BIOLOGICAL CHARACTERISTICS AND DRUG RESISTANCE PROFILE OF P. FALCIPARUM

Transcription

1 BIOLOGICAL CHARACTERISTICS AND DRUG RESISTANCE PROFILE OF P. FALCIPARUM ADDIMAS TAJEBE NIGATIE (BSc., MSc.) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2017 Supervisors: Prof. Laurent Claude Renia, Main supervisor Associate Professor Bruce Malcolm Russell Co-supervisor Examiners: Associate Professor Tan Shyong Wei Kevin Associate Professor Lisa Ng Fong Poh

2 Declaration I hereby declare that this thesis is my original work and has been written by me in its entirety. I have duly acknowledged all the sources of information used in the thesis. This thesis has also not previously been submitted for any degree at any university. Addimas Tajebe Nigatie 22 August 2017 i

3 Acknowledgements I would like to express immense gratitude to my supervisor, Prof. Laurent Renia, for his invaluable guidance, supervision, suggestions, and encouragement throughout this work. I also would like to thank my co-supervisor, Dr. Bruce Russell, for his support and cosupervision. It is my pleasure to forward my wholehearted acknowledgement to Dr. Rossarin Suwanarusk and Dr. Lee Wenn Chyau for their incredible and tireless support, coaching, close monitoring and mentoring during my laboratory work. Lastly, I would like to forward my deepest gratitude to my family for their support and encouragement at every instance. ii

4 Table of Contents Contents Pages Declaration... i Acknowledgements... ii Table of Contents... iii Summary... ix List of tables... xii List of figures... xiii CHAPTER ONE Introduction... 1 CHAPTER TWO Literature review Introduction Drug-resistant Plasmodium parasites Chloroquine resistant P. falciparum Mode of action Mechanism of resistance Chloroquine resistance and fixation Reversal of chloroquine resistant parasites... 7 iii

5 2.3 Chloroquine resistant P. vivax Sulfadoxine/pyrimethamine resistant P. falciparum Mode of action Mechanism of resistance Mefloquine resistant P. falciparum Mode of action Mechanism of resistance Lumefantrine resistant P. falciparum Mode of action Mechanism of resistance Artemisinin resistant P. falciparum Mode of action Mechanism of resistance Combination therapy and prevention of drug resistance Drug shifting and re-introducing used drug Biological characteristics of P. falciparum Rosetting of P. falciparum History of rosette discovery and rosetting Plasmodium species Prevalence of rosetting and associations with severe malaria iv

6 Postulated roles of rosetting Rosetting and blood group Rosette disruption and reversal Factors affecting rosette formation Autoagglutination, endothelial cytoadhesion and rosetting Autoagglutination Endothelial cytoadhesion Rosetting receptors and ligands for P. falciparum and P. vivax Ligands and receptors for P. falciparum Rosetting ligands of P. falciparum Rosetting ligands and receptors of P. vivax Host receptors for cytoadhesion and rosetting Glycosaminoglycans and sulfated glycoconjugates The aim of the study Specific aims CHAPTER THREE Material and methods Method Study design v

7 3.3. Culturing of P. falciparum strains Parasite synchronization and concentration Sorbitol synchronization Percoll synchronization Cryopreservation of parasites Thawing of cryopreserved parasites In vitro drug assay Drug plate preparation and assay design In vitro microtest and parasite susceptibility to anti-malarial drugs Ring stage sensitivity assay Drug plate preparation and assay design Parasite incubation Harvesting the culture In vitro rosetting assay Wet mount preparation Statistical analysis CHAPTER FOUR Results In vitro parasite culture and synchronization vi

8 4.2 Drug sensitivity profiling of P. falciparum Microscopic observation of drug-treated and drug-free parasites Growth inhibition and chloroquine sensitivity profiling of P. falciparum Growth inhibition and artesunate sensitivity profiling of P. falciparum Growth inhibition and artesunate sensitivity of ARS-233 and MKT Growth inhibition and sensitivity profiling of 3D7 strain Growth inhibition and sensitivity profiling of Dd2 strain Growth inhibition and sensitivity profiling of FVT201 strain Growth inhibition and sensitivity profiling of MKK183 strain Comparison of sensitivity of parasites to the four anti-malarial drugs Sensitivity profiling of P. falciparum ring stages Sensitivity profiling of 3D7 and Dd2 strain ring stages Phenotyping and sensitivity profiling of 3D7 and Dd2 strain ring stages An in vitro rosetting phenotype of P. falciparum CHAPTER FIVE Discussion Drug sensitivity profiling of P. falciparum Ring stage sensitivity profile In vitro rosetting phenotype of P. falciparum vii

9 CHAPTER SIX Conclusion and recommendation Conclusion Recommendation Reference viii

10 Summary Background: Malaria remains an important public health problem worldwide. Malaria is a vector-borne infectious disease transmitted by the Anopheline mosquito. Plasmodium is the etiological agent of malaria. There are six Plasmodium species which are infectious for humans. P. falciparum and P. vivax are the major etiological agents for malaria. Both are responsible for high morbidity and mortality worldwide. Global control of malaria and eradication efforts are challenged by the emergence of drug-resistant malaria parasites. Although the emergence of drug-resistant parasites has been reported from time to time, the biological characteristics and drug sensitivity profile of parasites remain unclear. Therefore, there is a need for thorough research to fill this gap. Aim: To study the biological characteristics and drug sensitivity profile of P. falciparum in vitro Method: In vitro biological characterization and drug sensitivity profiling of both laboratory-adapted strains and field isolates were conducted. We did in vitro drug sensitivity profiling of parasites using four types of anti-malarial drugs. We used chloroquine sensitive and resistant falciparum strains as quality control. A modified in vitro drug sensitivity assay was conducted on 3D7 and Dd2 ring stage parasites using high concentrations of artesunate. In addition, an in vitro rosetting assay was also conducted using 3D7 lab strain and FVT201 field isolate. Results: In vitro drug sensitivity profiling of parasites showed a statistically significant sensitivity difference (P<0.05). The 3D7 strain was sensitive to chloroquine, whereas the Dd2, MKT1116, ARS-272 and ARS-233 strains were resistant to chloroquine (IC 50 >100nM). Both chloroquine resistant and sensitive strains were sensitive to artesunate ix

11 (IC 50 <10nM). A different pattern of resistance was observed for the field isolates, MKK183 and FVT201. The MKK183 isolate showed resistance to chloroquine (IC 50 >100nM) and mefloquine (IC 50 >30nM), whereas the FVT201 isolate showed resistance only to mefloquine (IC 50 >30nM). All of the parasites were sensitive to lumefantrine (IC 50 <150nM) and artesunate (IC 50 <10nM) respectively. However, the 3D7 strain showed a significant level of sensitivity (P<0.05) to lumefantrine when compared to the FVT201 and Dd2 strains. In the modified in vitro sensitivity assay, the parasites showed a different sensitivity profile among the ring stage subpopulations. 11-hour-old and 16-hour-old ring stage parasites were more sensitive than 6-hourold ring stage parasites. There was also a difference in sensitivity between 3D7 and Dd2 ring stage parasites at concentrations of 100nM and 200nM. We also did a rosetting phenotyping assay in vitro using two P. falciparum strains. The P. falciparum strains showed significant differences of rosetting rate in vitro (P<0.05). Conclusion: Both laboratory-adapted and field strains showed different drug sensitivity profiles, and all parasites were sensitive to both lumefantrine and artesunate. Ring stage parasites showed a sensitivity profile to artesunate. The 6-hour-old ring stage parasites had reduced sensitivity to artesunate compared to late ring stage parasites. P. falciparum strains (3D7 and FVT201) formed rosetting in vitro. There was a different rosetting rate between the two strains. Recommendation: In vitro drug sensitivity profiling of parasites and associated molecular markers should be done to establish appropriate drug combinations for better treatment. Artesunate-induced drug dormancy should be correlated with clinical data and molecular markers for monitoring of current anti-malarial treatment efficacy. In addition, the role of x

12 rosetting phenotype and severe malaria should be studied so that anti-rosetting antibodies or vaccines can be developed. Outcomes: In general, the study will contribute to a better understanding of both the biological characteristics and drug sensitivity profiles of falciparum parasites. This will also help to correlate both clinical and laboratory data and establish a drug-resistant malaria surveillance system. Key words: P. falciparum, drug sensitivity, in vitro, rosetting xi

13 List of tables Table 1. Anti-malarial drug-resistant polymorphisms Table 2. A summary of anti-malarial drug compound and their target Table 3. Discoveries of rosette forming Plasmodium species Table 4. The effect of anti-malarial drugs on each P. falciparum strains Table 5. The effect of artesunate on different stages of parasites xii

14 List of figures Figure1.Resensitization of chloroquine resistant parasites by chlorpheniramine... 8 Figure 2. Sorbitol synchronization of ring stage parasites Figure 3. Percoll concentration of matured parasites Figure 5. Microscopic observation of drug-free and drug-treated parasite growth Figure 6. Growth inhibition and sensitivity profiling parasite to chloroquine Figure 7. Growth rate of Plasmodium strains exposed to artesunate Figure 8.Growth inhibition of ARS-233 and MKT1116 strains Figure 9.In vitro growth inhibition and sensitivity profile of 3D7 strain Figure 10. In vitro growth inhibition of Dd2 strain Figure 11.In vitro growth inhibition of FVT201 strain Figure 12. In vitro growth inhibition of MKK183 strain Figure 13.Comparison of IC 50 values of four anti-malarial drugs against four parasites. 57 Figure 14. The 3D7 strain at different ring stage subpopulation Figure 15. The Dd2 strain of different ring stage subpopulations Figure 16.The phenotype of artesunate-treated 3D7 strain at 6-hour ring stage Figure 17.Phenotype of artesunate-treated Dd2 strain at 6-hour ring stage Figure 18.Sensitivity profile of 3D7 and Dd2 ring stages to artesunate xiii

15 Figure 19. In vitro rosetting and rosetting rate of P. falciparum strains xiv

16 Acronyms ACT Artemisinin combination drugs PVMDR1 Plasmodium vivax multidrug resistance 1 PVCRT-O PfK13 PFMDR PFCRT CQ AS LUM MQ CQR SVMNT CVMNK CVIET Plasmodium vivax chloroquine transporter gene Plasmodium falciparum kelch propeller gene Plasmodium falciparum multidrug resistance Plasmodium falciparum chloroquine resistance transporter gene Chloroquine Artesunate Lumefantrine Mefloquine Chloroquine resistance Serine, valanine,methionine,threonine and asparagine Cysteine, valanine,methionine,asparagine and lysine Cysteine, isoleusine, valanine, glutamic acid and threonine FY*A Fy glycoprotein allele A FY*B Fy glycoprotein allele B CR1 Complement receptor 1 ICAM VCAM CD TSP-1 CHO Intercellular adhesive molecule Vascular cell adhesion molecule Cluster domain Thrombospondin-1 Chinese hamster ovary xv

17 HABP1 Hyaluronan binding protein 1 PECAM-1 DV CQH DHPS DHFR SNP Platelet endothelial cell adhesion molecule-1 Digestive vacuole Protonated chloroquine Dihydropteroate synthase Dihydrofolate reductase Single nucleotide polymorphism PfEMP-1 Plasmodium falciparum erythrocyte membrane protein -1 PfRh4 Plasmodium falciparum rhoptry 4 CRDS CSA VAR2CSA Curdlan sulfate Chondroitin-4- sulfate A Variable recptor2 chondroitin-4- sulfate A CD36 Cluster of differentiation 36 ICAM-1 Intercellular adhesion molecule -1 CD31 Cluster of differentiation 31 VCAM Vascular cell adhesion molecule CD35 Cluster of differentiation 35 CD236R Cluster of differentiation 236 receptor TSP-1 Thrombospondin-1 PECAM-1 Platelet endothelial cell adhesion molecule-1 IgM Immunoglobulin M xvi

18 DBL-1 Duffy binding like domain 1 xvii

19 CHAPTER ONE 1. Introduction Malaria is a vector-borne infectious disease that accounts for high mortality and morbidity worldwide. The World Health Organization (WHO) reported in 2015 that 214 million malaria cases occurred worldwide; 89% of the cases were from sub-saharan Africa. Of these, 438,000 deaths were reported; 91% of these fatal cases were from sub-saharan Africa. (1) Plasmodium is the etiological agent of malaria. (2) To date, there are six medically important malaria parasites which are infective for humans. These are P. falciparum, P. vivax, P. malariae, P. ovale, P. knowlesi and P. cynomolgi. (3). P. knowlesi and P. cynomolgi are zoonotic malaria found in southeast Asia. (4)(5)(6)(7) Among all human infecting Plasmodium species, P. falciparum and P. vivax are responsible for most of global malarial mortality and morbidity. P. falciparum predominates in Africa (8) while P. vivax is responsible for most of the malarial burden outside of Africa. (9) Sub-Saharan Africa is the most vulnerable region with the highest malaria-associated death toll. Children and pregnant women in this region are the groups most susceptible to severe malaria due to P. falciparum. (10) On the other hand, P. vivax is not endemic to many African countries, and vivax malaria is commonly considered as benign, but vivax malaria can cause severe anemia. P. vivax infection have been reported from four countries in West Africa; three countries in Central Africa, two countries in Southern Africa, three countries in East Africa. The six countries in the Horn of Africa, including Sudan, Eritrea, Djibouti, Ethiopia, Somalia, and South Sudan, have relatively moderate to higher number of cases (8). Unlike other African countries, Ethiopia has both P. falciparum and P. vivax infections, although P. falciparum is the leading cause of most of the severe malaria cases. The mechanism of infection by P. falciparum and P. vivax 1

20 is different. The infection mechanism of vivax malaria is associated with human Duffy antigen polymorphism. (11)(12) There are two types of Duffy polymorphic antigens on human erythrocytic membranes which are encoded by the FY*A and FY*B alleles. (13) Therefore, the pathogenesis of P. vivax is mediated by the interaction between parasite Duffy binding proteins (antigens) and host Duffy antigen receptor chemokine /DARC. (14) In contrast, P. falciparum follows Duffy-independent infection mechanism. P. falciparum is usually considered to be a severe form of malaria, whereas P. vivax is associated with benign malaria, but P. vivax can also increase the risk of anemia due to cytoadhesion and rosette formation. (15) P. falciparum most commonly causes a severe form of malaria due to sequestration and cytoadhesion in such peripheral tissues as the brain and placenta, (16)(17) and the severity of the disease is contributed by host parasite factors. (18) In this work, we tried to study the biological characteristics of P. falciparum. We also studied the drug sensitivity profile of P. falciparum laboratory-adapted strains and field isolates. 2

21 CHAPTER TWO 2. Literature review 2.1 Introduction Plasmodium falciparum is the most severe form of malaria and causes the highest death toll. This is due to the complex nature of the parasite. Biological characteristics of the parasite, such as sequestration and endothelial cytoadhesion, and the rosetting phenotype of the parasite are associated with the severity of malaria caused by P. falciparum. Furthermore, the parasite adaptation mechanism to many antimalarial drugs and development of a drugresistant phenotype have been a challenge for malaria control and eradication. In this study, we did an in vitro biological characterization (rosetting) and drug sensitivity profiling of both laboratory-adapted P. falciparum strains and field isolates. 2.2 Drug-resistant Plasmodium parasites Plasmodium parasites develop resistance to anti-malarial drugs. Drug-resistant parasites are spread worldwide. A number of anti-malarial drugs have been used for both treatment and prophylaxis. Chloroquine was the first antimalarial drug deployed worldwide for malaria treatment. Chloroquine was a cheap and fairly accessible antimalarial drug and was used in many malaria-endemic countries. In fact, not only the low price of chloroquine but its good safety profile favored chloroquine for massive usage since the earliest times. However, chloroquine resistant P. falciparum first emerged in the 1960s in Southeast Asia, in the 1970s in South America and East Africa, (19) and in the 1980s in other parts of Africa. (20) As a result, malaria treatment shifted from chloroquine (a first-generation anti-malarial drug) to a second generation of anti-malarial drugs such as sulphadoxine-pyrimethamine (SP) in the 1970s. (21) The human Plasmodium parasite also developed resistance to sulphadoxine- 3

22 pyrimethamine. Malaria treatment has changed again to artemisinin, artemisinin derivatives, and their combination or partner drugs. Currently, artemisinin and artemisinin derivatives with their partner drugs are the primary drugs for P. falciparum malaria treatment worldwide. For instance, the use of chloroquine was discontinued in Ethiopia in 2004 because P. falciparum became resistant to chloroquine. (22) However, chloroquine is still in use for vivax malaria treatment. Studies report that P. vivax has also developed resistance to chloroquine. Chloroquine resistant P. vivax has been found in Southeast Asia, (23) China, (24) South America (25) and Africa (Ethiopia (22) ). Although chloroquine resistant P. vivax was reported in Ethiopia, (26) the drug is still recommended for vivax malaria treatment. This situation can expose P. falciparum to chloroquine in co-endemic areas Chloroquine resistant P. falciparum Chloroquine resistant P. falciparum was highly prevalent worldwide. The introduction of artemisinin, artemisinin derivatives and their partner drugs, which are effective for treatment of both chloroquine resistant and sensitive strains, has reduced the prevalence. The mode of action of chloroquine and its resistance mechanism are well established. The drug was used for long-term treatment of P. falciparum and then withdrawn. After being withdrawn chloroquine possibly reuse with modifications or reversal of resistant P. falciparum to chloroquine sensitive strains. The drug shifting from old drug to the new drug with new treatment regimen by itself can reverse the direction of chloroquine resistance selection pressure and can reverse parasite sensitivity Mode of action The mechanism of action of chloroquine has been the intense focus of research for decades, and evidence supports the notion that the principal target is the heme detoxification pathway 4

23 in the parasite digestive vacuole (DV), where the parasite degrades erythrocytic hemoglobin to obtain amino acids essential for the parasite s growth. In the process, the parasite polymerizes the liberated toxic heme monomers to an inert form of biocrystals of hemozoin, which is not toxic to the parasite. Chloroquine is a diprotic(cqh 2+ 2 ) weak base with (pka of 8.1 and 10.2). The relative proportions of the neutral, monoprotonated (CQH + ), and diprotonated (CQH 2+ 2 ) species vary with ph. Therefore, a higher proportion of the drug remains unchanged in the neutral ph of the blood. This allows chloroquine to diffuse freely across the parasite s membranes. However, when chloroquine encounters the acidic parasite digestive vacuole, it becomes diprotonated and unable to transverse across the vacuolar membrane. When the weak base enters the acidic environment of the parasite s digestive vacuole (ph ~5), the equilibrium is shifted toward the CQH 2+ 2 species, which is unable to diffuse across the membrane and instead becomes trapped. (27) As a result it accumulates in the digestive vacuole; chloroquine binds to hematin, a heme dimer. This interaction prevents the detoxification of free heme, leading to the buildup of heme monomers that permeabilize the parasite s membrane, resulting in eventual drug accumulation and death of the parasite due to chloroquine- induced toxicity. (28) Mechanism of resistance The chloroquine resistance mechanism of P. falciparum is well established. Chloroquine resistant P. falciparum parasites show genetic polymorphism at the membrane transporter gene (Pfcrt). (29) The polymorphisms at the Pfcrt K76 gene and its adjoining region of Pfcrt (72,74,75), (30) which represents (C72S, M74I, N75E, and N75K), (31) are associated with chloroquine resistance. These polymorphisms occur due to point mutations on wild type Pfcrt K76 or CVMNK) haplotypes. Those polymorphisms have different geographical 5

24 associations and distribution. In Papua New Guinea (SVMNT) haplotypes are commonly found, and the (CVIET) haplotypes are spread throughout Southeast Asia and Africa. The SVMNT/CVMNT and CVMET/CVMNT haplotypes are found in South America (in Brazil/Peru and in Ecuador/Colombia) respectively. (31) The second well established chloroquine resistance mechanism is the multidrug resistant gene (Pfmdr1) polymorphism and its amplification or copy number variation in P. falciparum parasites. (32) The amino acid changes at Y184F, S1034C, N1042D, and D1246Y of the Pfmdr1 gene are also proposed to modulate multidrug response in P. falciparum. (31) Single amino acid substitutions at the (Pfcrt-K76T) and (Pfmdr1-N86Y) positions are a strong predictor of chloroquine resistant P. falciparum. Chloroquine resistant parasites are characterized by an efflux of chloroquine from the parasite s vacuole, provided that the parasite reduces chloroquine-induced toxicity. In contrast, chloroquine sensitive parasites accumulate chloroquine in their food vacuole and hence the drug affects the heme polymerization process inside the parasite s vacuole. The polymerization of heme to hemozoin pigment is the key step in the parasite s survival mechanism, because heme is toxic to the parasite. Although hemoglobin degradation provides an amino acid for the intracellular parasite, it also produces heme, which is toxic to the growing parasite. Therefore, the parasite s heme toxicity survival mechanism works by converting heme to a nontoxic crystalline structure called hemozoin. (33) Chloroquine resistance and fixation Chloroquine resistance was highly prevalent worldwide before the introduction of the new antimalarial drug effective at killing chloroquine resistant parasites. As a result, the prevalence of chloroquine resistant parasites has been reduced in areas where artemisinin and artemisinin derivative drugs are employed with appropriate partner drugs. However, there is 6

25 still fixation of molecular markers in some areas which are associated with chloroquine resistant P. falciparum. In Africa, where artemether-lumefantrine combination therapy has been used, molecular markers such as Pfcrt (K76) and Pfmdr1 (N86) have been selected. Clinical studies showed that artemether lumefantrine-treated parasites were selected for wild type Pfcrt K76 and Pfmdr1 N86. Artemisinin is effective in killing both wild type and mutant parasites, whereas its partner, lumefantrine, selects for wild type. Therefore, the parasite genotype is converted to a restored chloroquine sensitive parasite phenotype and chloroquine can be used once more. (34) Reversal of chloroquine resistant parasites The mechanism of parasite resistance to chloroquine relied on the efflux of the drug out of the food vacuole, thereby decreasing the concentration of the drug inside the parasite s digestive vacuole. This mechanism is carried out by transporter membrane proteins. Chemosensitizing of the transporter membrane protein can change the structure and/or conformation of the protein; as a result, chloroquine resistance can be reversed in parasites. Chemosensitizers such as verapamil and desipramine are used to reverse resistant parasites to chloroquine sensitivity and potentiate the efficacy of chloroquine, despite the fact that chemo sensitizers are not yet used in vivo. Treating chloroquine resistant parasites with verapamil reverses chloroquine sensitivity by acting on the membrane ion channel of chloroquine resistant parasites. (35) Another chemo sensitizer or reversal called desipramine also reverses chloroquine resistant parasites ex vivo. (36) Chlorpheniramine and its chemical analogs can reverse chloroquine resistant parasites to sensitive parasites by inhibiting chloroquine transport activities in the digestive vacuole. (37) 7

26 Figure1.Resensitization of chloroquine resistant parasites by chlorpheniramine The inhibition of Pfrct (CQR) occurs by proton donation of chlorpheniramine to chloroquine and the diprotic chloroquine accumulates inside the digestive vacuole and inhibits hemozoin formation, enhancing parasite killing. This figure was adapted from (37). 2.3 Chloroquine resistant P. vivax P. vivax resistance to chloroquine has been proposed to result from modifications of membrane transport channel proteins such as Pvcrt-o and Pvmdr1. (23) Any structural changes or mutations in these channel proteins have been proposed to cause a change in transmembrane permeability of the parasite s vacuole and thus a reduction in parasite toxicity. Plasmodium parasites develop resistance to antimalarial drugs through mutations, copy number variations or other modifications of drug targets. Copy number variations, mutations, and genetic polymorphisms are reported as drug resistance mechanisms for P. falciparum. (38) A study in Indonesia and Thailand has also found that Pvmdr1 polymorphism and amplification are associated with antimalarial susceptibility. Isolates from Indonesia with a single copy and Pvmdr1 mutation at (Y976F) have shown resistance to chloroquine, but low IC 50 values for mefloquine, amodiaquine and artesunate. In contrast, P. vivax isolates from Thailand with amplification of Pvmdr1 had lower susceptibility to mefloquine, amodiaquine 8

27 and artesunate but higher susceptibility to chloroquine. (39) Therefore, in areas where P. falciparum and P. vivax are co-endemic, the use of chloroquine and other anti-malarial drugs would have different selective pressure with regard to Pvmdr1 polymorphism and amplification. A study in Ethiopia has shown a high prevalence of Pvcrt-o- and Pvmdr1 mutations, (40) but it was not determined if these mutations were associated with resistance or treatment failure. Another study in southern Ethiopia also found a high incidence of chloroquine treatment failure in P. vivax positive patients. (41) Therefore, use of chloroquine as a first line drug for P. vivax treatment might be an indicator of incomplete withdrawal of chloroquine selective pressure on P. falciparum. This situation occurs due to the co-endemic nature of the species, misdiagnosis of the parasite, and mistreatment of patients. 2.4 Sulfadoxine/pyrimethamine resistant P. falciparum After the development of parasite resistance to chloroquine in Southeast Asia and East Africa, sulfadoxine/pyrimethamine was introduced in the 1960s as the second generation of anti-malarial drugs. (42) This drug shifting was made due to the decline of chloroquine efficacy for the treatment of P. falciparum. Sulphadoxine/pyrimethamine was also challenged by the spread of resistant parasites Mode of action This group of antimalarial drugs targets the parasite s folate pathway, is called an antifolate anti-malarial drug and is traditionally grouped into two types. Type-1 antifolates (sulfonamides and sulfones) target the dihydropteroate synthase (DHPS) while type-2 antifolates (pyrimethamine, biguanides and triazine metabolites, quinazolines) target dihydrofolate reductase (DHFR) (43). 9

28 2.4.2 Mechanism of resistance The resistance mechanism to this drug is described as single point mutation, which causes single amino acid substitution on the genes encoding for the two enzymes dihydrofolate reductase and dihydropteroate synthase. Treatment with sulphadoxine/pyrimethamine selects for DHFR variants [(I51),(R59), and (S108N)] and for DHPS variants [(436S), (G437), and (E540)]. (44) A study showing a two-point mutation at DHFR (S108N and C59R) and a onepoint mutation at DHPS (S436A or A437G) revealed an increased in vitro resistance against pyrimethamine but did not correlate sufficiently with in vivo treatment failure in children. (45)(46) 2.5 Mefloquine resistant P. falciparum Mefloquine is a 4-methanol quinoline and was introduced in the 1970s. (28) Mefloquine has a long half-life of days. (28) The long-time sustainability of mefloquine metabolite in vivo supports its chemo suppressive property, providing prophylaxis for areas with a high transmission rate of malaria. (47) Mode of action The mechanism of action of mefloquine was shown to be involved in the heme polymerization inhibition process, but heme polymerization inhibition is lower than that of chloroquine due to the weak basicity of mefloquine and reduced diffusion to the parasite s compartments. (43) Mefloquine is also a much less potent enhancer of the peroxidase activity of heme than chloroquine. (48) Mefloquine also interferes with the transport of solutes to the digestive vacuole of the parasite. (49) 10

29 2.5.2 Mechanism of resistance Mefloquine resistant parasites are characterized by amplification of the multidrug resistant (Pfmdr1) gene and Pgh1 protein expression. (32) Resistance to mefloquine by the multidrug resistant Pfmdr1 gene showed cross-resistance to halofantrine and quinine. (50) On the other hand, a reduced Pfmdr1 copy number showed the heightened susceptibility of parasites to lumefantrine, artemisinin, halofantrine mefloquine and quinine. (51) This study also showed that knockdown parasite lines with a low Pfmdr1 copy number had no change on chloroquine sensitivity. In another study, mefloquine selected parasite lines also showed an inverse relationship between the level of chloroquine resistance and increased Pfmdrl gene copy number. (50) The mechanism of mefloquine resistance has also a negative correlation with parasite sensitivity to other anti-malarial drugs. 2.6 Lumefantrine resistant P. falciparum Lumefantrine is a hydrophobic lipophilic anti-malarial drug which is described as an artemisinin partner drug in treating malaria. Lumefantrine has a longer half-life, and its bioavailability is increased by coadministration of fat in adults. (52) The improved bioavailability of lumefantrine increases the anti-malarial activity of the drug and improves the cure rate Mode of action The mode of action of lumefantrine remains constant due to its longer half-life and effective cure rate. (53) Lumefantrine targets the schizont stages of erythrocytic parasites, but the exact mechanism of action is not well defined. This drug is believed to halt the formation of hematin by binding with heme. Hematin crystallization is the main mechanism of heme 11

30 detoxification by the parasite to survive anti-malarial drug pressure. This parasite survival mechanism is targeted by anti-malarial drugs and effectively disrupts parasite physiology Mechanism of resistance The resistance mechanism and associated molecular markers to lumefantrine have not been established so far. However, there are reports showing that lumefantrine tolerance/resistance is associated with parasite Pfmdr1 gene amplification, and polymorphism at amino acid N86Y was found in in vivo resistance to lumefantrine in Africa. (54) A single Pfcrt transporter gene polymorphism at (K76) was found to be associated with in vivo selection by lumefantrine. 2.7 Artemisinin resistant P. falciparum Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties produced by the plant Artemisia annua. (55) Artemisinin, artemisinin derivatives, and their partner drugs are effective against P. falciparum. (53) Therefore, artemisinin drugs are deployed for antimalaria treatment worldwide Mode of action The mode of action of artemisinin has been the subject of debate and is still unclear. There have been different propositions about this effective anti-malarial drug. Artemisinin is thought of as a pro-drug, where its reductive cleavage releases carbon-centered free radicals. The free radicals generated from the cleavage of artemisinin react with many of the parasite s proteins and induces parasite killing. (56) Artemisinin is considered as a pro-drug and activation of artemisinin releases free radicals which kill the parasite. The free radicals kill the parasite in a number of ways, but the mechanism of activation and parasite killing is unclear. The first proposed activation of artemisinin is via the free ferrous ion derived. The 12

31 second argument is about activation of artemisinin by haem. The haem which is derived from both parasite biosynthesis at the early ring stage, and hemoglobin degradation in the late stages, is crucial for the mode of action of artemisinin and the killing of the parasite. With artemisinin there is a risk of high rate recrudescence due to its short half-life, but artemisinin and its derivatives have a swift killing rate of parasites and reduce parasitemia. The mode of action of artemisinin is not well defined so far. The known mechanism of the artemisinin endoperoxide drug works by the generation of free radicals, and the free radicals kill the parasite. Artemisinin also targets the P. falciparum phosphatidylinositol-3-kinase (PfPI 3 K). The Pfk13 point mutation (C508Y) was associated with increased PfPI 3 K. (57) The study found that an elevated PI 3 P, the lipid product of PfPI 3 K, was associated with resistant parasites Mechanism of resistance There are reports about the emergence of artemisinin resistant P. falciparum. Artemisinin resistant P. falciparum is characterized by mutations at the (PfK13)-propeller gene, which is strongly associated with extended parasite clearance time. Thus, (PfK13) mutations have been used as resistance molecular markers. (58) A recent study in Thailand showed that the PfK13 mutation is characterized by a change in a single amino acid at C580Y. (59) This mutation was also found in Vietnam, western Cambodia and Myanmar. (60) Based on C580Y mutation prevalence, resistant parasites spread in Southeast Asia. (61)(30)(63) The PfK13 gene polymorphism was found in sub-saharan Africa, but it was different from that found in Southeast Asia. (64) In some countries, a similar mutation was found ( Kenya, Tanzania, Mali, Gambia, Gabon, Ghana, Congo DRC and Cote d'ivoire, Madagascar, Cameroon and Nigeria (64) ). African PfK13 polymorphisms are different from Southeast Asian, the exception being 13

32 (PfK13, P533L), which is found in both regions. (65) A non-artemisinin resistant polymorphism (A578S) was also found in Asia (Cambodia-Thailand, (63) Bangladesh, (66) India (67) ) and Africa (Uganda, (68) Kenya, (69) Equatorial Guinea (70) and Comoro Island (58) ). PfK13 polymorphisms were found in Uganda, (68) Angola and Mozambique, (71) Kenya, (72) Mali, (73) Senegal (74) and Tanzania. (65) A study in southwestern Ethiopia found a mutation at the PfK13 propeller gene (N531I), but it was not associated with treatment failure. (75) Another study conducted in three different regions of Ethiopia did not find any mutations in the PfK13 propeller gene associated with a resistance phenotype or treatment failure. (65) These polymorphisms are considered to be a strong predictor of treatment failure and/or the emergence of resistant parasites. Artemisinin resistance mechanisms, mode of action and target molecules have yet to be characterized. There is doubt about the exact mechanism of action against blood stage parasites. Biochemical studies have shown that artemisinin is activated by haem. (76) The heme produced from the parasite s biosynthesis pathway and host s hemoglobin digestion is used to activate artemisinin and kill intra-erythrocytic parasites. (76) Artemisinin has a potent inhibitory effect on (PfPI3K), but increased PfPI3K has been seen in resistant parasites with (C580Y) mutation. (57) The mechanism of artemisinin resistance is controversial, (77) because artemisinin sensitive parasites also showed PfK13 polymorphism. (64) The parasite s intrinsic behavior and biological modifications may contribute to the parasite s development of resistance to artemisinin. (78) Therefore, a deeper understanding of parasite biology and artemisinin s mechanism of action is crucial in uncovering the drug resistance mechanism of the parasite. 14

33 2.8 Combination therapy and prevention of drug resistance Combination therapy is the newest method of malaria treatment utilized in both emergency situations and to prevent the spread of drug-resistant parasites in endemic areas. Combination therapy has been introduced globally to prevent the spread of drug-resistant parasites. Artemisinin and artemisinin derivative combination therapy is a new strategy for malaria treatment and is recommended for treatment in areas where malaria is endemic. (79) A combination of antimalarial drugs, each with a different half-life and pharmacokinetic property, can effectively cure malaria. Partner drugs like artemisinin reduce parasitemia rapidly, while a partner drug with an extended drug metabolite in the blood can eliminate the parasite and prevent recrudescence. Combination drugs such as artesunate-mefloquine and artemether-lumefantrine treatments are deployed in Southeast Asia (Thailand, Cambodia) (80) and Africa. (81) The characteristics of artesunate and artemether are a short half-life and fast killing during the erythrocytic stage of the parasite, whereas mefloquine and lumefantrine both have an extended half-life and prevent recrudescence after antimalaria treatment. Therefore, combination therapy is recommended for effective parasite clearance and high cure rate, which promotes malaria eradication. (81) Antimalarial drug combination therapy was started after the worldwide spread of parasites resistant to first- and second-generation antimalarial drugs (chloroquine and sulfadoxine/pyrimethamine respectively). 2.9 Drug shifting and re-introducing used drug Chloroquine and sulphadoxine pyrimethamine have long been used for treatment of malaria. These anti-malarial drugs are now no longer in use for P. falciparum malaria due to the spread of drug-resistant parasites. These drugs have therefore been banned from deployment and have been replaced by new antimalarial drugs such as artesunate and lumefantrine with a 15

34 new treatment strategy. The drug pressure has, therefore, been changed from chloroquine to sulphadoxine/pyrimethamine, then to artemisinin and artemisinin derivatives and their partner drug. This drug shifting may change the mechanism of parasite selection for adaptation, and there may be a reappearance of parasites sensitive to the old drugs. This can happen for several reasons. The first reason is the complete eradication of resistant parasite phenotypes through the massive killing of newly introduced potent antimalarial drugs. A second reason is the difference in drug target on the parasite between the new and old drugs, leading the parasite to new adaptation routes or mechanisms. A third reason is that the combination of antimalarial drugs potentiates the effectiveness of the partner drug and assures complete parasite clearance. The final reason is the introduction of new antimalarial drugs which target multiple parasite biomolecules or pathways. Malaria parasites dynamically evolve along with the persistence of anti-malarial drug pressure. A change in treatment pressure or drug affects the parasite s evolutionary process. Therefore, the old drugs can be reintroduced after a long period of withdrawal. Chloroquine was the first drug which was withdrawn from use for treatment. Since then sulphadoxine/pyrimethamine was the second drug banned from being used. Based on molecular markers, as a key indicator of the parasite phenotype, the prevalence of drug-resistant-associated molecular phenotyping SNPs helps to reintroduce an old and previously used drug. The prevalence of Pfcrt 76T in Malawi, Kenya, and Tanzania was high while chloroquine was in use, but recently its prevalence has dropped or disappeared due to the banning of chloroquine. The decline in chloroquine resistance, (Pfcrt, 76T), occurs due to the withdrawal or apparent use of chloroquine. Chloroquine resistance has disappeared in Malawi since Tanzania has shown a significant downward prevalence of Pfcrt 76T, and it is declining in Kenya as 16

35 well. (82)(83) Therefore, it is possible that the drug chloroquine could be used again to treat malaria. The use of artemisinin combination therapy (ACT) has increased throughout Africa (84) and elsewhere. Therefore, chloroquine with an appropriate partner drug might be used in the future, possibly being limited to target the Plasmodium species. (82)(85) Table 1 Antimalarial drug-resistant polymorphisms Drugs Target Selected amino acid References gene changes(polymorphisms) Chloroquine PFCRT C72S, M74I, N75E, N75K, Veiga, et al., 2016 PFMDR1 K76T N86Y, Y184F, S1034C, N1042D, D1246Y Li, et al., 2014 Mefloquine PFMDR1 N86Y, 1034S, 1042N, Ibraheem, et al., N sulphadoxine DHPS S436A and A437G Jelinek, et al.,1998 Pyrimethamine DHFR (I51), (R59), (S108N), Jelinek, et al.,1998 C59R Lumefantrine PFMDR1 N86Y, Y184F, S1034C, Sisowath, et al., 2007 N1042D, D1246Y Artesunate PFK13 C508Y, Y493H, M476I, Huang, et al., 2015 R539T, I543T The parasite biology and biological process are critical for effective anti-malarial drug targeting. The design of anti-malarial drug targets should consider the parasite's unique 17

36 biological feature. The most anti-malarial drugs,which have been used so far targeted a number of structural biomolecules and biological pathways in the parasite. The following drug targets such as parasites' digestive vacuole, nucleic acid metabolism(pyrimidine synthesis, purine salvage for DNA/RNA), folate pathways(86), hemoglobin metabolism, phospholipids' metabolism, artemisinin receptors(87), mitochondrial electron transport channels, and reduction and oxidation process have been reported so for (86)(88)(89)(90)(91)(92). Table 2. A summary of anti-malarial drug compound and their target Drugs Original compound Mode of action Reference Quinine quinoline inhibition of membrane Petersen, et al.,2011 methanol trafficking, heme Mungthin, et al.,1998 detoxification Chloroquine 4-aminoquinoline inhibits accumulation of hemozoin and heme Petersen, et al.,2011 Mungthin, et al.,1998 Mefloquine detoxification 4-methanolquinoline inhibits accumulation of hemozoin, binds with parasite phosphatidylinositol Petersen, et al.,2011 Mungthin, et al.,1998 Halofantrine phenanthrene heme detoxification Mungthin, et al.,1998 methanol Amodaquine 4-aminoquinoline inhibiting heme polymerization Petersen, et al.,2011, Mungthin, et al.,

37 Sulphadoxine sulfonamide dihydropteroate synthase Olliaro,1999 Pyrimethamine anti-protozoa dihydrofolate reductase Olliaro,1999 Proguanil biguanide dihydrofolate reductase inhibitor and enhance Srivastava, et al.,1999 atovaquone activity Primaquine 8-aminoquinoline clears hypnozoites, non specific interaction with lipid Petersen, et al.,2011 and Basso, et al.,2011 membranes, oxidative stress Atovaquone naphthoquinone inhibits mitochondrial Nosten and White, 2007 function targeting the cytochrome b 1 complex Artesunate artemisinin Parasite DNA damage, heme detoxification, oxidative stress, alkylation of proteins Nosten and White, 2007,Olliaro,etal.,2001, Gupta, et al., 2016 and other unknown mode of actions Dihydro- artemisinin Parasite DNA damage, heme Nosten and White, artemisinin detoxification, oxidative 2007,Olliaro, et al.,2001, stress, alkylation of proteins Gupta, et al., 2016 and other unknown mode of actions 19

38 artemether artemisinin Parasite DNA damage, heme detoxification, oxidative stress, alkylation of proteins Nosten and White, 2007, Olliaro, et al.,2001, Gupta, et al., 2016 and other unknown mode of actions Lumefantrine benflumetol binding with heme and inhibit Ezzet, et al.,1998 hematin formation Piperaquine bisquinoline inhibits accumulation of Nosten and White,2007 hemozoin Biological characteristics of P. falciparum The biological characteristics of the Plasmodium parasite determine both the pathobiology and pathogenesis of malaria. Severe malaria is associated with the biological characteristics of the blood stage of the parasites. For instance, sequestration, autoagglutination, endothelial cytoadhesion, and rosetting are the key biological characteristics of the erythrocytic stages of Plasmodium parasites Rosetting of P. falciparum Plasmodium parasites form rosette during the intra-erythrocytic cycle. Rosetting is a phenomenon in which uninfected red blood cells bind around infected red blood cells. Plasmodium parasite-infected erythrocytes form rosettes with uninfected erythrocytes to survive in vivo by sequestering infected erythrocytes in the microvasculature and avoiding splenic clearance mechanisms.(93) 20

39 History of rosette discovery and rosetting Plasmodium species The first discovery of rosetting was made by David et al.(94) in P. fragile-infected toque monkey erythrocytes. The study reported that uninfected erythrocytes showed agglutinations to infected red blood cells in vitro, and the phenomenon was referred as rosetting. Another study on P. falciparum-infected human erythrocytes found rosette formation in vitro.(95) The study found that rosette formation had occurred during the trophozoite to schizont stages but that the ring stage parasite did not form rosettes. The rosetting phenomenon was also discovered in P. vivax,(96) P. oval(97) and P. malaria.(98) All four human-infecting Plasmodium species form rosettes.(98) A study in Thailand found that P. ovale-infected blood formed a rosette with uninfected red blood cells in a way similar to P. falciparum and P. vivax rosetting in uninfected red blood cells. The study showed that 24 hours after culturing P. ovale, the pigment containing parasites formed a rosette, but uninfected red blood cells did not form a rosette.(97) Another clinical case in Bangladesh found a severe case of P. malariae with rosette formation.(99) Table 3. Discoveries of rosette forming Plasmodium species Authors Method Plasmodium species Host David, et al.,1988 In vitro P. fragile Toque Monkey Handunnetti, et al.,1989 In vitro P. falciparum Human Udomsangpetch, et al.,1995 ex vivo P. vivax Human Angus, et al.,1996 ex vivo P. ovale Human Lowe, et al.,1998 wet mount P. malariae Human 21

40 Prevalence of rosetting and associations with severe malaria The phenotypic characteristics of Plasmodium parasite isolates are very crucial in parasite pathobiology and disease prognosis. Rosetting is a known Plasmodium parasitic phenotype that occurs during the erythrocytic cycle of the parasite. Rosetting and its association with disease severity is fundamental in understanding parasite evolution and pathogenesis. In addition, an understanding of the intrinsic relationship between rosette formation and endothelial cytoadherence could be of paramount importance in parasite biology and disease severity. Rosetting parasites are found worldwide and have been associated with severe malaria. However, in some places, rosetting has not been associated with severe malaria. There are contradictions in the reports about the role of rosettes in severe malaria. Severe malaria in Africa is characterized by rosette formation but not with severe malaria in Southeast Asia (reviewed by (100)). A study of Malian children showed that P. falciparum rosetting is a virulence factor associated with severe malaria, but it is unclear whether rosetting is associated with all clinical forms of severe malaria, including cerebral malaria.(101) Analysis of subcategories of severe malaria (coma, severe anemia, non comatose neurological impairment, repeated seizures or a small heterogeneous group with signs of renal failure or jaundice) also showed high levels of rosetting.(101) Another study in Kenya found a correlation between higher rosette frequency and increasing severity of disease.(102) The study stated that rosetting was not associated with cerebral malaria and other forms of severe malaria. Rosette formation did not reflect an invasion advantage of the parasite to uninfected erythrocytes. It was believed that rosette formation might have a role in microvasculature obstruction and restriction to macrophage phagocytosis to contribute to pathogenesis.(103) A study in Thailand has found that rosette formation and disease severity 22

41 did not correlate.(104) Rosetting of P.vivax showed no associations with fever, thrombocytopenia, anemia or reticulocytosis in patients.(96) Rosette formation occurs in Plasmodium vivax preferentially to normocytes but does not facilitate merozoite invasion.(103)(105) This implies that rosette formation and merozoite invasion are independent phenomena. Another study showed that P. malariae form rosettes not associated with parasite pathogenicity.(98) Postulated roles of rosetting There are many hypotheses that have been put forward about rosetting in Plasmodium parasites. It has been postulated that rosetting of uninfected erythrocytes enhances merozoite invasion. Rosetting does not give multiplication or growth advantages to the parasites and does not affect the invasive efficiency of the parasite.(106) Rosette formation makes PfEMP1 inaccessible to antibodies and clearance by the immune system, providing an immune evasion mechanism.(107) The spontaneous binding of infected erythrocytes to uninfected erythrocytes to form rosettes is a property of some strains of Plasmodium falciparum linked to malaria with severe complications.(108) One hypothesis states that complement receptor1 deficiency reduces rosetting. The study showed the role of CR1 association in rosette formation. The CR1- dependent rosetting mechanism demonstrated occurs commonly in P. falciparum isolates.(109) A study done in Papua New Guinea showed that CR1-deficient red blood cells show reduced P. falciparum rosetting, and the group hypothesized that "if rosetting is a direct cause of malaria pathology, CR1-deficient individuals should be protected against severe disease." CR1 polymorphism conferred protection against severe malaria,(110) which indirectly implicates the involvement of CR1 in P. falciparum pathogenesis. The study also 23

42 found that individuals with genetic disorder α-thalassemia are protected from severe malaria.(110) CR1 is the host s erythrocyte receptor for PfRh4, a major P. falciparum ligand essential for sialic acid independent invasion.(111) Rosetting and blood group P. falciparum rosetting is determined by ABO blood group. Rosetting, a parasite virulence phenotype associated with severe malaria, is reduced in blood group O erythrocytes when compared with groups A, B, and AB.(112) Parasite isolates from blood group 'O' rosette less than isolates from other blood groups. Rosetting is associated with severe malaria and influenced by blood group. Rosetting also plays a role in the pathogenesis of severe malaria.(102) Therefore, the protection effect of severe malaria is due to a reduction in rosetting. Antibodies against PFEMP1 binding inhibit rosette formation and protect against severe malaria.(113) The ABO blood group antigens are expressed not only on erythrocytes but also expressed on endothelial cells, platelets and serum proteins. The ABO blood group of a malaria patient determines the development of the disease.(106) Larger rosettes are formed when parasites grow in the red blood cells of a preferred blood group, and small size rosettes form in blood group O more frequently than in blood group A, B or AB. This implies that the size of the rosette reveals the match between a specific parasite and its preferred blood group. The ABO blood group also determines rosette reversal or disruption.(114) The reversal effect could indicate that infections from a blood group A individual to another blood group might result in severe malaria or vice versa. 24

43 Rosette disruption and reversal Plasmodium falciparum rosettes disrupt via enzymes due to the trypsin sensitivity of the parasite ligand and the neuraminidase sensitivity of receptors. Sialylated proteins from uninfected red blood cell are factors for rosetting, because sialic acid interacts with the parasite.(115) A study in Thailand showed that the P. vivax rosette is sensitive to both heparin and trypsin.(116) According to this study, plasma obtained from malaria positive patients reversed rosette formation, and plasma obtained from healthy donors did not reverse rosette formation in all isolates. This implies that rosette reversal via plasma-derived factors are originated from the parasite or host biomolecules during infection. Plasma from convalescing patients reverses rosetting increasingly from the time of admission through recovery, and Plasmodium vivax rosette-reversing activity appears to have species-specific components.(116) Factors affecting rosette formation Parasite density is an important factor in determining the frequency of both autoagglutination and rosette formation. Antimalarial drugs reduce rosetting. Artesunate, artemether and quinine reduce both cytoadhesion and rosetting in vivo and in vitro drug exposure in P. falciparum. Artesunate is more effective than quinine in reducing rosetting. Exposure of the parasite to artesunate for less than 2 hours both in vivo and in vitro reduced both cytoadherence and rosetting by >50%, while quinine reduces rosetting in more than 4 hours of drug exposure in vivo but does not reduce cytoadherence.(117) This indicates that artesunate is more potent than quinine in reducing the pathology of severe malaria. Another study of African P. falciparum isolate showed inhibition of rosettes in vitro by Curdlan 25

44 sulfate (CRDS), which is a sulfated glycoconjugates compound that is chemically similar to such known rosette-inhibiting drugs as heparin.(108) Autoagglutination, endothelial cytoadhesion and rosetting There are three types of adhesion phenotypes (endothelial cytoadhesion, rosetting and platelet-mediated clumping of infected red blood cells) that occur during Plasmodium parasite infection (reviewed by (100)). Cytoadhesion is an adhesion of infected red blood cells to endothelial cells, whereas rosetting refers to an adhesion or binding of uninfected red blood cells to infected red blood cells. The other adhesive phenotype is platelet-mediated clumping of infected red blood cells to each other. The main difference among the three phenomena is the type of cell engaged to form agglutinate, rosette and endothelial cytoadhesion. Therefore, the adhesion of an infected cell to another infected cell forms an autoagglutination, while the adhesion of an infected red blood cell to an endothelial cell results in endothelial cytoadhesion. The adhesion of an uninfected red blood cell to an infected cell forms rosettes Autoagglutination Autoagglutination of infected red blood cells is caused by the clumping of platelets and infected red blood cells.(118) Plasmodium falciparum-infected red blood cells adhere to each other and form large autoagglutinates. This phenotype is common in field isolates and is strongly associated with severe malaria.(119) Noninfected red blood cells form aggregates in the form of rouleax, and the aggregation is reversible.(120) The activation of platelets causes red blood cell aggregation, which leads to clot formation.(120) 26

45 Endothelial cytoadhesion Endothelial cell adhesion is the adhesion of infected erythrocytes with different host receptors. Platelet endothelial cell adhesion molecule-1/cd31 mediates adhesion of infected erythrocytes to the endothelial cell receptor.(121) The adhesion of an infected erythrocyte to the placenta is mediated by parasite-derived VAR2CSA allosteric adhesin to the host chondroitin-4- sulfate (CSA).(122) The type of receptor interaction determines the clinical outcome of malaria. The binding of parasites to the CD36 receptor is found to be higher in non severe disease but identical in all cerebral malaria patients.(123) The binding of parasite isolates to the ICAM-1 receptor is found to be higher in non anemic disease and cerebral malaria patients.(123) This indicates that the type of receptor, not the binding affinity or broadness of affinity, determines the clinical outcome of malaria. If a specific host receptor binds a broad spectrum of parasite isolates, it may not predict the clinical complexity of the disease Rosetting receptors and ligands for P. falciparum and P. vivax Mature intra-erythrocytic parasites produce parasite proteins which are exported to the surface of infected red blood cells. These proteins mediate an interaction between uninfected red blood cell surface protein and exported parasite proteins. Just as the rosetting rate is different for P. falciparum and P. vivax, the molecules involved in rosette formation are also different. In addition, the rosette formation rate is different across different Plasmodium intra-species. The more pathogenic strains or subspecies there are, the more likely they are to form rosettes.(124) 27

46 Ligands and receptors for P. falciparum Plasmodium falciparum forms rosettes and has cytoadhesive properties. The rosetting and cytoadhesion of P. falciparum to the erythrocyte is mediated by extracellular proteins, which are expressed by genes from the chromosomal telomere region, such as the VAR gene, STEVOR gene and RIF gene.(125) These proteins are exported from the intra-erythrocytic parasites to the surface of infected red blood cells and interact with uninfected red blood cell receptors or endothelial cells Rosetting ligands of P. falciparum The 3D7 genome contains 59 VAR, 149 RIF and 28 STEVOR genes.(126) These genes express parasite-derived proteins, which determine the pathogenesis and antigenicity of P. falciparum. Both cytoadherence and rosetting by P. falciparum are mediated by parasitederived infected erythrocyte surface proteins but have been studied as separate entities. P. falciparum strains expressing rosetting ligand, PFEMP1, cytoadhere to the brain endothelial cell line, which shows that cytoadhesion and rosetting are not separate phenomena.(93) Although both cytoadhesion and rosetting could occur in a parasite isolate, the ligands and receptors which mediate cytoadhesion are different from rosette mediating ligands and receptors. In contrast, antibodies against an N-terminal PFEMP1 variant (NTS-DBL1α and DBL2γ domains) inhibit and reverse cytoadhesion in P. falciparum.(93) The PFEMP1 ligand, NTS-DBL1α, binds to complement receptor1 (CD35) to form P. falciparum rosettes.(111) Rosetting, the adhesion of P. falciparum infected erythrocytes to uninfected erythrocytes, is a virulent parasite phenotype associated with the occurrence of severe malaria.(127) Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs and is mediated by a subset of P. falciparum membrane protein1 (PfEMP1) variant adhesions 28

47 expressed on the infected host cell surface.(128). Rosetting has been associated with severe disease, but its functional significance and the host receptors and parasite ligands involved are only partially known.(129) CD36 is the erythrocyte receptor which mediates rosetting in P. falciparum.(130) The receptors ICAM and VCAM also form rosettes. Rosette formation in P. falciparum is also associated with complement receptor1 (CR1). A specific parasite ligand region at PFEMP1 (NTS-DBL1α)(93) and a specific complement receptor1 region from the host cell(109) interact with each other to form rosettes in P. falciparum. The STEVOR gene expresses from P. falciparum-infected red blood cells and binds to uninfected red blood cells independently of Pfemp1 to form rosettes.(131) This study has shown that antibodies against the STEVOR gene inhibited merozoite invasion. This raises the issue of whether rosette formation may indirectly facilitate merozoite invasion. The STEVOR gene expression on the IRBC leads to PfEMP1-independent rosette formation, and Glycophorin C is also a host receptor.(131) The repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (IRBCs) and bind to red blood cells preferentially of blood group A to form large rosettes.(132) The study shows that the group A antigen is the major receptor of A-RIFIN, which forms large rosettes. RIFINs are localized in the parasitophorous vacuole, Maurer's cleft and IRBC membrane surface, and both RIFIN and PFEMP1 up regulated during rosette selection.(132) Rosetting ligands and receptors of P. vivax A study in Thailand showed that rosette formation of P. vivax is dependent on the divalent cations (Ca 2+/ Mg 2+ ) and is highly sensitive to trypsin and heparin. Rosette reversal was also independent of blood group.(116) In another study, host blood group, parasitemia and 29

48 reticulocyte count did not correlate with P. vivax resetting.(105) The human blood group CD35 was involved in P. falciparum rosetting, but it was not involved in P. vivax resetting.(105) Plasmodium vivax also forms rosettes preferentially to normocytes, and Glycophorin C (CD236R) mediates P. vivax rosette formation. However, rosette formation does not facilitate merozoite invasion in either P. vivax(105) or P. falciparum.(103) P. vivax infection increases the rigidity of P. vivax-infected red blood cells due to rosetting, but the ligands responsible for rosetting are not clear yet. (126) P. vivax-infected reticulocytes have shown cytoadherence to various endothelial receptors such as VCAM, ICAM, and E- selectin.(133) Host receptors for cytoadhesion and rosetting P. falciparum erythrocyte membrane protein1 (PFEMP1) has a fundamental role in antigenic variation and cytoadhesion. The CD36 receptor is the most common binding agent, followed by ICAM.(134) CD36 binding is lower in severe malarial anemia, and ICAM binding is also lower in cerebral malaria. Hematocrit and parasite adhesion are associated with lower adhesion to CD36 and ICAM in children with severe anemia.(135) P. knowlesi binds to endothelial receptors such as ICAM and VCAM, but it does not bind to CD36 at a significant level.(136) There are also other receptors involved in P. falciparum cytoadhesion, such as Endothelial Leukocyte Adhesion Molecule 1 (E-selectin), Vascular Cell Adhesion Molecule 1 (VCAM-1), CHO-receptor X, Hyaluronan Binding Protein 1 (HABP1), Platelet Endothelial Cell Adhesion Molecule-1 (CD31/PECAM-1), Thrombospondin-1 (TSP-1), CSA and Fractalkine.(134) Both ICAM and CD36 mediate P. falciparum cytoadhesion in complicated and uncomplicated malaria.(137) The two adhesive molecules ICAM and CSA have shown adhesive properties in P. vivax.(138) Another study in Papua New Guinea on 30

49 CR1-deficient individuals showed that rosette formation by P. falciparum was reduced and individuals were protected from severe malaria.(110) The complement receptor-deficient cells have reduced rosetting, which protects against severe malaria.(110) Complement receptor1 has a specific region for binding to P. falciparum-infected erythrocytes to form rosettes.(109) Rosetting, which forms by unifected red blood cells and infected red blood cells, can adhere to endothelial cells, bind to the CD36 receptor, immunoglobulins and the blood group A antigen.(139) Multiple serum factors also act as a bridging molecule for rosette formation.(140) Immunoglobulin (IgM) binds to rosette-forming infected erythrocytes and stabilizes rosette formation in P. falciparum in vitro.(141) Glycosaminoglycans and sulfated glycoconjugates Glycosaminoglycans and sulfated glycoconjugates disrupt rosetting.(142) Biomolecules such as heparin, heparan sulfate, hyaluronic acid, dextran sulfate, chondroitin sulfate and fucoidan disrupt rosetting. Fucoidan is the most effective in disrupting rosettes, while chondroitin sulfate disrupts rosettes less effectively. The rest of the glycosaminoglycans, such as heparin, heparin sulfate, dextran sulfate and dermatan sulfate, disrupt rosettes intermediately.(142) Duffy binding-like domain 1 (DBL-1) binds to N-sulfated heparin and heparan sulfate, which disrupts rosetting. DBL-1 binds specifically to erythrocytes, and heparan sulfates bind to endothelial cells.(143) Sulfated glycoconjugates are more effective for rosette disruption, while anionic glycosaminoglycans such as hyaluronic acid and chondroitin sulfate (A, B, C) have no effect on rosette formation.(144) Rosette-disrupting sulfated glycoconjugates also inhibit merozoite reinvasion of erythrocytes.(144) Cytoadhesion is inhibited by heparan sulfate, but heparan sulfate and heparan-like molecules are involved in rosetting;(145) 31

50 however, heparin and other sulfated oligosaccharides can disrupt rosettes.(128) The blood antigens are determinants for rosetting in clinical P. falciparum isolates.(146) In general, rosetting is a Plasmodium parasite adhesive phenotype which occurs in many Plasmodia spp. Rosetting is associated with pathological conditions caused by severe malaria. The association between severe malaria and P. falciparum rosette formation is more pronounced in sub-saharan Africa, but the association of disease severity, rosetting phenotype of the parasite and the driving factors are not yet clear in this particular region. Rosetting in Plasmodium parasite is a multifaceted biological characteristic of the parasite. Different rosetting parasite ligands and host receptors are involved during rosette formation in the intraerythrocytic cycle of the parasite. Host blood group antigens are a key determinant factor for the rosetting phenotype of the parasite. In addition, glycosaminoglycans and sulfated glycoconjugates are involved in rosette formation and rosette disruption The aim of the study The main aim of this study was to determine the in vitro biological characteristics and drug sensitivity profile of P. falciparum strains Specific aims I. To describe the biological characteristics of P. falciparum II. To observe the rosetting phenotype of P. falciparum strains in vitro III. To assess the drug sensitivity profile of P. falciparum laboratory strains and field isolates 32

51 CHAPTER THREE 3. Material and methods 3.1. Method 3.2. Study design This research was designed to study the biological characteristics and drug sensitivity profile of P. falciparum laboratory-adapted strains and field isolates. The biological characteristics of parasites such as in vitro rosetting phenotype were studied. The rosetting parasite phenotype was studied for both laboratory-adapted falciparum strains and field isolates. This helps us to describe the phenotypic characteristics of parasites and to establish correlation with the in vivo biological characteristics of the parasite. The phenotypic characteristics of Plasmodium parasite play a significant role in the better understanding of parasite pathobiology and pathogenesis. This in vitro description of the parasite phenotype (rosetting) simulates parasite pathogenecity in patients. In addition, in vitro drug sensitivity profiling of P. falciparum laboratory strains and field isolates was studied. This in vitro drug sensitivity profiling of both laboratory-adapted parasites and field isolates is key in comparing the sensitivity of field isolates and laboratory-adapted parasites to commercially available antimalarial drugs. The in vitro sensitivity of the parasite helps us to understand the mechanism of drug resistance development and associated parasite phenotype. Ring stage hypersensitivity and phenotyping were conducted on laboratory-adapted falciparum strains. Ring stage parasite hypersensitivity mimics the mechanism of artemisinin-induced parasite dormancy and revival of the quiescent parasites in vitro. This assay was designed to study parasite recrudescence and parasite phenotype related to artemisinin resistance. 33

52 3.3. Culturing of P. falciparum strains P. falciparum strains were obtained from LR Lab and cultured in vitro. After culturing of the parasites for a few cycles, stage synchronization was done using 5% sorbitol or mature stage parasites were concentrated using the percoll gradient method (65%/35%) and sub-cultured for further synchronization. The ring stage parasites were then cryopreserved for in vitro drug assay. The field isolates also obtained from LR Lab had previously been collected from Thailand Parasite synchronization and concentration Parasite synchronization and concentration are crucial steps in vitro parasite culturing for an assay. Therefore, either sorbitol synchronization or percoll gradient concentration of mature stage parasites was used for the preparation of parasites for an in vitro drug assay and rosetting assays Sorbitol synchronization The sorbitol synchronization method was used to prepare tightly synchronized young ring stage parasites for drug assay. 5% sorbitol was used to synchronize cultured parasites. The parasite culture, which had more than 80% young ring stage, was subjected to 5% sorbitol treatment for synchronization. Sorbitol kills mature blood stage parasites (trophozoites and schizonts) while the ring stage parasite survives. In order to treat parasites with 5% sorbitol, the culture was taken out of the incubator and spun down at 1800rpm for 5 minutes. The supernatant was removed, and the pack cells were resuspended to 20% hematocrit with 5% sterile sorbitol. The culture was then incubated for 13 minutes at 37 0 C in an incubator. After incubation, the cells were re-centrifuged at 1800rpm for 5 minutes, and the supernatant was removed. The pack cells were washed twice with pre-warmed complete RPMI1640. Finally, 34

53 the pack cells were transferred to a 25cm 2 culturing flask with 10ml complete pre-warmed RPMI medium and incubated. Giemsa-stained thin smears were prepared before and after sorbitol treatment. The thin smear before sorbitol treatment helped us to observe the proportion of young ring stage parasites compared with matured schizonts. The percentage or proportion of ring stage parasites that is more than 1.5% can be treated by sorbitol. The thin smear after sorbitol treatment helped us to control parasitemia at every life cycle during subsequent culturing or cryopreservation. Figure 2. Sorbitol synchronization of ring stage parasites The cultures were treated with sorbitol to obtain tightly synchronized ring stage parasites. Young ring stage parasites survived sorbitol treatment, whereas the mature stage parasites died Percoll synchronization Percoll gradient centrifugation is used for concentration of mature parasites (late trophozoites/schizonts). The percoll gradient was made by making 65%(4ml) /35%(4ml) percoll layer, and then 4ml of the resuspended matured parasite culture was overlaid. This layer of percoll cell culture was centrifuged at 2000rmp for 15 minutes. After centrifugation, the supernatant was removed carefully without disturbing the schizont band which had formed at the top of the 35% percoll layer. The fine band (schizonts) was then taken by pipette and transferred into a new sterile Falcon tube (15ml). Uninfected blood was added 35

54 and washed with complete medium. After washing, the parasite was incubated in an in vitro culture system for further growth cycle. The procedure was repeated until highly synchronized parasites were obtained. On the other hand, the bottom layer (65% percoll layer), which had both uninfected red blood cells and young ring stage parasites, was washed and re-incubated without the addition of fresh blood. The synchronized parasites which were obtained from the schizont were cryopreserved when they were tiny ring stage parasites. Figure 3. Percoll concentration of matured parasites The 35% percoll layer created a schizont band, whereas the 65% percoll layer created the bottom layer, where the trophozoite stage of the parasite was found. The last bottom layer of percoll contained ring stage parasites and uninfected red blood cells Cryopreservation of parasites After synchronization, the parasite was cryopreserved using glycerolyte solution as described elsewhere with modification.(147) To put it briefly, the culture suspension was centrifuged at 1800rpm for 5 minutes, and the blood pack was measured. Glycerolyte solution was then added (0.33 times the volume of infected blood) drop by drop. Glycerolyte solution was again added (1.33 times the volume of blood pack) drop by drop and gently mixed. Finally, the blood-glycerolyte solution was measured (0.4-1ml volume) and aliquoted to labeled cryotubes. The tubes were placed in an isopropanol master cooler and frozen at C for 18 hours. The tubes were transferred to liquid nitrogen and stored until the drug assay. 36

55 3.6. Thawing of cryopreserved parasites The cryopreserved parasites were thawed using a standard NaCl gradient method, as described in detail in Kosaisavee et al.(148) Briefly, the cryotubes were removed from the liquid nitrogen tank and warmed within a closed container for one minute in a water bath at 37 o C. The tubes were gently agitated for one minute. The thawed blood was measured using a 1ml pipette and transferred to a 50ml sterile Falcon tube. NaCl solution was added to the thawed blood using a blunt end needle and syringe. First, 12% NaCl (0.2 times the volume of infected blood) was added drop by drop and left for 5 minutes inside the biosafety cabinet. Second, 1.6% NaCl (10 times the volume of infected blood) was quickly added drop by drop. Third, 0.9% NaCl (10 times the volume of infected blood) was quickly added drop by drop. When each drop of NaCl solution had been added, the mixture was agitated continuously using the right hand. Finally, the mixture was centrifuged at 1800rpm for 5 minutes. The supernatant was removed. The blood pack was washed with incomplete RPMI1640. After washing, the pellet was resuspended in 10ml of P. falciparum malaria culture medium (RPMI 1640 supplemented with 2.4 g/l d-glucose and Albumax II). The blood medium mixture (BMM) was then incubated in an in vitro culture system (5% CO 2, 5% O 2 and 90% N 2 ) at 37.5 C for 2 hours before being used for a drug susceptibility test (this preincubation step helps the parasite to equilibrate to normal cellular function post-thawing before being exposed to an anti-malarial drug). Finally, the BMM was transferred to the predosed plate for drug assay. 37

56 3.7. In vitro drug assay Drug plate preparation and assay design Drug plates were prepared from four antimalarial drugs (chloroquine diphosphate, artesunate, mefloquine and lumefantrine) which were from Sigma-Aldrich, USA. A stock solution was prepared for each drug using the appropriate solvent. Briefly, the drug powder was measured as ( g or 2.5 to 5mg) and dissolved in an equal volume of the appropriate solvent to obtain 1mg/ml stock solution. Different concentration ranges were prepared from each stock solution. The drug concentration range was adapted from Quashie et al.(149) and Woodrow et al.(150) with modifications. The concentration ranges were indicated in nanomolar, CQ (11.32 to nm, g/mol), MQ (3.64 to nM, g/mol), LUM (4.56 to nm, g/mol) and AS (0.68 to 88.45nM, 384.4g/mol). Chloroquine diphosphate and mefloquine were dissolved in ultrapure water, while lumefantrine was dissolved in DMSO (100%) and artesunate was dissolved in 70% ethanol. The two-fold serial dilution of each drug was made in 70% absolute ethanol. Then 25ul of each dilution was dispensed into sterile microplate in triplicate using a multichannel pipette. The microplates were left in the working biosafety cabinet overnight to dry the drugs. The microplates were then sealed with Top Seal-A Plus sealing tape (Perkin Elmer) and covered with microplate covers. Finally, the plates were stored at 4 o C until being used. Eight wells (7+1) per strip were used in triplicate per plate for each drug where the drug-free control wells were included in the first rows of the microplates. The assay was performed three times using different batches of parasite strains. Chloroquine resistant and sensitive strains were included in the assay for quality control. 38

57 3.7.2 In vitro microtest and parasite susceptibility to anti-malarial drugs An in vitro microtest (48-hour schizont maturation) assay was performed on laboratoryadapted falciparum strains and field isolates respectively on predosed microplates (microtest TM tissue culture plate 96 well, Becton Dickinson, USA). The plates were prepared as described above. After pre-incubation, percentage parasitemia (1%) and hematocrit (2%) were adjusted using uninfected fresh blood (group type O) and complete culture medium (RPMI1640, supplemented with Albumax II). Then 200µl of blood media mixture was dispensed into the pre-dosed drug plates. The plates were incubated in an in vitro culture system (5%CO 2, 5%O 2 and N 2 90% supply). Thick smears were done as described by Russell et al.(151) After the treated parasites were incubated for approximately 42 hours, the culture was harvested, and incubation was stopped when 80% of the ring stage parasites had developed to mature schizonts from the drug-free well. Thick smears were then prepared and dried from each well, and the slides were stained with 5% Giemsa for 13 minutes. The stained slides were dried and examined by light microscope under oil immersion objective. During the microscopic observation, only mature schizonts with 3 well-defined chromatin dots were counted. The number of schizonts per 100 asexual parasite stages was counted from each dried smear(eight concentration points per slide) Ring stage sensitivity assay A ring stage sensitivity assay was performed using two laboratory-adapted falciparum strains (3D7 and Dd2). These two strains have different genetic backgrounds. The first strain is sensitive to chloroquine, while the second strain is chloroquine resistant. This ring stage sensitivity assay was designed to assess the difference in sensitivity to artesunate of the ring stage populations. 39

58 3.7.4 Drug plate preparation and assay design Drug plates were prepared from a small amount of artesunate powder (0.002 gram). Sterile Falcon tubes (15ml) were prepared and labeled. A small amount of artesunate powder was measured and dissolved in 70% absolute ethanol to get a stock concentration of 1mg/ml. Then a high concentration of artesunate was prepared from the stock solution (100, 200 and 700nM) by dispensing a different amount of the stock solution on labeled microplates. The pre-dosed microplates were left overnight in a biosafety cabinet to dry the dispensed drug solution. The microplates were sealed using the top seal, and the prepared drug plates were stored in a refrigerator at 4 o C until being used. This experiment was performed in triplicate with both negative and positive controls. The negative control was three wells containing infected red blood cells without drug, and the positive control was the wells with drug and without washing of the drugs prior to harvesting (72 hours) Parasite incubation Parasite incubation was done after thawing and adjustment of both percentage parasitemia (0.5%) and hematocrit (2%). This percentage parasitemia was performed by diluting from higher parasitemia to 0.5%. This dilution allowed the expansion of the parasite for two full cycles of up to 72 hours for reinvasion of surviving merozoites after drug exposure and to prevent high parasitemia from the drug-free control well. The parasite was thawed, resuspended and incubated for 40 minutes to revive the thawed parasites and to enable the physiological activity of the parasite to adjust. Then the culture was taken out of the incubator and centrifuged. The packed cell was resuspended to the required volume. The parasitemia and hematocrit were adjusted to 0.5% parasitemia and 2% hematocrit using complete RPMI1640 respectively. Then 200ul of the culture suspension 40

59 was added to the pre-dosed drug plates. The plates were incubated in an in vitro culture system for 5 hours (first round). After 5 hours of drug exposure, the first plate was washed with complete RPMI1640, and the supernatant was removed. Finally, the washed pack cell was resuspended with 200ul of pre-warmed complete RPMI1640. After washing and resuspension of the packed cell with complete culture medium, the culture was re-incubated to allow the parasites to grow after drug exposure. After 24 hours of incubation, the medium was replaced. The same procedure was used for the rest of the ring stage parasites. The positive controls (the first rows from each 96-well microplate ) was not washed rather it was incubated for 72 hours under drug exposure Harvesting the culture In this experiment, parasites were incubated for two consecutive full asexual cycles to allow the surviving or dormant parasites to re-grow in the second cycle after drug exposure. Therefore, parasite harvesting was done after reincubation of the culture for 72 hours. During this incubation time, quiescent or dormant parasites were expected to survive and develop to mature stages. The phenotypes of surviving parasites were observed under a light microscope from Giemsa-stained thick smear, and photos were taken. In general, P. falciparum strains are sensitive to artesunate at 100nM, 200nM and 700nM concentrations in the standard WHO microtest with an incubation period of 48 hours. We therefore expected that ring stage parasites would also be sensitive to artesunate with minimal or insignificant variation within 5 hours exposure, regardless of their age and strain variation In vitro rosetting assay A rosetting assay was done from cryopreserved parasites as described by Lee et al. (2014). First, parasites were thawed and grown in 10% type matching serum supplemented 41

60 RPMI1640 for a few cycles and synchronized. Rosetting mature parasites (predominantly schizonts) were prepared at 4% parasitemia and 3% hematocrit. Finally, wet mount was prepared from the culture for microscopy. This direct wet mount was made from the culture for the rosetting assay Wet mount preparation A wet mount was prepared in an Eppendorf tube (0.6ml). The tubes were labeled, and 5µl of 100% double-filtered Giemsa stain was transferred into Eppendorf tubes. Then 95µl of the culture suspension was transferred to Eppendorf tubes. The cells were stained for 15 minutes. Meanwhile, clean, dry glass slides were prepared and labeled. After staining, 7.6µl of the stained culture suspension was placed on a glass slide and covered with a 22x32MM cover slip. Then the slides were observed under an oil immersion objective lens for rosettes. Both rosetting and non rosetting infected erythrocytes were counted, excluding ring stage infected erythrocytes. The counting was made up to 200IRBC. Photos of the rosettes were taken. The rosetting rate (RR) was calculated using the formula: (RR=(Rosette frequency)x 100) 200IRBC 3.9. Statistical analysis All statistical analyses were done using Graph Pad Prism 7. The graphs represent both the 50% inhibitory concentration and percentage growth inhibition of drugs against parasites. First, microscopic data were obtained from Giemsa-stained thick smears. Then the data were recorded on spreadsheets. The records were transferred to Graph Pad Prism 7 and used to calculate the IC 50 values for each drug. First, the drug concentration was transformed to log scale, and the dose inhibition-variable slope (four parameters) was used to obtain IC 50 values 42

61 and percentage growth inhibition curves. A nonparametric analysis (Kruskal-Wallis Test) was used to compare the IC 50 values within the group for the four anti-malarial drugs against each parasite strain. Then the Mann Whitney U Test was used to compare the differences between the drugs and between the strains for each of the antimalarial drugs. 43

62 CHAPTER FOUR 4. Results 4.1 In vitro parasite culture and synchronization Plasmodium falciparum laboratory strains were grown in vitro and maintained for several cycles. The parasites were synchronized and cryopreserved at tiny ring stages for in vitro drug assay and rosetting assay at different times. This in vitro parasite culture was done for a few weeks to prepare the parasites for the in vitro biological characterization and drug sensitivity assays (fig.4, A). We applied both synchronization and concentration methods and obtained a single stage of the cultured parasites (fig.4, B&C). 5% sorbitol was used to synchronize the culture, and highly synchronized tiny ring stage parasites were obtained (fig.4, B). On the one hand, the percoll gradient was used to concentrate mature stages of the parasite (fig.4, C). Concentrated late trophozoite/schizonts (80%) were obtained. Figure 4. In vitro parasite culture and synchronization A representative figure of a three-week in vitro cultured 3D7 strain (A). A minimum of one and half weeks (5-6 parasite life cycle)were used for synchronization of parasite. If the initial parasitemia were very low during thawing a maximum of three weeks were used. 5% sorbitol was used to synchronize the culture, and highly synchronized tiny ring stage parasites were obtained (B). Percoll gradient was used to concentrate mature parasite stages (C). The schizonts separated from the mixed stage parasites were resuspended with fresh blood (O-blood group type) and cultured further to get ring stage parasites for the drug assay. 44

63 4.2 Drug sensitivity profiling of P. falciparum In vitro drug sensitivity profiling of P. falciparum parasites was done using four antimalarial drugs to determine the drug sensitivity profiles of both lab-adapted falciparum strains and field isolates. After exposure of the parasites to anti-malarial drugs, parasite phenotyping was done using a light microscope, and the following results were obtained Microscopic observation of drug-treated and drug-free parasites The growth inhibition potential of different anti-malarial drugs was used to test the sensitivity profile and growth rate of the parasites. The growth of Plasmodium parasite under drug exposure arrested at tiny ring stage due to drug inhibition potential (B). On the other hand, the growth of Plasmodium parasite under drug-free conditions showed normal physiological conditions and reached to schizont stages (A). Comparison of the parasite phenotype from thick smear clearly shows a morphological variation between two conditions. The parasite remained at tiny ring stage when it was exposed to a high concentration of anti-malarial drugs (B), whereas the parasite grew to mature stages under normal physiological conditions (drug-free conditions) (A). Figure 5. Microscopic observation of drug-free and drug-treated parasite growth (A) Growth of parasites from tiny ring stage to mature schizont in drug-free conditions. (B) Inhibited or arrested parasite growth at tiny ring stages due to antimalarial drug treatment. 45

64 4.2.2 Growth inhibition and chloroquine sensitivity profiling of P. falciparum Chloroquine was used to study the in vitro sensitivity profiles of P. falciparum strains. A few falciparum strains were treated with chloroquine using the standard microtest assay, and the results were obtained from an independent microscopic counting (P 1 and P 2 ). Independent microscopic observation of thick smears reduces technical bias during thick smear microscopy. The growth inhibition and sensitivity profiling of each parasite was obtained (fig.6, A-C). The 50% inhibitory concentration (IC 50 values) of the drugs were found to be different (fig.6, D). The 3D7 strain was sensitive to chloroquine (IC 50 <100nM), whereas the other two falciparum strains (MKT1116 and ARS-233) were resistant to chloroquine (IC 50 >100nM (fig.6, D)). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between groups. There was a statistically significant difference between the groups in chloroquine sensitivity (P<0.05). 46

65 Figure 6. Growth inhibition and sensitivity profiling of parasite to chloroquine This in vitro experiment was performed in triplicate and two independent microscopic counting was performed. Three of the P. falciparum strains were exposed to chloroquine, and their in vitro parasitic growth rate was determined (A-C). The 3D7 strain was sensitive to chloroquine, whereas the other two falciparum strains (MKT1116 and ARS-233) were resistant to chloroquine (D). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between the groups. The stars on the figure represent, * P<0.05, **P<0.01, ***P<0.01, and **** indicate P < Growth inhibition and artesunate sensitivity profiling of P. falciparum The four falciparum strains were exposed to artesunate, and their growth rate was determined (fig.7, A-D). The 50% inhibitory concentration (IC 50 values) of each drug was found to be different (fig.7, E). All four falciparum strains (3D7, ARS-233, MKT1116 and ARS-272) were found to be sensitive to artesunate with IC 50 values below the reference values (IC 50 <10nM). The parasites' sensitivity profiles were compared to their IC 50 values and showed a difference in sensitivity within the groups. The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between the 47

66 groups. The stars on the figure represent, ns=p>0.05, *=P <0.05, **P<0.01,***P<0.01, and **** indicate P < Figure 7. Growth rate of Plasmodium strains exposed to artesunate This in vitro experiment was performed in triplicate and two independent microscopic counting was performed. Four different falciparum strains were treated with artesunate, and parasitic growth rate was determined (A-D). The 3D7 strain was sensitive to chloroquine, whereas the other three strains were resistant to chloroquine (D). These four falciparum strains (3D7, ARS-233, MKT1116 and ARS-272) were exposed to artesunate, and the parasites' sensitivity profile was compared with the IC 50 values of each parasite. All of the parasites were sensitive to artesunate, despite the fact that their IC 50 values vary between strains. The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between the groups. The stars on the figure represent, ns*p>0.05, P <0.05, **P<0.01, ***P<0.01, and **** indicate P < Growth inhibition and artesunate sensitivity of ARS-233 and MKT1116 Two P. falciparum strains (ARS-233 and MKT1116) were treated with a higher concentration range of artesunate ( ng/ml), and their growth rate was determined from microscopic counting of thick smears (fig.8, A-B). The sensitivity profiles of these two falciparum strains were compared. Both strains had been tested earlier and had been found to be chloroquine resistant, but the parasites were sensitive to artesunate below the cutoff 48

67 values (IC 50 <10nM). Despite the fact that their IC 50 values vary between MKT1116 and ARS-233 strains, the concentration range was higher by one extra point from the previous artesunate concentrations. The drug sensitivity profiles of both MKT1116 and ARS-233 were found to be different. Their sensitivity profiles were compared based on their IC 50 values, and these two strains showed a significant difference (P<0.05) in their sensitivity to artesunate (fig.8). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between the groups. The stars on the figure represent, ns*p>0.05, P <0.05, **P<0.01, ***P<0.01, and **** indicate P < Figure 8.Growth inhibition of ARS-233 and MKT1116 strains This in vitro experiment was performed in triplicate and two independent microscopic counting was performed. The ARS-233 (A) and MKT1116 (B) strains were exposed to a higher concentration range of artesunate, and the parasites' growth rate was determined. The concentration range of artesunate was higher by one extra point from the previous concentrations being used. The MKT116 and ARS-233 sensitivity profiles were compared 49

68 based on their IC 50 values (fig 8., C), and both strains showed statistically significant differences in their sensitivity to artesunate (P<0.05). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between the groups. The stars on the figure represent, ns*p>0.05, P <0.05, **P<0.01, ***P<0.01, and **** indicate P < Growth inhibition and sensitivity profiling of 3D7 strain The growth inhibition and sensitivity of the 3D7strain was tested in vitro against four antimalarial drugs (chloroquine, mefloquine, lumefantrine and artesunate). The growth rate of the 3D7 strain was determined for each drug based on Giemsa-stained thick smears and microscopic counting (fig.11, A-D). The growth of the parasite was inhibited at higher concentrations of each drug. The 50% inhibitory concentration (IC 50 values) of each drug against the 3D7 strain was obtained. When we compare the 50% inhibitory concentration (IC 50 values) of each drug, there was a statistically significant difference (P<0.05). Although the level of sensitivity was different for each drug, the parasite was found to be sensitive to the four anti-malarial drugs (chloroquine IC 50 <100nM, mefloquine IC 50 <30nM, lumefantrine IC 50 <150nM and artesunate IC 50 <10nM). The strain is inherently sensitive to chloroquine, and it was also found to be sensitive to the other three anti-malarial drugs. Statistical analysis was performed using the Kruskal Wallis Test, followed by the Mann Whitney U Test, for comparison of each drug against the 3D7 strain. The stars on the figure represent, ns=p>0.05, *P <0.05, **P<0.01, ***P<0.01, and **** indicate P <

69 Figure 9.In vitro growth inhibition and sensitivity profile of 3D7 strain The 3D7 strain was exposed to chloroquine (A), mefloquine (B), lumefantrine (C) and (D), and the growth rate of the 3D7 strain was determined for each drug based on Giemsa-stained thick smears and microscopic counting. The in vitro experiment was performed in triplicate independently. The 50% inhibitory concentration (IC 50 values) of each drug was determined. 3D7 is inherently sensitive to chloroquine, and it was also found to be sensitive to the other three anti-malarial drugs (E). The strain showed a significant sensitivity difference to the drugs. The stars on the figure represent, ns=p>0.05, * P <0.05, **P<0.01, ***P<0.01, and **** indicate P < Growth inhibition and sensitivity profiling of Dd2 strain In vitro growth inhibition of the Dd2 strain was done using chloroquine, mefloquine, lumefantrine and artesunate. Three out of four anti-malarial drugs (mefloquine, lumefantrine and artesunate) inhibited the growth rate of the Dd2 strain, as determined by microscopic counting of Giemsa-stained thick smears and growth inhibition graphs (fig.11, A-D). The Dd2 strain is a known chloroquine resistant parasite and was also found to be chloroquine resistant, but it was sensitive to mefloquine, lumefantrine and artesunate. Statistical analysis was performed using the Kruskal Wallis Test, followed by Mann Whitney U Test, to compare the statistical difference between the groups. The stars on the figure represent, 51

70 ns=p>0.05, *P<0.05, **P<0.01,***P<0.01, and **** indicate P < Although the strain was found to be sensitive to lumefantrine, lumefantrine sensitivity was less than that of the other strains. Figure 10. In vitro growth inhibition of Dd2 strain The Dd2 strain was exposed to chloroquine (A), mefloquine (B), lumefantrine (C) and artesunate (D). The growth rate of the Dd2 strain was determined for each drug based on Giemsa-stained thick smears and microscopic counting. The in vitro experiment was performed in triplicate independently. The 50% inhibitory concentrations of chloroquine, mefloquine, lumefantrine, and artesunate were tested for the Dd2 strain. The strain was chloroquine resistant, whereas it was sensitive to the other three drugs (E). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between the groups. The stars on the figure represent, ns=p>0.05, *P<0.05, P <0.05, **P<0.01, ***P<0.01, and **** indicate P < Growth inhibition and sensitivity profiling of FVT201 strain The FVT201 falciparum strain was treated in vitro with four anti-malarial drugs (chloroquine, mefloquine, lumefantrine and artesunate), and the growth rate of this parasite was inhibited by each of the drugs except mefloquine, which showed low inhibition (fig.12, A-D). The FVT201 field isolate was exposed to chloroquine, mefloquine, lumefantrine and artesunate. The growth rate of the FVT201 field isolate was determined from microscopic 52

71 counting of Giemsa-stained thick smears (fig.12, A-D). The 50% inhibitory concentration (IC 50 values) of each drug against the FVT201 isolate showed statistically significant level of variation (P<0.05). This isolate was resistant to mefloquine (IC 50 >30nM), but it was sensitive to chloroquine (IC 50 <100nM), lumefantrine (IC 50 < 150nM) and artesunate (IC 50 <10nM) (fig.12, E). The statistical analysis was performed using the Kruskal Wallis Test, followed by the Mann Whitney U Test, to compare the statistical difference between the groups. The stars on the figure represent, ns = P>0.05, *=P <0.05, **=P<0.01,***=P<0.01, and **** indicate P < Figure 11.In vitro growth inhibition of FVT201 strain The FVT201 field isolate was exposed to chloroquine (A), mefloquine (B), lumefantrine (C) and artesunate (D). The growth rate of the FVT201 field isolate was determined for each drug based on Giemsa-stained thick smears and microscopic counting. The in vitro experiment was performed in triplicate independently. The 50% inhibitory concentration (IC 50 values) of each drug against the FVT201 isolate was determined and compared. FVT201 was mefloquine resistant, but sensitive to the other three drugs (E). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between the groups. The stars on the figure represent, ns = P>0.05, *=P <0.05, **=P<0.01, ***=P<0.01, and **** indicate P <

72 4.2.8 Growth inhibition and sensitivity profiling of MKK183 strain In vitro growth inhibition of the MKK183 field isolate was conducted using four types of anti-malarial drugs. The growth rate of the MKK183 field isolate was inhibited by all of the anti-malarial drugs, based on Giemsa-stained thick smears and microscopic counting (fig.13, A-D). However, inhibition of the parasite by two of the drugs, chloroquine and mefloquine, was very low. The 50% inhibitory concentration (IC 50 values) of each drug against the MKK183 strain showed a significant level of variation (P<0.05). The strain was resistant to chloroquine (IC 50 >100nM) and mefloquine (IC 50 >30nM) but sensitive to both lumefantrine (IC 50 <150nM) and artesunate (IC 50 <10nM). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare the statistical difference between the groups. The stars on the figure represent, ns = P>0.05, *=P <0.05, **=P<0.01, ***=P<0.01, and **** indicate P <

73 Figure 12. In vitro growth inhibition of MKK183 strain The MKK183 field isolate was exposed to chloroquine (A), mefloquine (B), lumefantrine (C) and artesunate (D). The growth rate of the MKK183 field isolate was determined for each drug based on Giemsa-stained thick smears and microscopic counting. The in vitro experiment was performed in triplicate independently. The 50% inhibitory concentration of chloroquine, mefloquine, lumefantrine, and artesunate were determined for the MKK183 strain. MKK183 was chloroquine and mefloquine resistant but sensitive to lumefantrine and artesunate (E). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare statistical differences between the groups. The stars on the figure represent, ns = P>0.05, *=P <0.05, **=P<0.01, ***=P<0.01, and **** indicate P < Comparison of sensitivity of parasites to the four anti-malarial drugs In vitro drug sensitivity profiling of four falciparum strains was conducted using four antimalarial drugs (chloroquine, mefloquine, lumefantrine and artesunate (fig.9-13, A-D), and their IC 50 values were compared within and between groups (fig.13, A-D). Based on their IC 50 values, all four strains showed statistically significant levels of variation (P<0.05). Both laboratory-adapted falciparum strains and field isolates were compared. The 3D7 strain was sensitive to all four drugs, whereas the Dd2 strain was sensitive to only three of the drugs (mefloquine, lumefantrine and artesunate). The 3D7 and Dd2 strains were sensitive to 55

74 artesunate and mefloquine, and there was no a statistically significant variation between the two strains (P>0.05,fig.19,B&D)). However, the 3D7 and Dd2 strains showed sensitivity variation to lumefantrine (fig.19, C). The field isolates FVT201 and MKK183 showed a significant difference in sensitivity to chloroquine (P=0.01). Even though the FVT201 strain was resistant to mefloquine (IC 50 <30nM), and the MKK183 strain was mefloquine sensitive (IC 50 <30nM), the two parasites did not show significantly different levels of sensitivity to mefloquine (P>0.05, fig.19, B)). On the other hand, FVT201 and MKK183 parasites were sensitive to lumefantrine (IC 50 <150nM) and artesunate (IC 50 <10nM). These parasites showed differences in sensitivity to artesunate that were statistically significantly (P<0.01, fig 19, D). In general, all four falciparum strains showed a significant level of difference in sensitivity to all four anti-malarial drugs (A-D). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used for comparison of the statistical differences between the groups. The stars on the figure represent, ns=p>0.05,*=p <0.05, **=P<0.01,***=P<0.01, and **** indicate P <

75 Figure 13.Comparison of IC 50 values of four anti-malarial drugs against four parasites Four different parasite strains were exposed to chloroquine(a), mefloquine (B), lumefantrine (C) and artesunate (D) at different times as described earlier. The sensitivity profiles of each parasite were compared per drug, and the parasites showed statistically significant differences in sensitivity (P<0.05, fig.13, A-D). The Kruskal Wallis Test, followed by the Mann Whitney U Test, was used to compare statistical differences between the groups. The stars on the figure represent, ns =P>0.05, *=P <0.05, **=P<0.01, ***=P<0.01, and **** indicate P < Table 4. The effect of anti-malarial drugs on each P. falciparum strains Anti-malarial Anti-malarial activity of drugs(ic 50 nm) against P. falciparum strains Drugs 3D7 Dd2 ARS-272 ARS-233 MKT1116 FVT201 MK183 Chloroquine ND Mefloquine ND ND ND Lumefantrine ND ND ND

76 Artesunate Sensitivity profiling of P. falciparum ring stages Ring stage parasites are proposed to be an essential stage in the parasite s surviving artemisinin and artemisinin derivative drug treatment. Artesunate induces parasite dormancy in vitro, and the parasite revives after removal of the drug. Phenotyping of the parasite at this stage of survival and prediction of parasite recrudescence in patients have been challenges so far. Therefore, in this study we conducted a modified in vitro sensitivity assay (ring stage sensitivity profile assay) and tried to identify the parasite phenotype after short-term artesunate exposure and 72 hours of parasite re-incubation. The study was conducted on highly synchronized ring stage parasites at three different developmental stages (0-6 hours, 11 hours and 16 hours of ring stage parasites). We hypothesized that parasites at different ages respond differently to 5 hours exposure of artesunate, and we found that each subpopulation showed different sensitivity to high artesunate concentrations (100nM, 200nM and 700nM). We used parasites with two different genetic backgrounds. One of the parasites tested was chloroquine sensitive (3D7), and the second parasite was chloroquine resistant (Dd2). There was a significant difference in sensitivity of these two strains to artesunate (fig.18, F) Sensitivity profiling of 3D7 and Dd2 strain ring stages 3D7 and Dd2 ring stage parasites were used to observe the in vitro sensitivity profile of ring stage parasites to artesunate. The 3D7 strain is intrinsically sensitive to chloroquine, whereas the Dd2 strain is chloroquine resistant, possessing multi-copy numbers of the Pfmdr1gene. The parasites were prepared at three different developmental stages. This is due to the fact that both the stage of the parasite and the time of exposure to artesunate are factors in the in 58

77 vitro activity of artesunate, therefore 6-hour-old parasites (fig. 14&15, A) were exposed to three different concentrations of artesunate (100nM, 200nM and 700nM) in vitro. The other two stages from both strains, 11-hour old ring stage and 16-hour old ring stage, were also exposed to the same concentration of artesunate under similar conditions. We considered that the three different concentrations of artesunate enabled us to observe the survival or recovery potential of young parasite stages with different survival rates. Earlier studies had shown that late ring stage parasites were more sensitive to artesunate than young ring stage parasites under similar conditions, artesunate concentration and exposure time. In other words, as the parasite develops to mature stage it becomes more susceptible to artesunate. Therefore, we tested three different stages of the parasite at three artesunate concentrations and observed the parasite phenotype in vitro. The first three photos were taken from the initial stage of the parasite from Giemsa-stained thin smear (fig. 14&15, A) at each sampling time point. Figure 14. The 3D7 strain at different ring stage subpopulation The 3D7 strain was thawed and incubated for 45 minutes to obtain young ring stage parasites (A, 6 hour old ring) sampled for assay. Then, the remaining culture was re-incubated for an extra five hours (A, 11 hour old ring) and sampled for assay. Finally, the remaining culture was further incubated for an additional five hours (A, 16 hour old ring). 59

78 Figure 15. The Dd2 strain of different ring stage subpopulations The Dd2 strain was thawed and incubated for 45 minutes to obtain young ring stage parasites (A, 6 hours) and sampled for assay. Then the remaining culture was re-incubated for an extra five hours (A, 11 hour old ring) and sampled for assay. Finally, the remaining culture was further incubated for an additional five hours (A, 16 hour old ring) Phenotyping of 3D7 and Dd2 strains after short-term artesunate exposure The parasite phenotype is depicted in both treated and drug-free conditions. Parasites under drug-free conditions were fully grown from tiny ring stage to fully developed schizont (fig.15, A&B). The parasites undergoing a long period of artesunate exposure were used as a positive control. This group was represented by parasites which were exposed to three different concentrations of artesunate without washing off the drug for 72 hours. This condition arrested parasite growth completely, and the parasites remained at the ring stage in all the three concentration points (100nM, 200nM and 700nM)(fig.15, A). On the other hand, both 3D7 and Dd2 strains were exposed to high concentrations of artesunate (100nM, 200nM, and 700nM) for only five hours and further incubated for an extended time to allow the parasite to recover (72 hours). The phenotypes of treated and untreated parasites were observed from Giemsa-stained thick smears (fig.16&17, A & B). Although the growth was very slow, the parasites were able to recover after 72 hours of incubation at 100nM and 200nM (fig.16, B). The parasites did not recover at 700nM.There was also a different rate of recovery between 100nM and 200nM (fig.15, B). In general, parasite growth was slowed at 60

79 100nM and 200nM but arrested at 700nM, whereas the drug-free condition showed fully grown parasite phenotypes (schizonts).the parasite phenotype is depicted below from artesunate-treated, 6-hour 3D7 strain ring stages (fig.16&17, A&B). Figure 16.The phenotype of artesunate-treated 3D7 strain at 6-hour old ring stage The 3D7 strain was exposed to high concentrations of artesunate: 100nM, 200nM and 700nM (A&B). The experiment was performed in triplicate independently. In the first group, the parasite was exposed to 100nM, 200nM, and 700nM artesunate concentrations for 72 hours, and the drug was not washed off (A). The parasite did not recover and grew at 100nM, 200nM and 70nM artesunate exposure for two full cycles (72 hours). In the second group, similar parasite stages were exposed to 100nM, 200nM and 700nM artesunate for five hours, the drug washed off and the parasite re-incubated for 72 hours (B). The phenotypes of treated and untreated parasites were observed from Giemsa- stained thick smears(a&b). Figure 17.Phenotype of artesunate-treated Dd2 strain at 6-hour old ring stage The Dd2 strain was exposed to high concentrations of artesunate100nm (A), 200nM (B), and 700nM (C) for five hours and re-incubated for 72 hours (B). The phenotypes of treated and 61

80 surviving parasites were observed from Giemsa-stained thick smears. This experiment was performed in triplicate independently Phenotyping and sensitivity profiling of 3D7 and Dd2 strain ring stages The primary advantage of an in vitro assay is to resolve the limitation of in vivo drug response by the parasite and associated biological characteristics. Therefore, in vitro phenotyping and drug sensitivity profiling simplify this problem and enable us to characterize the parasite after anti-malarial drug treatment. Artesunate resistance and/or treatment failure are correlated with both delayed parasite clearance and ring stage dormancy. We treated two laboratory-adapted falciparum strains in vitro and phenotypically characterized (fig.18, A-F). The 3D7 and Dd2 ring stage parasites were grown under drug pressure and drug-free conditions. The drug-free parasites grew well, whereas the growth of treated parasites was affected at 100nM, 200nM and 700nM. However, the 3D7 parasite was able to recover at 100nM and 200nM artesunate concentrations (fig.18, A). On the other hand, the Dd2 ring stage parasite which was exposed to 200nM and 700nM artesunate was completely arrested, and the ring stage parasites did not show any growth phenotype when observed under light microscope (D). The sensitivity profile of ring stage parasites was compared between 3D7 and Dd2 strains. There was a statistically significant difference between the two strains of early ring stage populations (6 hours) and late ring stages populations (16 hours) (B&E). The 6-hour-old ring stage parasites recovered better than the 11-hour-old and 16-hour-old ring stage parasites of both strains, but the growth of the 3D7 strain was significantly different from that of the Dd2 strain at the 6-hour old ring stage(f). When we observe the 3D7 strain alone, there was a significance difference between the 6- hour ring stage and the 16-hour old ring stage, and the 11-hour and 16-hour old ring stage parasites (B). This comparison was not possible for the Dd2 strain, because the 11-hour old 62

81 ring stage and 16-hour old ring stages did not grow (E). The statistical analysis was performed using the Kruskal Wallis Test, followed by the Mann Whitney U Test. The stars on the figure represent, ns = P>0.05, *=P <0.05, **=P<0.01, ***=P<0.01, and **** indicate P < Figure 18.Sensitivity profile of 3D7 and Dd2 ring stages to artesunate The 3D7 and Dd2 ring stage parasites were treated with three different concentrations of artesunate (A &D).The experiment was repeated three times, and the results were recorded thick smears microscopy. Both parasites were fully grown to schizont in drug-free conditions (A&D). The growth of the parasite was affected at 100nM, 200nM and 700nM artesunate concentrations; there was limited growth at 100nM and 200nM, while there was no growth at 700nM (A&D).The 3D7 strain recovered at 100nM and 200nM(A), whereas the Dd2 strain recovered only at 100nM (D).The sensitivity profile of each parasite stage was determined, and parasites at 6-hour old ring stage showed a significance difference in sensitivity to artesunate (B,C,E&F).The 3D7 strain showed a higher recovery rate at 100nM compared to the Dd2 strain at the 6-hour old ring stage (B&E). 63

82 Table 5. The effect of artesunate on different stages of parasites P. falciparum strains Activity of artesunate against different stage of P. falciparum strains(%) 6-hour old 11-hour old 16-hour old 3D Dd An in vitro rosetting phenotype of P. falciparum The rosetting phenotype of the P. falciparum strain is the spontaneous binding of uninfected erythrocytes to Plasmodium-infected erythrocytes. Rosetting occurs due to the interaction between the parasite and host biomolecules. We did an in vitro rosetting test on P. falciparum strains and found that the parasite formed rosetting in vitro at mature stages. In vitro rosetting of P. falciparum parasites was observed by light microscope from wet mount (fig. 19, B). The nonrosetting infected erythrocyte is also described at (fig.19, A). Three replicates of in vitro rosetting assays were performed. The rosetting phenotype and rosetting frequency were determined, and in vitro rosetting was observed (fig.19, B&C). 64

83 Figure 19. In vitro rosetting and rosetting rate of P. falciparum strains Nonrosetting uninfected erythrocyte and P. falciparum parasite-infected erythrocyte (A). Rosetting of uninfected erythrocytes by P. falciparum parasite-infected erythrocytes as observed in wet mount (B). The rosetting rate of P. falciparum strains was observed in vitro from Giemsa-stained wet mount (E). The experiment was done in triplicate, and the rosetting rates were compared. 65