Dissertations / Theses on the topic 'Trypanosomiasis; Sleeping sickness'

Poverty and disease are bound together in rural communities of sub-Saharan Africa (SSA) exacerbated by weak social services and conflict. The infectious disease burden in SSA combines the neglected tropical diseases (NTDs) and the 'big three' (malaria, HIV/AIDS and tuberculosis), so-called because they attract more global attention and hence funding. NTDs include human African trypanosomiasis (HAT or sleeping sickness), first noticed by the outside world during the slave trade era and later in the 2-th century by widespread epidemics of disease across the tsetse fly belt. HAT describes two diseases: i) Gambian HAT caused by Trypanosoma brucei gambiense is characteristically chronic with an infectious period lasting up to three years and ii) Rhodesian HAT caused by T.b. rhodesiense is an acute disease, killing its victim within weeks of infection. The two diseases are frequently considered together as both are transmitted by tsetse flies, the parasites are morphologically indistinguishable and the associated diseases are both fatal if left untreated. However, the two diseases are clinical, epidemiologically and geographical distinct, each requiring different control strategies. Under field conditions, where microscopy is the basic diagnostic tool, differentiation is simply by geographical location of the patient; the Great Rift Valley separates the Gambian disease present in West and Central Africa, from East and southern Africa's Rhodesian disease. Control strategies are also distinct; while the Belgian and French colonial strategies to control the disease were patient-centred, the British colonial powers in East Africa were motivated by the effect of tsetse borne diseases on animal health. Towards the end of the colonial ear, both types of disease were heading for elimination but during the immediate post-colonial era in the 1960s, political instability compromised the rigid HAT control programs that had been put in place. For zoonotic Rhodesian sleeping sickness, complex tsetse control programmes proved difficult to maintain and to justify economically; for Gambian sleeping sickness the generalised breakdown of medical services allowed the disease to return, sometimes to devastating levels. The millennium development goals (MDGs) set out in 2000, highlighted specific challenges and opportunities for national and global development. HAT impacts national health goals of national development plans and MDGs and impedes rural development of SSA. NTDs were not addressed directly by MDGs but the World Health Organization (WHO) has reaffirmed its commitment not only to control of HAT but also to eliminate it as a public health problem by 2020. Currently there are 25 countries reporting HAT to WHO, and while the overall prevalence of HAT across Africa continues to fall, epidemics have been recorded, particularly from central Africa, South Sudan and Uganda. Uganda is uniquely, the only country affected by both T.b. gambiense and T.b. rhodesiense and until the present study, there was no evidence to suggest that the two parasite species co-existed in Uganda. The development of a new control paradigm for T.b. rhodesiese in South East Uganda has lowered the incidence of human infections and, more importantly, halted the northerly spread of this parasite. However, recurring epidemics in several established and new disease foci in central Uganda highlight the difficulties involved in eliminating this disease. The present study assesses past and present HAT control strategies centred on Dokolo, Kaberamaido and Soroti Districts located at the centre of Uganda. These districts are highly endemic for T.b. rodesiense, they represent the region of concern for overlap with T.b. gambiense foci in central Uganda, and are the current focus of the Stamp out Sleeping sickness control initiative. The point prevalence of T. brucei s.1 in cattle reservoir from villages with (out) reported human disease located at specific distances to Otuboi, Chagwere and Ochero cattle markets, was evaluated before and six months after trypanocidal treatment, to assess the transferrable impact of zoonotic T.b. rhodesiense to the human population. Overall, the proportion of T. brucei s.1 in cattle dropped significantly from 22% at baseline to 9% six months after trypanocide treatment (P < 0.05, Chi-square + 17.92, 95% C.I. + 1.71 to 4.49). All villages located in sub-counties that received at least 80% treatment coverage had a drop in T. brucei s.1 prevalence from 30.4% (95%, C.I + 22.8 to 38.0) before treatment was done, to 12.9% (95%, C.I. + 7.4 to 18.4) six months after treatment. More specifically, impact on human infective T.b. rhodesiense was also halved. In fact only three cattle were detected with the parasite six months after treatment compared with six from those sampled as baseline. This study also utilises documented cases between 2009 and 2012 to assess the current HAT reporting system for monitoring and evaluating transmission dynamics of the disease. Using a questionnaire, capacity and preparedness of healthcare professionals to respond to disease epidemics was assessed. The point prevalence of sleeping sickness in the three districts in 2009 was determined by screening volunteers. Microscopic examinations detected trypanosomes in four volunteers (4/5311 or 0.075 %) while PCR detected significantly more infections (24, p < 0.001). Multiplex PCR showed that ten of the Trypanozoon infections were T.b. rhodesiense while nested PCR identified four infections as T.b. gamiense, indicating that the distribution of the two forms of sleeping sickness overlaps in Uganda. Second phase investigations followed up the PCR positive cases; these people were screened again, together with members of their homestead and the inhabitants of three neighbouring homes. Besides microscopy and PCR, study subjects were examined clinically for sleeping sickness and completed a questionnaire to assess community recognition of the disease. This extended screen revealed no new cases underlining the importance of stringent early screening that PCR techniques can provide. At local healthcare centres, 54% of reported sleeping sickness cases were diagnosed only at the late stage, indicating a weakness in early diagnosis and hence early reporting. Interviews with local health workers also revealed weaknesses in recognition of clinical signs and a gap in diagnostic capacity. While records at treating hospitals remain a useful indicator for targeting active foci of infection, improvement in capacity to diagnose HAT at an early stage should contribute both to rural health and disease control strategies and also towards WHO's 2020 target of elimination of HAT.

The tsetse transmitted fatal disease of humans, sleeping sickness, is caused by two morphologically identical subspecies of the parasite T. brucei; T. b. rhodesiense and T. b. gambiense. Current distributions of the two forms of disease are not known to overlap in any area, and Uganda is the only country with transmission of both. The distribution of Rhodesian sleeping sickness in Uganda has expanded in recent years, with five districts newly affected since 1998. This movement has narrowed the gap between Rhodesian and Gambian sleeping sickness endemic areas, heightening concerns over a potential future overlap which would greatly complicate the diagnosis and treatment of the two diseases. An improved understanding of the social, environmental and climatic determinants of the distribution of Rhodesian sleeping sickness is required to allow more effective targeting of control measures and to prevent further spread and possible concurrence with Gambian sleeping sickness. The work presented in this thesis investigates the drivers of the distribution and spread of Rhodesian sleeping sickness in districts of central and eastern Uganda which form part of the recent disease focus extension. The spatial distribution of Rhodesian sleeping sickness was examined in Kaberamaido and Dokolo districts where the disease was first reported in 2004, using three different methodologies. A traditional one-step logistic regression analysis of disease prevalence was compared with a two-step hierarchical logistic regression analysis. The two-step method included the analysis of disease occurrence followed by the analysis of disease prevalence in areas with a high predicted probability of occurrence. These two methods were compared in terms of their predictive accuracy. The incorporation of a stochastic spatial effect to model the residual spatial autocorrelation was carried out using a Bayesian geostatistical approach. The geostatistical analysis was compared with the non-spatial models to assess the importance of spatial autocorrelation, to establish which method had the highest predictive accuracy and to establish which factors were the most significant in terms of the disease’s distribution. Links between Rhodesian sleeping sickness and landcover in Soroti district were also assessed using a matched case-control study design. Temporal trends in these relationships were observed using an annually stratified analysis to allow an exploration of the disease’s dispersion following its introduction to a previously unaffected area. This work expands on previous research that demonstrated the source of infection in this area to be the movement of untreated livestock from endemic areas through a local livestock market. With regards to the comparison of regression frameworks, the two-step regression compared favourably with the traditional one-step regression, but the Bayesian geostatistical analysis outperformed both in terms of predictive accuracy. Each of these regression methods highlighted the importance of distance to the closest livestock market on the distribution of Rhodesian sleeping sickness, indicating that the disease may have been introduced to this area via the movement of untreated cattle from endemic areas, despite the introduction of regulations requiring the treatment of livestock prior to sale. In addition, several other environmental and climatic variables were significantly associated with sleeping sickness occurrence and prevalence within the study area. The temporal stratification of the matched case-control analysis highlights the dispersion of sleeping sickness away from the point of introduction (livestock market) into more suitable areas; areas with higher proportions of seasonally flooding grassland, lower proportions of woodland and dense savannah and lower elevations. These findings relate to the habitat preferences of the predominant vector species in the study area; Glossina fuscipes fuscipes, which prefers riverine vegetation. The findings presented highlight the importance of the livestock reservoir as well as the climatic and environmental preferences of the tsetse fly vector for the introduction of Rhodesian sleeping sickness into previously unaffected areas, the subsequent spread of infection following an introduction and the equilibrium spatial distribution of the disease. By enhancing the knowledge base regarding the spatial determinants of the distribution of Rhodesian sleeping sickness within newly affected areas, future control efforts within Uganda may be better targeted to decrease prevalence and to prevent further spread of the disease.

Trypanosome alternative oxidase (TAO) is the sole terminal oxidase responsible for the aerobic respiration of the parasite T. b. brucei. Specific strains of this parasite cause the neglected tropical disease Human African trypanosomiasis (HAT), and thus TAO is an interesting target for the potential treatment of this disease. Inhibition of TAO with the natural product inhibitors colletochlorin B or ascofuranone has been shown to clear infections of T. b. brucei in mice at high concentrations. However, these natural product inhibitors contain undesirable chemical functionality and have poor physicochemical properties, preventing adequate drug exposure to effectively treat HAT. Robust protocols for the expression and purification of recombinant TAO were developed, which enabled the development of biochemical assays to identify inhibitors of TAO function. Single point inhibition screening of the Medicines Malaria Venture 'kinetoplastid collection' of 400 compounds identified a range of micro-molar inhibitors of TAO. A program of chemical optimisation was carried out around the natural product inhibitor colletochlorin B, with the aim to improve the physicochemical properties and retain inhibitory potency against TAO. The structure activity relationships generated over the course of this exploration identified a dependency on high lipophilicity to retain potent TAO inhibition. The TAO inhibitors synthesised were also assessed for parasite growth inhibition and mammalian cell cytotoxicity to correlate inhibition data with cellular efficacy, in collaboration with Novartis. The physicochemical properties of these novel compounds showed improvement over the natural product colletochlorin B and prompted further assessment of leading compounds in advanced parasite kill kinetic and parasite clearance assays at Novartis. The data generated in these assays for compounds synthesised in this thesis determined that TAO inhibition results in a trypanostatic response, and not a preferred trypanocidal response in T. b. brucei.

The haptoglobin-haemoglobin receptor (HpHbR) is expressed by the African try- panosome, T. brucei, whilst in the bloodstream of the mammalian host. This allows ac- quisition of haem, but also results in uptake of trypanolytic factor 1, a mediator of in- nate immunity against non-human African trypanosomes. Here, the structure of HpHbR in complex with its ligand, haptoglobin-haemoglobin (HpHb), is presented, revealing an elongated binding site along the membrane-distal half of the receptor. A ~50° kink allows the simultaneous binding of two receptors to one dimeric HpHb, increasing the efficiency of ligand uptake whilst also increasing binding site exposure within the densely packed cell surface. The possibility of targeting this receptor with antibody-drug conjugates is ex- plored. The characterisation of the unexpected interaction between T. congolense HpHbR and its previously unknown ligand, haemoglobin, is also presented. This receptor is iden- tified as an epimastigote-specific protein expressed whilst the trypanosome occupies the mouthparts of the tsetse fly vector. An evolutionary pathway of the receptor is proposed, describing how the receptor has changed to adapt to a role as a bloodstream form-specific protein in T. brucei. Apolipoprotein L1 (ApoL1) is the pore-forming component of the trypanolytic factors. An expression and purification protocol for ApoL1 is presented here, and the functionality of the protein established. Initial attempts to characterise the pores and structure of ApoL1 are described.

A longitudinal epidemiological study of trypanosome infections in domestic livestock was carried out over thirteen months in Busia District, an endemic area of Rhodesian sleeping sickness. It was demonstrated that chemoprophylaxis of domestic livestock with Samorin ® (Rhône Poulenc, France) could virtually eliminate the reservoir of potentially human infective T. brucei spp. infections in domestic animals. In addition to being a potentially useful control measure during outbreaks of sleeping sickness, chemoprophylaxis showed a net economic benefit in the productivity of local cattle. The duration of chemoprophylaxis of domestic livestock with Ethidium ® (Laprovet, France) was much less, as was the economic benefit to livestock production, however the level of challenge was much less in this trial. From the relative preference of tsetse flies for each species of domestic animal and the prevalence and incidence of trypanosome infections, it was concluded that cattle and to a lesser extent pigs were the most important species of domestic livestock in the animal reservoir of Rhodesian sleeping sickness in Busia District. Of the risk factors investigated for cattle acquiring trypanosome infections, the most important was an existing trypanosome infection. The effect on the epidemiology of sleeping sickness of a greater than expected interaction between species of trypanosome infecting cattle offers a possible mechanism for the natural control of the animal reservoir of Rhodesian sleeping sickness. Various parasitological diagnostic techniques for the detection of trypanosome infections in cattle in the field were assessed. Maximum sensitivity of diagnosis and minimum bias in the relative importance of each species of trypanosome was achieved by selecting a suitable combination of diagnostic techniques.

Over the past two decades movement of cattle towards the north of Uganda has enabled the Trypanosoma brucei rhodesiense focus in south-eastern Uganda to spread into previously unaffected districts. This thesis brings together important epidemiological data regarding the impact of mass cattle drug treatment on the point prevalence of several different species of trypanosome in a newly endemic area of human sleeping sickness. Crucially the findings illustrate mass drug treatment is effective in reducing the prevalence of T. b. rhodesiense in cattle, thus minimising the reservoir potential of these animals in the epidemiology of human disease. During 2006 a control programme was launched to halt the northward spread of this zoonotic parasite. This programme, entitled ‘Stamping Out Sleeping Sickness’ (SOS) proposed to reduce the prevalence of Human African Trypanosomiasis (HAT) in the newly affected districts by reducing the prevalence of this parasite in the main animal reservoir of infection – domestic cattle. Cattle were mass treated using trypanocides to clear infections. Previous work demonstrated the prevalence of T. brucei s. l. and T. b. rhodesiense in cattle was higher in the districts of Dokolo and Kaberamaido than in the other SOS intervention districts (Selby 2011). To determine whether animals in these areas were also exposed to pathogenic cattle trypanosomes samples were screened for the presence of T. vivax and T. congolense savannah using PCR. Chapter three of this thesis determined the prevalence of these trypanosomes in cattle in these districts. Before treatment had taken place the prevalence of T. vivax was 2% (4/200, 95% CI 3.57 – 0.12%) in Dokolo and 7.3% (21/310, 95% CI 10.17 - 4.24 %) in Kaberamaido. The prevalence of T. congolense savannah at baseline was 3.5% (7/200, 95% CI 7.08–1.42 %) in Dokolo and 9.1% (21/230, 95% CI13.6–5.7 %) in Kaberamaido. Monitoring was conducted three, nine and 18 months post treatment and both pathogens were detected at all time points. The impact the treatment had on point prevalence varied by trypanosome species and between the two districts. Several clusters of villages in Dokolo and Kaberamaido continued to report cases of HAT after the initial SOS intervention due in part to their proximity to livestock markets (Batchelor et al., 2009). In 2008 re-treatment of these ‘high risk’ areas was undertaken. Monitoring was performed before and six months after treatment. Cattle blood samples were collected at 20 village sites from ten ‘case-positive villages’ (from which human sleeping sickness cases had been reported six months prior to June 2007) and from ten ‘case-negative villages’ (no reported human sleeping sickness cases six months prior to June 2007). These samples were screened for all of the aforementioned trypanosomes using species specific PCR protocols. Chapter five details the results of this screening, and assessed whether re-treatment in Dokolo and Kaberamaido was effective in reducing the prevalence of trypanosomiasis. The re-treatment had a dramatic effect, significantly reducing the point prevalence of overall trypanosomiasis in the 20 villages screened from 38.1% (95% CI = 40.5 – 35.79%) at baseline to 26.9% (95% CI 28.96 – 24.97, p < 0.0001) at six months. Looking at each species separately, point prevalence of three out of four detected species of trypanosome fell significantly, including T. b. rhodesiense, which was reduced to 25% of its baseline prevalence. Finally the two SOS treatment cycles were compared both statistically and spatially with emphasis on trends at village level and the occurrence of mixed infections.

This thesis deconstructs the social, ecological, and colonial elements of the 1900-1910 Human African Trypanosomiasis (African Sleeping Sickness) epidemic which affected British Uganda and Belgian Congo. This paper investigates the epidemic’s medical history, and the subsequent social control policies which sought to govern the actions of the indigenous population. In addition, this paper argues that the failure to understand and respect the region’s ecological conditions and local knowledge led to disease outbreaks in epidemic proportions. Retroactive policies sought to inflict western medical practices on a non-western population, which resulted in conflict and unrest in the region. In the Belgian Congo, colonial authorities created a police state in which violence and stringent control measures were used to manage the local population. In Uganda, forced depopulation in infected regions destabilized local economies. This thesis compares and contrasts the methods used in these regions, and investigates the effects of Germ Theory on Sleeping Sickness policy and social perceptions during the colonial period in Africa.

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The alkaloid cryptolepine (1) and eight synthetic analogues (2-8) were assessed for in vitro activities against Trypanosoma brucei. Four of the analogues were found to be highly potent with IC50 values of less than 3 nM and three of these were assessed against T. brucei brucei infection in rats. The most effective compound was 2,7-dibromocryptolepine (7); a single oral dose of 20 mg/Kg suppressed parasitaemia and increased the mean survival time to 13.6 days compared with 8.4 days for untreated controls. In addition, four huperzine derivatives (9-12) were shown to have in vitro antitrypanosomal activities with IC50 values from 303-377 nM.

Sleeping sickness, caused by Trypanosoma brucei, is a deadly disease that affects some of the poorest countries in sub-Saharan Africa. Although the disease prevalence is declining, strengthening of the current control efforts, including introduction of more adequate chemotherapeutic options, is needed to prevent the re-emergence of yet another epidemic. Nitroaromatic compounds, such as nifurtimox (in combination with eflornithine) and fexinidazole (in clinical trials), have been recently introduced for the treatment of the second stage of sleeping sickness. These compounds are believed to act as pro-drugs that require intracellular enzymatic activation for antimicrobial activity. Here, the role of the bacterial-like nitroreductase TbNTR as a nitrodrug activating enzyme is examined through overexpression and knock-out studies in T. brucei. Multiple attempts to purify soluble recombinant TbNTR from E. coli were unsuccessful, because the recombinant protein was found to be membrane associated. In keeping with the role of TbNTR in nitrodrug activation, loss of an NTR gene copy in T. brucei was found to be one, but not the only, mechanism that may lead to nitrodrug resistance. Furthermore, in the bloodstream form of T. brucei, resistance was relatively easy to select for nifurtimox, with no concurrent loss of virulence and at clinically relevant levels. More worryingly, nifurtimox resistance led to a decreased sensitivity of these parasites to other nitroaromatic compounds, including a high level of cross-resistance to fexinidazole. Conversely, generation of fexinidazole resistance resulted in cross-resistance to nifurtimox. Should these findings translate to the field, emerging nitrodrug resistance could reverse all recent advances in the treatment of sleeping sickness, made since the introduction of eflornithine 20 years ago. Therefore, all efforts should be made to ensure nitroaromatic drugs are used only in drug combination therapies against sleeping sickness, in order to protect them from emerging resistance.

Trypanosoma brucei is a protozoan parasite that is the causative agent of sleeping sickness in sub-Saharan Africa. T. brucei has a complex life cycle involving passage between a mammalian host and the tsetse fly. The parasite evades the mammalian immune system via expression of Variant Surface Glycoprotein (VSG) on the cell surface. VSG genes are expressed at telomeric expression sites and at these sites are a number of Expression Site Associated Genes (ESAGs). One unusual ESAG, ESAG9, is developmentally regulated: RNA for these genes accumulates during the transition from slender to stumpy cells in the mammalian bloodstream and cellassociated protein is only detected transiently in stumpy and differentiating cells. Transgenic cell lines were generated which ectopically express one or more members of the ESAG9 gene family. Biochemical and cytological analyses using these cell lines indicated that some members of this family are glycosylated and GPI-anchored, and also that one gene, ESAG9-K69, is secreted. ESAG9-K69 is also secreted by wild-type stumpy parasites. In vivo experiments with tsetse flies did not conclusively show whether ESAG9 proteins play a role in the establishment of a tsetse fly mid-gut infection by transgenic trypanosomes. However, In vivo and ex vivo experiments using the mouse model of trypanosomiasis indicated that expression of ESAG9 proteins may alter parasitaemia in the mouse and results in a significant decrease in the proportion of CD4+ T cells in the mouse spleen.

A class of linear diamidines was synthesized for the evaluation as a treatment of Human African Trypanosomiasis. These fused heterocyclic compounds are thiazole[5,4-d]thiazoles and are of interest because the parent compound, 2,5-Bis(4-amidinophenyl)-thiazolo[5,4-d]thiazole HCl salt, which is also called DB 1929, has exhibited a low nanomolar IC50 value against Trypanosoma brucei rhodesiense and has shown selectivity for binding to the human telomere G-quadruplex over that of DNA duplex. A fluoro and a methoxy derivative have been synthesized and are currently undergoing testing for activity and binding affinity. In addition, fluorescence studies of selected diamidines were done to study the effect of structural variation on fluorescence. This data is useful since it can determine what types of moieties are needed to yield a compound that will fluoresce in the higher wavelengths (500 nm and above) of the visible spectrum, which would be advantageous in determining the uptake of the drug in the trypanosome within the endemic areas of Africa with a simple microscope.

Dissertação de Mestrado Integrado em Medicina Veterinária
A Tripanossomose Africana (TA) é uma doença parasitária provocada por várias espécies de Trypanosoma, transmitidas por dípteros do género Glossina, vulgo moscas tsé-tsé. Esta doença afeta humanos e animais, tomando nos humanos o nome de Doença do Sono, e nos animais o nome de Nagana. O diagnóstico pode ser realizado por meio de técnicas de visualização parasitária, técnicas serológicas e técnicas moleculares. A terapêutica depende da fase da doença, da espécie do parasita e da espécie do hospedeiro, tendo em atenção a elevada toxicidade dos fármacos. Este facto aliado à inexistência de uma vacina eficaz surge como justificativa deste trabalho, o estudo de alternativas terapêuticas para a TA. Os objetivos deste trabalho foram a determinação da eficácia farmacológica do Benznidazol (BNZ), um antichagásico da família dos nitroimidazóis, bem como a determinação de uma dose infetante por via oral. Para este trabalho foram utilizados 25 murganhos (Mus musculus) BALB-C e 37 murganhos CD-1, e parasitas da espécie Trypanosoma brucei brucei estirpe GVR35 distribuídos por três ensaios. Em dois ensaios de eficácia farmacológica os animais foram infetados por via IP com 500 parasitas por animal. Foi então administrado 10mg/kg BNZ SID PO, durante 5 dias no primeiro ensaio e 11 dias no segundo. Foram medidas parasitémias, pesos, e taxas de sobrevivência. Na segunda experiência foram medidos ainda títulos de IgG total, IgM total e subclasses de IgG anti-T.b.brucei, parâmetros hematológicos e concentração das citocinas IL-4, IFN-γ, NO e TGF-β1. Num terceiro ensaio pretendeu-se determinar a dose infetante por via oral, e consistiu na administração de 500 parasitas por animal, em suspensão de PBS-Glucose a um grupo de animais e a administração de 2x105 parasitas por animal a outro grupo. A análise estatística foi realizada recorrendo aos testes Wilcoxon rank-sum, Correlação de Spearman, Análise de regressão linear, Mantel-Cox log-rank test e Two-way ANOVA. Os resultados dos ensaios revelaram que não foi possível estabelecer infeção por via oral até uma dose de 2x105 parasitas/animal em veículo aquoso. Foi também possível determinar que o BNZ foi ineficaz nos protocolos estudados não controlando significativamente a parasitémia nem aumentando a sobrevivência. Relativamente a achados hematológicos o tratamento falhou em controlar a anemia, evidenciando-se uma tendência significativa para a macrocitose no grupo tratado. Os animais tratados apresentavam maiores títulos de subclasses de IgG, especialmente de IgG2a e IgG3, assim como uma maior libertação de IFN-γ, com significância confirmada por teste estatístico (Two-way ANOVA). É possível concluir que embora o BNZ não seja um bom candidato para a terapêutica de TA, é um bom imunomodulador, estimulando uma resposta Th1. É também possível concluir que com uma dose inferior a 2x105 parasitas/animal em veículo aquoso não se desenvolve infeção por via oral.
ABSTRACT - TRYPANOSOMA BRUCEI BRUCEI MURINE EXPERIMENTAL MURINE INFECTION AND STUDIES ON PHARMACOLOCICAL EFFECTIVENESS OF BENZNIDAZOLE - African Trypanosomiasis (AT) is a parasitic disease caused by several species of Trypanosoma, transmitted by diptera of the Glossina genus, also known as the tsetse flies. This disease affects humans and animals, in humans takes the name of Sleeping Sickness, and in animals takes the name of Nagana. Diagnosis can be performed by parasite visualization, serology and molecular techniques. The treatment depends on the stage of the disease, the species of parasite and host species, knowing that all the drugs for AT are very toxic. With this knowledge, and due to the lack of an effective vaccine, the justification of this work is to find new therapeutic approaches for AT. The objectives of this study were to determine the pharmacological effectiveness of Benznidazole (BNZ), a nitroimidazole antichagasic drug, and ascertaining an infective dose for oral infection, that may be important in carnivores. For this purpose, 25 (Mus musculus) BALB-C and 34 CD-1 mice, and Trypanosoma brucei brucei strain GVR35 parasites were used in this study divided by three experiments. In two of these experiments the pharmacological effectiveness was tested. The animals were treated with 10mg/kg of BNZ, once a day for 5 days for the first experiment and 11 days for the last. Parasitemias, weight gain and survival rates were measured. In the final experiment, anti-T.b.brucei antibody titers, hematological parameters and concentration of cytokines (IL-4, IFN-γ, NO and TGF-β1) were also measured. In the remaining experiment, which tested an infective dose per os, two groups of mice were exposed, using a feeding probe, to a dose of 500 parasites per animal and 200 000 (2x105) parasites per animal, suspended in Glucosed PBS, respectively. Statistical analysis was performed using the Wilcoxon rank-sum test, Mantel-Cox log-rank test, Two-way ANOVA, Spearman’s correlation and Linear regression analysis. The results of these experiments revealed that it was not possible to establish oral infection with dose of up to 2x105 parasites per animal in an aqueous medium. It was also possible to determine that BNZ was ineffective in the protocols studied, due to a lack of control of parasitemia or a significant increase of host survival. As for hematological values the anemia was not controlled, showing a significant trend for macrocytosis in the treated group. Treated animals had higher titers of IgG subclasses, especially IgG2a and IgG3, as well as increased release of IFN-γ, with significance confirmed by statistical testing (Two-way ANOVA). It was concluded that although BNZ is not a good candidate for therapy of AT, it is a good immunostimulator, enhancing a Th1 response. It is also possible to conclude that a dose 2x105 parasites per animal in an aqueous medium does not establish oral infection.

RESUME

La Trypanosomiase Humaine Africaine (THA) appelée également « maladie du sommeil» est une maladie parasitaire provoquée par un protozoaire du genre Trypanosoma dont deux sous-espèces (T. brucei gambiense et T. brucei rhodesiense) sont pathogènes à l’homme. La stratégie de lutte contre cette maladie est essentiellement basée sur le dépistage précoce et le traitement des malades, complété avec le contrôle du vecteur. Cependant, l’utilisation du service de dépistage de la THA par les communautés exposées représente un défi majeur. L’adhésion aux campagnes de dépistage actif avec des équipes mobiles spécialisées était en-dessous de 50% dans certains villages endémiques fin des années nonante. De surcroît, l’utilisation des services de santé fixes en RDC est si faible que ceci compromet le dépistage passif dans les formations sanitaires fixes. Notre hypothèse est que cette faible utilisation des services de santé pourrait elle-même être due à un problème d’acceptabilité du dépistage et traitement de la THA par les communautés vivant dans les zones de transmission de la THA. Tout ceci compromet l’élimination de la THA comme problème de santé publique, un but que s’est fixé la communauté internationale d’ici 2020.

Ce travail a comme objectif d’explorer cette dimension socioculturelle de la maladie qui est souvent négligée dans le contrôle de la THA et générer une meilleure connaissance de ces aspects.

Nous avons réalisé cinq études en total pour adresser la question de la sous-utilisation des services de dépistage et traitement de la THA par les communautés et sa relation avec l’acceptabilité des services. Nous avons d’abord développé une première étude qui évalue les résultats du traitement de la THA en analysant rétrospectivement les données de routine du programme de contrôle de la THA pour l’année 2006 à 2008. Ensuite, nous avons réalisé trois études qualitatives par focus group (groupe focalisé) et entretiens individuels pour documenter la dimension socioculturelle de la lutte contre la THA. D’abord une étude qui a exploré les perceptions sur la THA dans la communauté, suivi par une étude qui explore les perceptions sur le traitement de la THA et une autre qui se concentre sur les pratiques diagnostics des professionnels de santé face à un syndrome neurologique en contexte de ressources limitées.

Une cinquième étude combine une enquête-ménage avec des focus groups et des entretiens individuels pour explorer les perceptions de la communauté sur la santé en général et les services de santé.

Nous avons comparé les obstacles à l’utilisation des services de dépistage et traitement de la THA identifiés dans ce travail avec les messages de sensibilisation sur la THA utilisés au programme de contrôle de la THA en RDC et nous avons développé des recommandations stratégiques.

L’évaluation des indicateurs de performances sur l’issue de traitement montre que le taux de suivi post-thérapeutique est faible dans son ensemble :25 % pour le premier suivi de six mois et moins d’un pourcent des patients revient pour la dernière visite de contrôle au mois 24. Nous avons aussi observé dans cette étude un taux d’échec au mélarsoprol et à la pentamidine respectivement de 30% et de 22 % au Kasaï Oriental qui sont cependant difficilement interprétables, car le dénominateur est incomplet. Comme très peu de patients reviennent au contrôle post-thérapeutique, cette proportion est probablement biaisée vers ceux qui sont en échec de traitement.

L’étude de perception de la THA montre que la maladie est bien connue dans les communautés vivant dans les zones à risque. Par contre, plusieurs obstacles au dépistage et traitement de la THA ont été identifiés. Les plus importants sont :la toxicité des médicaments de la THA, les obstacles financiers, l’inadéquation entre le programme de dépistage des équipes mobiles et les occupations des communautés, les interdits qui accompagnent le traitement de la THA, le manque de confidentialité et la peur de la ponction lombaire.

L’étude sur la perception du traitement de la THA a montré que le mélarsoprol est perçu comme un médicament toxique et est surnommé « médicament des interdits ». Par contre, le régime NECT est perçu comme un nouveau médicament moins toxique qui a rendu les interdits liés au mélarsoprol obsolètes sauf un seul, celui de ne pas avoir de rapport sexuel pendant la période de traitement et de suivi post thérapeutique qui est de 6 mois. Les interdits ont été instaurés de manière empirique par les professionnels de santé et les communautés pour mitiger les effets indésirables du mélarsoprol. Leur violation pourrait entrainer des conséquences graves et mortelles. Ces interdits sont fortement ancrés dans les croyances de la communauté et constituent aujourd’hui un obstacle au dépistage et traitement.

L’étude sur les pratiques diagnostiques des professionnels de santé en matière de syndrome neurologique en contexte de ressources limitées a montré qu’en zone rurale le diagnostic est principalement clinique. Les obstacles perçus au diagnostic de confirmation sont essentiellement d’ordre financier puisque le patient doit tout financer de sa poche. Autres obstacles évoqués sont le manque d’outils de diagnostic et la perception de la communauté qui voit le clinicien comme un devin (petit dieu) ou oracle capable de « deviner » directement la maladie sans passer par un processus diagnostique de laboratoire.

L’étude sur les perceptions de la santé et des services de santé a montré que les capacités de travailler (82%) et les capacités de se mouvoir (66%) sont les signes de bonne santé les plus perçus. 90% des responsables des ménages perçoivent positivement la santé de leur ménage. Les opinions sur le service de santé sont partagées.

Les études présentées dans ce travail ont généré des nouvelles connaissances sur la dimension socioculturelle de la THA. L’analyse des messages de sensibilisation sur la THA utilisés par le programme de contrôle de la THA en RDC en termes de comparaison avec les obstacles au dépistage et traitement de la THA identifiés dans ce travail montre que ces aspects socioculturels bien qu’étant des véritables goulots d’étranglements dans la dynamique de la lutte contre la THA ne sont pas bien ciblés par la communication sur la THA.

Les perspectives des communautés exposées au risque de la THA doivent être adressées par un dialogue continu entre professionnels de santé et communautés adapté aux réalités locales. Ainsi il sera possible d’améliorer de manière opérationnelle les stratégies d’information, éducation et communication, et de façon plus large, le dépistage et traitement de la THA en intégrant la dimension socioculturelle de la THA dans la politique de lutte contre la THA.

SUMMARY

Human African Trypanosomiasis (HAT), also known as “sleeping sickness” is a parasitic disease caused by protozoa of the species Trypanosoma. There are two types that infect humans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. The strategy used to control sleeping sickness consists of early case detection and treatment of patients, together with vector control. Meanwhile, utilization/access to HAT screening by the affected communities remains a major challenge. Adherence to active screening programs with mobile units was below 50% in certain endemic villages end of the 90’s. Moreover, utilization of fixed health facilities in DRC is so low that it compromises passive case finding. Our hypothesis is that this low utilization of health services is caused by a problem of acceptance of case detection and treatment of HAT by the communities living in the HAT transmission zones. This compromises the target of the international community to eliminate HAT as a public health problem by 2020. This thesis wants to explore and tries to generate more knowledge on the socio-cultural aspect that is often neglected in the control of HAT.

We conducted five studies to address the lack of community participation in HAT screening and treatment activities and the relation with acceptance of these services.

The first study evaluated the results of HAT treatment by retrospectively analyzing data of the routine HAT control program for the period 2006-2008.

Afterwards we performed three qualitative studies consisting of focus group discussions and individual interviews to document the socio-cultural dimension of the fight against HAT. The first study explored the community perceptions regarding sleeping sickness. The second study explored the perceptions regarding HAT treatment and a third study focused on diagnostic practices of health professionals in low-resource settings facing a neurological syndrome.

The fifth study consists of a household survey, focus group discussions and individual interviews to explore community perception regarding health in general and health services. We compared the identified barriers to screening and treatment of HAT with awareness messages on sleeping sickness used by the HAT control program in DRC and we developed strategic recommendations. The evaluation of performance indicators for treatment showed that compliance with post-treatment follow-up is very poor: 25% for the first post-treatment follow-up examination at six months and less than 1% of the patients returns for the final examination at 24 months. In this study we also observed a treatment failure rate of respectively 30% and 22% for melarsoprol and pentamidine in Kasai-Oriental. However, these date are difficult to interpret because of an incomplete denominator. As only few patients return for follow-up visits, this proportion is probably biased towards those in treatment failure.

The study on the perception of sleeping sickness shows that the disease is well known amongst the communities living in the endemic areas. However, several screening and treatment barriers were identified. The most important are: drug toxicity, financial barriers, the incompatibility between the itineraries of the mobile screening teams and the local communities’ activities, the prohibitions related to HAT treatment, lack of confidentiality and fear of lumbar punctures. The study on the perceptions regarding HAT treatment show that melarsoprol is perceived as a toxic drug and is nicknamed the ‘taboo drug’. On the other hand the NECT regime is perceived as the new drug that is less toxic and that has abolished all the taboos of melarsoprol with the important exception of sexual intercourse during the treatment period and the post-treatment follow-up period of six months.

The prohibitions have been established empirically by healthcare providers and communities to mitigate the side effects of the melarsoprol regimen. Violating these restrictions is believed to cause severe and sometimes mortal complications. Communities adhere strictly to these prohibitions and this constitutes a barrier for HAT screening and treatment.

The study focusing on diagnostic work-up of neurological syndromes in low-resource settings by health care providers has shown that in rural areas diagnosis is usually clinical. Barriers to confirmation of diagnosis are mainly related to the purchasing power of the patient. Other reported barriers are a lack of diagnostic tools and the communities’ perceptions associated with the care provider. Clinicians are perceived as diviners being able to directly identify the cause of the illness without using laboratory tests. The study regarding the perceptions on health and health services has shown that ability to work (82%) and ability to move (66%) are the most perceived signs of good health. 90% of the household responsibles positively perceive the health of their family. The opinions on the health services are divided.

The studies presented in this thesis have generated new insights on the socio-cultural dimension of HAT. The analysis of the awareness messages on HAT in DRC compared with the reported HAT screening and treatment barriers have shown that

although these sociocultural aspects are real bottlenecks in the dynamic of the fight against HAT, they are not targeted by the communication on HAT.

The prospects for communities at risk of HAT should be addressed through continuous dialogue between health professionals and communities adapted to local realities.

It will thus be possible to operationally improve the information strategies, education and communication, and more broadly, screening and treatment of HAT by integrating the socio-cultural dimension in the fighting policy against sleeping sickness.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Human African trypanosomiasis (sleeping sickness) is a devastating disease which is endemic in parts of sub-Saharan Africa. It is caused by the protozoan parasite T. brucei, which are transmitted by the bite of infected tsetse flies. Although the disease is fatal if left untreated, there is a lack of safe, effective and affordable drugs available; therefore new drugs are urgently needed. The aim of the work presented in this thesis is to develop novel trypanocidal compounds. It is divided into two parts to reflect the two distinct strategies employed to achieve this aim. The first part focuses on the inhibition of glycophosphoinositol (GPI) anchor synthesis by inhibiting the Zn2+-dependent enzyme, GlcNAc-PI de-N-acetylase. Trypanosomes have a variable surface glycoprotein (VSG) coat, which allows them to evade the human immune system. The GPI anchor attaches the VSG to the cell membrane; therefore inhibiting GPI synthesis should expose the parasite to the immune system. Initially, large substrate analogues were synthesized. These showed weak inhibition of the enzyme. Zinc-binding fragments were screened, and small molecule inhibitors based on salicylhydroxamic acid were then synthesized. These compounds showed modest inhibition, but the excellent ligand efficiency of salicylhydroxamic acid indicates this may be a promising starting point for further inhibitors. The second part details the P2 strategy. The P2 transporter is a nucleoside transporter unique to T. brucei, which concentrates adenosine. The transporter also binds and selectively concentrates compounds that contain benzamidine and diaminotriazine P2 motifs, which can enhance the potency and selectivity of these compounds. The sleeping sickness drugs melarsoprol and pentamidine contain P2 motifs. Compounds comprising a P2 targeting motif, a linker and a trypanocidal moiety were synthesized. Initially, a diaminotriazine P2 motif was attached to a trypanocidal tetrahydroquinoline (THQ) protein farnesyl transferase (PFT) inhibitor, with limited success. The P2 strategy was also applied to a non-selective, trypanocidal, quinol moiety. The quinol moiety was attached to diaminotriazine and benzamidine P2 motifs, and an increase in selectivity for T. brucei over MRC5 cells was observed.

RESUME

La Trypanosomiase Humain Africaine (THA) demeure un problème de santé publique pour plusieurs pays en Afrique subsaharienne. Le contrôle de la THA est basé essentiellement sur la stratégie de dépistage actif suivi du traitement des personnes infectées. Le dépistage actif est réalisé par des unités mobiles spécialisées, bien que les services de santé fixes jouent un rôle important en détectant « passivement » des cas. Le dépistage reposait jadis sur la palpation ganglionnaire mais, depuis le développement du test d’agglutination sur carte (CATT), trois possibilités se sont offertes aux programmes de contrôle à savoir: i) continuer avec la palpation ganglionnaire ii) combiner la palpation ganglionnaire avec le CATT iii) recourir au CATT seul. Certains programmes comme celui de la République Démocratique du Congo (RDC) ont opté pour la combinaison en parallèle de la palpation ganglionnaire avec le CATT. Toute personne ayant une hypertrophie ganglionnaire cervicale et/ou un CATT positif est considéré comme suspecte de la THA. Elle sera soumise aux tests parasitologiques de confirmation à cause de la toxicité des médicaments anti-THA. Les tests parasitologiques classiques sont l’examen du suc ganglionnaire (PG), l’examen du sang à l’état frais (SF), la goutte épaisse colorée (GE). La sensibilité de cette séquence a été estimée insuffisante par plusieurs auteurs et serait à la base d’une grande perte de l’efficacité de la stratégie dépistage-traitement. D’autres techniques de concentration ont été développées comme la mini-Anion Exchange Concentration Technique (mAECT), la Centrifugation en Tube Capillaire (CTC) et le Quantitative Buffy Coat (QBC), mais ces techniques de concentration ne sont pas utilisées en routine.

En RDC, une interruption des activités de contrôle en 1990 a eu comme conséquence une réémergence importante de la maladie du sommeil. Depuis 1998 les activités de contrôle ont été refinancées de manière structurée.

Ce travail vise deux buts à savoir le plaidoyer pour la continuité des activités de contrôle et la rationalisation des stratégies de contrôle. Nous avons évalué l’évolution de la maladie du sommeil en rapport avec le financement, son impact sur les ménages ainsi que la communauté. L’exercice de rationalisation a porté sur les outils de dépistage et de confirmation. Nous avons d’abord évalué la validité des tests, leur faisabilité ainsi que les coûts et ensuite nous avons effectué une analyse décisionnelle formelle pour comparer les algorithmes de dépistage et pour les tests de confirmation.

Pendant la période de refinancement structurel de la lutte contre la THA en RDC (1998-2003), le budget alloué aux activités a été doublé lorsqu’on le compare à la période précédente (1993-1997). Le nombre des personnes examinées a aussi doublé mais par contre le nombre des nouveaux cas de THA est passé d’un pic de 26 000 cas en 1998 à 11 000 en 2003. Le coût par personne examinée a été de 1,5 US$ et celui d’un cas détecté et sauvé à 300 US$. Pendant cette période, les activités ont été financées par l’aide extérieure à plus de 95%. Cette subvention pourrait laisser supposer que l’impact de la THA au niveau des ménages et des communautés est réduit mais lorsque nous avons abordé cet aspect, il s’est avéré que le coût de la THA au niveau des ménages équivaut à un mois de leur revenu et que la THA fait perdre 2145 DALYs dans la communauté. L’intervention par la stratégie de dépistage-traitement a permis de sauver 1408 DALYs à un coût de 17 US$ par DALYs sauvé. Ce coût classe l’intervention comme « good value for money ».

Le recours au CATT seul s’est avéré comme la stratégie la plus efficiente pour le dépistage actif. Le gain marginal lorsque l’on ajoute la palpation ganglionnaire en parallèle est minime et n’est pas compensé par le coût élevé lié à un nombre important des suspects soumis aux tests parasitologiques. Les techniques de concentration ont une bonne sensibilité et leur faisabilité est acceptable. Leur ajout à l’arbre classique améliore la sensibilité de 29 % pour la CTC et de 42% pour la mAECT. Le coût de la CTC a été de 0,76 € et celui de la mAECT de 2,82 €. Le SF a été estimé très peu sensible. L’algorithme PG- GE-CTC-mAECT a été le plus efficient avec 277 € par vie sauvée et un ratio de coût-efficacité marginal de 125 € par unité de vie supplémentaire sauvée. L’algorithme PG-GE-CATT titration avec traitement des personnes avec une parasitologie négative mais un CATT positif à un seuil de 1/8 devient compétitif lorsque la prévalence de la THA est élevée.

Il est donc possible dans le contexte actuel de réduire la prévalence de la THA mais à condition que les activités ne soient pas interrompues. Le recours à un algorithme recourant au CATT dans le dépistage actif et à la séquence PG-GE-CTC-mAECT est le plus efficient et une efficacité de 80%. La faisabilité et l’efficacité peut être différent d’un endroit à l’autre à cause de la focalisation de la THA. Il est donc nécessaire de réévaluer cet algorithme dans un autre foyer de THA en étude pilote avant de décider d’un changement de politique. Le recours à cet algorithme implique un financement supplémentaire et une volonté politique.

SUMMARY

Human African Trypanosomiasis (HAT) remains a major public health problem affecting several countries in sub-Saharan Africa. HAT control is essentially based on active case finding conducted by specialized mobile teams. In the past the population screening was based on neck gland palpation, but since the development of the Card Agglutination Test for Trypanosomiasis (CATT) three control options are available to the control program: i) neck gland palpation ii) CATT iii) neck gland palpation and CATT done in parallel .Certain programs such as the one in DRC opted for the latter, combining CATT and neck gland palpation. All persons having hypertrophy of the neck gland and/or a positive CATT test are considered to be a HAT suspect. Confirmation tests are necessary because the screening algorithms are not 100 % specific and HAT drugs are very toxic. The classic parasitological confirmation tests are lymph node puncture (LNP), fresh blood examination (FBE) and thick blood film (TBF). The sensitivity of this combination is considered insufficient by several authors and causes important losses of efficacy of the screening-treatment strategy. More sensitive concentration methods were developed such as the mini Anion Exchange Concentration Techniques (mAECT), Capillary Tube Centrifugation (CTC) and the Quantitative Buffy Coat (QBC), but they are not used on a routine basis. Main reasons put forward are low feasibility, high cost and long time of execution.

In the Democratic Republic of Congo, HAT control activities were suddenly interrupted in 1990 and this led to an important re-emergence or the epidemic. Since 1998 onwards, control activities were financed again in a structured way.

This works aims to be both a plea for the continuation of HAT control as well as a contribution to the rationalization of the control strategies. We analyzed the evolution of sleeping sickness in the light of its financing, and we studied its impact on the household and the community. We aimed at a rationalization of the use of the screening and confirmation tools. We first evaluated the validity of the tests, their feasibility and the cost and we did a formal decision analysis to compare screening and confirmation algorithms.

The budget allocated to control activities was doubled during the period when structural aid funding was again granted (1998-2003) compared with the period before (1993-1997). The number of persons examined per year doubled as well but the number of cases found peaked at 26 000 in 1998 and dropped to 11 000 in the period afterwards. The cost per person examined was 1.5 US$ and per case detected and saved was 300 US$. The activities were financed for 95 % by external donors during this period. This subvention could give the impression that the impact of HAT on the household and the household was limited but when we took a closer look at this aspect we found that the cost at household level amounted to one month of income and that HAT caused the loss of 2145 DALYs in the community. The intervention consisting of active case finding and treatment allowed to save 1408 DALY’s at a cost of 17 US$ per DALY, putting the intervention in the class of “good value for money”.

The use of CATT alone as screening test emerged as the most efficient strategy for active case finding. The marginal gain when neck gland palpation is added is minor and is not compensated by the high cost of doing the parasitological confirmation test on a high number of suspected cases. The concentration methods have a good sensitivity and acceptable feasibility. Adding them to the classical tree improves its sensitivity with 29 % for CTC and with 42 % for mAECT. The cost of CTC was 0.76 US$ and of mAECT was 2.82 US$. Sensitivity of fresh blood examination was poor. The algorithm LNP-TBF-CTC-mAECT was the most efficient costing 277 Euro per life saved and a marginal cost effectiveness ratio of 125 Euro per supplementary life saved. The algorithm LNP-TBF-CATT titration with treatment of persons with a negative parasitology but a CATT positive at a dilution of 1/8 and more becomes competitive when HAT prevalence is high.

We conclude that it is possible in the current RDC context to reduce HAT prevalence on condition that control activities are not interrupted. Using an algorithm that includes CATT in active case finding and the combination LNP-TBF-CTC-mAECT is the most efficient with an efficacy of 80 %. Feasibility and efficacy may differ from one place to another because HAT is very focalized, so it is necessary to test this novel algorithm in another HAT focus on a pilot basis, before deciding on a policy change. Implementation of this algorithm will require additional financial resources and political commitment.

Doctorat en Sciences de la santé publique
info:eu-repo/semantics/nonPublished

Procyclic culture forms of Trypanosoma brucei species and antibodies to these parasites were used in developing antibody-detection and antigen-detection assays for diagnosis of African human sleeping sickness. An agglutination assay using live procyclic trypanosomes--the Procyclic Agglutination Trypanosomiasis Test (PATT) was developed for detecting anti-trypanosome antibodies in the sera of trypanosome-infected vervet monkeys and humans. Antibodies to procyclic surface antigens were detected by the PATT in sera of vervet monkeys as early as 7 days post-infection with T. b. rhodesiense. Positive agglutination titres were obtained with sera from monkeys with active, untreated infections and with sera taken soon after successful drug cure. Similar positive agglutination results were also observed using the PATT with sera from T. b. gambiense-infected patients from Cote d'Ivoire and Sudan and with documented sera from T. b. rhodesiense-infected patients from Kenya. No agglutination reactions were observed with preinfection sera from vervet monkeys, with sera from uninfected Canadians or with sera from Americans working in endemic areas. Together these results confirm the diagnostic value of using procyclic trypanosomes to detect anti-trypanosome antibodies in human African sleeping sickness. A double antibody sandwich ELISA using monoclonal antibodies and polyclonal rabbit antibodies to the surface membrane antigens of procyclic trypanosomes was developed. This assay detected circulating trypanosomal antigens in the sera of trypanosome-infected mice and in the sera from parasite-infected patients. However, limited success was obtained with this sandwich ELISA when tested on a larger repertoire of sera from infected humans. Rabbit antibodies made against whole lysates of T. b. rhodesiense procyclics were then employed in an antigen-trapping sandwich ELISA. The results demonstrated the effectiveness of this sandwich ELISA in revealing the infection status of vervet monkeys or humans infected with either T. b. rhodesiense or T. h. gambiense. Trypanosomal antigens were detected in the sera of parasitologically confirmed monkeys and patients but not in preinfection sera nor in control sera from uninfected North Americans. The PATT and the sandwich ELISA exhibited higher sensitivities than the currently employed diagnostic assay for human sleeping sickness, the Card Agglutination Trypanosomiasis Test (CATT), when tested with sera of parasitologically-confirmed humans. The sandwich ELISA was superior to the antibody-detecting PATT and CATT in monitoring trypanocidal drug-treated patients. The overall sensitivity of the PATT and sandwich ELISA was 94.3% and 97.4% and the specificity was 84.5% and 95.5%, respectively. These results thus confirm the diagnostic value of these tests for the diagnosis of human African sleeping sickness. Identification of diagnostically useful antigens was attempted in order to facilitate the adaptation of these diagnostic assays to a simpler format for field application. Pooled sera obtained from trypanosome-infected patients was used as a probe to detect trypanosome antigens separated by high performance liquid chromatography, immunoaffinity and immunoblotting techniques. Most of the antigens were detected in the higher molecular weight range (>62 Kd). Immunization of mice with the target antigens yielded six trypanosome-specific monoclonal antibodies. In a double antibody sandwich ELISA, these antibodies were successful in trapping circulating parasite antigens in sera from trypanosome-infected mice as early as 3 days post-infection. Some of these antigens have been partially biochemically characterized. Trypanosomal antigens were also detected by these antibodies in the urine of infected mice. The antigen-capture sandwich ELISA using either the selected monoclonal antibodies or the rabbit anti-procyclic whole lysate antibodies gave similar results with sera from trypanosome-infected mice, human sleeping sickness patients and uninfected humans from North America and Kenya. The results showed that these MAbs and their antigens were useful in the diagnosis of African human sleeping sickness.
Graduate

This dissertation is a history of sleeping sickness research work and prevention programs during the German colonial period in East Africa, focusing on the regions around Lake Victoria and Lake Tanganyika. It examines efforts to study and prevent sleeping sickness, analyzing how both fit into the social, political, and economic dynamics of African life. It covers two phases of German colonial attention to epidemic sleeping sickness between 1902 and 1914: an initial phase of research and scientific expeditions from 1902 to 1906, then a period dominated by the introduction of disease prevention measures in affected areas from 1907 to 1914. Highlighting the local complexity and far-reaching impact of sleeping sickness, I show that sleeping sickness research and prevention emerged from the intersection of tropical medicine expertise, African mobility, and German colonial and African politics. Sleeping sickness, and subsequent efforts toward its treatment and prevention, redefined the boundaries of political power and social influence within African communities during a crucial period of change in the region. By creating new arenas of engagement between African communities and European scientists, specifically in newly-built sleeping sickness camps and among the African medical auxiliaries employed in them, sleeping sickness work created economic relationships, reshaped social and political hierarchies, and set new ground rules for African agriculture and trade. Kings, chiefs, and colonial scientists contended with African communities' demands for treatment, their resistance to examination, and their claims on the use of land and waterways. Further, inter-colonial sleeping sickness research and subsequent prevention programs played a pivotal role in the development of tropical medicine, strengthening disciplinary boundaries and defining the trajectory of future research. My work weaves together narratives of research and disease prevention from metropolitan Europe and East Africa, in contrast to strictly colonial and national histories of health and medicine that have preceded it.

Trypanosomes are hemoflagellated protozoan parasites causing chagas disease in South America, Leishmaniasis throughout the world, and African sleeping sickness in humans and nagana in animals in Africa. About 55 million people and 25 million cattle have been estimated to be at risk of contracting African sleeping sickness or nagana respectively. Once injected into the blood stream via the bite of a tsetse fly, the parasite evades the host's immune response by repeatedly changing its surface antigens, thus making the development of a vaccine seem impossible. Furthermore, chemotherapy existing today can be toxic, suggesting that novel methods to prevent diseases caused by trypanosomes are essential. All parasites of the Trypanosomatidae family contain unique microtubular structures called the subpellicular microtubules. Microtubules are made of tubulin and of microtubule associated proteins (MAPs). Unlike other microtubules, the subpellicular microtubules are crosslinked to one another and to the plasma membrane. The unique structure of the subpellicular microtubules has been attributed to unique trypanosome subpellicular MAPs which stabilize the microtubule polymers and crosslink them to one another. Three unique types of subpellicular MAPs have been identified: MARP, which is a high molecular mass MAP that stabilizes microtubules, p52 that is a 52kDa MAP which crosslinks microtubules, and pI5, which is a I5kDa protein which bundles microtubules. Because trypanosome MAPs have been shown to be unique to these parasites, these molecules could serve as useful target sites for therapy. In this study pI5 was cloned and sequenced and shown to contain highly organized, nearly identical tandem repeats with a periodicity of 10 amino acids, rich in positively charged and in hydrophobic amino acids. It was shown that pI5 can also bind phospholipids, suggesting that it may not only bundle the microtubule polymer through its positively charged amino acids but may also crosslink the microtubules to the plasma membrane through its hydrophobic regions, thus contributing to the stable structure of the subpellicular microtubules. To test for the efficiency of pI5 as a vaccine candidate, the recombinant pI5 was cloned into an adenovirus, which was used as a vaccine delivery system for pI5. Mice were vaccinated with the native purified pI5, with the expressed recombinant pI5 and with the adenovirus containing the recombinant pI5 gene (Ad-pI5). The results indicated that pI5 protected 100% of the animals vaccinated with the recombinant molecule (8/8), and 87% of the animals vaccinated with the native protein (7/S), while none of the control animals were protected. Animals that were vaccinated with the Ad-pI5 were protected but so were the control animals vaccinated with an adenovirus containing the lacZ gene. We have shown that vaccination with the adenovirus is associated with an elevated CDS+ T cell response which is known to be trypanostatic (S6), suggesting that animals vaccinated with Ad-pIS may have been protected not only by the specific anti-plS response but also by non specific immunity that was induced by the adenovirus itself. The source of the native and recombinant pI5 was from a different strain of T. brucei that was used for challenge. Since the subpellicular microtubules are common to all members of the Trypanosomatidae family, pI5 may ultimately serve as a common target for therapy to all types of diseases caused by trypanosomes.
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.

In recent years the transmission of the Human African Trypanosomiasis could be significantly reduced. The reported cases in 2016 reached a historic low level of 2184 cases and these achievements can be ascribed to intense control and surveillance programmes.118 However, most of the reported cases (>1000 in 2015) occurred in the Democratic Republic of the Congo and thus, need to be treated adequately. In particular, when the parasites have traversed the blood-brain barrier (BBB), treatment proved to be even more difficult. In addition, the number of cases always came in waves due to many reasons, e.g., development of resistances. Thus, it can be expected from experiences of the past that the number of cases will increase again. Hence, novel chemical entities are desperately needed in order to overcome the drawbacks which are associated with the current treatment options. Our drug discovery approach included an initial drug repurposing strategy combined with a phenotypic screening. S. Niedermeier found novel active compounds derived from commercial fluoroquinolones. The most promising hit compound was further developed by G. Hiltensperger resulting in the lead quinolone amide GHQ168 (IC50 = 0.047 µM). This doctoral thesis is about new insights into the SAR of the quinolone amides and the enhancement of the lead compound. Special consideration was given to the fluorine atom in the quinolone amides and how certain fluorine substitution patterns influence the antitrypanosomal activity, physicochemical properties and pharmacokinetics (i.e. ‘fluorine walk’). Moreover, the ability of the compound class crossing the BBB should be investigated. This feature is inevitable necessary in order to potentially treat African sleeping sickness stage II. The Gould-Jacobs protocol was predominantly used for the synthesis of the quinolone core. Since former SAR studies mainly concentrated on the variation in positions 1, 3 and 7, quinolone scaffolds (2a-i) with diverse substitution patterns regarding positions 5, 6, 7 and 8 were synthesised in this thesis. The resulting quinolone amides were evaluated for their antitrypanosomal activity. Voluminous residues in position C-5 resulted in diminished activities (compounds 13, 16 and 18) and solely small-sized moieties were tolerated. In particular the fluorine atom in position 5 revealed beneficial trypanocidal effects as shown for compounds 6 (IC50 = 0.05 µM), 8 (IC50 = 0.04 µM), and 24 (IC50 = 0.02 µM). Furthermore, having fluorine only in position 5 of the quinolone core could considerably reduce the cytotoxic effects (CC50 >100 µM, SI = >2000 for 6). Hence, the 5-fluoro-substituted quinolone amides were considered superior to GHQ168. Regarding the C-6 position all other moieties (e.g., H in 9, OCH3 in 10, CF3 in 12) except of a fluorine atom decreased the activity against Trypanosoma brucei brucei. A double fluorination in C-6 and C-8 was not beneficial (IC50 = 0.06 µM for 7) and a single fluorine atom in C-8 even showed a negative effect (IC50 = 0.79 µM for 5). The logP value is considered a surrogate parameter for lipophlicity and thus, affecting permeability and solubility processes. In particular the fluorine atom influences the lipophilicity due to versatile effects: Lipophilicity is increased by additional fluorine atoms on aromatic rings (7, 23) and reduced by fluorine atoms at an alkyl chain (49), respectively. Additionally, the 5-fluoro-substituted quinolone amides (6, 8, and 24) could prove the contrary effect of decreasing lipophilicity when the aromatic fluorine substituent is in vicinity to a carbonyl group. For the most promising drug candidates 6, 23, and 24 the respective metabolites and the metabolic turnover were investigated by C. Erk. In comparison to GHQ168 the hydroxylation of the benzylamide was prevented by the para-fluorine atom. Hence, half-life was extended for compound 23 (t1/2 = 6.4 h) and N-desalkylation was the predominant pathway. Moreover, the respective fluorine substitution pattern of the quinolone core affected the metabolism of compound 6. The 5-fluoro-substituted quinolone amide was less prone for biotransformation (t1/2 = 7.2 h) and half-life could even be further prolonged for compound 24 (t1/2 = 7.7 h). Due to the most appropriate safety profile of compound 6, this particular drug candidate was considered for in vivo study. Its poor solubility made a direct intraperitoneal administration unfeasible. Thus, an amorphous solid dispersion of 6 was generated using the spray-drying method according to the previous protocol. Unfortunately, the required solubility for the predicted in vivo study was not achieved. Furthermore, the compound class of the quinolone amide was evaluated for its ability for brain penetration. The methanesulfonyl precursor 48 was synthesised and subsequently radiofluorinated in the group of Prof. Dr. Samnick (Department of Nuclear Medicine, University Hospital of Würzburg). The labelled compound [18F]49 was administered to mice, and its distribution throughout the body was analysed using positron emission tomography and autoradiography, respectively. The autoradiography of the murine brains revealed medium to high concentrations of [18F]49. Therefore, the quinolone amides are generally suitable for treating Human African Trypanosomiasis stage II. A scaffold hopping approach was performed starting from the quinolone amides and concluding with the compound class of pyrazoloquinolin-3-ones. The intramolecular hydrogen bond between the sec. amide and the C-4 carbonyl moiety was replaced by a covalent bond. The two compound classes were comparable regarding the antitrypanosomal activity to some degree (IC50 = 7.9 µM (EK02) vs. 6.37 µM (53a)). However, a final evaluation of 59 was not possible due to poor solubility
Die Verbreitung der Afrikanischen Schlafkrankheit konnte in den vergangenen Jahren deutlich verringert werden. Die dokumentierten Fallzahlen aus 2016 erreichten ein historisch niedriges Niveau, was auf eine engmaschige Kontrolle und Überwachung zurückzuführen ist. Dennoch gibt es nach wie vor zahlreiche Krankheitsfälle (>1000 Fälle im Jahr 2015 für die Demokratische Republik Kongo), die entsprechend behandelt werden müssen. Die Therapie wird insbesondere dann erschwert, wenn die Parasiten die Blut-Hirn-Schranke überwunden haben. Außerdem treten die Krankheitsfälle aus mehreren Gründen, wie beispielsweise durch Resistenzentwicklung immer wieder schubweise auf. Die Erfahrungen aus der Vergangenheit zeigen, dass die Fallzahlen jederzeit wieder ansteigen können. Deshalb sind neue Arzneistoffe dringend notwendig, um die Nachteile der aktuellen Behandlungmöglichkeiten umgehen zu können. Unsere Suche nach neuen Wirkstoffen beinhaltete eine anfängliche Umwidmung eines zugelassenen Arzneistoffes in Verbindung mit einem Phänotyp-basierten Screening. S. Niedermeier entdeckte neue aktive Verbindungen, die sich von handelsüblichen Fluorchinolonen ableiteteten. Die vielversprechenste Substanz wurde von G. Hiltensperger zum Chinolonamid GHQ168 (IC50 = 0.047 µM) weiter optimiert. Diese Arbeit befasst sich mit neuen Erkenntnissen zur Struktur-Wirkungs Beziehung der Chinolonamiden und mit der Verbesserung der Leitsubstanz. Besondere Berücksichtigung fanden dabei die Fluor-Substitutionen an den Chinolonamiden. Es sollte untersucht werden, inwiefern gewisse Fluorsubstitutionsmuster die antitrypanosomale Wirkung, physiko-chemische Eigenschaften und Pharmakokinetik beeinflussen („Fluor Walk“). Außerdem sollte ermittelt werden, ob diese Substanzklasse die Blut-Hirn-Schranke überwinden kann. Dieses Merkmal muss unabdingbar vorhanden sein, um die Afrikanische Schlafkrankheit in Stufe II potentiell behandeln zu können. Das Gould-Jacobs-Verfahren wurde hauptsächlich für die Synthese des Chinolongrundgerüstes angewandt. Da sich die vorausgegangene Analyse der Struktur-Wirkungs Beziehungen vornehmlich auf das Variieren der Substitutenten in den Positionen 1, 3 und 7 konzentrierte, wurden in dieser Arbeit Chinolone (2a-i) mit diversen Substitutionsmustern in den Positionen 5, 6, 7 und 8 synthetisiert. Die erhaltenen Chinolonamide wurden auf ihre antitrypanosomale Aktivität untersucht. Voluminöse Reste in der Position C-5 verursachten verringerte Aktivitäten (Verbindungen 13, 16 and 18), d.h. nur kleine Reste waren hinnehmbar. Vor allem ein Fluor-Atom in Position 5 wirkte sich günstig auf die antitrypanosomale Wirkung aus, was mit Verbindungen 6 (IC50 = 0.05 µM), 8 (IC50 = 0.04 µM), und 24 (IC50 = 0.02 µM) gezeigt werden konnte. Des Weiteren reduzierte sich die zytotoxische Wirkung (CC50 >100 µM, SI = >2000 für 6), wenn sich das Fluor-Atom nur in Position 5 des Chinolongrundgerüsts befindet. Deshalb wurden die Chinolonamide mit Fluor in Position 5 gegenüber GHQ168 als überlegen erachtet. In Bezug auf Position 6 zeigen die Reste (z. B. H in 9, OCH3 in 10, CF3 in 12), mit Ausnahme des Fluor-Atoms, eine verringerte Aktivität gegenüber Trypanosoma brucei brucei. Eine zweifache Fluorsubstitution in C-6 und C-8 war nicht vorteilhaft (IC50 = 0.06 µM für 7) und ein einfaches Fluor-Atom in C-8 zeigte einen negativen Effekt (IC50 = 0.79 µM für 5). Der logP Wert wird als Surrogatparameter der Lipophilie betrachtet und wirkt sich somit auf Permeabilität- und Löslichkeitsprozesse aus. Insbesondere das Fluor-Atom beeinflusst die Lipophilie durch vielfältige Effekte: die Lipophilie wird durch zusätzliche Fluor-Atome am Aromaten erhöht (7, 23), beziehungsweise durch Fluor-Atome an einer Alkylkette verringert (49). Zusätzlich konnte für die 5-fluoro-substituierten Chinolonamide (6, 8, 24) der paradoxe Effekt gezeigt werden, dass die Lipophilie verringert wird, sobald ein aromatischer Fluorsubstituent in unmittelbarer Nähe zu einer Carbonylgruppe steht. Für die vielversprechensten Wirkstoffkandidaten 6, 23 und 24 wurden die jeweiligen Metabolite und der metabolische Umsatz von C. Erk untersucht. Im Vergleich zu GHQ168 wurde einer Hydroxylierung des Benzylamid-Restes durch eine para-Fluor-Substitution vorgebeugt. Dadurch wurde die Halbwertszeit der Verbindung 23 verlängert (t1/2 = 6.4 h) und eine N-Desalkylierung war der vorrangige Abbauweg. Außerdem wirkte sich das entsprechende Fluorsubstitutionsmuster auf den Metabolismus von Substanz 6 aus. Das 5-fluoro-substitutierte Chinolonamid war weniger anfällig für Biotransformationen (t1/2 = 7.2 h) und die Halbwertszeit konnte für die Substanz 24 sogar noch weiter verlängert werden (t1/2 = 7.7 h). Aufgrund des geeigneten Sicherheitsprofils der Verbindung 6, wurde für diesen Wirkstoffkandidaten eine In-vivo-Studie in Betracht gezogen. Die schlechte Wasserlöslichkeit machte jedoch eine direkte intraperitoneale Gabe unpraktikabel. Deshalb wurde durch Sprühtrocknung der Verbindung 6, gemäß der früheren Vorgehensweise, eine „amorphous solid dispersion“ erzeugt. Die benötigte Löslichkeit für die vorausberechnete In-vivo-Studie wurde dabei leider nicht erreicht. Darüber hinaus wurde die Substanzklasse der Chinolonamide hinsichtlich ihrer Fähigkeit, ins Gehirn zu gelangen, untersucht. Dazu wurde die Methansulfonyl-Vorstufe 48 in der Gruppe von Prof. Dr. Samnick (Institut für Nuklearmedizin, Universitätsklink Würzburg) synthetisiert und mit [18F]Fluor markiert. Die markierte Verbindung [18F]49 wurde anschließend in Mäuse injiziert und dessen Verteilung im Körper mittels Positronen-Emissions-Tomographie, beziehungsweise mittels Autoradiographie analysiert. Die Autoradiographie der Mäusegehirne zeigte mittlere bis hohe Konzentrationen von [18F]49. Demnach sind die Chinolonamide generell dafür geeignet, die Afrikanische Schlafkrankheit in Stufe II zu behandeln. Ein “Scaffold Hopping”-Ansatz wurde für die Chinolonamide angestrebt und ergab schließlich die Substanzklasse der Pyrazolochinolin-3-one. Die intramolekulare Wasserstoffbrückenbindung zwischen dem sek. Amid und der Carbonylgruppe in C-4 wurde durch eine kovalente Bindung ersetzt. Die beiden Substanzklassen waren im Ansatz, bezogen auf ihre antitrypanosomale Wirkung, miteinander vergleichbar (IC50 = 7.9 µM (EK02) vs. 6.37 µM (53a)). Dennoch konnte eine abschließende Bewertung aufgrund mangelnder Löslichkeit nicht stattfinden