Use of molecular and bioinformatics tools for developing methods of epidemiological identification of trypanosomes
, PhD thesis, School of Environment and Life Sciences.
Human African trypanosomiasis (HAT), also known as sleeping sickness, has been a major health problem for populations in Africa caused by the Trypanosoma brucei spp parasite. Although, the current number of the reported cases is on the decrease, more efforts are required to try to control or eliminate the disease. The recent advances in molecular techniques have contributed towards identifying taxonomic groups at all levels (species, subspecies, populations, strains and isolates). Commonly, field samples are collected and stored using Whatman FTA cards. Many molecular epidemiological tools are available for detection and strain typing in trypanosomes. These tools include nested ITS-PCR, which is based on size variation of the ITS genes and MGE-PCR, which is based on variations in position of mobile genetic elements (MGEs). Although commonly used, these tools have not been fully validated. For example, the ITS-PCR has not been used or validated against blood samples obtained from sleeping sickness patients in Angola. Furthermore, the MGE-PCR system has not been evaluated for use directly from FTA cards.
The aim of this thesis is to develop improved molecular tools to assist diagnostic and epidemiological studies. In order to improve the molecular diagnostic use of Whatman FTA cards, an extraction method based on Chelex was investigated. Using Chelex to extractT. brucei DNA from FTA cards, followed by a nested ITS-PCR detection system, allowed parasite DNA detection to 1ng/µl. To evaluate this tool on field samples, ITS-PCR amplification was carried on DNA eluted by Chelex extraction from 36 FTA cards spotted with blood from Angolan patients tested positive for trypanosomiasis by the card agglutination test (CATT). Twenty four of these samples were successfully PCR amplified using mammalian tubulin primers. Of these 24 samples, 11 (= 45.8%) were confirmed as trypanosome positive utilising a specific ITS-PCR based approach. As such, this indicates that further work is necessary to improve the PCR-based reliability of diagnosis.
To this end, an MGE-PCR approach was used to attempt parasite strain identification.. Although the MGE-PCR was found to be more sensitive than ITS-PCR in amplification of DNA from FTA cards, the resulting sequence data was not able to confirm that the amplicons were of trypanosome origin and hence further analysis, or approaches, are required.
With a view to developing new diagnostic tools a bioinformatic analysis of mobile elements inserted in RHS/pseudogenes in the T.brucei genome was carried out. The aims of this were to locate variable regions of these genes that could be used as detailed markers for trypanosome strain identification. Sequences of the RHS genes were retrieved from the T.brucei brucei and T. brucei gambiense genomes to investigate positional diversity of MGEs within this family of genes. Differences were found in the presence/absence of RIME elements in one RHS gene between the two subspecies. More detailed investigation of all RHS gene classes in T. b. brucei showed six classes of RHS gene types and within each class, individual sequences showed evidence of insertion by MGEs. In some specific instances, evidence of pre-insertion, insertion and subsequent removal of MGEs was seen. This enabled a temporal evolutionary sequence of events to be interpreted. As such, the RHS genes offer the opportunity to develop specific molecular epidemiological tools for investigating the evolution of MGEs in field samples and to carry out temporally informed epidemiological tracking of isolates.
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