Factors influencing the spread and selection of drug resistance in Human African Trypanosomiasis

Nalunkuma Kazibwe, Anne J. (2008) Factors influencing the spread and selection of drug resistance in Human African Trypanosomiasis. PhD thesis, University of Glasgow.

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A growing problem with drug resistance in Human African Trypanosomiasis has necessitated the implementation of screening programmes to monitor for its spread. This thesis describes the study of several factors that can influence the selection and propagation of drug resistance in T. brucei.
Human African Trypanosomiasis (HAT) is caused by T. brucei gambiense and T. brucei rhodesiense. The few drugs used for the treatment of the disease are either toxic, cause severe side effects or suffer from parasite resistance. The T. brucei P2 transporter, which is encoded by the gene TbAT1, mediates uptake of melaminophenyl arsenicals and diamidines. Reduced P2 uptake is associated with drug resistance. A number of point mutations found in a laboratory derived melarsoprol resistant T. brucei stock (STIB 777R) allowed development of a PCR/RFLP based molecular method to identify resistance alleles. By 1999, 20-30% of patients treated in Omugo, NW Uganda were failing to respond to melarsoprol. PCR/RFLP analysis indicated that mutant alleles accounted for 58.5% of those in circulation. Melarsoprol was withdrawn in 2001 and by 2003 mutant TbAT1 alleles accounted for only 14% of those in circulation in NW Uganda. The current study aimed to determine the incidence of the PCR/Sfa NI TbAT1 mutant alleles in 2006, some five years after melarsoprol had been withdrawn as first-line treatment. Successful molecular analysis of 91 of 132 (68.9%) T. b. gambiense field isolates from Omugo and Moyo in NW Uganda indicated the presence of only TbAT1 wild type alleles. Mutant alleles thus appear to have disappeared. This may be the result of parasite fitness cost following the withdrawal of melarsoprol as a stage II first-line drug from Omugo health centre, Arua, since 2001. This apparent instability of TbAT1 mutants in the field may be exploited for rational or alternating use of melarsoprol and eflornithine (DFMO) to ensure a longer life for eflornithine, delaying the onset of resistance.
Insight into the overall population structure of the T. b. gambiense from Omugo, Arua (N=54) and Moyo (N=17) was obtained using mini/microsatellite marker analysis. Genetic diversity was observed to be more intra than inter regional. Multilocus genotype data analysis revealed the Omugo, Arua, population was genetically distinct from the Moyo population (Nei’s genetic distance=0.176). The evidence indicated surprisingly little genetic exchange with an excess in homozygosity (Fis >0) and alleles in linkage disequilibrium (P<0.05) within the Omugo, trypanosome population. This excess in homozygosity may be due to population sub-structuring, trypanosome inbreeding, or migration of patients. The latter is likely occurring from the neighbouring T. b. gambiense endemic disease focus in Southern Sudan. The findings suggested that the T. b. gambiense from Arua is not panmictic, clonal or epidemic but there is some level of genetic exchange.
The possibility that T. b. gambiense can infect animals raises the prospect that wild or domestic animals may act as a reservoir and that a veterinary link to gambiense Human African Trypanosomiasis exists. Treatment of animals for babesiosis and trypanosomes with diminazene, uptake of which is mediated through TbAT1/P2 could select for P2-defective drug resistant trypanosomes, thereby threatening control of the human disease as well. Species detection by PCR for animal and human trypanosomes in dog isolates (N=190) from the tsetse fly endemic Jos Plataeu, Nigeria did not reveal T. b. gambiense, but multiple infections with T. brucei (95%), T. vivax (89%), and subspecies T. congolense forest (54%) and savannah (50%) were detected. The dogs were also infected with other parasites, including Babesia canis (22%) and Hepatozoon canis (16%). Multiple infections can make correct diagnosis difficult and the infections are likely to be missed by the less sensitive microscopy method.
The trypanocidal action of the diamidine group of trypanocides, diminazene, pentamidine and furamidine (DB75) are principally mediated through the TbAT1/P2. In addition, pentamidine is taken up by two additional T. brucei transporters called High Affinity Pentamidine Transporter (HAPT1) and the Low Affinity Pentamidine Transporter (LAPT1). DB75 also has a secondary unknown route. Loss of TbAT1/P2 leads to significant resistance to DB75 and diminazene but not pentamidine. Identification of other markers of resistance is necessary to determine if other routes of drug entry do exist apart from P2 and whether these can be exploited for the delivery of new trypanocides into the trypanosomes. Adaptation of the T. brucei tbat1 knock-out cell line to higher concentrations of diminazene by in vitro selection for resistance led to loss of HAPT1. The resultant phenotype was similar to the previously characterised pentamidine resistant clone B48, but more resistant to diminazene and DB75. The adapted line was still capable of accumulating 1 µM radiolabelled diminazene suggesting both HAPT1 and LAPT1 as possible routes for diminazene uptake. Adaptation of the T. brucei tbat1 knock-out cell line to a high concentration of DB75 over the same 6 months period did not lead to increased resistance.
Overall the project has confirmed an important role for tbat1/P2 in development of resistance to melarsoprol in the field. Importantly, it appears that removal of the selection pressure of melarsoprol leads to a loss of tbat1 alleles associated with resistance in a population of trypanosomes capable of genetic exchange in NW Uganda. Although evidence for a dog reservoir for T. b. gambiense in Nigeria was lacking in this study, a risk of selecting resistance in animals must remain high on any list of consideration. I have further shown that the diamidine drug, diminazene, used in veterinary medicine also appears to enter T. brucei via the HAPT1 transporter, as well as the P2 transporter. Loss of HAPT1 through selection with diminazene leads to high level pentamidine resistance, which could indicate a further risk in selection of human infectious trypanosomes also resistant to drugs like pentamidine.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: human African trypanosomiasis, drug resistance, population genetics
Subjects: Q Science > QR Microbiology > QR180 Immunology
Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Supervisor's Name: Barrett, Professor Michael
Date of Award: 2008
Depositing User: Dr Anne Kazibwe/ A J N K Nalunkuma Kazibwe
Unique ID: glathesis:2008-381
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Sep 2008
Last Modified: 10 Dec 2012 13:18
URI: https://theses.gla.ac.uk/id/eprint/381

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