The purpose of this study was to describe the characteristics and findings of antimalarial drug efficacy studies conducted in Ethiopia and to use the findings to formulate recommendations for antimalarial drug efficacy monitoring and use of evidence to inform antimalarial treatment policy for the Ethiopian setting. This study reviewed 44 antimalarial efficacy studies conducted in Ethiopia from 1974 to 2011. The analysis of results indicated that chloroquine as the first-line antimalarial drug for the treatment of malaria due to Plasmodium falciparum had a 22% therapeutic failure in 1985. Chloroquine was replaced with sulfadoxine-pyrimethamine in 1998, more than 12 years later, when its therapeutic failure had reached 65%. Sulfadoxinepyrimethamine at the time of its introduction had a treatment failure of 7.7%; it was replaced after seven years in 2004 by artemether-lumefantrine; by then its treatment failure had reached 36%. The WHO recommends the replacement of a first-line antimalarial drug when more than 10% of treatment failure is reported. The replacement drug should have a therapeutic efficacy of more than 95%; while the change itself should be completed within two years. The prolonged delay to replace failing antimalarial drugs in Ethiopia seems to have been influenced mainly by the lack of systematic antimalarial drug efficacy data collection and pragmatic use of the data and evidence gathered.Almost eight years after its introduction, isolated studies show that the efficacy of artemether-lumefantrine has decreased from 99% in 2003 to around 96.3% in 2008. Though this decrease is not statistically significant (chi-square 1.5; P=0.22) and has not reached the threshold of 10%, it is plausible that its efficacy may drop further. This is mainly due to regulatory provisions in the country that allow marketing of oral artemisinin mono-therapies that are not recommended for malaria treatment, use of less effective antimalarial combination drugs in the neighboring countries and widespread drug quality problems. The situation calls for and this study recommends the establishment of stringent drug efficacy monitoring and early warning system and alignment of the antimalarial drug regulatory practices with recommendations of the WHO. / Health Studies / D. Litt. et Phil. (Health Studies)

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: In Africa alone, Plasmodium, the causative agent of malaria is estimated to kill a child, under the age of five every thirty seconds140. The ability of the parasite to rapidly attain resistance, has resulted in immunity of the parasite to all, except one group of frontline drugs. The need to develop novel drugs, vaccines and prevention strategies that are accessible and affordable for third world countries is of the utmost importance to prevent needless human suffering and death. The glycolytic pathway is an attractive drug target since it is the principal source of ATP for the parasite. Many of the glycolytic enzymes have been studied and proposed as drug targets, but the importance of these enzymes for the function of the pathway as a whole has not been considered. It is known, from the frameworks of metabolic control analysis, that control of the flux and metabolite concentration can be divided among the individual steps. Differential control analysis of Plasmodium and erythrocyte glycolysis may reveal potential drug targets. These analyses require a detailed kinetic model of Plasmodium glycolysis, and the feasibility of constructing and validating such a model was the aim of this study. In this work we determined the feasibility of constructing and validating a detailed kinetic model for the Plasmodium falciparum glycolytic pathway. Whether the construction and validation of this kinetic model was feasible or not was decided on the basis of the ability to: i) culture and isolate sufficient asexual parasites for enzymatic and steady state assays , ii) obtain kinetic parameters such as Km and Vmax for each glycolytic enzyme, either from literature or experimentally, iii) measure glycolytic fluxes, iv) determine glycolytic intermediate concentrations, v) construct a kinetic model from the kinetic parameters and vi) validate it with steady state glycolytic fluxes and metabolite concentrations Each of the above criteria were successfully addressed. In summary, the kinetic parameters and glycolytic fluxes that were measured experimentally, were used to construct and partially validate a detailed kinetic model, respectively. Further validation of the model by means of steady state metabolite concentrations was shown to be possible with the development of a suitable protocol to measure the glycolytic intermediate concentrations. The model presented in this work may play an important role in drug target identification and improving the current understanding of host-parasite interactions and glycolytic regulation. / AFRIKAANSE OPSOMMING: Plasmodium, die parasiet wat malaria veroorsaak, is in Afrika alleen elke dertig sekondes verantwoordelik vir die afsterwe van ’n kind jonger as vyf jaar. Die parasiet se vermoë om vinnig weerstand op te bou het daartoe gelei dat Plasmodium weerstandbiedend is teen byna alle nuwe teen-malaria middels, behalwe vir ’n enkele toonaangewende groep. Die ontwikkeling van nuwe malaria teen-middels is van uiterste belang om lyding te voorkom. ’n Goeie teiken vir teen-malaria middels is die glikolitiese padweg omdat die metaboliese padweg essensieël is vir die produksie van ATP, die energiebron van die parasiet. Desondanks die feit dat meeste van die glikolitiese ensieme al goed bestudeer en as teiken voorgestel is, is dit steeds onduidelik hoe hierdie ensieme saam funksioneer om die metaboliese weg, as geheel, tot stand te bring. Metaboliese kontrole analise het aangetoon dat die glikolitiese beheer verdeel is tussen die onderskeie glikolitiese ensieme, m.a.w. geen enkele ensiematiese stap het volledige beheer oor die fluksie van die glikolitiese padweg nie. Die afsonderlike analise en vergelyking van Plasmodium - en rooibloedselglikolise met behulp van differensiële metaboliese kontrole analise sal moontlik gebruik kan word om gasheervriendelike teikens vir nuwe middels aan te toon. So ’n analise benodig ’n omvattende kinetiese model van Plasmodium glikolise. Derhalwe was die doel van hierdie studie om vas te stel hoe uitvoerbaar dit is om ’n kinetiese model van Plasmodium glikolise te konstrueer en te valideer. Die uitvoerbaarheid van die konstruksie en validering van die kinetiese model was geasseseer op grond van die vermoë om: i) parasietkulture te kweek en genoegsame parasiete, wat in die aseksuele fase is, te isoleer sodat ensiembepalings en bestendige toestand-bepalings gedoen kan word, ii) kinetiese parameters soos Km - en Vmax-waardes vir elke glikolitiese ensiem, hetsy vanuit literatuur of eksperimentele werk, te verkry, iii) glikolitiese fluksie te meet, iv) glikolitiese intermediaatkonsentrasies te bepaal, v) ’n kinetiese model van die bepaalde kinetiese parameters op te stel en vi) die model te valideer met glikolitiese flukswaardes en metaboliet- konsentrasies wat in die bestendige toestand verkry is. Elk van die bogenoemde kriteria was met sukses in hierdie studie aangespreek. Ter opsomming, die eksperimenteel bepaalde kinetiese parameters en glikolietiese flukswaardes was gebruik om onderskeidelik ’n gedetaileerde kinetiese model te konstrueer en gedeeltelik te valideer. Daar was getoon dat verdere validering van die model deur middel van bestendige toestand metabolietkonsentrasies moontlik is met die ontwikkeling van ’n geskikte protokol om glikolitiese intermediaatkonsentrasies te meet. Die model, soos opgestel in hierdie studie, kan moontlik ’n belangrike rol speel om teikens vir nuwe malaria teen-middels te identifiseer en om gasheer-parasiet interaksies en glikolitiese regulering beter te verstaan.

Background The years 1985 to 1988 and 1997 to 2001, were periods of high morbidity and mortality due to malaria in KwaZulu-Natal, South Africa. One reason for the increased burden of disease was the emergence of drug resistant Plasmodium falciparum. The parasite was resistant initially to chloroquine and then to sulphadoxine-pyramethamine, the medication of choice for the treatment and prevention of malaria in different periods of time. The changing epidemiology of malaria in Mrica was exacerbated by policy makers not making timely and rational change to the failing malaria drug regimens to newer and effective ones. Purpose ofthe study This study was conducted to determine the impact of delayed introduction of sulphadoxine-pyramethamine (Fansidar®) and artemether-lumefantrine (Coartem®) as a first-line drugs for malaria in KwaZulu-Natal from 1985 to 1988 and 1997 to 2001 respectivel y, Study Design Observational, Analytic, Ecological Method The incidence of malaria in KwaZulu-Natal was compared during different phases of the period when chloroquine was the first line treatment. The baseline phase (1982 to 1984) was taken when chloroquine correctly should have been used and this was compared with the delayed phase (1985 to 1988), when it should have been replaced by of sulphadoxinepyramethamine. During the second period sulphadoxine-pyramethamine was the first line treatment of malaria, the baseline phase (1993 to 1996) when it correctly should have been used was compared to the delayed phase (1997 to 2001) of introduction of the alternate treatment of malaria with artemether-Iumefantrine. Ethical approval for this study was obtained from the Biomedical Research Ethics Committee, of the University of KwaZulu-Natal. Statistical Methods The relative association of malaria infection during the chloroquine baseline and change phases and the sulphadoxine-pyrametharnine baseline and change phases were compared with statistical significance at 0.05. Results The risk of malaria infection was 4.5 times (Incidence Risk Ratio = 4.5; 95% Confidence Interval: 4.1 to 5.0; P < 0.0001) higher in chloroquine change phase relative to the baseline phase. During the sulphadoxine-pyrametharnine period, the malaria risk was 3.5 times greater (Incidence Risk Ratio = 3.50; 95% Confidence Interval: 3.40- 3.60; p < 0.0001) in the change phase. In the chloroquine period, the malaria mortality risk was 9.1 times higher (95% Confidence Interval: 2.1 to 38.5; p=0.0003) and the case fatality rate was increased 1.3 times more (95% Confidence Interval: 1.0 to 1.7; p< 0.001) in the change period. The risk of death during the sulphadoxine-pyramethamine change phase was 4.8 times (95% Confidence Interval: 3.3 to 7.0; p<O.OOl) and case fatality rate of2 times (95% Confidence Interval: 1.5 to 2.7; p <0.001) relative to the baseline phase. Conclusions The dramatic change in the malaria epidemiology in Africa in recent times was exacerbated by delay in replacing first line failing antimalarial drugs. The establishment of sentinel sites for assessing drug resistance or failure and the application of World Health Organisation standards in drug resistance studies will go a long way to achieving the Roll Back Malaria target by 2010. / Thesis (MMed.)-University of KwaZulu-Natal, Durban, 2007.

[Truncated abstract] Malaria is the most important parasitic disease of man. Of the five species known to infect humans, Plasmodium falciparum causes most deaths and illness, especially when it affects children and pregnant women living in highly endemic areas of the rural tropics. Pharmacological therapies for malaria must be optimised for these groups and must be practical for administration in critically ill patients in remote settings. The clinical studies in this thesis evaluated the clinical pharmacology of modern antimalarial treatments in a Melanesian population exposed to highly endemic malaria. The clinical studies were conducted between March 2001 and June 2007, with final data analysis completed by mid-2008. They aimed to evaluate key pharmacokinetic, parasitological, host genetic and socio-cultural determinants of treatment effectiveness in children with uncomplicated and severe malaria and in pregnant women. A multi-centre study of children with uncomplicated malaria evaluated the efficacy of four treatment regimens, including three artemisinin combination treatments. PCR corrected recrudescence rates by day 42 were 81.5%, 85.4%, 88.0% and 95.2% for chloroquine + sulphadoxine-pyrimethamine, artesunate + sulphadoxine-pyrimethamine, dihydroartemisinin-piperaquine (DHA-PQ) and artemether-lumefantrine (AL), respectively. Determinants of efficacy in the DHA-PQ group included day 7 piperaquine (PQ) levels and baseline parasitaemia. Therefore, the worse than expected efficacy in this group may have been partly due to the high parasitaemias commonly seen in this population. ... Preliminary data suggested a protective effect of the erythrocyte polymorphism caused by the glycophorin C mutation against cerebral malaria. These studies also evaluated key pharmacokinetic, host genetic and socio-cultural determinants of the likely effectiveness of a novel pharmaceutical approach using artesunate suppositories for severe malaria. These demonstrated favourable absorption characteristics, clinical efficacy, safety and patient/community acceptability. Contrary to previous data, no evidence was found to suggest that the pharmacokinetic profiles or efficacy of artemisinin derivatives are likely to be compromised by a high prevalence of thalassaemia in this population. However, their highly variable bioavailability raises questions regarding the consistency of therapeutic response. Given the favourable efficacy and socio-cultural acceptability of rectal artesunate demonstrated in these studies, the PNG Ministry of Health has decided to add artesunate suppositories to its national pharmacopoeia and incorporate them into standard treatment recommendations. A final study compared the pharmacokinetics of chloroquine, sulphadoxine and pyrimethamine in pregnant, versus non-pregnant women. This demonstrated significantly lower concentrations of all three drugs and active metabolites in the pregnant group, due to a combination of effects on either volume of distribution, clearance and elimination half-life. It suggests that significant dosage alterations are necessary to optimise therapy in pregnant women.

The purpose of this study was to describe the characteristics and findings of antimalarial drug efficacy studies conducted in Ethiopia and to use the findings to formulate recommendations for antimalarial drug efficacy monitoring and use of evidence to inform antimalarial treatment policy for the Ethiopian setting. This study reviewed 44 antimalarial efficacy studies conducted in Ethiopia from 1974 to 2011. The analysis of results indicated that chloroquine as the first-line antimalarial drug for the treatment of malaria due to Plasmodium falciparum had a 22% therapeutic failure in 1985. Chloroquine was replaced with sulfadoxine-pyrimethamine in 1998, more than 12 years later, when its therapeutic failure had reached 65%. Sulfadoxinepyrimethamine at the time of its introduction had a treatment failure of 7.7%; it was replaced after seven years in 2004 by artemether-lumefantrine; by then its treatment failure had reached 36%. The WHO recommends the replacement of a first-line antimalarial drug when more than 10% of treatment failure is reported. The replacement drug should have a therapeutic efficacy of more than 95%; while the change itself should be completed within two years. The prolonged delay to replace failing antimalarial drugs in Ethiopia seems to have been influenced mainly by the lack of systematic antimalarial drug efficacy data collection and pragmatic use of the data and evidence gathered.Almost eight years after its introduction, isolated studies show that the efficacy of artemether-lumefantrine has decreased from 99% in 2003 to around 96.3% in 2008. Though this decrease is not statistically significant (chi-square 1.5; P=0.22) and has not reached the threshold of 10%, it is plausible that its efficacy may drop further. This is mainly due to regulatory provisions in the country that allow marketing of oral artemisinin mono-therapies that are not recommended for malaria treatment, use of less effective antimalarial combination drugs in the neighboring countries and widespread drug quality problems. The situation calls for and this study recommends the establishment of stringent drug efficacy monitoring and early warning system and alignment of the antimalarial drug regulatory practices with recommendations of the WHO. / Health Studies / D. Litt. et Phil. (Health Studies)

Malaria is a disease that causes great burden in public health worldwide. It was estimated that in 2011 there were 3.3 billion people at risk of acquiring malaria. According to data from the Ministry of Public Health of Ecuador, malaria incidence has shown a 99.9% steady decrease since year 2000. This study evaluated the effect of timely treatment on circulating gametocyte and malaria incidence rates. All cases reported in the province of Esmeraldas, Ecuador from July 2012 to March 2015 and to the national headquarters between February 2012 and December 2014 were studied. The effect of early treatment on: 1) follow-up gametocytemia at an individual level (cases reported in Esmeraldas); and, 2) incidence rates at a population level (within Esmeraldas and within Ecuador) was evaluated using a retrospective cohort and an ecologic study design, respectively. A total of 193 cases from the province of Esmeraldas were included in the retrospective cohort study. Patients were classified into three groups depending on time to treatment (i.e. how many days elapsed from symptoms onset to treatment) as follows: 1) early treatment for those treated within 2 days; 2) late treatment for those treated between 3 to 7 days; and, 3) extremely late for those receiving treatment after 7 days. A consistent association between time to treatment and follow-up gametocytemia was found in different regression models including logistic (adjusted OR = 0.20 and 0.28 for early and late treatment, respectively, p < 0.05), linear (parameter estimate = 0.018, p < 0.05) Poisson log linear (parameter estimate 0.103, p < 0.05), and negative binomial (parameter estimate = 0.111, p < 0.05). Extremely late patients had higher follow-up gametocytemia levels during follow-up visits 1 and 2. A survival analysis showed that extremely late treated patients tend to clear gametocytes later than the other two treatment arms (p > 0.05). Finally, there was a positive association between time to treatment and a period of transmisibiltiy, which was estimated based on the potential number of days that a patient has gametocytemia (p < 0.05). Population-level associations between time to treatment and malaria incidence rates were assessed through a two-tiered ecologic study: nationwide for Ecuador and provincewide for Esmeraldas.. A parish-level anlaysis of malaria among all parishes in the province of Esmeraldas revealed that malaria transmission differs widely within each population-level treatment arm classification group. A repeated measures negative binomial regression showed that there is a positive association between malaria incidence rate in subsequent periods and mean time to treatment, follow-up gametocytemia and a negative association with malaria incidence rate in the previous period (p < 0.05 for all associations). The nationwide analysis confirmed that there is wide variation in malaria incidence rate within each population-level treatment arm classification group. Although the World Health Organization (WHO) recommends timely malaria treatment there seems to be lack of peer-reviewed published evidence evaluating the association of time to malaria treatment with follow-up gametocytemia and incidence rates, especially in Latin America. This study has important public health implications. Firstly, there seems to be no clear definition for early malaria treatment. In this study, consistent evidence of the association between time to treatment and, specifically, early malaria treatment (i.e. malaria within 2 days of symptoms onset) with follow-up gametocytemia and period of transmissibliity is provided. Evidence provided here can serve as a basis for future research in other countreis facing similar conditions. Additionally, this information can serve to better inform public health policy, especially regarding the definition of early treatment and, thus, setting goals to accomplish early treatment among malaria infected patients. Noteworthy, Esmeraldas and Ecuador face significant challenges not only to achieve but to maintain malaria elimination, if achieved. These challenges arise from favorable local environmental conditions and to certain vulnerabilities like proximity to neibhoring areas with high malaria incidence, susceptibility to receive migration, specially refugees fleeing armed conflict, socio-economic disadvantages and remoteness of some parishes were malaria remains active. Moreover, the integration of the current national malaria control program into the organizational strucutre of the Ministry of Health may impose additional challenges like: 1) differential prioritization of other diseases, 2) lack of clear guidance about the role of the current malaria-dedicated personnel in the MoH strucutre; and, 3) lack of specification about on whom would accountability for malaria control rely. All these vulnerabilities should be properly addressed if malaria elimination is to be achieved in Ecuador. Finally, further research is required to confirm whether these trends and association are replicable across different populations, countries and continents. If these associations are similar or even stronger in other populations, then better malaria control programs informed in evidence-based definition of early malaria treatment could certainly be planned and implemented to achieve malaria elimination and control in other regions of the world.

Ph.D. / Background: Most recent data have shown a slight reduction in the incidence of malaria in Nigeria. However, cases of malaria in children younger than five years of age have continued to escalate amidst ‘simple’ and ‘effective’ treatment options. The realisation of the Millennium Development Goals (MDGs) – to halve the burden of malaria by 2015 – is becoming increasingly unrealistic in Nigeria following the alarming rates of malaria in children. Apart from the ecological and environmental factors, socio-cultural and behavioural factors might be responsible for the staggering cases of malaria in children in local communities in Nigeria. It was against this background that a sociological study of health care service utilisation was conducted among caregivers of children with malaria. The study investigated the perceived threat of malaria; how the local understanding of malaria affects the recognition of signs and symptoms, perceived aetiology, treatment-seeking patterns and the use of insecticide treated nets (ITNs). The socio-generational changes in the healthcare seeking behaviour between young and older mothers as well as differences in the patterns of health care service utilisation between rural and urban subjects were also interrogated. Method: This study adopted a qualitative research design using complementary methods. A total of 40 semi-structured interviews, 20 in-depth interviews and four focus group discussions (FGDs) were conducted with caregivers and health workers. The respondents included young and older parents between the ages of 25 and 80 years whose children or wards below the age of five had manifested malaria symptoms at one time or another. A purposive sampling procedure was used to select sample for the study. The study was conducted in two selected rural areas; Okanle and Fajeromi; and one urban centre; Ilorin, Kwara State of Nigeria. Findings: The research indicated that the perceived aetiology, symptoms and treatment of malaria in children were largely influenced by the socio-cultural patterns of the communities studied. The study found that the first line of treatment for children with malaria in the communities of study was usually home treatment using traditional herbal medicines. The use of modern health care facilities is usually seen as the last resort. The traditional beliefs about causes of malaria, affordability and trust in herbal medicines, on the one hand, were found to be responsible for the widespread use of herbal medicines in the treatment of malaria in children. On the other hand, poor service delivery, lack of money, attitudes of medical personnel, mixed feelings about the efficacy of modern medicines and lack of trust in the community health centres were some factors found to be responsible for delays in seeking modern health care services when children have malaria. More importantly, the decision to seek treatment from either traditional or modern sources was largely influenced by the network of informal social interaction and social support at household and community levels. In addition, the study also found some changes in the patterns of health care seeking behaviour of young and older caregivers but generally found no differences in the patterns of health care seeking behaviour between rural and urban participants. Finally, the study found that the majority of the respondents were not aware of the effectiveness of the ITNs. Consequently, there was a high dependence on the use of traditional preventive measures which included a local leaf known as “ewe-efon” translated as “mosquito leaf”. Apart from the perceived corruption and mismanagement at the level of distribution of the ITNs, lack of appropriate knowledge about the effectiveness of the ITNs was discovered to be responsible for the widespread non-acceptance of the ITN in the prevention of malaria in children.

The repeated emergence of Plasmodium falciparum resistance to first-line antimalarial drugs necessitates understanding the underlying resistance mechanisms to detect and monitor resistance in the field and to inform drug discovery efforts. With the advent of the FRG NOD human liver-chimeric (huHep) mouse model for P. falciparum genetic crosses, interest has renewed in harnessing this forward genetics tool to study traits including drug resistance. The antimalarial quinine (QN) is of particular interest as it has retained efficacy over 400 years as parasite resistance has been slow to develop against the drug, likely due to a multifactorial mechanism of which only several genes have been partially implicated. Chloroquine (CQ) is a former first-line drug for P. falciparum (that is still in use for P. vivax), and it’s phasing out has been associated with the recent emergence of CQ-sensitive P. falciparum parasites. While the CQ resistance transporter (PfCRT) is known to be the primary driver of resistance, studies have provided evidence for secondary modulators of CQ, of which only the multidrug resistance protein 1 (PfMDR1) transporter has been identified. This thesis addresses the hypotheses that additional mediators are involved in the parasite resistance mechanism to QN and that genes other than pfmdr1 modulate parasite resistance to CQ. In chapter 3, we present the P. falciparum genetic cross that we conducted between the QN- and CQ-sensitive African NF54 and QN- and CQ-resistant Cambodian Cam3.II parasites in huHep mice, in collaboration with Dr. Photini Sinnis’s laboratory at Johns Hopkins University. By applying different selective conditions to cross progeny bulk pools prior to cloning these bulks, we were able to recover 120 unique recombinant progeny from this cross. We observed minimal overlap in the progeny genotypes obtained from CQ and QN pressure, suggesting distinct mechanisms for parasite resistance to these drugs. Bulk progeny selection and progeny clone-based QN linkage mapping approaches identified quantitative trait loci (QTLs) on chromosomes 7 and 12, as well as minor QTLs on other chromosomes, consistent with a multifactorial resistance mechanism. We applied the latter approach to investigate parasite response to CQ and its active metabolite monodesethyl-CQ (md-CQ) and identified a novel chromosome 12 QTL in addition to pfcrt. Interestingly, while the chromosome 12 QTLs overlapped, the chromosome 7 QTL for high-grade QN resistance did not contain pfcrt. In chapter 4, we used bioinformatic approaches, whole-genome sequence data from our cross and field isolates, and literature review to identify the drug/metabolite transporter 1 (DMT1) as the top candidate of the chromosome 7 QTL, and S-adenosylmethionine mitochondrial carrier protein (SAMC), hydroxyethylthiazole kinase (ThzK), and ATP-dependent zinc metalloprotease (FtsH1) as the top candidates for the chromosome 12 QTLs. By harnessing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing methodologies (SNP-editing, knockout, and tagging), we obtained evidence favoring DMT1 as a marker of QN resistance and localized this transporter to structures associated with vesicular trafficking, PVM, lipid bodies, and the lysosome-like digestive vacuole. We also harnessed SNP-editing and identified FtsH1 as a potential mediator of QN resistance and a modulator of CQ and md-CQ resistance. QN, mefloquine, and lumefantrine belong to the same aryl-amino alcohol class, and we found that QN is structurally more similar to mefloquine than lumefantrine. We also showed that QN can partially inhibit heme detoxification. While conducting the work outlined in chapters 3 and 4, we identified an unmet need for quickly identifying clonal recombinant progeny and validating parasite identity, which inspired the study presented in chapter 5. We developed a genotyping method that can assess drug resistance-conferring SNPs directly from P. falciparum culture or infected blood as well as a multiplexed microsatellite genotyping method with five broadly informative markers. Both methods were applied in chapter 3 to identify clonal recombinant progeny, and the SNP genotyping method was used in chapter 4 to validate gene editing and progeny identity. We also tested the resolution, sensitivity, time, and cost of each method as well as whole-genome sequencing and recommended the ideal application for each genotyping method. Our data demonstrate that DMT1 is a novel marker for QN resistance, and a new chromosome 12 locus associates with CQ response, of which ftsh1 is a potential candidate. In chapter 6, we discuss the potential mechanisms by which DMT1 is involved in QN resistance, the potential impact of our findings, and future experiments that can further characterize the QN and CQ resistance mechanisms and the functional role of these candidate genes.

This study is based on a survey of over 900 respondents living in Tigray, Ethiopia and is intended to assist the Ethiopian government as well as other interested parties in analyzing the factors affecting the incidence of malaria in Tigray and those affecting people's choice of health care provider. / Two models were designed to answer these two questions. The first was a multinomial logit model in which socio-economic indicators were related to the incidence of malaria. The second model was specified as a conditional logit model aimed at determining people's choice between seeking treatment at a hospital/clinic or at a pharmacy/community health worker. / Economic development is the key to eradication of the major parasitic diseases, particularly malaria. An interesting result was obtained on the gender of the respondent; women in Tigray are less likely to report having had malaria than men. / Education level was found to be positively correlated with the likelihood of choosing the Hospital/Clinic option over the Pharmacy/Community Health Worker. / The Hospital/Clinic option was less likely to be chosen with increasing cost of treatment. (Abstract shortened by UMI.)

Background Malaria is a major threat to global public health, with adverse health effects. Nigeria alone accounts for 25% of the global burden of malaria. Children are especially vulnerable to malaria, and if the disease is not treated it could have fatal consequences. Mothers have an important role in ensuring that adequate and timely treatment is given to the child. Aim The aim of this study was to investigate whether there was an association between maternal level of education and the treatment with antimalarial drugs in malaria positive children under-5 in Nigeria. Methods This study was a cross-sectional study that utilized Demographic and Health Surveys (DHS) data from the 2015 Nigeria Malaria Indicator Survey. Data on 2’622 malaria positive children were used, and a logistic regression analysis was conducted to determine the association with maternal level of education. Results The mothers with a higher level of education had two times higher odds (OR 2.31, CI 1.62- 3.32) of making sure their child received treatment with antimalarial drugs, compared to the mothers with no education. With an increase of 38% (OR 1.38, CI 1.11-1.71) in the odds for the child receiving treatment with antimalarial drugs if the mother has primary education and an increase of 51% (OR 1.51, CI 1.24-1.84) if the mother has secondary education compared to mothers with no education. Conclusion Mothers with a higher level of education waere more likely to make sure that their child received treatment with antimalarial drugs, compared to the mothers with no education.