Acute and chronic sleeping sickness are fatal neglected tropical diseases caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense respectively (members of the sub-genus Trypanozoon). Accurate diagnostics are needed to guide treatment since the symptoms of disease are non-specific and the drugs that are used for treatment are too toxic to be administered to unconfirmed cases. Tests need to be simple enough to confirm clinical diagnosis of sleeping sickness in poorly-resourced, peripheral health centres and for use as epidemiological tools to detect T. b. rhodesiense in the zoonotic reservoirs of infection. This study focuses upon LAMP (loop-mediated isothermal amplification) as a novel diagnostic for sleeping sickness that may serve to bridge the gap between the need for sensitive, specific molecular diagnostics on the one hand and ‘field-friendly’ diagnostics on the other. Here, two previously published LAMP assays for Trypanozoons were compared to classic PCR based methods for the diagnosis of Trypanozoon infection status in 428 cattle blood samples. The results did not support the use of LAMP as an improved system for surveillance of T. b. rhodesiense in the zoonotic cattle reservoir. T. b. rhodesiense and T. b. gambiense subspecies specific LAMP assays were evaluated against traditional reference subspecies specific PCR tests, using DNA purified from 86 cryopreserved trypanosome isolates. Novel LAMP assays for these subspecies were also designed and evaluated. Both the published and novel assays for T. b. rhodesiense (targeting different regions of the SRA gene) were sensitive, specific and reliable when applied to purified DNAs, but were less consistent on field samples. The novel T. b. gambiense LAMP (targeting TgsGP) was sensitive and specific but this was not the case for the published LAMP assay (targeting the 5.8S rRNA gene). However reliability may be less than optimal for LAMP TgsGP. Finally, simple endpoint readout methods for LAMP were evaluated. The colour change reagent hydroxynaphthol blue was identified as the best currently available method taking cost, ease of use and reliability into consideration. In 2009 the number of reported sleeping sickness cases fell below 10,000 for the first time in 50 years. Improved LAMP diagnostics could facilitate the diagnosis of sleeping sickness and support the continued fight against this neglected, but deadly disease.

During the bloodstream stage of the Trypanosoma brucei lifecycle, the parasite exists in two different states: the proliferative slender form and the non-proliferative, transmissible, stumpy form. The transition from the slender to stumpy form is stimulated by a density-dependent mechanism and is important in infection dynamics, ordered antigenic variation and disease transmissibility. The slender to stumpy transition and the contribution of stumpy formation to within-host dynamics have been difficult to analyse, however, because cell-type specific markers have been restricted to imprecise morphological criteria. PAD1 is a recently identified stumpy-specific protein which acts as a molecular marker for stumpy formation and a functional marker for transmission. Here, the control of stumpy-specific gene expression via the 3’UTR has been analysed, identifying that there are repressive elements in the 3’UTR preventing inappropriate expression during the slender life stage. Further, both pleomorphic and monomorphic transgenic reporter cell lines utilising the PAD1 3’UTR have been created that report on stumpy formation in vitro and these have been used for the analysis of stumpyinducing chemical compounds. Finally, a sensitive and accurate qRT-PCR assay has been developed and optimised that faithfully reports both parasitaemia and stumpy formation throughout host infection. Using a chronic infection rodent model, stumpy levels have been monitored on the basis of conventional morphological and cell cycle assays, as well as by qRT-PCR for PAD1 expression. The results define the temporal order of events that result in the generation of stumpy forms early in a parasite infection and thereafter describe the dynamics of slender and stumpy forms in chronic infections extending over several weeks. This quantitative data has allowed the mathematical modelling of transmission competence in trypanosome infections, suggesting dominance of transmission stages throughout infection.

Undergraduate honors paper--Mount Holyoke College, 2007. Dept. of Biological Sciences. / Includes bibliographical references (leaves 46-50).