direct interactions between GFP-DCs and the parasites; further analyses are ongoing. Taken together, the present in vivo investigations not only reveal a motile behavior of DCs at the brain surface and in the upper cortical layers, but also suggest a relevant role of brain DCs in African trypanosome infection. In particular, these cells could play a role in the transition from immune resistance to immune tolerance during this severe brain infection

Two-photon microscopy investigation of brain dendritic cells in inflamed brain

Human African trypanosomiasis, also known as sleeping sickness, is a severe disease caused by the parasite Trypanosoma brucei (T.b.), in which systemic infection evolves into meningoencephalitis1. One of the most effective strategies adopted by several parasites to attack the host immune system is to interfere with dendritic cells (DCs)2, which play a key role of immune surveillance. In African trypanosomiasis, DCs have been analyzed in peripheral organs3,4 but have not been hitherto examined in the brain. We recently demonstrated that thy1GFP-M transgenic mice represent a novel tool for the study of brain DCs because they express green fluorescent protein (GFP) not only in neurons but also in DCs5. Here, we investigated in vivo, by two-photon microscopy, DCs and their interaction with T.b. in infected thy1GFP-M mice at different time points during the meningoencephalitic stage of the disease