Proteomic Selection of Immunodiagnostic Antigens for Human African Trypanosomiasis and Generation of a Prototype Lateral Flow Immunodiagnostic Device

Background

The diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens.

Methodology/Principal Findings

We took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device.

Conclusions/Significance

Using a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use.     

Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs)

The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials.     

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.     

The Burden of Human African Trypanosomiasis

Human African trypanosomiasis (HAT, or sleeping sickness) is a protozoan parasitic infection caused by Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense. These are neglected tropical diseases, and T.b. rhodesiense HAT is a zoonosis. We review current knowledge on the burden of HAT in sub-Saharan Africa, with an emphasis on the disability-adjusted life year (DALY), data sources, and methodological issues relating to the use of this metric for assessing the burden of this disease. We highlight areas where data are lacking to properly quantify the impact of these diseases, mainly relating to quantifying under-reporting and disability associated with infection, and challenge the HAT research community to tackle the neglect in data gathering to enable better evidence-based assessments of burden using DALYs or other appropriate measures.     

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.     

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.     

Purine metabolism in the bloodstream forms of trypanosoma gambiense and Trypanosoma rhodesiense

Bloodstream forms of Trypanosoma brucie gambiense and Trypanosoma brucei rhodesiense are incapable of de novo purine synthesis. Purine bases are converted directly to ribonucleotides and with the exception of guanine, are stable. Guanine is incorporated directly into ribonucleotides and also deaminated to xanthine. Purine ribonucleosides are hydrolyzed rapidly; these reactions may limit their incorporation since purine bases label the nucleotide pools more efficiently than do ribonucleosides. The apparent order of salvage efficiency for ribonucleosides is adenosine>inosine>guanosine>xanthosine for both organisms. T. b. gambiense salvages purine bases in the same order, while T. b. rhodesiense salvages purine bases in the order hypoxanthine>adenine>guanine>xanthine.     

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.     

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

BackgroundHuman African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense remains highly prevalent in west and central Africa and is lethal if left untreated. The major problem is that the disease often evolves toward chronic or asymptomatic forms with low and fluctuating parasitaemia producing apparently aparasitaemic serological suspects who remain untreated because of the toxicity of the chemotherapy. Whether the different types of infections are due to host or parasite factors has been difficult to address, since T. b. gambiense isolated from patients is often not infectious in rodents thus limiting the variety of isolates.Conclusions/SignificanceWhereas trypanosome characterisation assigned all these isolates to the homogeneous Group I of T. b. gambiense, they clearly induce very different infections in mice thus mimicking the broad clinical diversity observed in HAT due to T. b. gambiense. Therefore, these murine models will be very useful for the understanding of different aspects of the physiopathology of HAT and for the development of new diagnostic tools and drugs.     

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

Background

Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.

Methodology/Principal Findings

We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.

Conclusions/Significance

The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.     

Trypanosome alternative oxidase, a potential therapeutic target for sleeping sickness, is conserved among Trypanosoma brucei subspecies

Trypanosoma brucei rhodesiense and T. b. gambiense are known causes of human African trypanosomiasis (HAT), or “sleeping sickness,” which is deadly if untreated. We previously reported that a specific inhibitor of trypanosome alternative oxidase (TAO), ascofuranone, quickly kills African trypanosomes in vitro and cures mice infected with another subspecies, non-human infective T. b. brucei, in in vivo trials. As an essential factor for trypanosome survival, TAO is a promising drug target due to the absence of alternative oxidases in the mammalian host. This study found TAO expression in HAT-causing trypanosomes; its amino acid sequence was identical to that in non-human infective T. b. brucei. The biochemical understanding of the TAO including its 3 dimensional structure and inhibitory compounds against TAO could therefore be applied to all three T. brucei subspecies in search of a cure for HAT. Our in vitro study using T. b. rhodesiense confirmed the effectiveness of ascofuranone (IC₅₀ value: 1 nM) to eliminate trypanosomes in human infective strain cultures.     

Epidemiology of Sleeping Sickness in Boffa (Guinea): Where Are the Trypanosomes?

Human African Trypanosomiasis (HAT) in West Africa is a lethal, neglected disease caused by Trypanosoma brucei gambiense transmitted by the tsetse Glossina palpalis gambiensis. Although the littoral part of Guinea with its typical mangrove habitat is the most prevalent area in West Africa, very few data are available on the epidemiology of the disease in such biotopes. As part of a HAT elimination project in Guinea, we carried a cross-sectional study of the distribution and abundance of people, livestock, tsetse and trypanosomes in the focus of Boffa. An exhaustive census of the human population was done, together with spatial mapping of the area. Entomological data were collected, a human medical survey was organized together with a survey in domestic animals. In total, 45 HAT cases were detected out of 14445 people who attended the survey, these latter representing 50.9% of the total population. Potential additional carriers of T. b. gambiense were also identified by the trypanolysis test (14 human subjects and two domestic animals). No trypanosome pathogenic to animals were found, neither in the 874 tsetse dissected nor in the 300 domestic animals sampled. High densities of tsetse were found in places frequented by humans, such as pirogue jetties, narrow mangrove channels and watering points. The prevalence of T. b. gambiense in humans, combined to low attendance of the population at risk to medical surveys, and to an additional proportion of human and animal carriers of T. b. gambiense who are not treated, highlights the limits of strategies targeting HAT patients only. In order to stop T. b. gambiense transmission, vector control should be added to the current strategy of case detection and treatment. Such an integrated strategy will combine medical surveillance to find and treat cases, and vector control activities to protect people from the infective bites of tsetse.     

Identification of peptide mimotopes of Trypanosoma brucei gambiense variant surface glycoproteins

Background

The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests.

Methodology/Principal Findings

PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05).

Conclusions/Significance

We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.     

Gambiense Human African Trypanosomiasis and Immunological Memory: Effect on Phenotypic Lymphocyte Profiles and Humoral Immunity

In mice, experimental infection with Trypanosoma brucei causes decreased bone marrow B-cell development, abolished splenic B-cell maturation and loss of antibody mediated protection including vaccine induced memory responses. Nothing is known about this phenomenon in human African trypanosomiasis (HAT), but if occurring, it would imply the need of revaccination of HAT patients after therapy and abolish hope for a HAT vaccine. The effect of gambiense HAT on peripheral blood memory T- and B-cells and on innate and vaccine induced antibody levels was examined. The percentage of memory B- and T-cells was quantified in peripheral blood, prospectively collected in DR Congo from 117 Trypanosoma brucei gambiense infected HAT patients before and six months after treatment and 117 controls at the same time points. Antibodies against carbohydrate antigens on red blood cells and against measles were quantified. Before treatment, significantly higher percentages of memory B-cells, mainly T-independent memory B-cells, were observed in HAT patients compared to controls (CD20+CD27+IgM+, 13.0% versus 2.0%, p<0.001). The percentage of memory T-cells, mainly early effector/memory T-cells, was higher in HAT (CD3+CD45RO+CD27+, 19.4% versus 16.7%, p = 0.003). After treatment, the percentage of memory T-cells normalized, the percentage of memory B-cells did not. The median anti-red blood cell carbohydrate IgM level was one titer lower in HAT patients than in controls (p<0.004), and partially normalized after treatment. Anti-measles antibody concentrations were lower in HAT patients than in controls (medians of 1500 versus 2250 mIU/ml, p = 0.02), and remained so after treatment, but were above the cut-off level assumed to provide protection in 94.8% of HAT patients, before and after treatment (versus 98.3% of controls, p = 0.3). Although functionality of the B-cells was not verified, the results suggest that immunity was conserved in T.b. gambiense infected HAT patients and that B-cell dysfunction might not be that severe as in mouse models.     

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.     

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.     

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions

Background

At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests.

Methodology/Principal Findings

A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79.

Conclusions/Significance

We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.     

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.     

Estimating and Mapping the Population at Risk of Sleeping Sickness

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, persists as a public health problem in several sub-Saharan countries. Evidence-based, spatially explicit estimates of population at risk are needed to inform planning and implementation of field interventions, monitor disease trends, raise awareness and support advocacy. Comprehensive, geo-referenced epidemiological records from HAT-affected countries were combined with human population layers to map five categories of risk, ranging from “very high” to “very low,” and to estimate the corresponding at-risk population.

Results

Approximately 70 million people distributed over a surface of 1.55 million km² are estimated to be at different levels of risk of contracting HAT. Trypanosoma brucei gambiense accounts for 82.2% of the population at risk, the remaining 17.8% being at risk of infection from T. b. rhodesiense. Twenty-one million people live in areas classified as moderate to very high risk, where more than 1 HAT case per 10,000 inhabitants per annum is reported.

Discussion

Updated estimates of the population at risk of sleeping sickness were made, based on quantitative information on the reported cases and the geographic distribution of human population. Due to substantial methodological differences, it is not possible to make direct comparisons with previous figures for at-risk population. By contrast, it will be possible to explore trends in the future. The presented maps of different HAT risk levels will help to develop site-specific strategies for control and surveillance, and to monitor progress achieved by ongoing efforts aimed at the elimination of sleeping sickness.     

Optimization of the electrophile of chloronitrobenzamide leads active against Trypanosoma brucei

We previously reported the phenylchloronitrobenzamides (PCNBs), a novel class of compounds active against the species of trypanosomes that cause Human African Trypanosomiasis (HAT). Herein, we explored the potential to adjust the reactivity of the electrophilic chloronitrobenzamide core. These studies identified compound 7d that potently inhibited the growth of trypanosomes (EC₅₀ = 120 nM for Trypanosoma b. brucei, 18 nM for Trypanosoma b. rhodesiense, and 38 nM for Trypanosoma b. gambiense) without significant cytotoxicity against mammalian cell lines (EC₅₀ > 25 μM for HepG2, HEK293, Raji, and BJ cell lines) and also had good stability in microsomal models (t_1/2 > 4 h in both human and mouse). Overall these properties indicate the compound 7d and its analogs are worth further exploration as potential leads for HAT.