Academic literature on the topic 'Antimalarials Testing'

Abstract Objectives To evaluate Plasmodium malariae susceptibility to current and lead candidate antimalarial drugs. Methods We conducted cross-sectional screening and detection of all Plasmodium species malaria cases, which were nested within a longitudinal prospective study, and an ex vivo assessment of efficacy of a panel of antimalarials against P. malariae and Plasmodium falciparum, both PCR-confirmed mono-infections. Reference compounds tested included chloroquine, lumefantrine, artemether and piperaquine, while candidate antimalarials included the imidazolopiperazine GNF179, a close analogue of KAF156, and the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. Results We report a high frequency (3%–15%) of P. malariae infections with a significant reduction in ex vivo susceptibility to chloroquine, lumefantrine and artemether, which are the current frontline drugs against P. malariae infections. Unlike these compounds, potent inhibition of P. malariae and P. falciparum was observed with piperaquine exposure. Furthermore, we evaluated advanced lead antimalarial compounds. In this regard, we identified strong inhibition of P. malariae using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drug currently in clinical Phase IIb testing. Finally, in addition to GNF179, we demonstrated that the Plasmodium PI4K-specific inhibitor KDU691 is highly inhibitory against P. malariae and P. falciparum. Conclusions Our data indicated that chloroquine, lumefantrine and artemether may not be suitable for the treatment of P. malariae infections and the potential of piperaquine, as well as new antimalarials imidazolopiperazines and PI4K-specific inhibitor, for P. malariae cure.

It is important to discover novel antimalarial pharmacophores because of the widespread emergence of Plasmodium falciparum isolates resistant to the available drugs. Secondary metabolites derived from microbes associated with marine invertebrates are a valuable resource for the discovery of novel drug leads. However, the potential of marine microbes as a source of antimalarials has not been explored. We investigated the promise of marine microorganisms for the production of antimalarial activities by testing 2365 diverse microbial extracts using phenotypic screening of a multidrug resistant chloroquine resistant P. falciparum strain. We conducted counter screening against mammalian cells for the 317 active extracts that exhibited more than 70% inhibition at 1 µg/mL. The screen identified 17 potent bioactive leads from a broad range of taxa. Our results establish that the marine microbiome is a rich source of antiplasmodial compounds that warrants in depth exploration.

ABSTRACTEx vivoassay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-termin vitroculture andex vivoantimalarial susceptibility assays are relatively cumbersome, relying onin vivopassage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stagePlasmodium berghei,P. yoelii, andP. vinckei vinckeiusing a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

(1) Background: Malaria control has strongly benefited from the implementation of rapid diagnostic tests (RDTs). The malaria RDTs used in Burkina Faso, as per the recommendation of the National Malaria Control Program, are based on the detection of histidine-rich protein-2 (PfHRP2) specific to Plasmodium falciparum, which is the principal plasmodial species causing malaria in Burkina Faso. However, there is increasing concern about the diagnostic performance of these RDTs in field situations, and so constant monitoring of their accuracy is warranted. (2) Methods: A prospective study was performed in the health district of Nanoro, where 391 febrile children under 5 years with an axillary temperature ≥37.5 °C presenting at participating health facilities were subjected to testing for malaria. The HRP2-based RDT and expert microscopy were used to determine the diagnostic performance of the former. Retrospectively, the correctness of the antimalaria prescriptions was reviewed. (3) Results: Taking expert malaria microscopy as the gold standard, the sensitivity of the employed RDT was 98.5% and the specificity 40.5%, with a moderate agreement between the RDT testing and microscopy. In total, 21.7% of cases received an inappropriate antimalarial treatment based on a retrospective assessment with expert microscopy results. (4) Conclusion: Malaria remains one of the principal causes of febrile illness in Burkina Faso. Testing with HRP2-based RDTs is inaccurate, in particular, due to the low specificity, which results in an over-prescription of antimalarials, with emerging antimalarial drug resistance as an important risk and many children not being treated for potential other causes of fever.

Objective.The antimalarial drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have been used for decades to treat rheumatic diseases. CQ is still beneficial for the management of malaria during pregnancy. A growing body of research suggests that antimalarials are safe during pregnancy. There have been concerns about adverse longterm effects, mainly retinal toxicity, in offspring of women exposed to antimalarials during pregnancy. Our objective was to review the published evidence on safety of antimalarials during pregnancy, focusing on ocular toxicity in the offspring.Methods.Ovid Medline, Embase, and Cochrane Library databases were searched for the period from their inception to May 2010 inclusive with no restrictions on language or year of publication. Randomized controlled trials (RCT) and observational studies examining the safety of CQ or HCQ during pregnancy and reporting on visual function or ocular toxicity in the offspring of exposed women at any point of the followup were eligible for inclusion. The quality of evidence was assessed according to established criteria (the GRADE approach).Results.Twelve studies with a total of 588 offspring born to mothers treated with CQ or HCQ during pregnancy met the inclusion criteria. Five studies with a total of 251 exposed children reported no clinical visual abnormalities in any case. In an RCT on malaria prophylaxis, visual acuity in 251 infants exposed to CQin uterodid not differ from the placebo group. Detailed ophthalmological examination was performed in 4 studies and normal results were reported in all children (n = 59). Electro-physiological testing using electroretinogram was performed in 3 small cohorts and results were normal in all but 6 infants aged 3–7 months. All 6 children had normal fundoscopy before 4 years of age. Heterogeneity in comparison groups and in outcome measures precluded formal metaanalysis.Conclusion.Current evidence suggests no fetal ocular toxicity of antimalarial medications during pregnancy. The clinical significance of early electroretinogram anomalies reported in a small subset of infants remains to be established. Larger followup studies are warranted to confirm low risk of ocular toxicity in children following antenatal exposure to antimalarial medications.

Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, ‘Calxinin’. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials.

Background Schistosomiasis is highly prevalent in Africa. Praziquantel is effective against adult schistosomes but leaves prepatent stages unaffected—which is a limit to patient management and elimination. Given the large-scale use of praziquantel, development of drug resistance by Schistosoma is feared. Antimalarials are promising drugs for alternative treatment strategies of Schistosoma infections. Development of drugs with activity against both malaria and schistosomiasis is particularly appealing as schistosome infections often occur concomitantly with malaria parasites in sub-Saharan Africa. Therefore, antiplasmodial compounds were progressively tested against Schistosoma in vitro, in mice, and in a clinical study. Results Amongst 16 drugs and 1 control tested, pyronaridine, methylene blue and 5 other antimalarials were highly active in vitro against larval stage schistosomula with a 50% inhibitory concentration below 10 μM. Both drugs were lethal to ex vivo adult worms tested at 30 μM with methylene blue also active at 10 μM. Pyronaridine treatment of mice infected with S. mansoni at the prepatent stage reduced worm burden by 82% and cured 7 out of 12 animals, however in mice adult stages remained viable. In contrast, methylene blue inhibited adult worms by 60% but cure was not achieved. In an observational pilot trial in Gabon in children, the antimalarial drug combination pyronaridine-artesunate (Pyramax) reduced S. haematobium egg excretion from 10/10 ml urine to 0/10 ml urine, and 3 out of 4 children were cured. Conclusion Pyronaridine and methylene blue warrant further investigation as candidates for schistosomiasis treatment. Both compounds are approved for human use and evidence for their potential as antischistosomal compounds can be obtained directly from clinical testing. Particularly, pyronaridine-artesunate, already available as an antimalarial drug, calls for further clinical evaluation. Trial registration ClinicalTrials.gov Identifier NCT03201770.

ObjectivesTo examine the health-seeking behaviour and cost of fever treatment to households in Ghana.DesignCross-sectional household survey conducted between July and September 2015.SettingKassena-Nankana East and West districts in Upper East region of Ghana.ParticipantsIndividuals with an episode of fever in the 2 weeks preceding a visit during routine health and demographic surveillance system data collection were selected for the study. Sociodemographic characteristics, treatment-seeking behaviours and cost of treatment of fever were obtained from the respondents.ResultsOut of 1845 households visited, 21% (393 of 1845) reported an episode of fever. About 50% (195 of 393) of the fever cases had blood sample taken for testing by microscopy or Rapid Diagnostic Test, and 73.3% (143 of 195) were confirmed to have malaria. Of the 393 people with fever, 70% (271 of 393) reported taking an antimalarial and 24.0% (65 of 271) took antimalarial within 24 hours of the onset of illness. About 54% (145 of 271) of the antimalarials were obtained from health facilities.The average cost (direct and indirect) incurred by households per fever treatment was GH¢27.8/US$7.3 (range: GH¢0.2/US$0.1–GH¢200/US$52.6). This cost is 4.6 times the daily minimum wage of unskilled paid jobs of Ghanaians (US$1.6). The average cost incurred by those enrolled into the National Health Insurance Scheme (NHIS) was GH¢24.8/US$6.5, and GH¢50/US$11.6 for those not enrolled.ConclusionsPrompt treatment within 24 hours of onset of fever was low (24%) compared with the Roll Back Malaria Programme target of at least 80%. Cost of treatment was relatively high when compared with average earnings of households in Ghana and enrolment into the NHIS reduced the cost of fever treatment remarkably. It is important to improve access to malaria diagnosis, antimalarials and enrolment into the NHIS in order to improve the case management of fever/malaria and accelerate universal health coverage in Ghana.

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

Malaria is a life-threatening disease which is responsible for roughly one million deaths annually. Previous successes in attempting to eradicate the disease have only been short lived, owing to the increased development of resistance in the parasite. There is a continued need for novel compounds which act at novel therapeutic targets, with the Plasmodium falciparum cytochrome bc1 complex (Pfbc1) representing one such target. Its inhibition halts the biochemical generation of ATP, thus resulting in parasite cell death. Work described in this thesis was concerned with utilising molecular modelling, synthesis and biological testing to develop novel antimalarial compounds, which selectively inhibit this target. The structural details of a number of compounds known to be active or inactive against Pfbc1 were used in combination with six different ligand based virtual screening techniques, and applied to the ZINC lead like library of compounds to identify potential chemotypes active against malaria. These methods included fingerprint similarity searching, principal component analysis, and naïve Bayesian classification. The hits from each of these methods were merged and formed part of a consensus analysis in which compounds identified across several methods were deemed of more interest than those which appeared less frequently. Each molecule was given a score based on its occurrence in the virtual screening methods and also its physicochemical properties. Compounds were filtered to remove those with unfavourable chemical properties, or which contained known toxicophores. 19 compounds were ultimately purchased and tested in vitro against the 3D7 strain of the malaria parasite. 5 of the compounds reported single digit µM IC50 values, with each containing novel structural chemotypes. The lead candidate contained a benzothiazole core, and reported an IC50 value against 3D7 of 4.53 ± 1.86 µM. Additional testing showed the compounds to be inactive against bovine bc1, which is promising as strong bovine bc1 inhibition has been shown to be indicative of cardiotoxicity in humans. Molecular docking was extensively employed to rationalise the activity of Pfbc1 inhibitors such as atovaquone and HDQ. A number of quinolone containing compounds were also subject to docking, with key observations made with regard to interactions thought to be crucial to their antimalarial activity. The hits from LBVS were also the focus of docking, further supporting their potential as Pfbc1 inhibitors. QSARs were developed for a series of 4-aminoquinoline compounds which had been tested against both the NF54 and K1 strains of malaria. MLR, PLS and kNN machine learning methods were investigated, with molecular descriptors contained within valid models interpreted. Significant models were identified and shown to have strong predictive abilities for both strains. QSAR models were similarly developed for a series of thiazolide compounds with activity against hepatitis C. SVM was found to give a significant model which was able to predict the cell safety of the thiazolide derivatives. The rational design of the novel pyrroloquinolone chemotype led to the synthesis of 7 synthetic analogues to investigate its SAR, via alkylation and Winterfeldt oxidation reactions. The compounds reported 3D7 activity values between 75 nM and 1.02 µM, with molecular docking supporting their potential for Qo binding and thus Pfbc1 inhibition.

Vacuolar-type H+-pumping pyrophosphatases (V-H+-PPases) are primary active transporters that utilise the energy from PPi (inorganic pyrophosphate) hydrolysis to translocate H+ ions across a biological membrane. These proteins have been well characterised in plant cells where they function in parallel with the ubiquitous vacuolar-type H+-pumping ATPase (V-H+-ATPase) to generate proton gradients across the plant vacuole membrane. V-H+-PPases have also been identified on the plasma membrane and intracellular organelle membranes of several protozoan parasites responsible for causing severe disease in humans. The identification of V-H+-PPases in these disease-causing agents, and the absence of this class of proton pump in human cells, has exciting implications for its potential as an antiparasitic drug target. The P. falciparum genome encodes two genes, PfVP1 and PfVP2, that encode putative K+-sensitive (type I) and K+-independent (type II) V-H+-PPases respectively. Furthermore, V-H+-PPase activity in P. falciparum has been demonstrated in the asexual blood-stage parasite, both in terms of PPi-induced acidification of subcellular parasite compartments and PPi hydrolysis activity in parasite membrane preparations. V-H+-PPase activity in the blood-stage parasite was further characterised in this study. PPi-induced acidification of the parasite digestive vacuole (DV; a large lysosome-like organelle within the parasite cytosol) was sensitive to Ca2+, which is known to inhibit V-H+-PPase activity, but was insensitive to the V-H+-PPase type-specific inhibitor aminomethylenediphosphonate (AMDP). PPi hydrolysis in membrane-permeabilised, intact cells was also sensitive to Ca2+. This study also confirmed previous observations of K+ sensitivity of both H+ translocation and PPi hydrolysis, consistent with the presence of a K+ sensitive, type I V-H+-PPase in the intraerythrocytic parasite. This study specifically investigated the role of the putative type II V-H+-PPase, PfVP2, using PfVP2 knockout (KO) parasites. Although PfVP2 was not active on the DV membrane, it extruded H+ ions out of the parasite cytosol in the D10 strain of P. falciparum (only apparent when the parasite V-H+-ATPase was inhibited with the V-H+-ATPase inhibitor concanamycin A). PfVP2 KO parasites also displayed a small but significant decrease in PPi hydrolysis compared to wild type (WT) parasites in the D10 strain, consistent with PfVP2 hydrolysing PPi to Pi (inorganic phosphate). Despite these results supporting the presence of a functional PfVP2 protein in the intraerythrocytic-stage parasite, PfVP2 gene disruption was not associated with a fitness cost or increased sensitivity to the antiplasmodial agents concanamycin A, chloroquine and artemisinin in both the 3D7 and D10 strains. The subcellular localisation of PfVP2 was also investigated in this study. PfVP2, tagged with the green fluorescent protein (GFP) localised to punctate structures within the intraerythrocytic parasite cytosol. Disruption of PfVP2-GFP by brefeldin A (a compound known to interfere with the secretory pathway) and partial co-localisation of PfVP2-GFP with the P. falciparum Golgi Reassembly and Stacking Protein or PfGRASP, are consistent with a Golgi localisation of PfVP2.

The maintenance of a low intracellular [Na⁺] ([Na⁺]i) is a crucial aspect of cellular physiology. In mammalian cells this is achieved through the extrusion of Na⁺ via the well-characterised Na⁺/K⁺-ATPase. Approximately 12 hr after invasion of the human erythrocyte by the malaria parasite there is a profound increase in the permeability of the erythrocyte membrane to a wide range of solutes, including Na⁺. Na⁺ enters the infected erythrocyte via parasite-induced 'New Permeability Pathways' and there is, as a result, an increase in [Na⁺] in the erythrocyte compartment, with [Na⁺]i eventually reaching levels similar to those in the extra erythrocytic plasma (~130 mM). The parasite itself maintains a low [Na⁺]i. The resulting large inwardly-directed electrochemical Na⁺ gradient across the parasite plasma membrane energises the accumulation within the parasite of at least one essential nutrient (inorganic phosphate). The aim of this thesis was to characterise the mechanisms involved in Na⁺ regulation in the mature asexual 'trophozoite' stage of the human malaria parasite Plasmodium falciparum. The Na⁺-sensitive, fluorescent dye Sodium-binding BenzoFuran Isophthalate (SBFI) was used to measure [Na⁺]i in parasites functionally isolated from their host cells by saponin-permeabilisation of the host erythrocyte membrane. Under physiologically relevant conditions the resting [Na⁺]i in isolated trophozoites was estimated to be ~11 mM. Maintenance of [Na⁺]i was sensitive to the P-type ATPase inhibitor orthovanadate, consistent with Na⁺ extrusion being via a P-type Na⁺-ATPase, similar to the ENA (exitus natru; exit of sodium)-type ATPases that operate in some other protozoa, fungi and lower plants. ENA ATPases have been predicted to antiport H⁺ and the data obtained here are consistent with this being true of the P. falciparum Na⁺ extrusion system. The P. falciparum genome encodes a number of putative P-type ATPases; one of these, PfATP4, was found to share significant sequence similarities to ENA ATPases of other protozoa. A recent study showed that mutations in PfATP4 confer resistance to a newly-described class of antimalarials, the spiroindolones. The effect of the spiroindolones on ion regulation was therefore investigated. Several spiroindolones were shown to cause a profound disruption of [Na⁺]i regulation. In parasites with mutant PfATP4 there was both an impairment of Na⁺ regulation and a decrease in the spiroindolone-sensitivity of Na⁺ regulation. These results are consistent with PfATP4 being a Na⁺-ATPase and the target of the spiroindolones. The physiological role of another putative Na⁺ transporter, the Na⁺/H⁺-exchanger PfNHE was also investigated, as previous studies on its contribution to regulation of [Na⁺]i and intracellular pH (pHi) have been controversial. On the basis of a bioinformatics analysis it was predicted that the protein functions as an amiloride-insensitive, plasma membrane Na⁺-extruder, like its closely related plant homologues. However physiological studies revealed no significant role for such an NHE in either pHi or [Na⁺]i regulation in the P. falciparum trophozoite. This study constitutes a significant advance in our understanding of fundamental aspects of the cell physiology of the intraerythrocytic parasite, as well as shedding light on the mode of action of what promises to be an important new class of antimalarials, the spiroindolones.

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2017
Antimalarial drug resistance has always been an obstacle in the fight against malaria. Malaria parasites have developed resistance to most available antimalarial drugs and, more recently resistance to artemisinins, the first-line treatment for malaria, is emerging and spreading in Southeast Asia. Artemisinin resistance is characterized by delayed parasite clearance times observed in malaria patients. For now this resistance is considered to be partial by the WHO and artemisinin combination therapies remain as the mainstay of antimalarial treatment. In vitro assays are of paramount importance to detect and monitor drug resistance. Several in vitro drug assays exist, however their inherent characteristics, such as the use of radioactive or expensive reagents and their long turn-around times limit their application. The project developed in the context of this thesis aimed to develop a novel drug assay for Plasmodium spp. that would overcome some of the limitations of currently available drug assays. The underlying idea was to use hemozoin, a crystal produced by malaria parasites, to measure their own growth or maturation which would allow to detect drug effects. The hemozoin content increases as parasites mature inside the erythrocytes. Thus, hemozoin constitutes an optimal parasite maturation biomarker. Moreover, it is a birefringent crystal, and as such it is able to depolarize light. The resulting light depolarization can be easily detected by optical methods such as flow cytometry. It was previously shown by our laboratory that in a rodent model of malaria depolarization caused by hemozoin inside infected erythrocytes could be detected using a simple flow cytometric set-up. Moreover, the inhibitory effect of commonly used antimalarial drugs could also be determined after only 6-8 hours of incubation. The main objectives of the work developed during this thesis was to further develop the flow cytometric detection of hemozoin assay using P. falciparum in vitro cultures and to assess its performance ex vivo, in the field, using samples from malaria patients. A benchtop flow cytometer (Cyflow SL) was modified to allow the detection of light depolarization and it was used for all studies. Other commercially available cytometers (MoFlo, Accuri C6, Attune) were also easily adapted to detect light depolarization, showing that the measurement of this additional parameter can be accomplished in different instruments. In vitro cultures of P. falciparum were established and allowed to further investigate the potential of this novel method. Ring-stage synchronized cultures of P. falciparum were incubated with several antimalarial drugs (chloroquine, mefloquine, quinine, artemisinin, artesunate and pyrimethamine). Analysis of depolarizing events, corresponding to parasitized erythrocytes containing hemozoin, allowed the detection of parasite maturation. Furthermore, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 - 24 hours of incubation. The 50% inhibitory concentrations (IC50) obtained at 24 hours of incubation were comparable to previously reported values. However, these values were most of the times higher than the ones obtained with the already validated HRP2 ELISA assay. Indeed, IC50 values may differ considerably between assays. Different assays measure different parameters to assess parasite growth, at different time-points. Moreover, variations in parasite density and hematocrit as well as the stage-dependent action of antimalarial drugs, may influence these values. Altogether, explaining the differences in IC50 values that are commonly observed. The performance of the hemozoin detection assay in the field was assessed during a 6- month trial performed in Gabon, a malaria endemic country. The trial was conducted in the Centre de Recherches Médicales de Lambaréné – Albert Schweitzer Hospital. On site, an existing flow cytometer (Cyflow SL) was modified to detect light depolarization caused by hemozoin. A total of 46 samples from malaria patients were analyzed during this study. Blood samples were incubated with increasing concentrations of chloroquine, artesunate and artemisinin. The percentage of depolarizing cells was used as maturation indicator and measured at 24, 48 and 72 hours of incubation to determine parasite growth and drug effects. Analysis of ex vivo cultures of parasites obtained from blood samples of malaria patients showed four different growth profiles. The flow cytometric detection of hemozoin allowed to detect drug effects in 39/46 (85%) of samples. In 25 samples drug effects were measurable at 24 hours. In the remaining 14 samples parasite maturation was delayed, and thus drug effects were only detected at 48 hours of incubation. Obtained IC50 values showed that chloroquine-resistant parasites were still common and present in Lambaréné, Gabon but they were fully sensitive to artesunate and artemisinin. Finally, the usefulness of the hemozoin detection assay in the investigation of artemisinin resistance in vitro was also assessed. Artemisinin-resistant (MRA-1240) and sensitive (MRA-1239, 3D7) strains were cultured in vitro. Parasite maturation was determined based on the flow cytometric detection of hemozoin-containing cells. Two different drug assays were performed: 1) standard drug assay: where ring-stage parasites were continuously incubated with increasing concentrations of dihydroartemisinin (DHA) for 48 hours; and 2) pulse assay: where tightly ring-stage synchronized parasites were incubated with a single high-dose (700 nM) of DHA for 6 hours. Results showed that at 24 hours of incubation artemisinin-resistant parasites had increased IC50 values, in comparison to the artemisinin-sensitive strains (15 nM and 8 nM, respectively). Moreover, when parasites were exposed to a high-dose of DHA for 6 hours, increased survival rates associated with artemisinin resistance could be detected after only 30 hours of incubation. Interestingly, it was also observed that artemisinin-resistant parasites do not seem to enter dormancy, as it has been previously suggested by others. Microscopic assessment performed after 72 hours of incubation showed that parasites that survived to a 6-hour exposure to DHA were very close in terms of parasite development to the ones found in the drug free control. Further investigation using more artemisininresistant strains is required to determine whether increased IC50 values correlate with the delayed parasite clearance times observed in the patients; and if the underlying mechanisms of artemisinin resistance is or not related to dormancy. Overall, the work presented in this thesis shows that hemozoin detection by flow cytometry is an alternative, reagent-free and rapid drug assay that overcomes some of the limitations of currently available drug assays for P. falciparum. Moreover, it may also be a useful tool in the study of artemisinin resistance both in culture-adapted strains and, possibly in strains obtained directly from patients. Importantly, this work paves the way for the development and investigation of better tools to assess drug effects and monitor drug resistance in Plasmodium spp. Several novel hemozoin detection platforms are available or under development and should definitely be further explored for their potential to be used as antimalarial drug assays. Furthermore, combination of hemozoin detection with the measurement of other parameters, such as DNA and RNA content and parasite viability may even provide additional important information to reliably determine the developmental stage and metabolic status of parasites and, consequently, detect drug effects. Hopefully, this would lead to the development of an optimal antimalarial drug assay that could play an important role in the fight against malaria.

Malaria is a public health challenge that requires prompt treatment for those infected to make a full recovery. Treatment of malaria infection is to be started as soon as a diagnosis is confirmed. Antimalarial medications are administered to prevent and also to treat malaria. The type of medication used and the duration of therapy is dependent on the type of malaria-causing plasmodium species, the severity of the symptoms, geographical area where malaria infection occurred and the medication used to prevent malaria and whether there is pregnancy. Treatment of malaria from public health perspective is to reduce transmission of the infection to others, by reducing the infectious reservoir and to prevent the emergence and spread of resistance to antimalarial medicines. Medications used in the treatment of malaria infection come from the following five groups of chemical compounds: quinolines and aryl amino alcohols, antifolate, artemisinin derivatives, hydroxynaphthoquinones and antibacterial agents. The treatment of malaria is not initiated until the diagnosis has been established through laboratory testing. Artemisinin-based Combination Therapy (ACTs) has been used for the treatment of uncomplicated malaria. ACTs are also to enhance treatment and protect against the development of drug resistance. IV artesunate is used in the treatment of severe malaria, regardless of infecting species.

A wide deployment of malaria control tools have significantly reduced malaria morbidity and mortality across Africa. However, in the last five to seven years, there has been a resurgence of malaria in several African countries, raising the questions of whether and why current control mechanisms are failing. Since the first Plasmodium falciparum reference genome was published in 2002, few thousands more representing a broad range of geographical isolates have been sequenced. These advances in parasite genomics have improved our understanding of mutational changes, molecular structure, and genetic mechanisms associated with diagnostic testing, antimalarial resistance, and preventive measures such as vaccine development. In this chapter, we summarize the current progress on: (1) genomic characteristics of P. falciparum; (2) novel biomarkers and revolutionary techniques for diagnosing malaria infections; and (3) current vaccine targets and challenges for developing efficacious and long-lasting malaria vaccines.

As countries move from intense malaria transmission to low transmission there will be a demand for more sensitive tools and approaches in tracking malaria transmission dynamics. Surveillance tools that are sensitive in tracking real time infectious bites as well as infectious reservoir will be preferred to counting number of cases in the hospital or parasite prevalence. The acquisition and maintenance of anti-malarial antibodies is a direct function of parasite exposure, seroprevalence rates has been used as an efficient tool in assessing malaria endemicity and confirming malaria elimination. Plasmodium antibodies are explicit biomarkers that can be utilised to track parasite exposure over more extensive time spans than microscopy, rapid diagnostic testing or molecular testing and the conventional entomological inoculation rate. Seroprevalence studies can therefore help monitor the impact of malaria control interventions, especially when the parasite occurrence is low. As a result, antibody responses to Anopheles salivary proteins or Plasmodium species may potentially offer reliable information of recent or past exposure; recognise short-term or gradual changes in exposure to Plasmodium infection or to estimate individual-level exposure to infection. This book chapter will present about four studies we have conducted across eastern and western Africa on the efficiency of salivary gland proteins and antimalarial antibodies in tracking malaria transmission intensity. We hope that these could be used as surveillance tools in malaria elimination efforts.