A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 10⁴ tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.     

Focus-specific clinical profiles in human African Trypanosomiasis caused by Trypanosoma brucei rhodesiense

Background

Diverse clinical features have been reported in human African trypanosomiasis (HAT) foci caused by Trypanosoma brucei rhodesiense (T.b.rhodesiense) giving rise to the hypothesis that HAT manifests as a chronic disease in South-East African countries and increased in virulence towards the North. Such variation in disease severity suggests there are differences in host susceptibility to trypanosome infection and/or genetic variation in trypanosome virulence. Our molecular tools allow us to study the role of host and parasite genotypes, but obtaining matched extensive clinical data from a large cohort of HAT patients has previously proved problematic.

Methods/Principal Findings

We present a retrospective cohort study providing detailed clinical profiles of 275 HAT patients recruited in two northern foci (Uganda) and one southern focus (Malawi) in East Africa. Characteristic clinical signs and symptoms of T.b.rhodesiense infection were recorded and the degree of neurological dysfunction determined on admission. Clinical observations were mapped by patient estimated post-infection time. We have identified common presenting symptoms in T.b.rhodesiense infection; however, marked differences in disease progression and severity were identified between foci. HAT was characterised as a chronic haemo-lymphatic stage infection in Malawi, and as an acute disease with marked neurological impairment in Uganda. Within Uganda, a more rapid progression to meningo-encephaltic stage of infection was observed in one focus (Soroti) where HAT was characterised by early onset neurodysfunction; however, severe neuropathology was more frequently observed in patients in a second focus (Tororo).

Conclusions/Significance

We have established focus-specific HAT clinical phenotypes showing dramatic variations in disease severity and rate of stage progression both between northern and southern East African foci and between Ugandan foci. Understanding the contribution of host and parasite factors in causing such clinical diversity in T.b.rhodesiense HAT has much relevance for both improvement of disease management and the identification of new drug therapy.     

Clinical Profiles,Disease Outcome and Co-Morbidities among T. b. rhodesiense Sleeping Sickness Patients in Uganda

BackgroundThe acute form of Human African Trypanosomiasis (HAT, also known as Sleeping sickness) caused by Trypanosoma brucei rhodesiense has been shown to have a wide spectrum of focus specific clinical presentation and severity in East and Southern Africa. Indeed HAT occurs in regions endemic for other tropical diseases, however data on how these co-morbidities might complicate the clinical picture and affect disease outcome remains largely scanty. We here describe the clinical presentation, presence of co-infections, and how the latter impact on HAT prognosis.ConclusionsWe show a wide spectrum of sleeping sickness clinical presentation and disease outcome that was apparently not significantly influenced by concurrent infections. It would thus be interesting to determine the host and/or parasite factors that might be responsible for the observed diverse clinical presentation.     

Development of multiplex serological assay for the detection of human African trypanosomiasis

Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance.We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance.     

Discovery of Infection Associated Metabolic Markers in Human African Trypanosomiasis

Human African trypanosomiasis (HAT) remains a major neglected tropical disease in Sub-Saharan Africa. As clinical symptoms are usually non-specific, new diagnostic and prognostic markers are urgently needed to enhance the number of identified cases and optimise treatment. This is particularly important for disease caused by Trypanosoma brucei rhodesiense, where indirect immunodiagnostic approaches have to date been unsuccessful. We have conducted global metabolic profiling of plasma from T.b.rhodesiense HAT patients and endemic controls, using ¹H nuclear magnetic resonance (NMR) spectroscopy and ultra-performance liquid chromatography, coupled with mass spectrometry (UPLC-MS) and identified differences in the lipid, amino acid and metabolite profiles. Altogether 16 significantly disease discriminatory metabolite markers were found using NMR, and a further 37 lipid markers via UPLC-MS. These included significantly higher levels of phenylalanine, formate, creatinine, N-acetylated glycoprotein and triglycerides in patients relative to controls. HAT patients also displayed lower concentrations of histidine, sphingomyelins, lysophosphatidylcholines, and several polyunsaturated phosphatidylcholines. While the disease metabolite profile was partially consistent with previous data published in experimental rodent infection, we also found unique lipid and amino acid profile markers highlighting subtle but important differences between the host response to trypanosome infections between animal models and natural human infections. Our results demonstrate the potential of metabolic profiling in the identification of novel diagnostic biomarkers and the elucidation of pathogenetic mechanisms in this disease.     

Identification of Compounds with Anti-Proliferative Activity against Trypanosoma brucei brucei Strain 427 by a Whole Cell Viability Based HTS Campaign

Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC₅₀ value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC₅₀ values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.     

Clinical presentation of T.b. rhodesiense sleeping sickness in second stage patients from Tanzania and Uganda

Background

A wide spectrum of disease severity has been described for Human African Trypanosomiasis (HAT) due to Trypanosoma brucei rhodesiense (T.b. rhodesiense), ranging from chronic disease patterns in southern countries of East Africa to an increase in virulence towards the north. However, only limited data on the clinical presentation of T.b. rhodesiense HAT is available. From 2006-2009 we conducted the first clinical trial program (Impamel III) in T.b. rhodesiense endemic areas of Tanzania and Uganda in accordance with international standards (ICH-GCP). The primary and secondary outcome measures were safety and efficacy of an abridged melarsoprol schedule for treatment of second stage disease. Based on diagnostic findings and clinical examinations at baseline we describe the clinical presentation of T.b. rhodesiense HAT in second stage patients from two distinct geographical settings in East Africa.

Methodology/Principal Findings:

138 second stage patients from Tanzania and Uganda were enrolled. Blood samples were collected for diagnosis and molecular identification of the infective trypanosomes, and T.b. rhodesiense infection was confirmed in all trial subjects. Significant differences in diagnostic parameters and clinical signs and symptoms were observed: the median white blood cell (WBC) count in the cerebrospinal fluid (CSF) was significantly higher in Tanzania (134cells/mm³) than in Uganda (20cells/mm³; p<0.0001). Unspecific signs of infection were more commonly seen in Uganda, whereas neurological signs and symptoms specific for HAT dominated the clinical presentation of the disease in Tanzania. Co-infections with malaria and HIV did not influence the clinical presentation nor treatment outcomes in the Tanzanian study population.

Conclusions/Significance

We describe a different clinical presentation of second stage T.b. rhodesiense HAT in two distinct geographical settings in East Africa. In the ongoing absence of sensitive diagnostic tools and safe drugs to diagnose and treat second stage T.b. rhodesiense HAT an early identification of the disease is essential. A detailed understanding of the clinical presentation of T.b. rhodesiense HAT among health personnel and affected communities is vital, and awareness of regional characteristics, as well as implications of co-infections, can support decision making and differential diagnosis.     

A Literature Review of Economic Evaluations for a Neglected Tropical Disease: Human African Trypanosomiasis (“Sleeping Sickness”)

Human African trypanosomiasis (HAT) is a disease caused by infection with the parasite Trypanosoma brucei gambiense or T. b. rhodesiense. It is transmitted to humans via the tsetse fly. Approximately 70 million people worldwide were at risk of infection in 1995, and approximately 20,000 people across Africa are infected with HAT. The objective of this review was to identify existing economic evaluations in order to summarise cost-effective interventions to reduce, control, or eliminate the burden of HAT. The studies included in the review were compared and critically appraised in order to determine if there were existing standardised methods that could be used for economic evaluation of HAT interventions or if innovative methodological approaches are warranted. A search strategy was developed using keywords and was implemented in January 2014 in several databases. The search returned a total of 2,283 articles. After two levels of screening, a total of seven economic evaluations were included and underwent critical appraisal using the Scottish Intercollegiate Guidelines Network (SIGN) Methodology Checklist 6: Economic Evaluations. Results from the existing studies focused on the cost-effectiveness of interventions for the control and reduction of disease transmission. Modelling was a common method to forecast long-term results, and publications focused on interventions by category, such as case detection, diagnostics, drug treatments, and vector control. Most interventions were considered cost-effective based on the thresholds described; however, the current treatment, nifurtomix-eflornithine combination therapy (NECT), has not been evaluated for cost-effectiveness, and considerations for cost-effective strategies for elimination have yet to be completed. Overall, the current evidence highlights the main components that play a role in control; however, economic evaluations of HAT elimination strategies are needed to assist national decision makers, stakeholders, and key funders. These analyses would be of use, as HAT is currently being prioritized as a neglected tropical disease (NTD) to reach elimination by 2020.     

Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Background

The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) have been recently modified by coupling to oligochromatography (OC) for easy and fast visualisation of products. In this study we evaluate the sensitivity and specificity of the PCR-OC and NASBA-OC for diagnosis of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense human African trypanosomiasis (HAT).

Methodology and Results

Both tests were evaluated in a case-control design on 143 HAT patients and 187 endemic controls from the Democratic Republic of Congo (DRC) and Uganda. The overall sensitivity of PCR-OC was 81.8% and the specificity was 96.8%. The PCR-OC showed a sensitivity and specificity of 82.4% and 99.2% on the specimens from DRC and 81.3% and 92.3% on those from Uganda. NASBA-OC yielded an overall sensitivity of 90.2%, and a specificity of 98.9%. The sensitivity and specificity of NASBA-OC on the specimens from DRC was 97.1% and 99.2%, respectively. On the specimens from Uganda we observed a sensitivity of 84.0% and a specificity of 98.5%.

Conclusions/Significance

The tests showed good sensitivity and specificity for the T. b. gambiense HAT in DRC but rather a low sensitivity for T. b. rhodesiense HAT in Uganda.     

The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense

Targett G. A. T. and Wilson V. C. L. C. 1973. The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense. International Journal for Parasitology, 3: 5–11. A simple test for distinguishing between the morphologically identical subspecies Trypanosoma brucei rhodesiense, which is infective to man, and T. brucei brucei, which by definition is not, has been described. This test, the blood incubation infectivity test (BIIT), is based on absolute differences in the infectivity to rats of the subspecies after exposure to human blood, and was applied to strains which are preserved in the laboratory as stabilates. Five T. brucei brucei strains were BIIT negative since their infectivity was destroyed by incubation in normal human blood but only five of the nine T. brucei rhodesiense strains tested were consistently BIIT positive. The other four gave equivocal results, indicating that the resistance of T. brucei rhodesiense strains to the trypanocidal effect of human blood can change, probably as a result of maintenance in the laboratory.     

Identification of sVSG117 as an Immunodiagnostic Antigen and Evaluation of a Dual-Antigen Lateral Flow Test for the Diagnosis of Human African Trypanosomiasis

Background

The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.

Methodology/Principle Findings

Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.

Conclusion/Significance

In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use.     

A Four-Point Screening Method for Assessing Molecular Mechanism of Action (MMOA) Identifies Tideglusib as a Time-Dependent Inhibitor of Trypanosoma brucei GSK3β

BackgroundNew therapeutics are needed for neglected tropical diseases including Human African trypanosomiasis (HAT), a progressive and fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. There is a need for simple, efficient, cost effective methods to identify new molecules with unique molecular mechanisms of action (MMOAs). The mechanistic features of a binding mode, such as competition with endogenous substrates and time-dependence can affect the observed inhibitory IC₅₀, and differentiate molecules and their therapeutic usefulness. Simple screening methods to determine time-dependence and competition can be used to differentiate compounds with different MMOAs in order to identify new therapeutic opportunities.Conclusions/SignificanceAltogether this work demonstrates a straightforward method for determining molecular mechanisms of action and its application for mechanistic differentiation of two potent TbGSK3β inhibitors. The four point MMOA method identified tideglusib as a mechanistically differentiated TbGSK3β inhibitor. Tideglusib was shown to inhibit parasite growth in this work, and has been reported to be well tolerated in one year of dosing in human clinical studies. Consequently, further supportive studies on the potential therapeutic usefulness of tideglusib for HAT are justified.     

Population vulnerability and disability in Kenya's tsetse fly habitats

Background

Human African Trypanosomiasis (HAT), also referred to as sleeping sickness, and African Animal Trypanosomaisis (AAT), known as nagana, are highly prevalent parasitic vector-borne diseases in sub-Saharan Africa. Humans acquire trypanosomiasis following the bite of a tsetse fly infected with the protozoa Trypanosoma brucei (T.b.) spp. –i.e., T.b. gambiense in West and Central Africa and T.b. rhodesiense in East and Southern Africa. Over the last decade HAT diagnostic capacity to estimate HAT prevalence has improved in active case-finding areas but enhanced passive surveillance programs are still lacking in much of rural sub-Saharan Africa.

Methodology/Principal Findings

This retrospective-cross-sectional study examined the use of national census data (1999) to estimate population vulnerability and disability in Kenya''s 7 tsetse belts to assess the potential of HAT-acquired infection in those areas. A multilevel study design estimated the likelihood of disability in individuals, nested within households, nested within tsetse fly habitats of varying levels of poverty. Residents and recent migrants of working age were studied. Tsetse fly''s impact on disability was conceptualised via two exposure pathways: directly from the bite of a pathogenic tsetse fly resulting in HAT infection or indirectly, as the potential for AAT takes land out of agricultural production and diseased livestock leads to livestock morbidity and mortality, contributing to nutritional deficiencies and poverty. Tsetse belts that were significantly associated with increased disability prevalence were identified and the direct and indirect exposure pathways were evaluated.

Conclusions/Significance

Incorporating reports on disability from the national census is a promising surveillance tool that may enhance future HAT surveillance programs in sub-Saharan Africa. The combined burdens of HAT and AAT and the opportunity costs of agricultural production in AAT areas are likely contributors to disability within tsetse-infested areas. Future research will assess changes in the spatial relationships between high tsetse infestation and human disability following the release of the Kenya 2009 census at the local level.     

Adenosine Kinase of T. b. rhodesiense Identified as the Putative Target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine Using Chemical Proteomics

Background

Human African trypanosomiasis (HAT), a major parasitic disease spread in Africa, urgently needs novel targets and new efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) exhibits specific antitrypanosomal activity with an IC₅₀ of 1.0 µM on Trypanosoma brucei rhodesiense (T. b. rhodesiense), the causative agent of the acute form of HAT.

Methodology/Principal Findings

In this work we show adenosine kinase of T. b. rhodesiense (TbrAK), a key enzyme of the parasite purine salvage pathway which is vital for parasite survival, to be the putative intracellular target of compound 1 using a chemical proteomics approach. This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity. Further chemical validation demonstrated that compound 1 interacts specifically and tightly with TbrAK with nanomolar affinity, and in vitro activity measurements showed that compound 1 is an enhancer of TbrAK activity. The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition.

Conclusions/Significance

The results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides.     

LAMP for human African trypanosomiasis: a comparative study of detection formats

Loop-mediated isothermal amplification (LAMP) is at the forefront of the search for innovative diagnostics for human African trypanosomiasis (HAT). Several simple endpoint detection methods have been developed for LAMP and here we compare four of these: (i) visualization of turbidity; (ii) addition of hydroxynaphthol blue before incubation; (iii) addition of calcein with MnCl₂ before incubation and (iv) addition of Quant-iT PicoGreen after incubation. These four methods were applied to four LAMP assays for the detection of human African trypanosomiasis, including two Trypanozoon specific and two Trypanosoma brucei rhodesiense specific reactions using DNA extracted from cryo-preserved procyclic form T. b. rhodesiense. A multi-observer study was performed to assess inter-observer reliability of two of these methods: hydroxynapthol blue and calcein with MnCl₂, using DNA prepared from blood samples stored on Whatman FTA cards. Results showed that hydroxynaphthol blue was the best of the compared methods for easy, inexpensive, accurate and reliable interpretation of LAMP assays for HAT. Hydroxynapthol blue generates a violet to sky blue colour change that was easy to see and was consistently interpreted by independent observers. Visible turbidity detection is not possible for all currently available HAT LAMP reactions; Quant-iT PicoGreen is expensive and addition of calcein with MnCl₂ adversely affects reaction sensitivity and was unpopular with several observers.     

Activity of diimidazoline amides against African trypanosomiasis

We identified several diimidazoline mono- and diamides that were as potent as pentamidine against Trypanosoma brucei rhodesiense in vitro. All of these were also less cytotoxic than pentamidine, but none was as effective as the latter in a T. brucei rhodesiense-infected mouse model. A single imidazoline may be sufficient for high antitrypanosomal activity provided that a second weak base functional group is present.     

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

Background

There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.).

Methodology/Principal Findings

Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.

Conclusions/Significance

Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.     

Cathepsin-L Can Resist Lysis by Human Serum in Trypanosoma brucei brucei

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.     

Kenyan purple tea anthocyanins and coenzyme-Q10 ameliorate post treatment reactive encephalopathy associated with cerebral human African trypanosomiasis in murine model

Human African trypanosomiasis (HAT) is a tropical disease caused by two subspecies of Trypanosoma brucei, the East African variant T. b. rhodesiense and the West African variant T. b. gambiense. Melarsoprol, an organic arsenical, is the only drug used to treat late stage T. b. rhodesiense infection. Unfortunately, this drug induces an extremely severe post treatment reactive encephalopathy (PTRE) in up to 10% of treated patients, half of whom die from this complication. A highly reproducible mouse model was adapted to assess the use of Kenyan purple tea anthocyanins and/or coenzyme-Q₁₀ in blocking the occurrence of PTRE. Female Swiss white mice were inoculated intraperitoneally with approximately 10⁴ trypanosome isolate T. b. rhodesiense KETRI 2537 and treated sub-curatively 21 days post infection with 5 mg/kg diminazene aceturate (DA) daily for 3 days to induce severe late CNS infection that closely mirrors PTRE in human subjects. Thereafter mice were monitored for relapse of parasitemia after which they were treated with melarsoprol at a dosage of 3.6 mg/kg body weight for 4 days and sacrificed 24 h post the last dosage to obtain brain samples. Brain sections from mice with PTRE that did not receive any antioxidant treatment showed a more marked presence of inflammatory cells, microglial activation and disruption of the brain parenchyma when compared to PTRE mice supplemented with either coenzyme-Q₁₀, purple tea anthocyanins or a combination of the two. The mice group that was treated with coenzyme-Q₁₀ or purple tea anthocyanins had higher levels of GSH and aconitase-1 in the brain compared to untreated groups, implying a boost in brain antioxidant capacity. Overall, coenzyme-Q₁₀ treatment produced more beneficial effects compared to anthocyanin treatment. These findings demonstrate that therapeutic intervention with coenzyme-Q₁₀ and/or purple tea anthocyanins can be used in an experimental mouse model to ameliorate PTRE associated with cerebral HAT.     

Discovery of 2-(1H-imidazo-2-yl)piperazines as a new class of potent and non-cytotoxic inhibitors of Trypanosoma brucei growth in vitro

The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compounds from our in-house compound collection was screened in vitro against the parasite leading to the identification of compounds with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compound led to the identification of compound 34 that shows low nanomolar parasite growth inhibition (T. brucei EC₅₀ 5?nM), is not cytotoxic (HeLa CC₅₀?>?25,000?nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC₅₀ 8120?nM, P. falciparum EC₅₀ 3624?nM).