African trypanosomes : a model for the improvement of molecular diagnosis of blood borne parasites
Abd-Alla, Heba Ahmed
Historically, diagnosis has relied on clinical signs of disease, microscopy and serological testing. However, these approaches have a number of drawbacks for example, differential diagnosis, low sensitivity (microscopy) and the inability to differentiate past from current infections (serology). In the past decade the use of molecular techniques, such as the polymerase chain reactions (PCR) have gained favour. Many research groups have used these techniques to study the molecular epidemiology of diseases in sub-Saharan Africa. Such methodologies rely on the detection of genetic materials and as such are reliant on the specificity of their components and the quality of the starting materials. It is the aim of this thesis is to demonstrate improvements that can be made to sample collection that will help to enhance the reliability of these tests and highlight the importance of the diagnostic parameters. The model that I will use to demonstrate these improvements are African trypanosomes, these are the causative agents of sleeping sickness in humans and nagana in animals, and are wide spread across much of sub-Saharan Africa. My work will be presented as three main sections: Firstly, a comparison of the suitability of various different approaches to cattle blood sample collection – including the genetic materials prepared directly in the field and the use of Whatman FTA®cards – in terms of the provision of appropriate materials for molecular screening will be presented. It was found that uneven distribution of genetic materials occurs across the surface of the FTA®cards due to the matrix chemistry. Therefore suggestions for improvements for the preparation of materials to be stored on these cards and their downstream application are made. Secondly, a comparison between the specificity of the pan-Trypanosoma ITS-PCR reaction and the species-specific reactions is made. The ITS-PCR has gained favour in recent years as it is reported to be capable of identifying a wide range of trypanosomes, as this is a single nested PCR reaction the reduction in time and cost has been very appealing to researchers in this field. My work suggests that this test is not reliable in terms of the accurate detection of trypanosomes species, and in fact on a direct comparison of 969 samples, 37 parasitic events where identified by this approach compared to 197 when species-specific tests were applied. Thirdly, based on my findings from the previous two chapters I present two case studies, the first of which looks to evaluate the impact on the prevalence of trypanosome species in cattle after drug treatment during the Ugandan, Stamp Out Sleeping sickness (www.sleepingsickness.com) campaign. The results of this case study highlight the importance of understanding the relationship that occurs between trypanosome species in mixed infections, my second case study therefore looks to quantifying the infection load of Trypanosoma brucei and T. congolense within the midgut of their insect vector (Glossina morsitans morsitans) using qPCR.