University of Ghana http://ugspace.ug.edu.gh THE EFFECTS OF LOCAL ANTIMALARIAL DRUGS ON PLASMODIUM FALCIPARUM GAMETOCYTES BY COURAGE KAKANEY, BSc Hons (10103244) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL PARASITOLOGY DEGREE. JULY, 2014 0 University of Ghana http://ugspace.ug.edu.gh DECLARATION This thesis is the result of research work undertaken by Courage Kakaney under the supervision of the following names listed below and all references stated have dully been acknowledged. COURAGE KAKANEY (Candidate 10103244) DR. LINDA AMOAH (Supervisor) DR. BETHEL KWANSA-BENTUM (Supervisor) DR. LANGBONG BIMI (Supervisor) i University of Ghana http://ugspace.ug.edu.gh DEDICATION I dedicate this thesis to the 2013/2014 academic year form 3 biology students of Adidome Senior High School, who had faith in me to continue as their elective biology teacher despite my postgraduate engagements. Their patience, joy and trust during the inconvenient meetings I had to arrange to prepare them for their final exams, gave me a lot of encouragement. This thesis is also dedicated to the loving memory of my mother, Bless Nayram Kpodo, who taught me that even the largest task can be accomplished if it is done one step at a time with the best of one’s ability. ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I would like to express my special appreciation and thanks to my supervisors, Dr. Linda Amoah of the Immunology Department, Noguchi Memorial Institute for Medical Research (NMIMR), Dr. Langbong Bimi and Dr. Bethel Kwansa-Bentum both of the Department of Animal Biology and Conservation Science, University of Ghana for their excellent guidance, advice and profound contribution throughout this work. I am also much grateful to Professor Ben Gyan, Head of Immunology Department, NMIMR for allowing me to work in the department. I am also very grateful to Dr. Fred Aboagye-Antwi, Department of Animal Biology and Conservation Science, Legon for his assistance in preparing me for parasite cultures. Very special thanks to Miss Andrea Arku and Mr. Kakra Dickson, both technicians at the Immunology Department for the training they gave me in parasite culturing and growth inhibition assays. I also want to thank Mrs. Ruth Ayanful of the Epidemiology Department, NMIMR for her assistance in reading out my drug assay plates. I am also indebted to Dr. Regina Appiah-Opong, Head of Chemical Pathology Department, NMIMR for permitting me use the facilities her department. I also want to thank Ebenezer Ofori- Attah (technician) and Abigail Aning (research assistant) at Chemical Pathology Department, NMIMR for their assistance in freeze drying my herbal samples. A special thanks to my students. Words cannot express how grateful I am for the patience, love, trust, they showed me throughout my study. The discussions, encouragements and support from course mates; Francis Apaatar, Nakie Nettey, Linda Owusua, Hannah Tetteh, Samuel Yeboah, Jennifer Suubaar is also very much appreciated. iii University of Ghana http://ugspace.ug.edu.gh My heartfelt thanks and gratitude to my family; especially my father, Emmanuel Kakaney, and sisters, Juliana Kakaney and Millicent Kakaney for their support and encouragement throughout the course of my study. My deepest gratitude goes to Merdiya Mohammed for her undying love throughout all these times. She was always there during hard times to encourage and support me forge ahead. Above all I give thanks to the Almighty creator for the strength and life given me to go through this course. iv University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ........................................................................................................................................... i DEDICATION .............................................................................................................................................. ii ACKNOWLEDGEMENT ........................................................................................................................... iii TABLE OF CONTENTS .............................................................................................................................. v LIST OF ILLUSTRATIONS ..................................................................................................................... viii LIST OF TABLES ........................................................................................................................................ x LIST OF APPENDICES .............................................................................................................................. xi LIST OF ABBREVIATIONS ..................................................................................................................... xii ABSTRACT ................................................................................................................................................ xv CHAPTER ONE ........................................................................................................................................... 1 GENERAL INTRODUCTION ..................................................................................................................... 1 1.1 Background to the study ..................................................................................................................... 1 1.2 Rationale of the study ......................................................................................................................... 3 CHAPTER TWO .......................................................................................................................................... 5 LITERATURE REVIEW ............................................................................................................................. 5 2.1. Malaria .......................................................................................................................................... 5 2.2. Causative agent and vectors .......................................................................................................... 7 2.3. Biology and life cycle of the parasite ............................................................................................ 8 2.3.1. Life cycle of the vector phase (the definitive host) ............................................................... 8 2.4 Clinical diagnosis .............................................................................................................................. 11 2.5 Clinical presentation of uncomplicated P. falciparum malaria ..................................................... 13 2.6 Clinical presentation of complicated P. falciparum malaria ......................................................... 15 2.7 Treatment of malaria ......................................................................................................................... 16 2.8 Antimalarial drug resistance ......................................................................................................... 17 2.9 Current orthodox antimalarial drugs ................................................................................................. 18 2.9.1 Quinolines .................................................................................................................................. 18 2.9.2 Antifolates .................................................................................................................................. 20 2.9.3 Artemisinins ............................................................................................................................... 21 v University of Ghana http://ugspace.ug.edu.gh 2.9.4 Antibiotics .................................................................................................................................. 24 2.10 Local antimalarial drugs.............................................................................................................. 24 Taabea herbal mixture ......................................................................................................................... 25 Masada mixture ................................................................................................................................... 25 Top fever syrup ................................................................................................................................... 26 Kingdom mixture ................................................................................................................................ 26 2.11 Herbal plants used in local antimalarial drugs ................................................................................ 28 2.11.1 Azadirachta indica ................................................................................................................... 28 2.11.2 Vernonia amygdalina ............................................................................................................... 29 2.11.3 Ocimum viride .......................................................................................................................... 30 2.11.4 Paullinia pinnata...................................................................................................................... 30 2.11.5 Moringa oleifera ...................................................................................................................... 30 2.11.6 Solanum torvum ....................................................................................................................... 31 2.12 Malaria transmission and epidemiology ......................................................................................... 31 2.12.1 Levels of endemicity ................................................................................................................ 32 2.14 Malaria eradication ......................................................................................................................... 33 CHAPTER THREE .................................................................................................................................... 36 MATERIALS AND METHODS ................................................................................................................ 36 3.2 Study design ...................................................................................................................................... 36 3.3 Herbal drug preparations ................................................................................................................... 36 3.4 Cultivation of Plasmodium falciparum cultures ............................................................................... 37 3.4.1 Culturing for asexual assay ............................................................................................................ 37 3.4.1.1 Sorbitol synchronisation for ring forms .................................................................................. 37 3.4.1.2 Plating of drugs for asexual assay ........................................................................................... 38 3.4.2 Culturing of gametocytes for drug assay ....................................................................................... 39 3.4.2.1 Purification of gametocytes .................................................................................................... 39 3.4.2.2 Plating of drugs for gametocyte assay .................................................................................... 40 3.5 Kinetics of herbal antimalarial drugs ................................................................................................ 41 3.6 Statistical analysis ............................................................................................................................. 41 3.7 Ethical clearance ............................................................................................................................... 41 RESULTS ................................................................................................................................................... 42 4.1. IC50s of herbal drugs ................................................................................................................... 42 vi University of Ghana http://ugspace.ug.edu.gh 4.2. Kinetics of the herbal drugs ........................................................................................................ 43 4.3. Early stage growth inhibition by herbal drugs ............................................................................ 52 4.4. Late stage growth inhibition by herbal drugs .............................................................................. 54 DISCUSSION AND CONCLUSION ......................................................................................................... 56 5.1 DISCUSSION ................................................................................................................................... 56 5.2 CONCLUSSION ............................................................................................................................... 64 RECOMMENDATIONS ............................................................................................................................ 65 REFERENCES ........................................................................................................................................... 66 APPENDIX 1 .............................................................................................................................................. 94 MATERIALS AND EQUIPMENT ........................................................................................................ 94 Equipment ........................................................................................................................................... 94 Materials ............................................................................................................................................ 95 APPENDIX 2 .............................................................................................................................................. 97 PREPARATION OF MEDIA AND REAGENTS ................................................................................. 97 Thawing mix ....................................................................................................................................... 97 Freezing mix ....................................................................................................................................... 97 7.5% sodium hydrogen carbonate (NaHCO3) ..................................................................................... 97 20% glucose ........................................................................................................................................ 97 Albumax .............................................................................................................................................. 97 Incomplete media (ICPM) or parasite wash media (PWM) ................................................................ 98 Complete medium ............................................................................................................................... 98 Complete media + 2%NHS ................................................................................................................. 98 Complete media + NAG ..................................................................................................................... 98 Preparation of 5% sorbitol .................................................................................................................. 98 APPENDIX 3 .............................................................................................................................................. 99 PLATE DESIGNS .................................................................................................................................. 99 Plate design for asexual assay ............................................................................................................. 99 Plate design for gametocyte assay .................................................................................................... 102 APPENDIX 4 ............................................................................................................................................ 104 ASEXUAL GROWTH INHIBITION CURVES .................................................................................. 104 8. APPENDIX 5 .................................................................................................................................... 110 MISCELLANEOUS PICTURES ......................................................................................................... 110 vii University of Ghana http://ugspace.ug.edu.gh LIST OF ILLUSTRATIONS Plate 1: Map showing trends in malaria incidence (World Health Organization, 2014) .............................. 6 Plate 2: Plasmodium falciparum merozoite showing apical complex and other cellular organelles (Cowman and Crabb, 2006). ......................................................................................................................... 8 Plate 3: The Plasmodium falciparum life cycle; a. asexual stage in human and b. sexual stage in mosquito (Wirth, 2002). .............................................................................................................................................. 11 Plate 4: Picture of ring stage Plasmodium falciparum ................................................................................ 43 Plate 5: Picture of a raptured schizont of Plasmodium falciparum ............................................................ 44 Plate 6: Picture of early stage Plasmodium falciparum gametocytes ........................................................ 45 Plate 7: Picture of late stage Plasmodium falciparum gametocytes .......................................................... 46 Figure 1: Effect of drug IC10s on the ring stage development ..................................................................... 47 Figure 2: Effects of drug IC10s on trophozoite development ...................................................................... 48 Figure 3: Effects of drug IC10s on schizont development ............................................................................ 49 Figure 4: Effects of drug IC10s on early gametocyte development ............................................................. 50 Figure 5: Effects of drug IC10s on late stage gametocyte development...................................................... 51 Figure 6: Inhibition of early stage gametocytes by drugs ........................................................................... 53 Figure 7: Inhibition of late stage gametocytes by drugs ............................................................................. 55 Figure 8: Growth inhibition curve for AS .................................................................................................. 104 Figure 9: Growth inhibition curve for KG .................................................................................................. 104 Figure 10: Growth inhibition of RT ............................................................................................................ 105 Figure 11: Growth inhibition curve of TF .................................................................................................. 105 Figure 12: Growth inhibition of CM .......................................................................................................... 106 Figure 13: Growth inhibition curve of MS ................................................................................................. 106 viii University of Ghana http://ugspace.ug.edu.gh Figure 14: Growth inhibition curve of AN ................................................................................................. 107 Figure 15: Growth inhibition curve of YF .................................................................................................. 107 Figure 16: Growth inhibition curve of HB ................................................................................................. 108 Figure 17: Growth inhibition curve of TB .................................................................................................. 108 Figure 18: Growth inhibition curve of AD ................................................................................................. 109 Plate 8: Plated drugs in an incubating chamber ....................................................................................... 110 Plate 9: Gametocytes after percoll purification ........................................................................................ 111 ix University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1: Some examples of local antimalarial drugs on Ghanaian market ................................................. 25 Table 2: The herbal drugs showed IC50s significantly higher than artesunate with p<0.05. ...................... 42 Table 3: Plate 1 for asexual assay ............................................................................................................... 99 Table 4: Plate 2 for asexual assay ............................................................................................................. 100 Table 5: Plate 3 for asexual assay ............................................................................................................. 100 Table 6: Plate 4 for asexual assay ............................................................................................................. 101 Table 7: Plate 5 for asexual assay ............................................................................................................. 101 Table 8: Plate 1 for gametocyte assay ...................................................................................................... 102 Table 9: Plate 2 for gametocyte assay ...................................................................................................... 102 Table 10: Plate 3 for gametocyte assay ................................................................................................... 103 x University of Ghana http://ugspace.ug.edu.gh LIST OF APPENDICES APPENDIX 1 Materials and Equipment……………………………………………………………….94 APPENDIX 2 Preparation of media and reagents……………………………………………………..97 APPENDIX 3 Plate design…………………………………………………………………………….99 APPENDIX 4 Asexual growth inhibition curve………………………………………………………104 APPENDIX 5 Miscellaneous pictures…………………………………………………………………110 xi University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS WHO World Health Organisation G6PD Glucose-6-phosphate dehydrogenase CSIR Center for Scientific and Industrial Research ACT Artemisinin based combination therapy OPD Outpatients’ department GMP Global Malaria Programme IC50 Inhibitory concentration of 50% IC10 Inhibitory concentration at 10% HIV/AIDS Human Immuno-Deficiency Virus / Acquired Immune Deficiency Syndrome GDP Gross Domestic Product RBM Roll Back Malaria MMV Medicines for Malaria Venture MVI Malaria Vaccine Initiative RBC Red Blood Cell RDT Rapid Diagnostic Test pLDH Parasite Lactate Dehydrogenase HRP-II Histidine Rich Protein II Ht Hematocrit AL Arthemether-lumefantrine AS Artesunate PQ Primaquine AQ Amodiaquine MQ Mefloquine SP Sulphodoxine-pyrimethamine DHA Dihydroartemisinin xii University of Ghana http://ugspace.ug.edu.gh PPQ piperaquine CQ Chloroquine Pgh1 P- glycoprotein homologue 1 Pfmdr1 Plasmodium falciparum multidrug resistance protein 1 Pfcrt Plasmodium falciparum chloroquine Resistance transporter pABA p-aminobenzoic acid DHPS dihydropteroatte synthase DHFR dihydrofolate reductase SERCA sarcoplasmic-endoplasmic reticulum calcium ATPase RNA Ribonucleic Acid TB Taabea Herbal Mixture MS Masada Mixture HB Herbaquin TF Top Fever Syrup KG Kingdome Mixture YF Yafo Fever Mixture CM Class Malakare AD Adutwumwa Malamix AN New Angel Herbal Mixture RT Rooter Mixture EIR Entomological inoculation rate ELISA Enzyme linked immunosorbent assay DDT Dichlorodiphenyltrichloroethane TB Tuberculosis VIMT Vaccines that interrupt malara transmission FDA Foods and Drugs Authority BSC Biosafety cabinet xiii University of Ghana http://ugspace.ug.edu.gh CPM Complete parasite media PWM Parasite wash media NAG N-Acetyl Glucosamine NHS Normal Human Serum Rpm Revolutions per minute Rcf Relative centrifugal force DMSO Dimethyl sulfoxide IRB Institutional review board NMIMR Noguchi Memorial Institute for Medical Research xiv University of Ghana http://ugspace.ug.edu.gh ABSTRACT The gametocytocidal effect of antimalarial drugs has become important in malaria research owing to the global quest to eradicate the disease. In Ghana and most parts of Africa, people still depend on herbal medications despite advances in western medicine. However, these drugs lack the necessary scientific assessment. The IC50s of 10 known herbal antimalarial drugs on the Ghanaian market were determined. The development of sexual stages of the Plasmodium falciparum was monitored and the growth inhibitory effect of the herbal drugs on gametocytes was also assessed in vitro. All tested herbal drugs showed either high activity (IC50<10µg/ml) or moderate activity (10 µg/ml 50% growth inhibition of early stage gametocytes at 7 µg/ml. Only Masada herbal gave > 50% growth inhibition of late stage gametocytes at 7 µg/ml. Though all the herbal drugs were potent antimalarials, only Masada and Yafo fever showed strong gametocytocidal effects and can therefore be recommended as an alternative to orthodox antimalarial treatment. xv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE GENERAL INTRODUCTION 1.1 Background to the study Malaria, a disease caused by Plasmodium spp. remains a major health problem in sub-Saharan Africa (World Health Organization, 2012a). It is responsible for an estimated 207 million cases worldwide and 627,000 deaths in 2012 with 80% of the cases and 90% of the deaths occurring in Africa (World Health Organization, 2014). The life cycle of the Plasmodium sp. shows the gametocyte stage as the infectious stage to the Anopheles sp., which as the vector, it habours and transforms the parasite into sporozoites, the infective stage to humans. Since the pathophysiology of malaria is mostly due to the asexual stages of the Plasmodium parasite within the human host (Miller et al., 2002), most antimalarial drugs target the asexual stages of the parasite, which is responsible for the clinical symptoms of malaria. All currently available antimalarial compounds were discovered on the basis of their activity against the asexually reproducing red blood cell stages of the Plasmodium spp. (Buchholz et al., 2011). However, for the transmission of parasites to be possible, the gametocyte stage of the parasite must be transmitted from the human host to the Anopheles mosquito. Compared to the asexual parasites, gametocytes are less sensitive to all the commonly used antimalarial drugs except for primaquine (Benoit-Vical et al., 2007). Primaquine is currently the only licensed antimalarial drug that is effective against late stage Plasmodium falciparum gametocytes but has certain drawbacks such as its tendency to cause acute haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency (Peatey et al., 2012). 1 University of Ghana http://ugspace.ug.edu.gh Due to the emergence of resistance against orthodox drugs coupled with unsuccessful attempts to develop vaccines based on asexual blood stage antigens makes it important to focus on gametocyte stages of the Plasmodium parasite to prevent transmission instead. The Bill and Melinda Gates Foundation in 2007 spearheaded the campaign for a global eradication of malaria, which will be possible through the elimination of malaria transmission(Buchholz et al., 2011). The elimination and a possible complete eradication of malaria can be achieved if the transmission cycle of the Plasmodium parasite is interrupted. The earlier mentioned problems with orthodox medication coupled with the high cost of western medical care as well as socio-cultural practices make more than 80% of populations in Africa prefer the use of local medicine (World Health Organization et al., 2009). A major problem about traditional medicine in Ghana is the lack of test on their efficacy and toxicity. That notwithstanding, the Council for Scientific and Industrial Research (CSIR) and Ghana Standards Authority conduct tests into herbal drugs that are licensed and sold on the Ghanaian market. Antimalarial herbal drugs used in Ghana are usually concoctions with one or more active plant extracts. Even though some individual active ingredients like Azadirachta indica have shown reduction in asexual parasitemia and gametocytes of Plasmodium falciparum (Udeinya et al., 2008), it is not known if a combination of these active ingredients could be synergetic. There is also little or no information about the concentration of the active ingredients and their mode of action. For now the efficacy of these local drugs are based on the adverts and the testimony of users. With the new worldwide agenda on the total eradication of malaria, local antimalarial drugs must be tested to see if they actually block the transmission of parasites. A major challenge in testing the gametocytocidal activity of compounds is the difficulty in developing an effective assay (Lelièvre et al., 2012). The gold standard which involves counting 2 University of Ghana http://ugspace.ug.edu.gh gametocytes under the microscope is labour intensive and time consuming (Peatey et al., 2012). To add further, Plasmodium falciparum mature gametocytes are hard to get in large quantities in in vitro cultures. This has resulted in sparse information on in vitro studies since most of the available information on gametocytocydal activity of existing antimalarials was obtained from field-based clinical trials (Butcher, 1997). The awakened interest in parasite transmission blocking has made it important to develop new reliable and effective methods in determining gametocytotoxicity. It therefore, informs on which of these local antimalarial drugs should be endorsed as a herbal alternative in championing the course for malaria eradication in Ghana. It also provides insight into prospects of developing new antimalarial drugs from herbal sources, citing the problems of isolated drug resistant of Artemisinin and its derivatives, the linchpin of current drug of choice (Phyo et al., 2012). 1.2 Rationale of the study In Ghana, malaria still accounts for 32.5% of outpatients’ department (OPD) cases despite efforts to curb its incidence (Sesay and Esena, 2013). In accordance with the World Health Organization (WHO) regulations, the first drug of choice for malaria is artemisinin-based combination therapy (ACT) (World Health Organization, 2012a). Artemisinin-based combination therapy has been known to induce some gametocytocidal effects (Broek et al., 2005). The use of ACTs coupled with vector control measures has dramatically reduced the morbidity and mortality of malaria worldwide (Chong et al., 2013). However resistance to ACT has been discovered in Cambodia and Thailand (Dondorp et al., 2010), which is feared to hamper the progress of malaria eradication by 2015. The resistance to ACTs will be a dangerous consequence to the global 3 University of Ghana http://ugspace.ug.edu.gh malaria control, because there might be no suitable drugs for replacements (World Health Organization, 2014). Artemisinin being a drug developed from the plant Artemisia annua (Miller and Su, 2011), like its predecessor quinine, gives the hope that other herbal compounds could also be developed into very potent antimalarials in the future. Studies on local antimalarial drugs have proven to be effective against the symptoms of malaria (Asase et al., 2010). However since gametocytes do not elicit clinical symptoms, the gametocytocidal effects of these local antimalarial drugs cannot be assessed by way of consumption by patients. It may be possible that some of these drugs promote gametocytogenesis, hence inhibiting the symptoms and enhancing malaria transmission. Based on the high patronage of local antimalarial drugs by majority of Ghanaians (Tabi et al., 2006), identifying the gametocytotoxicity of these drugs would help inform on the drug of choice for those who seek herbal treatment. This would in a long way help eradicate malaria in Ghana. 1.2.1 Aim of study The main aim of the study was to determine the gametocytocidal effects of local antimalarial drugs. The specific objectives are: 1. To calculate IC50 for selected local antimalarial drugs 2. To identify local drugs that promote gametocyte production 3. To identify local drugs that inhibited the growth of gametocytes 4 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.1. Malaria Malaria is the most important parasitic disease of human beings. In 2012, there were an estimated 207 million cases with 627 000 deaths (World Health Organization, 2014). More than 85% of malaria cases and 90% of malaria deaths occur in sub-Saharan Africa, mainly in young children (White et al., 2014). In Africa, a child dies from malaria every 30 seconds, which translates to 2880 children per day (Finkel, 2007). Malaria, together with Human Immunodeficiency Virus / Acquired Immuned Deficiency Syndrome (HIV/AIDS) and tuberculosis, is one of the world’s most vital public health challenge compromising developing countries and it accounts for up to an overwhelming 2.7 million deaths per year (Breman, 2001). Malaria is endemic in 108 countries inhabited by about 3 billion (approximately 40%) people. As such, the disease is largely responsible for the poor economic growth of these areas, which further contributes to more cases of malaria (Gardner et al., 2002). 5 University of Ghana http://ugspace.ug.edu.gh Plate 1: Map showing trends in malaria incidence (World Health Organization, 2014) Malaria is commonly associated with poverty and may also be a major hindrance to economic development (Teklehaimanot and Mejia, 2008). In countries with endemic malaria, the annual economic growth rates over a 25-year period were 1.5% lower than in other countries. This implies that the cumulative effect of the lower annual economic output in a malaria-endemic country was a 50% reduction in the per capita GDP compared to a non-malaria endemic country (Gallup and Sachs, 2001). Malaria is a complicated disease and its spread may be attributed to a variety of factors such as ecological and socio-economic conditions, displacement of large populations, agricultural malpractices that cause increase in vector breeding sites, parasite resistance to antimalarial drugs and vector resistance to insecticides. In 1998, The World Health Organization (WHO) 6 University of Ghana http://ugspace.ug.edu.gh established a global partnership called Roll Back Malaria (RBM) in an attempt to half the world’s malaria frequency by 2010 (Nabarro, 1999). Apart from RBM, a number of promising antimalarial drug and vaccine discovery projects have also been launched. This includes the Medicines for Malaria Venture (MMV) funded by a number of organizations including The Bill and Melinda Gates Foundation, for the development of novel antimalarial drugs. The later also contributed more than 300 million US dollars to the Malaria Vaccine Initiative (MVI) (Gross, 2003). 2.2. Causative agent and vectors Five species of Plasmodium are known to infect and cause clinical cases in human beings. They are Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale and Plasmodium knowlesi. The most important of these protozoans are Plasmodium falciparum and P. vivax because they cause majority of deaths. Plasmodium falciparum which is the most lethal of the plasmodium species happens to be the most common human malaria parasite in sub- Saharan Africa (Greenwood, 2009). Plasmodium ovale and P. malariae cause milder forms of malaria and are less fatal (Mueller et al., 2007). Plasmodium knowlesi is a zoonotic species prevalent is Southeast Asia, causing malaria in macaques but can also cause severe malaria in humans (Collins, 2012). Effective transmission of these parasites depends on the density, longevity, biting habits, and efficiency of the anopheline vector. Out of the more than 400 anopheline species, only 25 are good transmitters of malaria (Sinka et al., 2010). The most effective vector is the Anopheles gambiae complex found in Africa (Bass et al., 2007). Having the most lethal plasmodium species and most effective anopheline vector, puts sub-Saharan Africa in constant risk of high malaria mortality and morbidity. 7 University of Ghana http://ugspace.ug.edu.gh 2.3. Biology and life cycle of the parasite The Plasmodium is an intracellular Apicomplexan parasite. Parasites of the Apicomplexan phylum are characterized by the presence of a special apical complex that is involved in host-cell invasion; the apical complex is made up of the microneme, dense granules and rhoptries (Cowman and Crabb, 2006). Plate 2: Plasmodium falciparum merozoite showing apical complex and other cellular organelles (Cowman and Crabb, 2006). 2.3.1. Life cycle of the vector phase (the definitive host) The life cycle begins with a blood meal by the female anopheline mosquito. The female mosquito takes regular blood meals to help in the development of its eggs (Briegel, 1985). The peripheral blood taken by the mosquitoes contains gametocytes of plasmodium which end up in the mid-gut of the mosquito. The male and the female gametocytes move from the red blood 8 University of Ghana http://ugspace.ug.edu.gh cells and differentiate to microgametes (male) and macrogametes (female). Fertilization takes place when a microgamete penetrates a macrogamete to form a zygote (the only diploid stage during the life cycle of the Plasmodium parasite). The zygote transforms into an ookinete, an elongated mobile cell inside the mosquito mid-gut, about 18 hours after blood meal. The ookinete penetrates the gut wall and is attached to the outer aspect of the mosquito gut, then the ookinete differentiate to a spherical oocyst. In the oocyst, multiplication takes place (i.e. meiosis) and the parasite becomes haploid again. This stage of multiplication is called sporogony, where thousands of sporozoites are produced. When the oocyst is mature, it ruptures to release sporozoites which migrate and accumulate in the salivary glands and complete maturation. The average duration of the sporogony is about 2 weeks with a range of 1-5 weeks, depending on species and temperature. Inoculation of the sporozoites into a new human host continues the life cycle. 2.3.2. Life cycle of the human phase (intermediate host) Sporozoites enter blood with mosquito saliva; which serves as an anticoagulant during blood meal (Stark and James, 1996). Usually an infected mosquito injects on average 10-100 sporozoites per blood meal. The sporozoites take few minutes to an hour to reach the liver and invade liver cells (hepatocytes). In the hepatocytes, the sporozoites transform to trophozoites which grow and divide by schizogony, to produce thousands of infective merozoites, about 10,000 for P. vivax and 30,000 for P. falciparum. The duration of hepatic schizogony is about 5 days for P. falciparum, but 13 days for P. malariae (Vaughan et al., 2008). Clinical symptoms of malaria are not apparent during liver stage maturation. In P. vivax and P. ovale, sporozoites may become dormant in the hepatocytes, as hypnozoites, which reactivate at variable intervals (most 9 University of Ghana http://ugspace.ug.edu.gh often 3-35 weeks) to cause relapses of malaria (Krotoski, 1989). The hepatic schizonts rupture and merozoites are released into the blood stream and rapidly invade RBCs. In the red blood cell, the merozoites transform into trophozoites, feeding on the content of the red blood cell and form food vacuoles where haemoglobin is digested. After a period of growth, a mature trophozoite starts dividing (erythrocytic schizogony) and produces a new generation of merozoites. A mature schizont of P. falciparum contains approximately 16 merozoites. The erythrocytic cycle lasts for about 48 hours for P. falciparum, P. vivax and P. ovale, but 72 hours for P. malaria and only 24 hours for P. knowlesi. When this stage is reached, the schizonts rupture releasing merozoites and other parasite byproducts (pyrogenic molecules) into the bloodstream. The pyrogenic molecules are responsible for sequence of events leading to fever and other clinical symptoms and signs of malaria. Immediately after release, the merozoites invade uninfected RBCs and repeat the cycle of erythrocytic schizogony. Some merozoites differentiate into sexual stages known as gametocytes (gamegony)- as early as 4 days for P. vivax and 10 days for P. falciparum. Adherence of infected RBCs to endothelial cells in various organs is called sequestration, which is characteristic of P. falciparum infections. Sequestration plays an important role in pathophysiology of severe malaria particularly cerebral malaria and malaria during pregnancy (Miller et al., 1994). 10 University of Ghana http://ugspace.ug.edu.gh Plate 3: The Plasmodium falciparum life cycle; a. asexual stage in human and b. sexual stage in mosquito (Wirth, 2002). 2.4 Clinical diagnosis Prompt and accurate diagnosis of malaria is an essential part of malaria case management and a key to reducing morbidity and mortality. Accurate diagnosis is also important for differential diagnosis of other febrile illnesses including sepsis, meningitis and pneumonia in which delayed diagnosis and treatment may be a cause of mortality (Berkley et al., 1999). The diagnosis of malaria is based on recognizing clinical symptoms and confirmation with detection of parasites in the blood. In sub-Saharan Africa, many primary health care facilities lack diagnostic equipment, such as microscopes, and well-trained staff for high-quality laboratory diagnosis (Ngasala et al., 2008). Malaria diagnosis is therefore still based on clinical features such as fever and presence of anaemia (World Health Organization et al., 2009). However, symptoms and signs of malaria are nonspecific. It is common to have symptoms of malaria overlap with many 11 University of Ghana http://ugspace.ug.edu.gh other acute febrile illnesses and co-infections(Berkley et al., 1999; Källander et al., 2004; Luxemburger et al., 1998; O’Dempsey et al., 1993; Tahita et al., 2013), as a result malaria misdiagnosis and over-diagnosis are very common in sub-Saharan Africa (Amexo et al., 2004; Gwer et al., 2007; Reyburn, 2004). The need to confirm parasites in the blood before therapy has become very important because of resistance of P. falciparum to the available monotherapies such as chloroquine, which is now replaced with artemisinin-based combination therapy(World Health Organization et al., 2009). The laboratory methods of malaria diagnosis include light microscopy and parasite antigen detection also known as the rapid diagnostic tests (RDTs). 2.4.1 Light microscopy Laboratory diagnosis by microscopy examination of stained blood smears remains the gold standard for case management, epidemiological studies, clinical trials of antimalarial drugs and vaccines, and a quality assurance of other malaria diagnostics tests. The microscopy of Giemsa stained blood films has high sensitivity and specificity when used by well-trained staff, which can detect as low as 50 parasites/ µl blood under field conditions (Moody, 2002; Payne, 1988). Other advantages of microscopy include low cost, determination of parasite densities, distinction between parasite species, differentiation between parasite stages and the possible diagnosing of other diseases. However the accuracy and usefulness of microscopy depends on factors such as the quality of the microscope, reagents and experience of the technician (Makler et al., 1998). 12 University of Ghana http://ugspace.ug.edu.gh 2.4.2 Rapid diagnostic tests Rapid diagnostic tests use immune-chromatographic technology to detect antigens derived from malaria parasites (Wongsrichanalai et al., 2007). Malaria antigens currently used as diagnostic targets include histidine-rich protein II (HRP-II) specific for P. falciparum (Murray and Bennett, 2009). Another antigen is parasite lactate dehydrogenase (pLDH) for detection of Plasmodium species. Monoclonal antibodies against pLDH can differentiate between P. falciparum and P. vivax (Murray and Bennett, 2009). There is limited data on the use of RDTs to identify P. knowlesi, however findings from a study show that the pLDH antibodies that detect P. falciparum and P. vivax can also be used to detect and distinguish P. knowlesi (McCutchan et al., 2008). Plasmodium aldose enzyme can also be used as a universal antigen target for all malaria parasites(Murray et al., 2008). Unlike HRP-II, pLDH is cleared from the blood about the same time as the parasites following successful treatment (Moody et al., 2000; Wongsrichanalai et al., 2007). However both PLDH and aldolase are expressed in gametocytes, limiting their use in monitoring response to therapy (Mueller et al., 2007). Rapid diagnostic tests offer a good alternative to microscopy, they are simple to perform and do not require skilled personnel (Bell et al., 2001). However limitations to RDTs include high cost, lack of density determination, persistent positivity and low sensitivity (Murray et al., 2003). 2.5 Clinical presentation of uncomplicated P. falciparum malaria Malaria is an acute illness. The first common symptoms are nonspecific and include; fever, headache, joint aches, malaise, vomiting, diarrhoea, body weakness, chest pain and poor appetite. These clinical symptoms usually appear 10-15 days after a person is infected with sporozoites. The symptoms of malaria vary according to the state of immunity, species of infecting parasites, and the epidemiology of malaria (Bruce et al., 2000; Doolan et al., 2009). 13 University of Ghana http://ugspace.ug.edu.gh In P. falciparum malaria, fever starts after few days of prodromal symptoms which starts during the last days of the incubation period which ranges from 9 to 14 days. At the onset, the fever is irregular, but usually occurs daily. It may be intermittent or continuous, and shows no sign of periodicity until the illness has continued for a week or more. The symptoms present in the prodromal phase continue and increase, so configuring a flu-like syndrome. Anorexia, dyspepsia, epigastric discomfort, nausea, vomiting and watery diarrhoea are frequent and may be misdiagnosed as a gastrointestinal infection. A dry cough and an increase in the respiration rate may becoming noticeable, arising the suspicion of an acute respiratory infection. Other non- specific physical findings are tender hepatosplenomegaly, orthostatic hypotension, and some degree of jaundice. The pulse rate of the infected person may become rapid measuring 100 to 120 beats/min and the blood pressure may be low (90–100 mmHg, systolic) (Strickland, 2001). When periodic febrile paroxysms occur, they may be daily (quotidian), every third day (tertian) or at about 36-hour intervals (subtertian). Laboratory exams may show little or no anaemia in mild cases and in otherwise healthy individuals. When present, it is usually normochromic and normocytic. The reticulocyte count is normal or depressed despite haemolysis, and increases only after the parasitemia is cleared. Serum haptoglobin may be undetectable indicating haemolysis. However, the degree of anaemia may greatly vary and is not always an indication of the severity of the attack. The white cell count is usually normal, but leucopenia with a count of 3,000–6000 cells/μl may be observed. Thrombocytopenia is common (usually around 100,000/μl) and does not correlate with severity. Serum transaminases and lactic dehydrogenase usually are moderately elevated as well as bilirubin concentrations, both conjugated (hepatocyte disfunction and/or cholestasis) and unconjugated (haemolysis). Prothrombin and partial tromboplastin time may be prolonged while 14 University of Ghana http://ugspace.ug.edu.gh fibrinogen levels are usually elevated. C-reactive protein and procalcitonin value are usually increased in both uncomplicated and severe malaria with levels correlated with parasitemia and prognosis according with some studies (Ahiboh et al., 2008; Chiwakata et al., 2001; Uzzan et al., 2006). Hyponatremia may arise in some cases, a transient increase in serum creatinine and blood urea nitrogen may be observed. Urine analysis reveals albuminuria and urobilinogen. Examination of peripheral blood film mostly reveals the presence of early trophozoites (typical small ring forms), often numerous, with multiple infected red blood cells. Late trophozoites, as well as schizonts, are seldom observed, being their presence an indicator of very heavy infections and poor prognosis. Gametocytaemia usually occurs in the second week after the onset of parasitemia and may persist for some weeks after cure (Wernsdorfer and McGregor, 1988). Although the subject may not appear very ill, serious complications may develop at any stage. In non-immune people P. falciparum malaria may progress very rapidly to severe malaria unless appropriate treatment is started. If the acute attack is rapidly diagnosed and adequately treated, the prognosis of falciparum malaria is good, even if complications may still occur. The response to treatment is usually rapid with resolution of fever and most symptoms within 3 days (White, 2011). Recrudescence with renewal of clinical manifestation and/or parasitemia, due to persistent erythrocytic forms, may occur. 2.6 Clinical presentation of complicated P. falciparum malaria Clinical features of uncomplicated malaria may progress to severe malaria if a patient is untreated or treated with ineffective drugs. The WHO has developed specific diagnostic criteria 15 University of Ghana http://ugspace.ug.edu.gh for severe malaria(World Health Organization, 2010). A patient is classified to be suffering from severe malaria if one or more of the following features are present: severe anaemia (haemoglobin < 5 g/dL or Ht < 15%), a common feature in infants and young children in malaria endemic areas; cerebral malaria (unarousable coma), with a Blantyre coma scale < 3 (in children) and Glasgow coma scale < 10 (in adults), a common feature in adult and children in areas of low transmission; Prostration; Respiratory distress induced by metabolic acidosis. Other criteria include Convulsions (> 1 episode/ 24 hours); Hypoglycaemia (< 2.2 mmol/L); Extreme weakness; Acidosis (plasma bicarbonate < 15 mmol/L); Renal failure (serum creatine above normal range for age); Shock; Macroscopic haemoglobinuria; Spontaneous bleeding; Pulmonary edema; Clinical jaundice; Hyperlactataemia (venous lactic acid > 5 mmol/L); and Hyperparasitaemia (> 25000/µL or > 5% of RBCs) (Pasvol, 2005). For patients with severe malaria receiving treatment, case fatality rate can reach 36.5% and if untreated severe malaria is fatal in majority of cases(Olliaro, 2008). 2.7 Treatment of malaria Due to development and spread of P. falciparum resistance to conventional monotherapies such as chloroquine and sulphodoxine-pyrimethamine (Myint et al., 2004), the WHO has recommended treatment of malaria with antimalarials that have different modes of action and different biochemical targets in the parasite, with the preference falling on ACTs (Wells et al., 2009). Artemisinin derivatives are extremely potent antimalarials that can also decrease malaria transmission because of its considerable effect on gametocytes(Barnes et al., 2005; Price et al., 1996; Sutherland et al., 2005). The WHO currently recommends the following ACTs: arthemether-lumefantrine (AL), artesunate-amodiaquine (AS+AQ), artesunate-mefloquine (AS+MQ), artesunate-sulphodoxine-pyrimethamine (AS+SP) and dihydroartemisinin- 16 University of Ghana http://ugspace.ug.edu.gh piperaquine (DHA+PPQ) (World Health Organization, 2010). Many countries in Africa, including Ghana have recently adopted ACTs, which are being deployed for treatment of uncomplicated malaria. The treatment of severe malaria includes clinical assessment of the patient, specific antimalarial treatment, adjunctive therapy and supportive care(World Health Organization, 2012b) . The specific antimalarial treatments for severe malaria include the cinchona alkaloids (quinine and quinidine) and artemisinin derivatives (artesunate, artemether and artemotil). Quinine is also a drug of choice for malaria in the first trimester of pregnancy, and malaria in children weighing <5 kg. Artesunate suppositories, which can be administered rectally, are recommended for pre-referral management of severe malaria in areas where parenteral antimalarial treatment would not be feasible (World Health Organization, 2010). 2.8 Antimalarial drug resistance The spread of resistance to antimalarial drugs is a major obstacle to effective case management; Chloroquine resistance in Africa resulted in increased morbidity and mortality (Björkman and Bhattarai, 2005; Korenromp et al., 2003; Marsh, 1998; Trape, 2001; Zucker et al., 2003), hence a major setback to malaria eradication. The switch to ACTs to treat uncomplicated malaria looks to be facing a hindrance with reported cases of resistance on the Thai-Cambodia border (Dondorp et al., 2010). Preserving the efficacy of the antimalarial drugs in use is of critical importance to public health in malaria endemic areas. Widespread use of antimalarial drugs for all types of fever and exposure to sub-therapeutic doses of drugs due to non-compliance of recommended dosages contribute markedly to drug resistance and necessitate continual change in malaria treatment guidelines (Rajakaruna et al., 2008). 17 University of Ghana http://ugspace.ug.edu.gh In 1973, the WHO defined antimalarial drug resistance as ―the ability of a parasite strain to survive and/or multiply despite the proper administration and absorption of an antimalarial drug in the doses equal to a higher than those normally recommended but within the tolerance of the subject‖. This definition was modified in 1986 to include the phrase ―drug must gain access to the parasite or the infected red blood cell for the duration of the time necessary for its normal action‖ (Bruce-Chwatt, 1979). Recent development of techniques for in vitro drug-sensitivity testing, high-performance liquid chromatography and molecular biology have improved our understanding of mechanisms of drug resistance. In this definition it is important to consider pharmacokinetic properties of antimalarial drugs, which vary in different individuals. For instance lumefantrine has inter-individual variation in bioavailability up to 20 fold (Ezzet et al., 2000). Currently, to confirm true drug resistance the blood concentration of the drug and its metabolite must be considered, also to be considered are recrudescence of the parasites, results of in vitro drug sensitivity and genetic markers of resistance (Ezzet et al., 2000; Vestergaard and Ringwald, 2007). 2.9 Current orthodox antimalarial drugs Malaria has been fought against with several drugs that have been developed to curb its menace on mankind. These drugs have varied mechanisms, which interfere with essential life processes of the parasite for its survival. 2.9.1 Quinolines The bark of the Cinchona officinalis tree has been used for centuries to treat fever associated with malaria infection from which the active ingredient is quinine. It remained the antimalarial drug of choice until the 1940s, when other antimalarial drugs such as its derivative; chloroquine 18 University of Ghana http://ugspace.ug.edu.gh replaced quinine. Quinine, however, is still used to treat clinical malaria and not as a prophylaxis due to its side effects and poor tolerability. Chloroquine is a 4-aminoquinoline derivative of quinine and for many years it was the main antimalarial drug used in the treatment of malaria until parasite resistance developed in the 1950s (Payne, 1987). It remains the most popular antimalarial developed to date due to its safety, low cost and efficacy (Björkman and Bhattarai, 2005). Today, the widespread of resistance to the drug has rendered it useless as a therapeutic agent, even though it still shows some efficacy in the treatment of the other human malaria parasites(Trape et al., 1998). Despite years of research, the actual molecular mechanism of chloroquine action remains an elusive topic. It is believed that chloroquine accumulates in the food vacuole of the parasite and prevents haem detoxification (Bray et al., 2005). Chloroquine resistance in malaria parasites has been attributed to the reduced concentrations of the drug in the + + food vacuole possibly due to drug efflux, pH modification in the vacuole, the role of a Na /H exchanger and transporters (Bray et al., 2005; Foley and Tilley, 1998). In P. falciparum, two genes have been implicated in resistance, namely Pfmdr1 and Pfcrt, which encodes Pgh1 and PfCRT proteins, respectively. Both of these proteins are localized to the food vacuole membrane. Mutations in these genes could lead to small increases in the food vacuole pH thus reducing the amount of chloroquine that can accumulate, rendering the drug ineffective (Spiller et al., 2002). Alternatively, PfCRT may increase the efflux of chloroquine by directly interacting with the drug (Cooper et al., 2007). Resistance is associated with several mutations in the PfCRT protein, while the loss of a Lys residue at position 76 has been shown as the critical mutation rendering the P. falciparum parasites resistant to the drug. This residue is located within the first transmembrane segment of PfCRT and may therefore play an important role in the properties of the channel or transporter (Cooper et al., 2005). Mutations in the Pfmdr1 gene is associated with resistance to mefloquine, quinine and halofantrine (Price et al., 2004; Reed et al., 2000). 19 University of Ghana http://ugspace.ug.edu.gh A number of related aminoquinolines have since been developed and applied. These include the following;  Amodiaquine – effective against chloroquine resistant strains, possible cross-resistance with chloroquine, has safety limitation (Bathurst and Hentschel, 2006). ® Atovaquone – usually used in combination with proguanil (Malarone ), resistance reported in 1996, it has cost limitation (Bathurst and Hentschel, 2006). TM Lumefantrine – usually co-formulated with arthemeter (Co-Artem ) and is highly effective against multi-drug resistant P. falciparum (Falade et al., 2005).  Halofantrine (Halfan): resistance reported in 1992, it has cost and safety limitations (Bathurst and Hentschel, 2006).  ®Mefloquine (Lariam ) – resistance reported in 1982, it has cost and safety limitations (Bathurst and Hentschel, 2006).  Primaquine – used for its gametocytocidal effect (P. falciparum) and its efficacy against intra hepatic forms of all types of malaria, no resistance, safety limitations (Bathurst and Hentschel, 2006; Chauhan and Srivastava, 2001). 2.9.2 Antifolates The antifolates are some of the most widely used antimalarial drugs. However, their role in malaria prevention is hampered by the rapid emergence of resistance once the parasites are placed under drug pressure. The direct effect of folate biosynthesis inhibition is a reduction in the synthesis of the amino acids serine and methionine as well as in pyrimidines, which leads to decreased synthesis of DNA. The antifolate drugs target the intra-erythrocytic stages as well as gametocytes of P. falciparum (Olliaro, 2001). The antifolates can generally be divided into two 20 University of Ghana http://ugspace.ug.edu.gh classes; the type-1 antifolates mimic the p-aminobenzoic acid (pABA) substrate of dihydropteroate synthase (DHPS) and include the sulfonamides (sulfadoxine) and sulfone (dapsone). While the type-2 antifolates (pyrimethamine and proguanil) inhibit dihydrofolate reductase (DHFR) (Olliaro, 2001). Interestingly both of these classes of target proteins are arranged on separate bifunctional enzymes (hydroxymethyldihydropterin pyrophosphokinase/dihydropteroate synthase or PPPK/DHPS and dihydrofolate reductase/thymidylate synthase or DHFR/TS) (Ivanetich and Santi, 1990). In addition, malaria parasites are capable of in vivo folate salvage from the extracellular environment as well as synthesizing folate derivatives from simple precursors. The mechanism of exogenous folate uptake by a carrier-mediates process has important implications in increasing the sensitivity of the antifolate inhibitors and is being investigated as a novel drug target (Wang et al., 2007). Pyrimethamine is a diaminopyrimidine and is mostly used in combination with sulfadoxine (Fansidar™) or depose leading to the simultaneous inhibition of DHFR and DHPS. Pyrimethamine crosses the blood-brain barrier as well as the placenta. Resistance to sulfadoxine-pyrimethamine combination therapy, however, emerged rapidly due to the appearance of point mutations in the active sites of the target enzymes resulting in reduced drug binding capacity (Cowman and Lew, 1990; Plowe et al., 1998). 2.9.3 Artemisinins Artemisinin is a sesquiterpene lactone extracted from the leaves of Artemisia annua. It is a potent, fast acting blood schizontocide that allows efficacy against all Plasmodium stages (Klayman, 1985). Its efficacy is especially broad and shows activity against all the asexual stages of the parasites including gametocytes (Mutabingwa, 2005; Skinner et al., 1996). The effects of 21 University of Ghana http://ugspace.ug.edu.gh artemisinin on gametocytes make it a good candidate for the prevention of malaria transmission. Originally, the mechanism of action of artemisinin was thought to be mediated by the peroxide ring of the drug, which is cleaved and activated by ferrous iron in the heme stores into toxic free radicals that can subsequently damage intracellular targets via alkylation (Meshnick et al., 2003). Recently, however, this theory was challenged by evidence that artemisin exerts its inhibitory effects on the malarial sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) resulting in an alteration of intracellular calcium levels (Eckstein-Ludwig et al., 2003). The actual mechanism of action, however, remains elusive and there have been different results from various studies (Krishna et al., 2004). Several derivatives of artemisinin have been developed since artemisinin itself is poorly absorbed and include dihydroartemisinin, artesunate (sodium salt of the hemisuccinate ester of artemisinin), artemether (methyl ether of dihydroartemisinin) and arthemether (ethyl ether of artemisinin) (Frédérich et al., 2002). Currently, the WHO recommends artemisinin-based combination therapy (ACT) as the first-line treatment against malaria infections where resistance to other antimalarial drugs is prevalent. One of the obvious disadvantages of using ACT for malaria case-management in Africa is the increased cost involved in combining therapies. Even so, several reasons exist for combining antimalarial drugs with an artemisinin derivative, namely an increase in the efficacy of antimalarial drugs, a decrease in the treatment period and a reduced risk of resistant parasites arising through mutation (Kremsner and Krishna, 2004). Several ACTs that have been developed and may have entered clinical trials are listed below (Gelb, 2007):  Pyramax: artesunate and the 4-aminoquinoline pyronaridine  Co-Artem™: artemether and lumefantrine  Artekin™: dihydroartemisinin and the quinolone-based drug piperaquine 22 University of Ghana http://ugspace.ug.edu.gh  Lapdap™: artesunate, chlorproguanil and dapsone  ASAQ: artesunate and amodiaquine There are several reasons why the appearance of parasite resistance to artemisinin was originally thought to be unlikely or at least delayed. These were spelt out by Meshnick (2002) as follows; 1. Parasites are not exposed to the drug for prolonged periods due to the short half-life of the drug. 2. Artemisinin is gametocytocidal, which reduces the transmission potential and spread of the parasite and 3. The frequent use of artemisinin combined with other antimalarial drugs (ACT) specifically introduces delay in the onset of resistance Evidence for in vitro resistance to an artemisinin derivative, however, appeared in field isolates from French Guiana in 2005. The increased arthemether IC50 s were ascribed to the presence of a mutation in the SERCA PfATPase6 gene and was attributed to inappropriate drug use that exerted selection pressures, favouring the emergence of parasites with an arthemether-resistant in vitro profile. There have been reports of resistance to artemisinin in Western Cambodia (Noedl et al., 2008). Even though reduced in vitro drug susceptibility is not tantamount to diminished therapeutic effectiveness, it could lead to complete resistance and thus called for the rapid deployment of drug combinations (Jambou et al., 2005). Lapdap™, a combination of chlorproguanil (targets DHFR), dapsone (targets DHPS) and the artemisinin derivative artesunate, was introduced in 2003 as malaria therapeutics to replace sulfodoxine-pyrimethamine treatment in Africa (Edwards and Biagini, 2006). 23 University of Ghana http://ugspace.ug.edu.gh 2.9.4 Antibiotics Evidence from in vivo experiments and clinical studies has shown that several antibiotics such as tetracycline, doxycycline and minocycline are active against the P. falciparum parasite (Geary and Jensen, 1983). The tetracyclins are antibiotics that were originally derived from Streptomyces species, but are usually synthetically prepared. They interfere with aminoacyl- tRNA binding and therefore inhibit protein synthesis in the parasite’s apicoplast or mitochondrion (Dahl and Rosenthal, 2008). This is due to the presence of genes in the mitochondrion and apicoplast that encode prokaryote-like ribosomal RNAs, tRNAs and various proteins (Dahl et al., 2006). Doxycycline is a synthetic tetracycline derivative with a longer half- life than tetracycline. The disadvantage of these antibiotics as antimalarial drugs is the development of abdominal cramps, diarrhoea, vaginitis and photosensitivity during treatment (Andersen et al., 1998). In vitro studies have shown that the effectiveness of antibiotics used as antimalarial drugs is inhibited by the presence of iron (Pradines et al., 2001). 2.10 Local antimalarial drugs There are numerous local antimalarial drugs on the Ghanaian market. They are mostly made up of one or more herbal extracts. The Foods and Drugs Authority has licensed a number of them to be sold even in pharmacies. However one can still find some unlicensed local antimalarial drugs on the open market. Below are 10 local antimalarial drugs licensed by the FDA for malaria treatment. 24 University of Ghana http://ugspace.ug.edu.gh Table 1: Some examples of local antimalarial drugs on Ghanaian market Trade name Dosage Indications Ingredients Adults Children Taabea herbal mixture 2 tablespoonful 1 tablespoonful Jaundice, typhoid, malaria, Ocinum viride, Azadirachtha indica, three times daily three times daily menstrual pains, menstrual Paullinia pinnate, Tetrapleura disorder, candidiasis, general tetraptera, Theobroma cacao, body pains, loss of appetite Cymbopogon citratus, Moringa oleifera Masada mixture 2 tablespoonful 1 tablespoonful Malaria Cryptolepis sanguinolenta (30 ml) three (15 ml) three times daily times daily Herbaquin 5 tablespoonful 2 tablespoonful Malaria Cryptolepsis sanguinolenta, Alstonia (75 ml) twice a (30 ml) twice a boonei, Azadirachta indica, Monodora day day myristica, Xylopia aethiopica Prophylaxis dosage: 1-2 25 University of Ghana http://ugspace.ug.edu.gh tablespoonful three times daily Top fever syrup 2 tablespoonful 1 tablespoonful Malaria fever Azadirachta indica, Alstonia boonei (30 ml) three (15) three times times daily daily Kingdom mixture 3 tablespoonful 1 tablespoonful Malaria Nauclea latifolia, Phyllanthus (45 ml) three (15 ml) three fratemus, Cryptolepsis sanguinolenta times daily times daily Yafo fever mixture 4 tablespoonful (60 ml) two times Malaria Cryptolepsis sanguinolenta, daily Azadirachta indica Class malakare 2 tablespoonful 1 tablespoonful Malaria, typhoid, body pains, Carapa procera, Cryptolepsis (30 ml) three (15 ml) three headache, tiredness, stress sanguinolenta 26 University of Ghana http://ugspace.ug.edu.gh times daily times daily Adutwumwa malamix 2 tablespoonful 2 tablespoonful Malaria, typhoid Anthocleista nobilis, Vitex grandifolia, (30 ml) three (30 ml) two Phyllanthus fratemus times daily times daily New angel herbal 2 tablespoonful 1 tablespoonful Jaundice, menstrual pain, Cola gigantean, Solanum torvum, mixture (30 ml) three (15 ml) three malaria, fever, loss of Spathodea campanulata, Bombax times daily times daily appetite, body pains buonopozense, Vernonia amygdalina Rooter mixture 4 dessert 3 desert Malaria, typhoid, jaundice Aloe schweinfurthil, Khaya spoonful, three spoonful, three senegalensis, Pileostigma thornnigil, times daily times daily Cassia siamea 27 University of Ghana http://ugspace.ug.edu.gh 2.11 Herbal plants used in local antimalarial drugs The local antimalarial drugs are made of the extracts of herbal plants that have traditionally been used in the treatment of malaria. With more interest in the interest of herbal medicine, some of these herbal plants have been studied scientifically to ascertain their efficacy and curative properties. 2.11.1 Azadirachta indica Azadirachta indica, also known as neem or nimtree is a tree within the family Meliaceae. It is one of the two species of the genus Azadirachta. It is native to India, Pakistan and Bangladesh; it grows in tropical and semi-tropical regions. Neem-based products from Azadirachta indica have been used extensively in traditional medicine in India for over 2000 years (Thakurta et al., 2007). It has been widely used for the treatment of helminthic, fungal, bacterial, viral and protozoan infections. It is a major component in the treatment of diabetic, skin and hepatic disorders (Thakurta et al., 2007). In Ghana Azadirachta indica is mainly used in the treatment of malaria. The leaves are boiled and concoction drunk or used for steam bath (Asase et al., 2010). There is substantial scientific research evidence to support the therapeutic effects of Azadirachta indica. For example extracts from neem have been shown to inhibit the growth of bacteria (Biswas et al., 2002). There have also been reports of the antiviral activities of neem tree extracts on viruses in dengue fever (Parida et al., 2002) and HIV (Mbah et al., 2007; Udeinya et al., 2004). Susceptible to neem tree extracts are important protozoa; Trypanosome (Yanes et al., 2004) Leishmania (Tahir et al., 1998) and Plasmodium; both asexual stages and gametocytes (Udeinya et al., 2008). The gametocytocidal activity of neem and transmission blocking properties (Lucantoni et al., 2010), makes it a potential candidate for the eradication of malaria. 28 University of Ghana http://ugspace.ug.edu.gh Of equal importance is the study which showed that neem extracts protects neurons from apoptosis during cerebral malaria (Bedri et al., 2013). 2.11.2 Vernonia amygdalina Vernonia amygdalina is a perennial shrub of the Asteraceae family that grows in tropical Africa. It is commonly known as ―bitter leaf‖ due to the bitter taste of its leaves. Vernonia amygdalina typically grows to a height of 2-5 m. The leaves are elliptical and up to 20 cm long (Ijeh and Ejike, 2011). In West Africa the plant has been domesticated and is used for both nutritional and medical purposes (Igile et al., 1994). The leaves which are edible are used in preparing the popular bitter leaf soup and its leaf extracts are also used as a tonic. It contains 79.93 % moisture, 20.08% dry matter, 35.81g protein, 68.35g carbohydrates, 25.47g dietary fibre and 4.70g lipids per 100g of dry weight, it also contains important vitamins and minerals such ascorbic acid, carotenoid, calcium, iron, phosphorus (Ejoh et al., 2007). Other nutritional elements found are potassium, sulphur, sodium, manganese, copper, zinc, magnesium and selenium (Atangwho et al., 2009; Bonsi et al., 1995; Eleyinmi et al., 2008). Vernonia amygdalina has widely been used in traditional medicine as an antihelminthic, antimalarial and laxative herb. It was observed that a sick chimpanzee in the wild chewed the leaves of the Vernonia amygdalina plant and after some time returned its normal state (Jisaka et al., 1993) . This led to further research into the therapeutic effects of the plant. It was shown to affect the weight, liver, urine and faecal output, plasma and liver cholesterol concentrations in mice (Igile et al., 1994). The antidiabetic effects of aqueous extract of leaves of Vernonia amygdalina was reported (Akah and Okafor, 1992). Reports show that low concentrations of 29 University of Ghana http://ugspace.ug.edu.gh water-soluble leaf extracts of V. amygdalina, potently retards the proliferative activities of human cancerous cells (Gresham et al., 2008). In Ghana the leaves are used in treating cough (Akinpelu, 1999). The antiplasmodium activity of Vernonia amygdalina has also been reported against Plasmodium falciparum in vitro and in vivo (Omoregie et al., 2011), showing adequate clinical response in the treatment of uncomplicated malaria cases (Challand and Willcox, 2009). 2.11.3 Ocimum viride Ocimum viride also known as basil is a tender-growing aromatic annual herb native to West Africa (Danso-Boateng, 2013). Ocimum viride leaves are used for the treatment of malaria in Ghana (Shankar et al., 2012). Ocinmum sp. contains thymol (Keita et al., 2000), which gives the plant its known anti-fungal and antibiotic properties (Mbata and Saikia, 2005). 2.11.4 Paullinia pinnata Paullinia pinnata is an African tropical plant whose roots and leaves are used in traditional medicine for many purposes; notable among them is the treatment of erectile dysfunction (Zamble et al., 2006). Paullinia pinnata has also been investigated to possess antibacterial properties (Anani et al., 2000). The plant has also been traditionally used for dressing wounds; research has proved its wound healing properties and cyto-protective activities (Annan and Houghton, 2010). Paullinia pinnata also showed some anti-plasmodial effects against Plasmodium berghei (Maje et al., 2007). 2.11.5 Moringa oleifera Moringa oleifera which belongs to the family Moringaceae is a highly valued medicinal plant, distributed in many countries of the tropics and subtropics. It has so many nutritional and 30 University of Ghana http://ugspace.ug.edu.gh medicinal benefits (Anwar et al., 2007). Moringa has been used to combat malnutrition in developing countries especially among infants and nursing mothers(Fahey, 2005). Moringa is known to have anti-bacterial properties (Fahey et al., 2002) and anti-fungal properties (Chuang et al., 2007). The susceptibility of Helicobacter pylori to compounds derived from Moringa (Haristoy et al., 2005) makes it efficacious in the treatment of gastritis and ulcers. Moringa has also shown good prospects in the prevention of cancer (Guevara et al., 1999). Moringa is also a widely used natural antimalarial product (Gbeassor et al., 1990). 2.11.6 Solanum torvum Solanum torvum belongs to the family Solanaceae, commonly known as Turkey berry is native to and cultivated in tropical Africa and West Indies. The fruits are used in most parts of Africa as vegetable and for medicinal purposes (Jaiswal, 2012). Solanum spp. seeds have antioxidant properties (Waghulde et al., 2011). Solanum torvum contains a number of important active phyto-chemicals like isoflavonoid sulfate and steroidal glycosides (Arthan et al., 2002), chlorogenone and neochlorogenone (Carabot Cuervo et al., 1991). Flavonoid intake has been known to be inversely related to mortality from coronary heart disease in epidemiological studies (Hertog et al., 1993). Solanum sp. is a used as an antimalarial by the Ga-Adangbe people of Ghana (Asase et al., 2010) 2.12 Malaria transmission and epidemiology Vectors are the key determinants of malaria transmission despite other factors such as parasite strain and health of individual are also involved. The distribution and abundance of an effective 31 University of Ghana http://ugspace.ug.edu.gh vector depends on environmental conditions like optimum temperature, altitude, humidity, seasonal pattern of rainfall and suitable water for breeding of larval stages (Robert et al., 2003). The intensity of malaria transmission is assessed by how often people are bitten by anopheline mosquitoes infected with sporozoites. The entomological inoculation rate (EIR) is the number of infective mosquito bites received per person per unit time (Hay et al., 2000). Measurements of sporozoites index from mosquito samples can be determined either microscopically by dissecting the mosquitoes for their salivary glands or using immunological assays such as Enzyme-linked immunosorbent assay (ELISA) to detect Plasmodium-specific circumsporozoite antigens (Gilles and Warrell, 2002). The vectorial capacity is a transmission probability index, i.e. the expected number of humans infected per infected humans, per day, assuming perfect transmission efficiency (Smith and McKenzie, 2004) or number of new infections the population of a given vector would distribute per case per day at a given place and time, assuming conditions of non-immunity (World Health Organization, 2010). The vectorial capacity is an indicator of stability of transmission, and depends on relative density of female anophelines, probability of mosquito to take blood meal, proportion of mosquitoes surviving incubation period and probability of daily survival. The key factor in determining intensity of transmission is the probability of daily survival of mosquitoes (Gilles and Warrell, 2002) 2.12.1 Levels of endemicity Geographical areas can be classified by intensity of transmission based upon the percentage of children, aged 2-9 years with splenomegaly or malaria parasitemia (Ngasala, 2010): Holo-endemic: (>75%) transmission occurs all year long 32 University of Ghana http://ugspace.ug.edu.gh Hyper-endemic: (50-75%) intense, but with periods of no transmission during dry season Meso-endemic: (11-50%) regular seasonal transmission Hypo-endemic: (<10%) very intermittent malaria transmission Malaria transmission can also be classified as stable, if the populations are continuously exposed to a fairly constant rate of malaria inoculations (EIR > 10/ year) or unstable if (EIR < 5/ year) and when the level of acquired immunity determines patterns of disease (World Health Organization, 2010). Stable and high malaria transmission occurs in holoendemic and hyperendemic areas, such as in tropical Africa. Unstable and low to moderate transmission occurs in meso and hypoendemic areas, such as much of tropical Asia, Central Asia and Latin America (Hay et al., 2004). 2.14 Malaria eradication In the 1950s, the World Health Organization and other international organizations made a pledge to eradicate malaria (Pampana and Russell, 1956) based on two assumptions that would later be seen as false. That is firstly, human malarial infection could be eliminated by treatment with antimalarial drugs such as chloroquine and secondly, transmission could be eliminated with residual insecticides such as dichlorodiphenyltrichloroethane (DDT). Firstly, the prevalence of chloroquine resistance, which was boosted by massive chloroquine use during the malaria eradication campaign made it impossible to clear Plasmodium falciparum parasitemia in many regions of Southeast Asia and South America with chloroquine. Secondly, both insecticide resistance and exophilic transmission undermined the efficacy of residual insecticides. In addition, massive problems with logistics, planning, resource allocation, and a lack of operational research contributed greatly to the failure of malaria eradication (Bruce-Chwatt, 1979). 33 University of Ghana http://ugspace.ug.edu.gh Due to the major logistical problems involved, the malaria eradication campaign formulated by the World Health Organization in the 1950s focused primarily on Southeast Asia and South America (Pampana and Russell, 1956)rather than sub-Saharan Africa, where the intensity of transmission and the morbidity and mortality of malaria were greatest. Even though, biologically and technically, malaria is not an ideal candidate for eradication (Aylward et al., 2000; Henderson, 1987; Stuart-Harris, 1984), there have been renewed efforts in malaria eradication. Malaria eradication is the permanent reduction to 0 of the worldwide incidence of malaria infection caused by a specific agent; i.e. applies to a particular malaria parasite species (World Health Organization, 2010). During the past 5 years, there has been a substantial increase in international funding for malaria control through major international financing mechanisms such as the Global Fund to fight HIV, TB and Malaria, the US President’s Malaria Initiative and the World Bank’s Booster Programme (Mendis et al., 2009). This, together with a high level of political commitment in endemic countries, has resulted in increased coverage of malaria interventions in endemic areas, and a reduction in malarial disease and death in several countries, including several in sub-Saharan Africa where the burden of malaria is greatest. Inspired by these achievements and by the momentum created by global advocacy, the possibility of malaria eradication has been placed again on the agenda of global health (Feachem and Sabot, 2008; Okie, 2008; Tanner and de Savigny, 2008). Therefore after a lapse of almost 40 years, the malaria eradication has been re-introduced on the global health agenda inspired, by the Gates Malaria Forum in October 2007 (Greenwood, 2009; Roberts and Enserink, 2007). Although there is lack of sufficient knowledge, systems and tools to eradicate malaria today, there is hope in the window of political will and financial resources to achieve the goal of eliminating the disease globally (Tanner and de Savigny, 2008). 34 University of Ghana http://ugspace.ug.edu.gh Vaccines can be very important in the efforts to eradicate malaria. Current strategies in developing malaria vaccines are primarily focused on Plasmodium falciparum and are directed towards reducing morbidity and mortality (Bojang et al., 2001). However if malaria vaccines are to eliminate malaria, they will need to have an impact on malaria transmission (Kaslow et al., 1988). This has led to the introduction of ―vaccines that interrupt malaria transmission‖ (VIMT), which includes not only ―classical‖ transmission-blocking vaccines that target the sexual and mosquito stages but also pre-erythrocytic and asexual stage vaccines that have an effect on transmission (The malERA Consultative Group on Vaccines, 2011). Vaccines that interrupt malaria transmssion also include vaccines that target the vector to disrupt parasite development in the mosquito. Importantly, if eradication is to be achieved, malaria vaccine development efforts will need to target other malaria parasite species, especially Plasmodium vivax, where novel therapeutic vaccines against hypnozoites or preventive vaccines with effect against multiple stages could have enormous impact (Alonso et al., 2011). 35 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHODS 3.1 Chemicals, reagents and equipment The sources and manufacturers of reagents, buffers, solutions and equipment used in the study are shown in the Appendix. 3.2 Study design The study is a part of an ongoing project to identify local antimalarial drugs that will prevent malaria transmission and recommend them as alternative to the first line drug of choice in malaria treatment in Ghana. The study looked at the susceptibility of chloroquine resistant 3D7A Plasmodium falciparum asexual stage to ten known herbal antimalarial drugs on the Ghanaian market. The herbal drugs were selected based on their approval by the Foods and Drugs Authority and their availability on the Market. The study also looked at the gametocytogenic ability of the herbal drugs at very low concentrations. Finally the growth inhibition of gametocytes by the ten herbal drugs was studied. All experiments were done in vitro. 3.3 Herbal drug preparations Ten known local antimalarial drugs [Yafo (YF), Masada (MS), Adutwumwa malamix (AD), Kingdom herbal (KG), Herbaquin (HB), Class malakare (CM), Angel Herbal (AN), Top Fever (TF), Rooter Mixture (RT) and Taabea (TB)] (all in liquid form) that have been approved by the Foods and Drugs Authority (FDA) were obtained from various herbal shops. About 10 ml of each drug was transferred into a 50 ml falcon tube under sterile conditions. The drugs were kept 36 University of Ghana http://ugspace.ug.edu.gh in -80 freezer for 48 hours. The drugs were then freeze-dried (lyophilized) using the 6 LABCONCO Freezone . About 10 mg of each freeze-dried drug was dissolved in 10 ml of distilled water [giving a 10x initial concentration (1000 µg/ml)]. The drugs were sterile filtered with 0.2 µm filters and stored in a -20 freezer. 3.4 Cultivation of Plasmodium falciparum cultures Continuous P. falciparum asexual cultures of the chloroquine-sensitive 3D7 strain were maintained in vitro using a modified method of Trager and Jensen (1976). 3.4.1 Culturing for asexual assay Complete parasite media consisted of RPMI 1640 supplemented with Herpes, L-glutamine, + NaHCO3, Glucose, Gentamycin and Albumax II. Parasites were cultured in O RBCs and placed o in a 37 C incubator with 92.5% Nitrogen, 2% Oxygen and 5.5% Carbon dioxide. The culture was then maintained with daily media change and after 48 hours the parasitemia was reduced to 1%. 3.4.1.1 Sorbitol synchronisation for ring forms Culture was maintained to get ≥ 5% synchronous ring forms in parasitemia. This was done by treating the parasite culture with 5% sorbitol, which resulted in approximately 95% of the parasites in the ring stage of development after 48 hours (Lambros and Vanderberg, 1979). In brief, a culture that had ≥ 5% rings in parasitemia was selected to be synchronized. About 5% o sorbitol was warmed to 37 C in a water bath (Thomastat Shaker T-225). In the Biosafety cabinet [BSC (SterilGARD Hood, the BARKER COMPANY Inc.)], the culture was transferred into a 15 ml tube and centrifuged at 2000 rpm for 5 minutes using the Sanyo HARRIER 18/80 centrifuge. 37 University of Ghana http://ugspace.ug.edu.gh All the media above the pellet was removed, and then 5% sorbitol was added to the pellet. The pellet in sorbitol was mixed thoroughly and made to stand in an incubator (RS Biotech Galaxy S) o at 37 C for 10 minutes. After 10 minutes the culture was centrifuged at 2000 rpm for 5 minutes and transferred into the BSC where the sorbitol was removed using a serological pipette. Parasite wash media [PWM (CPM without Albumax)] was added to the culture and mixed well. The culture was centrifuged at 2000 rpm at 5 minutes. The supernatant was aspirated and replaced with CPM. The contents were mixed thoroughly and the culture was centrifuged at 2000 rpm for 5 minutes. The culture was washed again with CPM. The pellet from the culture was transferred 3 into a new T-75 cm flask with 25 ml CPM. About 20 µl of the culture was placed in an Eppendorf tube and was centrifuged at 3000 rpm for 30 seconds using a Labnet, Spectrafuge 24D . About 80% of supernatant was taken off and 5 µl of the pellet was placed on a labeled slide and a thin smear prepared to assess the synchronization. The culture was gassed for 60 o seconds and placed in the 37 C incubator. 3.4.1.2 Plating of drugs for asexual assay About 100 µl of 5 different concentrations of each drug was plated in a 4-fold serial dilution starting at 100 µg/ml. The required concentration of each drug was added in triplicates. About 100 µl of 1% parasitemia culture was added to each treated well. The plates were placed into an incubating chamber and gassed for 6 minutes. The incubating chamber was then placed into the o 37 C incubator for 72 hours. After 72 hours a thin smear was prepared from each well. The smears were fixed in 100% methanol and stained with 10% Giemsa for 15 minutes. The slides were dried and observed under a light microscope using a 100x oil immersion objective lens. The parasitemia for each drug concentration in triplicates was estimated. The parasitemia estimates were used to calculate percentage growth inhibition and IC50s. 38 University of Ghana http://ugspace.ug.edu.gh 3.4.2 Culturing of gametocytes for drug assay Gametocytes were generated by a modified method of Tanaka and Williamson (2011). Culture 3 was started at 0.2% parasitemia at 6% hematocrit in a T-75 cm flask (750 µl of RBCs in 12.5 ml o CPM) on day 1. The culture was maintained under standard culture conditions (37 C, 92.5% nitrogen, 2% oxygen and 5.5% carbon dioxide) with the preparation of thin smears to monitor parasite growth. Media was replaced with 12.5 ml CPM on day 3. On days 4-11 media was replaced daily with 25 ml of CPM to reduce hematocrit to 3%. On days 9 –11, the spent media was taken off and replaced with N-Acetyl Glucosamine (NAG) treated CPM (CPM + 50 mM NAG) to get rid of asexual parasites. On day 12, the culture was treated with 60% percoll to obtain synchronous gametocyte. The gametocytes were put back into culture and after 3-4 hours, the gametocytes were transferred unto drug coated 96-well tissue culture plates for early stage gametocyte (I-II) assay. Left over gametocytes was maintained with daily change of NAG treated CPM till day 14. On day 15 a thin smear was prepared to examine the stage of the gametocytes. Late stage gametocytes (III-V) were transferred unto drug coated 96-well plates for late stage gametocyte assay. 3.4.2.1 Purification of gametocytes This was performed to obtain a ≥80% purified gametocytes in culture (Knight and Sinden, 1982). 90% percoll was prepared by adding 3.6 ml of 100% percoll to 0.4 ml parasite wash medium (PWM) in 15 ml falcon tube. The 90% percoll was further diluted to 60% by adding 2 ml PWM. The culture was transferred into a 15 ml tube and centrifuged at 15000 rpm for 10 minutes. The supernatant was discarded and pellets were resuspended in CPM to a 25% haematocrit. The o falcon tube was tilted at 60 angle and a sterile Pasteur pipette was used to transfer the 60% 39 University of Ghana http://ugspace.ug.edu.gh percoll unto the culture slowly ensuring that there was no mixing. The volume of 60% percoll used was twice the volume of the culture. The tube was centrifuged at 1450 rcf for 10 minutes at room temperature without brake using the Sanyo HARRIER 18/80 centrifuge. Parasites were collected at interphase and transferred into a new 15 ml tube. The parasites collected were centrifuged at 1500 rpm for 10minutes. The supernatant was discarded and resuspended in 3 ml PWM. The tube was centrifuged at 1500 rpm for 10 minutes and supernatant was discarded. 3 Pellets were transferred into a T-25 cm flask with 5 ml of CPM with 50 mM N-Acetyl Glucosamine (NAG). About 20 µl of the culture was aliquot into an Eppendorf tube and centrifuged at 3000 rpm for 30 seconds using the Labnet, Spectrafuge 24D centrifuge. The o culture was flushed with gas mixture and placed back into the 37 C incubator. Thin smears were made from the aliquot in the Eppendorf tube. 3.4.2.2 Plating of drugs for gametocyte assay A 96-well tissue culture plate was filled with 10 µl of three different concentrations (100 µg/ml, 7 µg/ml and 1 µg/ml) of each drug to be tested with artesunate (200 µg/ml), primaquine (200 µg/ml), DMSO (1%) serving as controls. The required concentration of each drug was added in triplicate. Gametocytemia was adjusted to 5% by adding appropriate volume of RBCs to the culture. About 90 µl of culture (CPM + 2 µl of RBCs) was aliquot into each treated well. The plates were put into an incubating chamber and gassed for 6 minutes. The incubating chamber o was place into the 37 C incubator. After 72 hours, thin smears were prepared from each well and stained with 10% Giemsa. The slides were dried and observed unto the 100x immersion oil objective lens to estimate gametocytemia. 40 University of Ghana http://ugspace.ug.edu.gh 3.5 Kinetics of herbal antimalarial drugs Eleven different CPMs were prepared by treatment with the IC10s of the herbal drugs and artesunate. Twelve separate cultures (11 with drug treatments and 1 with no drug treatment) were o set up at 1% parasitemia at normal culture conditions (37 C, 92.5% Nitrogen, 5.5 % Carbon dioxide, 2% Oxygen) on day 1. The appropriate modified media was used to replace spent media daily from days 2-8. Thin smears were prepared daily for each culture treatment and observed under the light microscope. The parasitemia and stages of parasites were examined and recorded for each culture treatment. On days 9-14 media was changed every 48 hours and thin smears where prepared with each media change. The parasitemia and stages of the parasites were examined and recorded for each culture treatment. 3.6 Statistical analysis Data was analysed using Microsoft Excel 2010 and R-Excel 2.15.3. 3.7 Ethical clearance The study was approved by the Institutional Review Board (IRB) of Noguchi Memorial Institute for Medical Research (NMIMR). 41 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.1. IC50s of herbal drugs All tested herbal drugs showed IC50s < 12 µg/ml on asexual stages. Top fever showed the lowest IC50 value being 0.06 µg/ml and Taabea showed the highest IC50 value of 11.58 µg/ml. The table below gives the IC50s of the tested drugs in ascending order, using artesunate as a control drug. Table 2: The herbal drugs showed IC50s significantly higher than artesunate with p<0.05. DRUG IC 50 (µg/ml) ± S.D Artesunate 2.9e-2± 0.002 Top Fever 6.0e-2± 0.028 Masada 2.5e-1± 0.035 Class malakare 3.5e-1± 0.135 Yafo fever 5.3e-1± 0.065 Angel Herbal 1.31 ± 0.160 Rooter Mixture 2.80 ± 0.363 Herbaquin 5.86 ± 0.540 Adutwumwa malamix 7.49 ± 0.248 Kingdom herbal 1.107e+1± 1.010 Taabea 1.158e+1± 2.484 42 University of Ghana http://ugspace.ug.edu.gh 4.2. Kinetics of the herbal drugs After 14 days of monitoring the cultures to see the progress of the kinetics and dynamics in growth into the production of gametocytes. The cultures were treated with very low concentration i.e. IC10s of the herbal drugs to monitor the rate at which they produce gametocytes Plate 4: Picture of ring stage Plasmodium falciparum 43 University of Ghana http://ugspace.ug.edu.gh Plate 5: Picture of a raptured schizont of Plasmodium falciparum 44 University of Ghana http://ugspace.ug.edu.gh Plate 6: Picture of early stage Plasmodium falciparum gametocytes 45 University of Ghana http://ugspace.ug.edu.gh Figure 7: Picture of late stage Plasmodium falciparum gametocytes 46 University of Ghana http://ugspace.ug.edu.gh ND ART CM TF KG AN TB AD RT MS HB YF 8 7 6 5 4 3 2 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 No. of days in culture Figure 1: Effect of drug IC10s on the ring stage development 47 parasitemia (%) University of Ghana http://ugspace.ug.edu.gh ND ART CM TF KG AN TB AD RT MS HB YF 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 2 3 4 5 6 7 8 9 10 11 12 13 14 No. of days in culture Figure 2: Effects of drug IC10s on trophozoite development 48 parasitemia (%) University of Ghana http://ugspace.ug.edu.gh ND ART CM TF KG AN TB AD RT MS HB YF 2.5 2 1.5 1 0.5 0 2 3 4 5 6 7 8 9 10 11 12 13 14 No. of days in culture Figure 3: Effects of drug IC10s on schizont development 49 parasitemia (%) University of Ghana http://ugspace.ug.edu.gh ND ART CM TF KG AN TB AD RT MS HB YF 9 8 7 6 5 4 3 2 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 No. of days in culture Figure 4: Effects of drug IC10s on early gametocyte development 50 parasitemia (%) University of Ghana http://ugspace.ug.edu.gh ND ART CM TF KG AN TB AD RT MS HB YF 10 9 8 7 6 5 4 3 2 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 No. of days in culture Figure 5: Effects of drug IC10s on late stage gametocyte development 51 parasitemia (%) University of Ghana http://ugspace.ug.edu.gh 4.3. Early stage growth inhibition by herbal drugs All the drugs tested, except for Taabea, at 100µg/ml showed more than 50% inhibition of early stage gametocytes. Four drugs; Kingdom herbal, Masada, Yafo fever and Herbaquin at 7 µg/ml showed a more 50% inhibition for early stage gametocytes At 1µg/ml only Masada and Yafo gave a more than 50% early stage gametocyte inhibition. 52 University of Ghana http://ugspace.ug.edu.gh 120 100 80 60 40 20 0 drug concentration Figure 6: Inhibition of early stage gametocytes by drugs 53 % growth inhibition University of Ghana http://ugspace.ug.edu.gh 4.4. Late stage growth inhibition by herbal drugs Primaquine at 10µg/ml gave a 100% late stage gametocyte growth inhibition. At 100µg/ml, Yafo, Herbaquin, Masada, Top fever, Class malakare and Rooter mixture showed a more than 50% late gametocyte growth inhibition. Only Masada showed ≥ 50% inhibition of late stage gametocytes (III-V) at 7 µg/ml. with none of the herbal drugs show a more than 50% growth inhibition at 1µg/ml 54 University of Ghana http://ugspace.ug.edu.gh 120 100 80 60 40 20 0 Drug concentrations Figure 7: Inhibition of late stage gametocytes by drugs 55 % growth inhibition University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE DISCUSSION AND CONCLUSION 5.1 DISCUSSION Although artemisinin and its analogues have given much help in the treatment of chloroquine- resistant malaria, these drugs are often unavailable and/or unaffordable to many people who live in malaria prone areas. An alternative to manufactured drugs is the use of traditional medicines which are readily available for the treatment of malaria. The discovery of artemisinin and its derivative from the herbal plant Artemisia annua and in the last decade has increased interest in the potential of locally grown plants to provide new and more potent drugs in the treatment of malaria and its eradication. The anti-malarial activity of the herbal drugs tested was based on their IC50s on the asexual stage of the Plasmodium falciparum parasite. IC50 is a measure of how effective a drug is. It indicates how much of a particular drug is needed to inhibit a given biological process or microorganism growth by half. In other words, it is the minimal concentration of a drug that can reduce the growth of parasites by half. Eight of the herbal drugs showed high anti-malarial activity (IC50 ≤ 10 µg/ml) with 2 having moderate anti-malarial activity (between IC50 ≥ 10 µg/ml and IC50 ≤ 50 µg/ml) (Ramalhete et al., 2008; Sanon et al., 2013). It can therefore be said that the tested drugs were effective in killing malaria parasites. The IC10 of Top fever syrup gave the lowest parasitemia (0.29%) of rings on day 2 which corresponds to it having the lowest IC50 value. Taabea also being the least anti-malarial with the highest IC50 value gave the highest ring parasitemia (2.64%) on day two. Top fever syrup also had a delayed peak of 4.85% parasitemia on day 8 along with Kingdom herbal, New angel herbal and Yafo fever that also had their peaks on day 8 with parasitemias of 4.86%, 4.39% and 4.48% 56 University of Ghana http://ugspace.ug.edu.gh respectively. The IC10 of Artesunate gave the highest ring parasitemia of 7.24% but gave a comparatively lower schizont parasitemia of 1.52%, and could be attributed to the schizonticidal effects of artesunate (Li and Weina, 2010; Lin et al., 1987; Mohanty et al., 2004). All the herbal drugs had a schizont parasitemia < 0.5% on day 2, with Class malakare, Top fever syrup, Kingdom herbal, New angel herbal, Taabea, Rooter mixture, Masada, Herbaquin and Yafo fever giving 0% parasitemia. Herbaquin gave the highest schizont parasitemia of 2.17% on day 6. Comparing the two asexual graphs, the parasitemia for rings were higher than schizont parasitemia suggesting that the anti-malarial effects of the herbal drugs are likely to be schizonticidal. This is confirmed with reported schizonticidal effects of Azadirachta indica, a component of the herbal preparation (Dhar et al., 1998; Udeinya et al., 2008). Parasites treated with New angel herbal developed the highest early stage gametocytemia of 8.46% and 2.25%on days 9 and 14 respectively, this may be attributed to the fact that the herbal plants used in the preparation of New angel herbal do not have reported gametocytocidal activities despite Vernonia amygdalina being reported to have an anti-malarial effect (Clarkson et al., 2004; Kraft et al., 2003). There were no gametocytes in the presence of Taabea on day 14 however it gave the second highest late stage parasitemia of 7.41%. This could mean there is a rapid transformation from early to late stage gametocytes in the presence of Taabea. Even though Taabea is composed of Azadirachta indica which has reported gametocytocidal activities (Dhar et al., 1998; Lucantoni et al., 2010; MacKinnon et al., 1997; Udeinya et al., 2006), it is likely that the added herbal plants lowered the gametocytocidal effects of the neem plant. Parasite cultures treated with New angel herbal gave the highest late stage gametocytemia of 8.99% on day 14, this coupled with it being the herbal drug with the highest early stage 57 University of Ghana http://ugspace.ug.edu.gh gametocytemia would imply that New angel herbal is gametocytogenic. Parasite cultures treated with Rooter mixture gave the lowest late gametocytemia of 0.58% on day 14. Yafo fever and Top fever treated parasite cultures gave the second and third lowest late stage gametocyte levels of 1.00% and 1.54% respectively. These results suggest that these drugs when taken in low concentrations will reduce gametocyte production as compared to Herbaquin, Kingdom herbal, Taabea and New angel herbal. These herbal drugs at low concentrations in parasite cultures have higher gametocyte production than artesunate which is known to be gametocytocidal (Bhatt et al., 2006). At a concentration of 100 µg/ml, all herbal drugs gave a more than 50% inhibition of early stage gametocytes. Yafo fever at 100 µg/ml concentration gave the highest early stage gametocyte inhibition of 95.45%. Yafo fever also recorded the highest inhibition of early stage gametocytes at 7 µg/ml and 1 µ/ml with 83.78% and 72.13% respectively. Recording an IC50 at 0.53 µg/ml would imply that Yafo fever has good effect on asexual stages of the parasite and early stage gametocytocidal effects too. Masada inhibited early stage gametocytes by 91.04%, 57.11% and 51.81% at 100 µg/ml, 7 µg/ml and 1 µg/ml respectively. With Masada exhibiting an IC50 of 0.25 µg/ml, it would imply that it is has gametocytocidal effects on early stage gametocytes. Taabea showed the lowest inhibition for early stage gametocytes, having inhibitions of 49.26%, 9.62% and 6.39% at 100 µg/ml, 7 µg/ml and 1 µg/ml respectively. Yafo, Herbaquin, Masada, top fever, Masada, Class malakare and Rooter mixture gave a higher than 50% inhibition of late stage gametocytes at 100 µg/ml with Masada giving the highest inhibition of 87.91%. Masada also gave the highest late stage inhibition among the ten drugs at 60.86% at 7 µg/ml. Masada’s gametocytocidal activities can be linked to the only herbal plant 58 University of Ghana http://ugspace.ug.edu.gh that it is composed of Cryptolepsis sanguinolenta. Even though there are no reported gametocytocidal effects on this plant, it is has high antimalarial activity and cryptolepine, a compound derived from the plant is a very potent antimarial compound which is currently under study(Kumar et al., 2003; Wright, 2005; Yarnell and Abascal, 2004). At 1 µg/ml Yafo fever gave the highest late stage inhibition of 39.87%. Taabea gave the lowest late stage gametocyte inhibition (39.13%) at 100 µg/ml and 3.42% for 1 µg/ml. New angel herbal mixture gave a below 50% inhibition at 100 µg/ml, also giving the lowest value of 9.77% at 7 µg/ml and 4.25% at 1 µg/ml. Class malakare showed a high late stage gametocyte inhibition (79.93%) at 100 µg/ml but low inhibitions of 13.74% and 6.44% at 7 µg/ml and 1 µg/ml respectively. The trend in Class malakare could not be accounted for. Primaquine and artesunate both at 200 µg/ml (i.e. 0.77 µM for primaquine and 0.52µM for artesunate) showed a more than 50% growth inhibition of early and late stage parasitemia. This is in line with literature about the gametocytocidal activities of both orthodox drugs(Price et al., 1996). Artesunate has reported a much lower IC50 value 0f 108nM for Plasmodium falciparum gametocytes(Benoit-Vical et al., 2007). Increasing the concentration of Primaquine to 10 µg/ml (38.56µM) showed a 100% growth inhibition of late stage gametocytes. This is supportive of reported IC50 values of primaquine and artemisinin( from which artesunate is derived) of 17.6 µM and 1.0 µM, respectively(Chevalley et al., 2010). From table 1, it can de deduced that all the herbal drugs tested had an active to moderate antimarial activity (IC50 < 20µg/ml) and would be effective in the eliminating of asexual stage parasites. However, Figures 4-7 show New angel herbal mixture, kingdom herbal and Taabea to be gametocytogenic whiles Yafo fever and Masada have produce gametocytocidal effects due to the strong gametocytocidal effects of Cryptolepsis sanguinolenta and Azadirachta indica. 59 University of Ghana http://ugspace.ug.edu.gh Since all the drugs were highly or moderately active against asexual stages of P. falciparum, those that were not gametocytocidal will potentially be aiding the transmission of malaria parasites even after patients have been cleared of clinical symptoms. Top fever had the lowest IC50 value of 0.06 ± 0.028 µg/ml making it the most effective herbal drug on the asexual stages of Plasmodium falciparum. This drug is composed of two plants; Azadirachta indica and Alstonia boonei, which have been widely used in the treatment of malaria in Ghana (Asase and Oppong-Mensah, 2009; Asase et al., 2010). Alstonia boonei tested on the chloroquine-resistant FcB1/Colombia strain showed inactivity (IC50 > 50 µg/ml) (Zirihi et al., 2005). However another test using the same chloroquine-resistant FcB1/Columbia strain showed a moderate anti-malarial activity with an IC50 value of 12.3 ± 0.2 µg/ml (Guédé et al., 2010). Different plant varieties may have accounted for the different values in IC50 since both experiments used ethanol extracts of Alstonia boonei with similar drying techniques. Azadirachta indica extracts tested on chloroquine resistant DD2 and chloroquine sensitive 3D7 strains of Plasmodium falciparum showed high anti-malarial activity with IC50 values of 1.7 µg/ml and 5.8 µg/ml respectively (El Tahir et al., 1999). Against the chloroquine-resistant W2 Vietnam strain of Plasmodium falciparum, Azadirachta indica showed a high anti-malarial activity of 4.7 µg/ml (Hout et al., 2006). The high anti-malarial activity of Top fever could mainly be due to the Azadirachta indica constituent or a possible unreported anti-malarial synergy between Alstonia boonei and Azadirachta indica. Yafo fever, Masada mixture and Class malakare exhibited IC50 values ≤ 1µg/ml showing very high anti-malarial activities. All three drugs have the herbal plant Cryptolepsis sanguinolenta in them. Apart from Masada that is made up of only Cryptolepsis sanguinolenta, Yafo fever and 60 University of Ghana http://ugspace.ug.edu.gh Class malakare combined Cryptolepsis sanguinolenta with Azadirachta indica and Carapa procera respectively. Cryptolepsis sanguinolenta is used in the traditional treatment of malaria in West Africa (Tempesta, 2010). Carapa procera is not a known antimalarial drug but there has been reports of its anti-filarial properties (Titanji et al., 1990). In vitro studies of C. sanguinolenta alkaloid isolates Cryptolepine and Isocryptolepine gave IC50 ranging between 0.2 - 0.8 µM, making the plant a very potent anti-malarial drug (Cimanga et al., 1997; Grellier et al., 1996). The high antimalarial activity of these three herbal preparations can be attributed to the strong anti-malarial activity of Cryptolepsis sanguinolenta for Yafo fever and Class malakare. The potency of Masada mixture may be attributed to the combined anti-plasmodial effect of both Cryptolepsis sanguinolenta and Azadirachta indica. New Angel Herbal and Rooter Mixture showed a high anti-plasmodial activity with IC50 ≥ 1 µg/ml but ≤ 5 µg/ml. Both drugs have completely different herbal compositions. New Angel Herbal is made up of Cola gigantean, Solanum torvum, Spathodea campanulata, Bombax buonopozense and Vernonia amygdalina whiles Rooter Mixture is made up of Aloe schweinfurthil, Khaya senegalensis, Pileostigma thornnigil and Cassia siamea. Even though Cola gigantean and Solanum torvum have anti-microbial and anti-inflammatory properties (Agyare et al., 2012; Chah et al., 2000), there is no in vitro anti-plasmodial report on these plants. Experiments with ethanol extracts of Solanum torvum have shown high anti-plasmodial activity with IC50 of 7.5±1.25 μg/ml (Dhanabalan, 2008). Bombax buonopozense has shown anti- plasmodial activity in mice against Plasmodium berghei (Akuodor et al., 2012; Iwuanyanwu et al., 2013). Vernonia amygdalina when tested in vitro showed (5 < IC50 < 10 µg/ml) which is lower compared to the IC50 of New angel herbal (Tona et al., 2004). The anti-plasmodial activity for New Angel Herbal can be attributed to the high anti-malarial activity of Vernonia 61 University of Ghana http://ugspace.ug.edu.gh amygdalina. Despite a vast array of medicinal uses for related species of Aloe schweinfurthil (Eshun and He, 2004), there has been no report of the plant’s anti-plasmodial activity in vitro. In vitro studies with Khaya senegalensis, Pileostigma thonnigii and Cassia siamea have shown these plants to have high anti-malarial properties with their extracts giving IC50 <5 µg/ml (El Tahir et al., 1999), IC50 within 6.20 - 15.06 µg/ml range (Kwaji et al., 2010), and IC50 = 5 µg/ml (Ajaiyeoba et al., 2008) respectively. These IC50 values will be the basis for the high anti- malarial activity of Rooter mixture. Adutwumwa malamix and Herbaquin had 5 µg/ml < IC50 < 10 µg/ml. Herbal plants used in Adutwumwa malamix are Anthocleista nobilis, Vitex grandifolia and Phyllanthus fratemus. Apart from Vitex grandifolia that had no records of in vitro anti-malarial activity, extracts of related species of Phyllanthus fratemus when tested on chloroquine sensitive 3D7 strain of Plasmodium falciparum in vitro showed IC50s of 7.25 µg/mL (Bagavan et al., 2011) which corresponds to IC50 value attained in this study. When Anthocleista nobilis ethanoic extracts were tested on chloroquine resistant K1 Plasmodium falciparum strain in vitro, IC50 value of 10.8 µg/ml was obtained (Sanon et al., 2013). Therefore the active anti-malarial effect of Adutwumwa malamix can be attributed to Anthocleista nobilis and Phyllanthus fratemus. Herbaquin is made of Cryptolepsis sanguinolenta, Alstonia boonei, Azadirachta indica, Monodora myristica and Xylopia aethiopica. The anti-malarial activities of Azadirachta indica, Alstonia boonei and Cryptolepsis sanguinolenta contribute to the anti-malarial effect of Herbaquin (Cimanga et al., 1997; Grellier et al., 1996; Hout et al., 2006; Zirihi et al., 2005), since records show that Monodora mysritica and Xylopia aethiopica demonstrate only anti- bacterial and anti-fungal properties with no anti-malarial effect (Tatsadjieu et al., 2003). 62 University of Ghana http://ugspace.ug.edu.gh Kingdom herbal and Taabea were the two drugs that showed moderate anti-malarial activity of 11.07 µg/ml and 11.58 µg/ml respectively. In addition to Phyllanthus fratemus and Cryptolepsis sanguinolenta, Kingdom herbal includes Nauclea latifolia which in previous experiments by Ajaiyeoba et al. (2005) was far less active towards malaria parasites as it showed IC50 of 478.9 µg/ml. This is higher than the recorded IC50 for Kingdom herbal of which Nauclea latifolia is a constituent. Taabea is composed of Ocimum viride, Azadirachta indica, Paullinia pinnate, Tetrapleura tetraptera, Theobroma cacao, Cymbopogon citratus and Moringa oleifera. Ocimum viride is known for its anti-fungal and anti-bacterial properties (Hammer and Carson, 2011). Paullinia pinnate, Tetrapleura tetraptera and Theobroma cacao though have been used for traditional treatment of malaria, do not have IC50 records of their extracts (Batista et al., 2009; Gessler et al., 1994; Idowu et al., 2010). The moderate anti-malarial property of Taabea can be attributed to additional effects of Cymbopogon citratus which have a reported IC50 of 12.1 μg/ml (Melariri et al., 2011). Moringa oleifera could not have contributed to the moderate antimalarial activity of Taabea due to the reports of its relatively low anti-malarial activity as it an IC50 value of more than 50 was recorded by Köhler et al. (2002). Not all the herbal plants used in the herbal drugs have reported anti-malarial activity. This may be due to the fact that some of these drugs have indications for other ailments. Only five (Masada mixture, Herbaquin, Top fever syrup, Kingdom mixture and Yafo fever mixture) out of the ten drugs had indications for malaria alone. Only Masada out of the five had only one herbal plant for only malaria treatment. Masada mixture being made of only Cryptolepsis sanguinolenta whose extracts have high anti-malarial activity accounts for the drug’s potency (Kirby et al., 1995). Herbaquin, despite being indicative for only malaria, is composed of five plants out of which only three are known anti-malarial drugs. Taabea showed the lowest anti-malarial activity 63 University of Ghana http://ugspace.ug.edu.gh among the ten drugs also had the highest number of herbal plant composition and highest number of disease indications. 5.2 CONCLUSSION All drugs tested showed potent antimalarial activity, however only Yafo fever and Masada showed very strong gametocytocidal activities. Such drug effects will reduce the transmission of the plasmodium parasite among its hosts. Even though Taabea and New angel fever syrup had anti-malarial effects on the asexual stages of the parasite, they were found to promote gametocyte growth and as such must not be used alone. 64 University of Ghana http://ugspace.ug.edu.gh RECOMMENDATIONS Based on the data gathered from this experiment, advocacy for the use of Yafo fever and Masada to achieve malarial eradication should be encouraged and incorporated into malaria control programmes. 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The American journal of tropical medicine and hygiene 68, 386—390. 93 University of Ghana http://ugspace.ug.edu.gh APPENDIX 1 MATERIALS AND EQUIPMENT Equipment Bio safety cabinet (SterilGARD Hood, the BARKER COMPANY Inc.) o Water bath (Thomastat Shaker T-225) set at 37 C Centrifuge (Sanyo HARRIER 18/80 and Labnet, Spectrafuge 24D) Modular incubator chamber Incubator (RS Biotech Galaxy S) set at 37C Electric vacuum pump Light Microscope Refrigerator Freezer Weighing balance (AND EK-3000i) Vortex (Fisher Scientific) o Oven (Bakura, Clini oven TF-31) set at 70 C Double water distillation unit (Fistreem International Ltd) Cell counters 94 University of Ghana http://ugspace.ug.edu.gh 6 Freeze-drier (LABCONCO Freezone ) Materials Weigh boat 96 well Tissue Culture plate (CytoOne) 5ml serological pipettes 10ml serological pipettes 25ml serological pipettes 20µl pipette tips 200µl Pipette tips 1000µl pipette tips Pasteur pipettes Plastic aspirators Cryo vials Nalgene Filter unit 500ml 15ml centrifuge tubes 50ml centrifuge tubes 3 25 cm tissue culture flask 3 75 cm tissue culture flask 10ml Syringes 95 University of Ghana http://ugspace.ug.edu.gh Microscope slides Tissue Non-powdered Gloves 0.2µm Filters Slide staining trough Aluminium foil Biohazard disposable bags Blood Lancets ACD vacutaineers 96 University of Ghana http://ugspace.ug.edu.gh APPENDIX 2 PREPARATION OF MEDIA AND REAGENTS Thawing mix 3.5 grams of Sodium Chloride (NaCl) was weigh and transferred into a conical flask with 100ml of distilled water. The solution was made to dissolve and transferred into the biosafety cabinet (BSC). The solution was filtered into a two 50ml falcon tubes using a needle, syringe and 0.22µl o filter. The thawing mix was stored at 4 C. Freezing mix 4.2grams of sorbitol and 0.9 grams of NaCl were dissolved in 100ml of distilled. 72ml of the solution was added to 28ml of glycerol. The solution was sent into the BSC and sterile filtered o into falcon tubes using 0.22µm filters. The freezing mix was stored at 4 C. 7.5% sodium hydrogen carbonate (NaHCO3) 7.5g of Sodium Hydrogen Carbonate (NaHCO3) was weighed and dissolved into 100ml of distilled water in a conical flask. The solution was sent into the biosafety cabinet and filtered o using a 0.2µm filter. The solution was stored at 4 C. 20% glucose 20g of glucose was weighed and dissolved in 100ml distilled water in a conical flask. The solution was transferred into the biosafety cabinet and filtered using a 0.2µm filter. The solution o was stored at 4 C Albumax 0.2g of Hypoxanthine was dissolved in 1L of RPMI 1640 in a conical flask. 50g of Albumax powder was added to the solution and placed on a magnetic stirrer till everything dissolved. The 97 University of Ghana http://ugspace.ug.edu.gh albumax solution was transferred into the BSC and filtered using a 0.8µm filtering unit and an electric vacuum pump. The solution was filtered again using a 0.2µm filtering unit and an electric vacuum pump. The albumax was then aliquoted into 50ml falcon tubes. The aliquots o were stored below -20 C. Incomplete media (ICPM) or parasite wash media (PWM) In the BSC, 13.5ml of 7.5% Sodium hydrogen carbonate (NaHCO3), 5ml of 20%Glucose, 0.5ml of 50mg/ml gentamycin were added to 500ml of RPMI-1640(with L-glutamine). The solution is o mixed thoroughly and kept at 4 C. Complete medium 20ml of Albumax was added to 180ml of incomplete media. Complete media + 2%NHS 1ml of Normal Human Serum (NHS) was added to 49ml of CPM in a 50ml falcon tube. It was o stored at 4 C. Complete media + NAG 0.56g of N-Acetyl Glucosamine powder was weighed and dissolved in 50ml of CPM. The o solution was sterile filtered using a 0.2µm filter. It was stored at 4 C. Preparation of 5% sorbitol 5g of sorbitol was weighed and dissolved in 100ml of distilled water in a conical flask. The solution was transferred into the biosafety cabinet and filtered using a 0.2µm filter. It was stored o at 4 C 98 University of Ghana http://ugspace.ug.edu.gh APPENDIX 3 PLATE DESIGNS Plate design for asexual assay 1 2 3 4 5 6 7 8 9 10 11 12 A CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM YF YF YF HB HB HB AS AS AS B CPM (100µg/ml) (100µg/ml) (100µg/ml) (25µg/ml) (25µg/ml) (25µg/ml) (0.11µg/ml) (0.11 µg/ml) (0.11 µg/ml) RBC CPM YF YF YF HB HB HB AS AS AS C CPM (25µg/ml) (25µg/ml) (25µg/ml) (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (0.03µg/ml) (0.03 µg/ml) (0.03 µg/ml) RBC CPM YF3 YF3 YF3 HB HB HB D CPM (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) RBC CPM YF4 YF4 YF4 HB HB HB E CPM (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) IRBC CPM YF5 YF5 YF5 AS AS AS F CPM (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) (1.8µg/ml) (1.8µg/ml) (1.80µg/ml) IRBC CPM HB HB HB AS AS AS G CPM (100µg/ml) (100µg/ml) (100µg/ml) (0.45µg/ml) (0.45µg/ml) (0.45µg/ml) IRBC CPM H CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM Table 3: Plate 1 for asexual assay 99 University of Ghana http://ugspace.ug.edu.gh 1 2 3 4 5 6 7 8 9 10 11 12 A CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM MS MS MS AN AN AN AS AS AS B CPM (100µg/ml) (100µg/ml) (100µg/ml) (25µg/ml) (25µg/ml) (25µg/ml) (0.11µg/ml) (0.11 µg/ml) (0.11 µg/ml) RBC CPM MS MS MS AN AN AN AS AS AS C CPM (25µg/ml) (25µg/ml) (25µg/ml) (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (0.03µg/ml) (0.03 µg/ml) (0.03 µg/ml) RBC CPM MS MS MS AN AN AN D CPM (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) RBC CPM MS MS MS AN AN AN E CPM (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) IRBC CPM MS MS MS AS AS AS F CPM (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) (1.8µg/ml) (1.8µg/ml) (1.80µg/ml) IRBC CPM AN AN AN AS AS AS G CPM (100µg/ml) (100µg/ml) (100µg/ml) (0.45µg/ml) (0.45µg/ml) (0.45µg/ml) IRBC CPM H CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM Table 4: Plate 2 for asexual assay 1 2 3 4 5 6 7 8 9 10 11 12 A CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM TB TB TB AD AD AD AS AS AS B CPM (100µg/ml) (100µg/ml) (100µg/ml) (25µg/ml) (25µg/ml) (25µg/ml) (0.11µg/ml) (0.11 µg/ml) (0.11 µg/ml) RBC CPM TB TB TB AD AD AD AS AS AS C CPM (25µg/ml) (25µg/ml) (25µg/ml) (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (0.03µg/ml) (0.03 µg/ml) (0.03 µg/ml) RBC CPM TB TB TB AD AD AD D CPM (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) RBC CPM TB TB TB AD AD AD E CPM (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) IRBC CPM TB TB TB AS AS AS F CPM (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) (1.8µg/ml) (1.8µg/ml) (1.80µg/ml) IRBC CPM AD AD AD AS AS AS G CPM (100µg/ml) (100µg/ml) (100µg/ml) (0.45µg/ml) (0.45µg/ml) (0.45µg/ml) IRBC CPM H CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM Table 5: Plate 3 for asexual assay 100 University of Ghana http://ugspace.ug.edu.gh 1 2 3 4 5 6 7 8 9 10 11 12 A CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM TF TF TF CM CM CM AS AS AS B CPM (100µg/ml) (100µg/ml) (100µg/ml) (25µg/ml) (25µg/ml) (25µg/ml) (0.11µg/ml) (0.11 µg/ml) (0.11 µg/ml) RBC CPM TF TF TF CM CM CM AS AS AS C CPM (25µg/ml) (25µg/ml) (25µg/ml) (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (0.03µg/ml) (0.03 µg/ml) (0.03 µg/ml) RBC CPM TF TF TF CM CM CM D CPM (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) RBC CPM TF TF TF CM CM CM E CPM (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) IRBC CPM TF TF TF AS AS AS F CPM (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) (1.8µg/ml) (1.8µg/ml) (1.80µg/ml) IRBC CPM CM CM CM AS AS AS G CPM (100µg/ml) (100µg/ml) (100µg/ml) (0.45µg/ml) (0.45µg/ml) (0.45µg/ml) IRBC CPM H CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM Table 6: Plate 4 for asexual assay 1 2 3 4 5 6 7 8 9 10 11 12 A CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM KG KG KG RT RT RT AS AS AS B CPM (100µg/ml) (100µg/ml) (100µg/ml) (25µg/ml) (25µg/ml) (25µg/ml) (0.11µg/ml) (0.11 µg/ml) (0.11 µg/ml) RBC CPM KG KG KG RT RT RT AS AS AS C CPM (25µg/ml) (25µg/ml) (25µg/ml) (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (0.03µg/ml) (0.03 µg/ml) (0.03 µg/ml) RBC CPM KG KG KG RT RT RT D CPM (6.25µg/ml) (6.25µg/ml) (6.25µg/ml) (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) RBC CPM KG KG KG RT RT RT E CPM (1.56µg/ml) (1.56µg/ml) (1.56µg/ml) (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) IRBC CPM KG KG KG AS AS AS F CPM (0.39µg/ml) (0.39µg/ml) (0.39µg/ml) (1.8µg/ml) (1.8µg/ml) (1.80µg/ml) IRBC CPM RT RT RT AS AS AS G CPM (100µg/ml) (100µg/ml) (100µg/ml) (0.45µg/ml) (0.45µg/ml) (0.45µg/ml) IRBC CPM H CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM Table 7: Plate 5 for asexual assay 101 University of Ghana http://ugspace.ug.edu.gh Plate design for gametocyte assay 1 2 3 4 5 6 7 8 9 10 11 12 A CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM MS MS YF YF YF MS PQ PQ PQ B CPM (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (200ng/ml) (200ng/ml) (200ng/ml) CPM YF YF YF MS MS MS AS AS AS C CPM (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (200ng/ml) (200ng/ml) (200ng/ml) YF YF YF MS MS MS D CPM (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) DMSO DMSO DMSO CPM HB HB HB AN AN AN PQ PQ PQ IRBC CPM E CPM (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (10µg/ml) (10µg/ml) (10µg/ml) HB HB HB AN AN AN IRBC F CPM (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) RBC RBC RBC CPM HB HB HB AN AN AN IRBC G CPM (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) CPM H CPM CPM CPM CPM CPM CPM CPM C PM C PM CPM CPM CPM Table 8: Plate 1 for gametocyte assay 1 2 3 4 5 6 7 8 9 10 11 12 A CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM TB TB TB TF TF TF PQ PQ PQ B CPM (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (200ng/ml) (200ng/ml) (200ng/ml) CPM TB TB TB TF TF TF AS AS AS C CPM (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (200ng/ml) (200ng/ml) (200ng/ml) TG TG TG TF TF TF D CPM (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) DMSO DMSO DMSO CPM AD AD AD CM CM CM PQ PQ PQ IRBC CPM E CPM (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (100µg/ml) (10µg/ml) (10µg/ml) (10µg/ml) AD AD AD CM CM CM IRBC F CPM (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) (7µg/ml) RBC RBC RBC CPM AD AD AD CM CM CM IRBC G CPM (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) (1µg/ml) CPM H CPM CPM CPM CPM CPM CPM CPM C PM CPM C PM CPM CPM Table 9: Plate 2 for gametocyte assay 102 University of Ghana http://ugspace.ug.edu.gh 1 2 3 4 5 6 7 8 9 10 11 12 A CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM CPM KG KG KG PQ PQ PQ B CPM (100µg/ml) (100µg/ml) (100µg/ml) (200ng/ml) (200ng/ml) (200ng/ml) CPM KG KG KG AS AS AS C CPM (7µg/ml) (7µg/ml) (7µg/ml) (200ng/ml) (200ng/ml) (200ng/ml) KG KG KG D CPM (1µg/ml) (1µg/ml) (1µg/ml) DMSO DMSO DMSO CPM RT RT RT PQ PQ PQ IRBC CPM E CPM (100µg/ml) (100µg/ml) (100µg/ml) (10µg/ml) (10µg/ml) (10µg/ml) RT RT RT IRBC F CPM (7µg/ml) (7µg/ml) (7µg/ml) RBC RBC RBC CPM RT RT RT IRBC G CPM (1µg/ml) (1µg/ml) (1µg/ml) CPM H CPM CPM CPM CPM CPM CPM CPM C PM CPM C PM CPM CPM Table 10: Plate 3 for gametocyte assay 103 University of Ghana http://ugspace.ug.edu.gh APPENDIX 4 ASEXUAL GROWTH INHIBITION CURVES 100 y = 8.481ln(x) + 23.249 80 R² = 0.9785 60 40 20 0 0 500 1000 1500 2000 Drug concentration(ng/ml) Figure 8: Growth inhibition curve for AS 80 70 y = 8.2194ln(x) + 30.803 60 R² = 0.8695 50 40 30 20 10 0 0 20 40 60 80 100 120 Drug concentration(µg/ml) Figure 9: Growth inhibition curve for KG 104 Grwoth inhibition (%) Growth Inhibition (%) University of Ghana http://ugspace.ug.edu.gh 90 80 y = 6.7284ln(x) + 43.829 70 R² = 0.6825 60 50 40 30 20 10 0 0 20 40 60 80 100 120 Drug concentration (µg/ml) Figure 10: Growth inhibition of RT 80 70 y = 2.7619ln(x) + 56.34 60 R² = 0.9784 50 40 30 20 10 0 0 20 40 60 80 100 120 Drug concentration(µg/ml) Figure 8: Growth inhibition curve of TF 105 Growth inhibition(%) Growth inhibition(%) University of Ghana http://ugspace.ug.edu.gh 100 y = 6.4055ln(x) + 61.455 80 R² = 0.5652 60 40 20 0 0 20 40 60 80 100 120 Drug concentration(µg/ml) Figure 9: Growth inhibition of CM 120 y = 8.0333ln(x) + 62.516 100 R² = 0.904 80 60 40 20 0 0 20 40 60 80 100 120 Drug concentration (µg/ml) Figure 13: Growth inhibition curve of MS 106 Growth inhibition(%) Growth inhibition (%) University of Ghana http://ugspace.ug.edu.gh 80 70 y = 5.6248ln(x) + 49.026 60 R² = 0.7954 50 40 30 20 10 0 0 20 40 60 80 100 120 Drug concentration (µg/ml) Figure 14: Growth inhibition curve of AN 100 y = 7.1559ln(x) + 53.986 80 R² = 0.8725 60 40 20 0 0 20 40 60 80 100 120 Drug concentration (µg/ml) Figure 15: Growth inhibition curve of YF 107 Growth inhibition(%) Growth Inhibition(%) University of Ghana http://ugspace.ug.edu.gh 90 80 y = 9.596ln(x) + 33.932 70 R² = 0.9263 60 50 40 30 20 10 0 0 20 40 60 80 100 120 Drug Concentration (µg/ml) Figure 16: Growth inhibition curve of HB 90 80 70 y = 5.1217ln(x) + 38.712 60 R² = 0.3167 50 40 30 20 10 0 0 20 40 60 80 100 120 Drug concentration (µg/ml) Figure 17: Growth inhibition curve of TB 108 Growth inhibition(%) Growth inhinition(%) University of Ghana http://ugspace.ug.edu.gh 90 80 y = 8.213ln(x) + 33.847 70 R² = 0.8618 60 50 40 30 20 10 0 0 20 40 60 80 100 120 Drug concentration (µg/ml) Figure 18: Growth inhibition curve of AD 109 Growth inhibition(%) University of Ghana http://ugspace.ug.edu.gh 8. APPENDIX 5 MISCELLANEOUS PICTURES Plate 8: Plated drugs in an incubating chamber 110 University of Ghana http://ugspace.ug.edu.gh Plate 9: Gametocytes after percoll purification 111