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Title: African isolates of Plasmodium falciparum and their influence on drug response
Author: Mukadam, Rabia
ISNI:       0000 0001 3429 1609
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2007
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Drug resistance of Plasmodium falciparum malaria is a huge problem. In Africa, as in other malarious areas, many of the current drugs are already useless or are failing rapidly. Chloroquine (CQ), amodiaquine (AM), sulfadoxine / pyrimethamine' (SP) and mefloquine (MQ) are notable members of this growing list. The molecular, basis of drug resistance is reasonably well known for these drugs and amino acid changes in the parasite proteins PjCRT, PjMDRJ, DHFR and DHPS have been implicated. These polymorphisms have been associated with drug resistance in vivo from clinical studies, largely using treatment failure as an end point or from in vitro studies, largely using field lines that were originally isolated many years ago. These studies are informative but some important information is lacking. For example, very little is known about the . phenotype / genotype relationship of current African field isolates and little is known about their likely response to new antimalarials that are currently under development. In this thesis, I have attempted to address some of these issues by culture adapting and studying contemporary field isolates from Africa. I have obtained and culture-adapted a panel of 32 field isolates from study sites in Kenya and characterized their response to currently used antimalarials as well as analyzing them for genetic polymorphisms that are known to be associated with drug resistance. As expected, the well-characterized mutations at positions 5 I, 59 and J08 in the DHFR gene, K76T in PjCRT and mutations at position 86 and 1246 in PjMDRI were largely present, although some mutations were absent in some isolates. What was more surprising is that parasites harbouring mutations in PjCRT and or PjMDR J were almost fully sensitive to CQ and AM. This includes many isolates with the critical K76T PjCRT change. This finding was corroborated using in vitro studies of genetically modified (OM) parasite lines that were allelically-exchange in respect of PjCRT. It was found that OM lines harbouring mutant PjCRT alleles (initially highly chloroquine resistant) became almost fully sensitive to CQ after three months continuous culture in the absence of drug pressure. One possible explanation for this is that PjCRT mutations are necessary but not sufficient for the chloroquine-resistant phenotype and that other genes are modified (probably upregulated rather than mutated). In addition, I have studied the likely effects of existing genetic polymorphisms on the . response of parasite lines to new drugs or combinations using field lines or allelically exchanged OM lines. Co-trimoxazole (CT) is an antifolate combination commonly used to treat bacterial infections and thought by some to have possible clinical utility against malaria. I have shown that parasite response to CT is affected by mutations of DHFR but this mainly occurs in the first mutations step. The acquisition of two or more mutations in DHFR has little further effect on the response on the parasites to CT. This suggests that CT might be useful to treat malaria but only provided that high enough doses can be given. I have also examined the effect of PjCRT polymorphisms on the response of parasite lines to novel 4-aminoquinoline drugs. I have looked at short chain CQ analogues and AM analogues that are modified to avoid metabolic escape. Examples of both of these groups of compounds are currently under drug development. I found that these drugs retain most of their activity against GM lines containing mutant PjCRT. However, there was slight but significant'PjCRT-mediated cross-resistance of the new 4-aminoquinolines with CQ and Desethyl-AM (the main circulating AM metabolite. Cross-resistance remained slight when the lines were, subjected to CQ pressure (becoming highly CQ-resistant. Nonetheless, the possibility remains that African field lines may be primed for rapid resistance to new quinoline, by virtue of their largely CQresistant PjCRT haplotypes. Finally, I examined the effect ofPjCRT polymorphisms on the response to DB75, a novel bis-benzyl amidine, currently under drug development for use as a broad-spectrum anti protozoal. Here the findings are somewhat more encouraging; PjCRT mutations seem to make the parasites more susceptible to this new amidine drug. Furthermore, binding of radiolabeled DB75 to the hematin target was also increased by mutant PfCRT. In' conclusion, I present a detailed study of contemporary isolates ofP. falciparu111 from a typical African setting. The results have some interesting implications for the deployment of current drugs and of drugs currently under development.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available