This ethnographic study was conducted among the riverine people, also known as Ribeirinhos, in the state of Amazonas, Brazil, during four months of field research. The study focused on learning from Ribeirinhos’ experiences and practices of malaria. In this thesis, I argue that paying attention to Ribeirinhos’ experiences and diagnostic, treatment, and control practices of malaria can provide useful insights into blind spots in the current interventions to control the disease in Brazil. As this is a thesis by publications, the findings are presented in three manuscripts. The first manuscript focuses on how malaria is experienced by Ribeirinhos. It explores the embodiment of malaria, empirical strategies to distinguish it from other febrile sicknesses, misalignment between bodies and current biomedical diagnosis methods, “becomings” of bodies and experiences, and the vicissitudes of having the disease. The second manuscript examines experiences and treatment practices for vivax malaria highlighting the uses of pharmaceuticals, side effects of antimalarial drugs, and traditional treatments for malaria. The third manuscript describes Ribeirinhos’ perceptions of malaria-carrying mosquitoes, their everyday practices to manage these beings, and their experiences with control interventions, such as time monitoring recommendations (TMR), indoor residual spraying (IRS), and insecticide-treated nets (ITN). The three manuscripts clearly show that Ribeirinhos' lives are thoroughly entangled with Amazonian rivers and forests; malaria also takes part in these entanglements. Learning from their experiences and practices of malaria has provided information about the nuances, improvisations, and continuous negotiations required to coexist with the parasite and disease vectors.

Department of Chemistry / MSc (Chemistry) / Due to the increasing incidence of Plasmodium strains that are resistant to current frontline antimalarial drugs, malaria remains a global public health challenge. In recent years, the emergence of resistance to frontline antimalarial drugs including the more recently discovered artemisinin class drugs has become one of the greatest challenges of controlling malaria incidence and mortality. There is, therefore, an urgent need to develop novel targets and antimalarial drugs that are effective against drug-resistant malarial parasites. Recent studies have demonstrated that calcium dependent protein kinases (CDPKs) regulate a variety of biological processes in the malaria parasite Plasmodium falciparum and that CDPK4 is important for parasite development. The gene disruption of CDPK4 in Plasmodium berghei, which results in major defects in sexual differentiation of the parasite has highlighted the importance of CDPK4 in Plasmodium biology and suggests that it may be used as a target for therapeutic drugs. PfCDPK4 is expressed in the gamete/gametocyte stage, and this could make PfCDPK4 an essential target for malaria drug discovery. The structure of PfCDPK4 was used as a template in the discovery of malaria drug leads and in designing chemical compounds or inhibitors that will show anti-parasitic activity against the target molecule. The model structure of PfCDPK4 was generated through homology modelling, and model structure validation confirmed that the model structure of PfCDPK4 is of stereochemical quality. The molecular modelling approach of in silico screening was utilized in this research, wherein a large library of chemical compounds, some natural chemical compounds, and clinically approved kinase inhibitors were screened against the target molecule PfCDPK4. In silico screening of the Bio-Focus library against PfCDPK4 resulted in twenty-six compounds being identified; in vitro single screening at a concentration of 5 μM confirmed that three compounds exhibit moderate antimalarial activity against the NF54 strain of Plasmodium falciparum, with the percentage inhibition ranging between 42% and 47%.

Early diagnosis and prompt treatment is one of the malaria control strategies used to minimize malaria morbidity and mortality. One of the mechanisms to implement early diagnosis and prompt treatment is community access to diagnostic services and effective antimalarial drugs. However, in Ethiopia the health system is underdeveloped and much of the rural population has limited access to modern health services. Therefore, the Ethiopian government introduced the Health Extension Programme(HEP) which is a community-based health care delivery system aimed at accessing essential health services through its health extension workers (HEWs). Involvement of the HEWs in prescribing and dispensing antimalarial drugs is shown to have improved community access to antimalarial drugs. However, there is insufficient knowledge of HEWs compliance to malaria treatment guidelines and patient adherence of patients treated by HEWs. The objectives of this study has been to describe the HEWs practice in malaria treatment, to evaluate adherence of patients to antimalarial drugs, to explore the factors influencing the HEWs malaria treatment practice and patient adherence, and to develop the guidelines to support the HEWs in malaria treatment practice. A qualitative study design was used to study the HEWs practice in malaria treatment along with patient adherence. Data were collected using in-depth face-to-face interviews, focus group discussion and patient medical record review and were analysed according to Tesch’s steps. The study revealed that the HEWs adequately comply with malaria treatment guidelines during diagnosis of malaria, as well as during the prescribing and dispensing of antimalarial drugs. However, there are some factors influencing the performance of HEWs. These are: shortage of diagnostic kit/RDT, shortage of antimalarial drugs, patient pressure to obtain coartem, work load, and community beliefs with regard to antimalarial drugs effectiveness. This study also revealed that the HEWs follow up after treatment of patients and good community support systems improved patient adherence to antimalarial drug use. Factors negatively influencing patient adherence were identified to include: forgetfulness, fear of shortage of drugs, adverse drug effects, duration of treatment, rapid relief of malaria symptoms and inadequate awareness of the consequence of incomplete dosage. Guidelines were developed to support the HEWs in malaria treatment practice with the aim to improve patient adherence to antimalarial drugs / Health Studies / D. Litt. et Phil. (Health Studies)

<p>This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper.</p><p>Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. </p><p>Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil).</p><p>Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE.</p><p>Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.</p>

This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper. Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil). Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE. Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.

Malaria is a life-threatening vector borne infectious disease caused by protozoan parasites of the genus Plasmodium. More than 100 species of Plasmodium can infect numerous animal species such as reptiles, birds and various mammals. However, human malaria is caused by four Plasmodium species -Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, and occasionally by the simian malaria parasite, Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the major causative agents and P. falciparum is the most virulent. About 300-500 million malaria infections occur every year leading to over 1-2 million deaths, of which 75% occur in African children of less than 5 years infected with P. falciparum. In spite of major global efforts to eliminate this disease over the past few decades, it continues to persist as a major affliction of human-kind imposing serious health and economic burden, especially to the poor countries. In India, the present scenario is about 2 million malaria positive cases every year, with almost 50% being caused by P. falciparum. Although remarkable attempts have been made over the years to develop vaccines against sexual and asexual stages of malaria parasite, an effective vaccine is still not in sight and remains as a distant goal. Hence, highly potent, less toxic and affordable antimalarial drugs remain as a first line therapy for malaria. Unfortunately, these parasites have been evolving against every known antimalarial drug and many of these drugs have lost their potency due to rapid emergence and spread of drug resistant strains. With development of resistance against frontline antimalarials such as chloroquine and antifolates, artemisinin and its derivatives seem to be the only effective antimalarials. However, recent reports on the possible emergence of artemisinin resistant strains, have led to the implementation of artemisinin-based combination therapies as a strategy to prevent drug resistance. Also, this continuous emergence of drug resistance has necessitated the development of new antimalarial drugs to combat this disease. While, Anopheles mosquitoes transmit parasites that infect humans, monkeys and rodents, Culex and Aedes mosquitoes predominate in the natural transmission to birds, and vectors of reptilian parasites are largely unknown. Of the approximately 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, and 30 of which are of major importance. Ironically, the strategies implemented for controlling Anopheles, have also been hampered by insecticide resistance and other practical difficulties that exist in the scope of their applicability. In the past few years several milestones have been achieved in parasite genome, transcriptome and proteome studies, which could be exploited for the development of new drugs and drug targets. One such promising target includes the metabolic pathways of the malaria parasite which differ significantly from its human host. This thesis entitled “Unique Features of the Heme-Biosynthetic Pathway in Human Malaria Parasite, Plasmodium falciparum” unravels the unique biochemical features of heme-biosynthetic enzymes of P. falciparum, which have the potential for being drug targets. This pathway was first identified in this laboratory over 15 years ago. In the present study, five of the 7 enzymes of this pathway have been cloned, expressed, properties studied and sites of localization identified. With the knowledge on the first two enzymes coming from earlier studies, it is now possible to depict the unique hybrid pathway for heme biosynthesis in P. falciparum with full experimental validation.

This thesis entitled “Mechanism of Anticancer and Antimalarial Action of a Modulator of Heat Shock Proteins” describes the successful elucidation of the mechanism of anticancer and antimalarial action of 15-Deoxyspergualin (DSG). DSG, a relatively well known immunosuppressant and antitumor molecule has been demonstrated to kill the malaria parasite in vitro and in vivo (Midorikawa et al., 1997; Midorikawa et al., 1998). A highly polar molecule, DSG binds the carboxy terminal “EEVD” motif of heat shock proteins, Hsp70 and Hsp90, enhances the ATPase activity of Hsp70 (Nadler et al., 1992; Nadler et al., 1998), and modulates several seemingly unrelated cellular processes. DSG has also been demonstrated to inhibit protein synthesis and polyamine synthesis in cells (Kawada et al., 2002; Hibasami et al., 1991), and previously speculated to inhibit malaria parasite growth by inhibiting polyamine synthesis. The grim situation with regard to malaria infection and mortality, principally an offshoot of the emergence of chloroquine resistant strains of the causative agent of malaria - Plasmodium falciparum, calls for intense efforts towards developing efficacious antimalarial agents with few side effects. DSG, having been used already in graft rejection cases in man and demonstrated to potently inhibit malaria in mice (Midorikawa et al., 1997), offers promise in this regard. It was, therefore, of interest to solve the mystery of its mechanism of antimalarial action. Chapter 1 surveys literature related to DSG mechanism of action and presents the thesis objective. Chapter 1 also gives an overview of heat shock proteins and their role in cancer, and the biology of the malaria parasite (Plasmodium falciparum), the working of the principal metabolic pathways existing in it, and a description of processes related to the intriguing, relict plastid present in apicomplexans. The metabolic processes previously speculated to be targeted by DSG, and those later found to be involved in DSG mechanism of action – polyamine synthesis and transport, protein synthesis and apicoplast processes are dealt with in more detail. Though DSG has been speculated to kill the malaria parasite by inhibiting polyamine synthesis, that DSG could clear malaria infection in Plasmodium berghei infected mice did not corroborate with the observation that inhibitors of polyamine biosynthesis are incapable of inhibiting the malaria parasite in vivo probably because the parasites make do with polyamines salvaged from the host (Assaraf et al., 1984; Bitonti et al., 1987). On the other hand, DSG is known to bind heat shock proteins, and inhibit protein synthesis, and heat shock proteins are speculated to be involved in the activation of HRI (heme regulated inhibitor), a type of eIF2á kinase that phosphorylates the eukaryotic initiation factor, eIF2á in conditions of heme deficiency or other cellular stress. eIF2á phosphorylation leads to stalling of protein synthesis. It seemed likely that if HRI is activated upon sequestration of heat shock proteins by DSG, it would culminate in protein synthesis inhibition and ultimately, cell death. With the intention to investigate this line of thought, the PlasmodB database was mined for proteins essential to the existence of heme dependent protein synthesis in Plasmodium falciparum. Two Hsp70 proteins from Plasmodium falciparum, one with the carboxy terminal “EEVD” motif implicated in DSG binding, and one without, and an Hsp70 interacting protein were cloned and expressed in their recombinant form in Escherichia coli. The preliminary characterization of these heat shock proteins described in Chapter 2 revealed that they were functionally active. DSG did not inhibit either the chaperone activity of the Hsp70s or the interaction of Hsp70 with Hip, but stimulated their ATPase activity as anticipated. Chapter 3 gives a complete picture of the mechanism of protein synthesis inhibition by DSG in the standard protein synthesis system – reticulocyte lysate. The experiments carried out revealed that DSG inhibits protein synthesis precisely through the mechanism envisaged, i.e. through phosphorylation of HRI following sequestration of Hsp70. Experiments involving exogenous addition of heat shock protein to in vitro translation reactions confirmed this hypothesis. Moreover, DSG inhibited protein synthesis in cancer cells in vivo, too, and HRI knockdown cells were not affected by DSG. Interestingly, the Hsp70 levels in various cancer cell lines inversely correlated with the inhibitory activity of DSG, and modulation of Hsp70 levels through standard methods altered DSG inhibition of protein synthesis in these cells. It was thus confirmed that DSG did indeed inhibit mammalian cells through the pathway envisaged. Its previously reported antitumor property is probably through this outlined mechanism of interference with protein regulation. In the malaria parasite, too, DSG inhibited protein synthesis through eIF2 alpha phosphorylation following Hsp70 sequestration as outlined in Chapter 4. However, while the concentration of DSG required for inhibition of malaria parasite growth was in the nanomolar range, high micromolar concentrations of DSG were required to effect protein synthesis inhibition in the malaria parasite, indicating that yet another target for DSG existed in the malaria parasite. With protein synthesis no longer a candidate target of DSG, I looked into the previously implicated polyamine synthesis pathway. In the event of DSG inhibiting polyamine transport in addition to polyamine biosynthesis, it would be expected to clear malaria infection in vivo contrary to other inhibitors of polyamine biosynthesis. In Chapter 5, evidence for the polyamine synthesis pathway in the malaria parasite is provided. Experiments involving incorporation of radiolabeled precursors in the malaria parasite and in mammalian cells, however, revealed that only high micromolar concentrations of DSG inhibit polyamine synthesis. Polyamine transport was also studied in considerable detail in malaria parasite infected red blood cells. Though infected red blood cells demonstrated different kinetic parameters, implying that new polyamine transporters were employed by the parasite on the red blood cell upon infection, DSG did not potently inhibit polyamine transport, either. The mystery of the target of DSG in the malaria parasite was, however, close to solution, when the growth inhibition of the malaria parasite by DSG was studied carefully. DSG invoked “delayed death” – a phenomenon wherein death is invoked only one cycle after incubation with the inhibitor. “Delayed death” is typical of inhibitors that target apicoplast processes (Fichera and Roos, 1997). DSG did not inhibit either fatty acid synthesis or prokaryotic protein synthesis – processes that occur in the apicoplast, but effected a decrease in the amount of nucleus encoded proteins that are targeted to the apicoplast, suggesting that it inhibited the trafficking of nucleus encoded proteins to the apicoplast. Confocal microscopy of parasites transfected with GFP fusion protein confirmed these findings, and is described in Chapter 6. The thesis ends with a summary of the findings in Chapter 7. Apicoplast processes have always been considered to harbor immense potential in the development of antimalarial agents, thanks to the absence of an equivalent organelle and hence pathways, in the human host. Trafficking of nucleus encoded proteins to the apicoplast has remained unexplored however. The work done in this thesis not only serves to demystify DSG with regard to its mechanism of action, but also paves the way for further studies in this area of intracellular trafficking, which could help in the development of more efficacious antimalarial agents. It also adds a new dimension to previous work conducted with regard to the anticancer action of DSG. Appendix 1 revolves around inhibitors which target various apicoplast processes. Apicoplast processes have been conventionally linked to the intriguing but unfortunate (with respect to clinical application) “delayed death”. Results presented in this section demonstrate that not all apicoplast processes invoke “delayed death”. Inhibition of apicoplast processes such as fatty acid biosynthesis and heme synthesis evoke rapid death. Inhibitors designed to target these processes could, therefore, be highly efficacious.

This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural settings. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703

Les travaux de thèse présentés dans ce manuscrit portent sur l’étude chimique de plantes du genre Psiadia. Trois axes de recherche ont été menés parallèlement à savoir (1) une étude chimiotaxonomique à partir de 11 espèces du genre Psiadia endémiques de La Réunion, (2) un criblage biologique réalisé sur 16 espèces du genre Psiadia dont 11 endémiques de La Réunion et 5 endémiques de Maurice et (3) une étude phytochimique ciblée sur l’espèce Psiadia arguta endémique de Maurice. Le premier axe comportant l'étude chimiotaxonomique menée par une approche métabolomique avait pour objectif d'identifier des marqueurs chimiotaxonomiques. Cette étude a été effectuée à partir des analyses CG-SM et CG-DIF des composés volatils et des analyses RMN 1H des composés non volatils de 11 espèces endémiques de La Réunion récoltées sur différents lieux géographiques et au cours des saisons estivale et hivernale. Une analyse intra-espèce a permis d'étudier la variabilité saisonnière et/ou géographique de la composition chimique de chaque espèce. Une analyse inter-espèces a conduit à deux classifications différentes des 11 espèces selon leur composition en métabolites volatils et non volatils. Le deuxième axe avait pour objectif d'identifier parmi 11 espèces du genre Psiadia endémiques de La Réunion et 5 espèces endémiques de Maurice, les espèces présentant une ou des activités biologiques prometteuses. Les cibles biologiques choisies ont été le parasite Plasmodium falciparum responsable du paludisme, la lignée cellulaire humaine cancéreuse HeLa responsable du cancer du col de l'utérus et l'enzyme HRP (Horseradish peroxydase) intervenant dans la réponse inflammatoire. À l'issue de ce criblage, 5 espèces se sont révélées prometteuses : les espèces réunionnaises P. amygdalina et P. anchusifolia et les espèces mauriciennes P. arguta et P. lithospermifolia pour l'activité antiplasmodiale, ainsi que l'espèce réunionnaise P. dentata pour les trois activités testées. Le troisième axe consacré à une étude phytochimique de P. arguta réalisée par un fractionnement bioguidé de l'extrait brut a conduit à l'isolement et à l'identification de 16 terpénoïdes : 2 triterpènes et 14 diterpènes de structure labdane dont 4 sont de structure nouvelle. Cinq diterpènes se sont révélés particulièrement actifs contre le parasite P. falciparum : l'acétate de labda-13(E)-en-8α-ol-15-yle, l'acétate de labdan-8α-ol-15-yle, le 13-épi-sclaréol, le labda-13(E)-ène-8α,15-diol et le (8R,13S)-labdane-8,15-diol. Par ailleurs, une étude métabolomique menée par RMN 1H sur des plantules de P. arguta cultivées in vitro et acclimatées a permis l'étude des facteurs influençant la production de ces composés bioactifs. / The present work describes the chemical composition of the plant genus Psiadia and focuses on three research topics: (1) a chemotaxonomic study of 11 species endemic to Reunion island, (2) a biological screening of 16 Psiadia species among which 11 are endemic to Reunion and 5 are endemic to Mauritius and (3) a phytochemical investigation of Psiadia arguta, endemic to Mauritius. The aim of the chemotaxonomic study was to identify chemical markers by a metabolomic approach using GC-MS and GC-FID for volatiles compounds and 1H NMR for non-volatiles compounds. The 11 studied species were harvested in different locations and seasons in order to analyze the seasonal or geographical variability of the chemical profile of each species. This study led to two classifications of the 11 species in terms of the composition of volatiles and non-volatiles compounds. The objective of the second research topic was to identify within 11 species endemic to Reunion island and 5 species endemic to Mauritius, the most active species for the biological activities tested. The targeted activities were antiplasmodial against Plasmodium falciparum, anticancer against the human cancer cell lines HeLa and anti-inflammatory through the inhibition of the enzyme HRP (Horseradish Peroxidase). Four species, P. amygdalina and P. anchusifolia, endemic to Reunion, and P. arguta and P. lithospermifolia, endemic to Mauritius, were particularly active against P. falciparum. Besides, P. dentata (endemic to Reunion) displayed interesting antiplasmodial, anticancer and anti-inflammatory activities. The third research topic was devoted to a phytochemical investigation of P. arguta by a bioguided fractionation and led to purification and identification of 16 terpenoids: 2 triterpenes and 4 diterpenes including 4 new compounds. The evaluation of the antiplasmodial activity of all isolated compounds allowed to highlight activities of five diterpenes: labda-13(E)-en-8α-ol-15-yle acetate, labdan-8α-ol-15-yle acetate, 13-epi-sclareol, labda-13(E)-ene-8α,15-diol and (8R,13S)-labdane-8,15-diol. Furthermore, in order to identify factors influencing the production of bioactive compounds, P. arguta has been multiplicated using in vitro culture techniques and micropropagated plants were acclimatized.

Early diagnosis and prompt treatment is one of the malaria control strategies used to minimize malaria morbidity and mortality. One of the mechanisms to implement early diagnosis and prompt treatment is community access to diagnostic services and effective antimalarial drugs. However, in Ethiopia the health system is underdeveloped and much of the rural population has limited access to modern health services. Therefore, the Ethiopian government introduced the Health Extension Programme(HEP) which is a community-based health care delivery system aimed at accessing essential health services through its health extension workers (HEWs). Involvement of the HEWs in prescribing and dispensing antimalarial drugs is shown to have improved community access to antimalarial drugs. However, there is insufficient knowledge of HEWs compliance to malaria treatment guidelines and patient adherence of patients treated by HEWs. The objectives of this study has been to describe the HEWs practice in malaria treatment, to evaluate adherence of patients to antimalarial drugs, to explore the factors influencing the HEWs malaria treatment practice and patient adherence, and to develop the guidelines to support the HEWs in malaria treatment practice. A qualitative study design was used to study the HEWs practice in malaria treatment along with patient adherence. Data were collected using in-depth face-to-face interviews, focus group discussion and patient medical record review and were analysed according to Tesch’s steps. The study revealed that the HEWs adequately comply with malaria treatment guidelines during diagnosis of malaria, as well as during the prescribing and dispensing of antimalarial drugs. However, there are some factors influencing the performance of HEWs. These are: shortage of diagnostic kit/RDT, shortage of antimalarial drugs, patient pressure to obtain coartem, work load, and community beliefs with regard to antimalarial drugs effectiveness. This study also revealed that the HEWs follow up after treatment of patients and good community support systems improved patient adherence to antimalarial drug use. Factors negatively influencing patient adherence were identified to include: forgetfulness, fear of shortage of drugs, adverse drug effects, duration of treatment, rapid relief of malaria symptoms and inadequate awareness of the consequence of incomplete dosage. Guidelines were developed to support the HEWs in malaria treatment practice with the aim to improve patient adherence to antimalarial drugs / Health Studies / D. Litt. et Phil. (Health Studies)