THE DEVELOPMENT OF A NEW PLANT-BASED CULTURE …



THE DEVELOPMENT OF A NEW PLANT-BASED CULTURE MEDIUM FOR PLASMODIUM FALCIPARUM, IN VITRO STUDIES ON THE ANTIMALARIAL ACTIVITIES OF FOUR COMMONLY USED MEDICINAL PLANTS IN NIGERIAN AND SOME ASPECTS OF THE IMMUNOLOGICAL IMPLICATIONS OF THE USE OF INSECTICIDES TREATED CURTAINS FOR THE PREVENTION OF MALARIA IN CHILDREN.

BY

ADEROUNMU ADEOLA OMOTAYO

THE DEVELOPMENT OF A NEW PLANT-BASED CULTURE MEDIUM FOR PLASMODIUM FALCIPARUM, IN VITRO STUDIES ON THE ANTIMALARIAL ACTIVITIES OF FOUR COMMONLY USED MEDICINAL PLANTS IN NIGERIAN AND ASPECTS OF THE IMMUNOLOGICAL IMPLICATIONS OF THE USE OF INSECTICIDES TREATED CURTAINS FOR THE PREVENTION OF MALARIA IN CHILDREN.

A thesis submitted (but NEVER defended) in partial fulfilment of the requirement for the degree of Doctor of Philosophy (Ph. D) in Medical Parasitology of the University of Lagos

By ADEROUNMU Adeola Omotayo

B.Sc., M.Sc. UNILAG

SEPTEMBER 2003

DECLARATION

I hereby declare that this thesis titled:

"The development of a new plant-based culture medium for plasmodium falciparum, in vitro studies on the antimalarial activities of four commonly used medicinal plants in nigerian and aspects of the immunological implications of the use of insecticides treated curtains for the prevention of malaria in children,"

is an original research carried out by me, Aderounmu Adeola Omotayo in the Department of Medical Microbiology and Parasitology, College of Medicine of the University of Lagos, Lagos, Nigeria and the Department of Immunology, Stockholm University, Sweden.

DEDICATION

"This project is dedicated to the African children suffering morbidity and mortality due to malaria and other preventable childhood diseases"

ACKNOWLEDGEMENT

The University of Lagos, School of Post-Graduate Studies granted the permission for this research to be carried out at Stockholm University.

UNESCO-American Society of Microbiologists and the International Centre for Culture and Science (ISC) in Switzerland provided the scholarship that facilitated my stay in Stockholm.

I am grateful to Professor A.F Fagbenro-Beyioku for her valuable guidance and support right from the days of my MSc programme at idiaraba and during the time away from home.

My sincere gratitude to Professor Klavs Berzins of the Department of Immunology, Stockholm University, (SU), for being a very kind man and at the same time for being my host and supervisor. I will never forget you. I appreciate the tutelage of Dr. Ahmed Bolad during all the time spent in and out of the laboratory.

I am grateful to Dr. Bolaji Thomas for providing a useful lead and for wonderful words of encouragements.

Special thanks to:

Professor Marita Troye-Blomberg, the Head of my department at Stockholm University

Margareta Hagstedt for her assistance in the Laboratory

All my seniors and friends in CMUL, Ninan, Bola, Amisu, Sola, Gbenga, Emeka

All my seniors and friends at SU, Jacob, Ben, Sallah, Nina-Maria, Gawa, Masashi, Manijeh, Shiva,

I remain indebted to my parents, Reverend and Mrs S. A. Aderounmu and to my siblings, Tosin, Wale, Ayoola, Yemi and Ayodele.

TABLE OF CONTENTS

Declaration

Certification

Acknowledgement

Table of contents

Chapter 1

Introduction and Literature review

Chapter 2

Material and methods

Chapter 3

Results

Chapter 4

Discussion

Chapter 5

Summary

REFERENCES

ABSTRACT

Malaria infection has been a major worldwide cause of death for centuries. An estimated 300-500 cases each year cause 1.5 to 2.7 million deaths, more than 90% in children under 5 years of age in Africa. The laboratory cultivation of malaria parasites is an important tool for the understanding of parasite biology, biochemistry, molecular biology, immunology and pharmacology. The requirements for human serum and the general high cost of existing approaches used for the cultivation of malaria parasites pose technical limitations to the application of these methods in many poor countries where malaria is endemic. In the first part of this study, short-term cultivation of Plasmodium falciparum, F32 strian, was achieved in a medium containing plant exudate and mice liver extract following earlier cultivation of the parasites in medium containing human serum. The parasitaemia increased from 4.0% to 7.6% on day 4 and the addition of hypoxanthine (0.02-0.04µM) to the new medium enhanced parasite growth as 9% parasitaemina was observed at 48 hours in a separate culture well containing the new medium and hypoxanthine. All the asexual stages of the erythrocytic phase of the parasite life cycle were seen throughout the duration of the cultivation. The results obtained in this study probably represents the first successful attempt to cultivate P. falciparum in vitro in a plant-based media supplemented with animal extract. In addition, the essence of the ongoing research is to develop an inexpensive malaria culture system that will enhance malaria research in poorer countries mostly afflicted with the scourge of the infection of P. falciparum. It is also known that the spread of multidrug resistance to P. falciparum, which is on the increase, has contributed to malaria specific mortality. This has highlighted the irrepressible need to develop new antimalarial drugs from natural products. Such drugs need be not only available but also affordable, safe to use and effective for developing countries where malaria undoubtedly remains one of the worst scourges contributing to mortality. In this study, the crude organic and aqueous extracts of four commonly used herbal plants from Nigeria have been evaluated for antimalarial activity against P.falciparum in vitro. These plants which include Cymbopogon giganteus, Azadirachta indica, Enantia chlorantha and Morinda lucida inhibited parasite growth efficiently in terms of reduced number of infected ring stages of the parasite. It is shown here that E.chlorantha-known to contain alkaloids, lignin, saponins and tannin-with crude extract inhibition of 68.9% and 54.0% at 500µg/ml as organic and aqueous respectively, is a promising antimalarial plant. When combined, lower concentrations of these extracts gave higher efficacy. The results justify the ethnomedicinal use of these four plants for the treatment of fever and malaria. Apart from chemotherapy, another malaria control strategy is currently being monitored closely is the use insecticides treated nets (ITNs) and curtains. These chemical barriers form a promising preventive measure against the deadly Plasmodium malaria parasite but concerns have been raised that the use of ITN may place young children at an increased risk of developing severe malaria at an older age by delaying the development of acquired immunity to malaria. Exposure to infection is regarded as a prerequisite for the natural development of immunity to malaria and protection against the blood stages of the parasite seems to rely on immunoglobulins (Ig)G, specifically on IgG1 and IgG3 antibodies. Interventions to reduce or eliminate malaria in children living in high transmission areas could lead to loss of these malaria antibodies resulting to rebound mortality in later childhood. This study has been conducted to compare the immunological responses in children from 2 villages in the vicinity of Ouagadougou in Burkina Faso. Enzyme-linked immunosorbent assay, (ELISA) analyses of plasma samples from asymptomatic children, aged 3-7 years, living in villages with and without insecticides treated curtains, (ITC) show no clear difference in the protective immunoglobulin levels in the two groups of children. In addition, PCR analyses of the DNA from these children show no distinction in the multiplicity of infection. These results probably indicate that the use of ITC does not interfere with the acquisition of antimalarial immunity associated with childhood in malaria endemic areas. 

CHAPTER 1

INTRODUCTION

Plasmodium falciparum infection annually causes an estimated 500 million infections with 1.5-2.7 million deaths, more than 90% in children under 5 years of age in Africa (Good, 2001; Sach and Malaney, 2002). Efforts to prevent and control the disease have been hampered by the development of resistance to insecticides in the mosquito vector and to therapeutic agents in the parasite. Antimalarial regimens are difficult and there is yet no effective vaccine against malaria (Rozmajzl et al., 2001). Malaria is endemic in Nigeria and the population at highest risk includes children, pregnant women and the non-immune. Not less than 25% of infant deaths and 20% of maternal mortality cases in Nigeria are attributed to malaria. In addition, more than 65% of Nigeria's population of 100-120 million people experiences at least one attack of malaria each year (Aderounmu, 1999, M.Sc thesis).

Cultivation of malaria parasites is an important tool for the understanding of parasite biology, biochemistry, molecular biology, immunology and pharmacology (Ringwald et al., 1999). Since Trager and Jensen establised the in vitro culture of P.falciparum in 1976, the medium used has been complemented by the addition of 10% human serum. The requirements for human serum pose technical limitations to the application of this method (Asahi and Kanazawa, 1994: Asahi et al., 1996). For many reasons, it would be advantageous to replace human serum in the culture medium. In malaria-endemic regions, local sera may contain antimalarial drugs or immune factors that render them useless for culturing the parasites (Siddiqui and Palmer, 1980; Divo and Jensen, 1982a; Jensen et al.,1982). A few reports however have shown that African donors can support the growth of laboratory adapted strains of parasites and fresh isolates and that acute phase homologous serum may be useful for the continuous in vitro culture of reference strain (Oduola et al., 1992; Binh et al., 1997 and Ringwald et al., 1999).

It has generally been accepted that nonimmune human serum is required for optimal parasite growth. However the requirement for a regular supply of nonimmune human serum entails difficulties in conducting research in most of the African continent, where malaria transmission occurs at a high level throughout the year. Nonimmune human type AB-positive serum is relatively scarce and expensive in countries where malaria is not endemic (Divo and Jensen, 1982a; Ringwald et al., 1999).

Furthermore, serum from donors living in malaria-free areas differ considerably in their ability to support parasite growth and therefore, it is recommended that several units of serum from different donors be pooled together to reduce the batch-to-batch differences in the support of parasite growth (Divo and Jensen, 1982b; Jensen, 1988). It cannot also be excluded that drugs not directed against the malaria parasites may nevertheless influence the development of the parasites. More problems include blood type compatibility and risks associated with the handling of infectious agents (Lingnau et al., 1994). In addition, any widely used vaccine should not be grown in human serum when there is a real possibility of contamination with infectious agents (Divo and Jensen, 1982a).

A number of successful attempts to replace the human serum components of the medium used for the in vitro cultivation has been reported (Siddiqui and Richmond-Crum,1977; Ifediba and Vanderberg, 1980; Sax and Rieckamn 1980; Divo and Jensen, 1982b; Willet and Canfield 1984; Asahi and Kanazawa 1994; Oduola et al., 1985; Lingnau et al., 1993, 1994; Ofulla et al., 1993,1994). All outlined the obvious disadvantages of drawing experimental data from parasites grown in serum supplemented medium due to batch-to-batch invariabilities problems with availability and cost in some places and the probability of contracting viral infections. Parallel comparisons of studies from different laboratories can therefore be easily standardised once this important variable has been excluded. These investigators have used both commercial and defined formulations to mimic the growth requirements supplied by the human serum though no thorough identification has been carried out as to which factor(s) of human serum are necessary for growth (Ofulla et al., 1993; Asahi et al., 1996; Binh et al.,1997 and Flores et al.,1997). The use of various animal sera and serum factions as substitutes for human sera have had only limited success hence the attempts to find a suitable sustitute for human serum are continuing.

Importance of the culture system to malaria research

The methods for cultivation of the erythrocytic stages of P.falciparum reported by Trager and Jensen (1976) have been usefully applied in nearly all aspects of research on malaria: chemotherapy, drug resistance, immunology and vaccine development, pathogenesis, gametocytogenesis and mosquito transmission, genetics, the basis for resistance of certain red cells, cellular and molecular biology and biochemistry of the parasites and their relationship with their host erythrocytes (Trager and Jensen, 1997).

Chemotherapy

It was obvious that cultures of P. falciparum could be used for the direct testing of blood schizonticides against the parasites and hence to screen new antimalarial drugs (Trager, 1987). Malaria cultures provide accessible forms of the target organism itself, developing within its natural host cell, the erythrocyte, but apart from the intact host. It is possible to expose the parasite to any desired concentration of drug for any desired time and access the extent of inhibition. Particularly important in this context has been the development of a rapid measurement of the antimalarial activity of a large number of compounds using microdilution method. In view of the striking success of artemisinin, natural products, especially extracts of medicinal plants provide a logical starting point (Kirby, 1996). In vitro studies are particularly useful for attempts to discover the mode of antimalarial compounds.

Drug resistance

This can be easily demonstrated and quantitated in cultures. Moreover, a short-term in vitro test can be applied to a large number of clinical isolates to determine the prevalence of drug-resistant malaria (Ringwald et al., 1996). Cultures have been useful in attempts to determine the genetic and biochemical basis for drug resistance.

Vaccine development

A prerequisite for the ultimate goal of a vaccine against malaria is a method for in vitro propagation of the parasites (WHO, 1975). The main use of cultures in relation to development of malaria vaccines has been the identification of target antigens for both the asexual erythrocytic stages and for those of the sexual stages (Kaslow et al., 1992; Feng et al., 1993). They have also been used for the in vitro assessment of immunity, especially to test for the antibodies that inhibit merozoite invasion. In addition, the cultures have been used to provide gametocytes to infect mosquitoes. The mosquitoes in turn are used for both studies on transmission blocking immunity and to infect volunteers in clinical vaccination trials. The cultures have proved useful for tests of vaccines against sporozoites and pre-erythrocytic stages as well as for those against the asexual erythrocytic stages.

Pathogenesis, protein export and take-over of erythrocytes

The phenomenon of sequestration of late-stage parasites in the capillaries of deep organs is responsible for the severe manifestations of falciparum malaria including, in particular, cerebral malaria (Turner, 1997). Sequestration is mediated by attachment of trophozoite- and schizont-infected red cells via the knobs to endothelial cells of small blood vessels. The formation of knobs requires the production by the parasite of at least 3 proteins that are exported from the parasite to reach the host erythrocyte plasma. When the cultures made all stages of P.falciparum readily available, Langreth et al., (1978) found that the knobs appeared on the infected cells at a time corresponding to the time of sequestration.

Cell biology: the mitochondrion

Electron microscopy of cultured parasites firmly established the presence of a moderately cristae mitochondrion visible in all developmental stages, although those from the gametocytes had notably more cristae (Langreth et al., 1978). Classical inhibitors of mitochondrial function rapidly shut down protein and nucleic acid biosynthesis suggesting an energy-yielding role for the mitochondrion (Ginsburg et al., 1986). Further studies have suggested that the parasite mitochodrion is a tempting target for new chemotherapeutics (El Wakeel, et al., 1985; Gringras and Jensen, 1993).

Innate resistance: mutant human erythrocytes providing protection against severe malaria

Cultures of P.falciparum show the cellular basis for the great selective advantage of heterozygotes (SA) over homozygotes (AA) with regard to the severe malaria (Marsh, 1992). SA individuals show a 90% protection from cerebral and other severe forms of malaria. With reduced oxygen (5%), as might occur in venules partially blocked with adherent cells, the development of the parasite is notably inhibited in SA erythrocytes. Other haemoglobinopathies show a marked geographical correlation with a high incidence of malaria but the cellular basis for the resistance to malaria conferred by these has been harder to demonstrate in culture (Trager and Jensen, 1997).

Gametocytes and gametocytogenesis in cultures: transmission of cultured parasites through mosquitoes

It is possible to produce in cultures, gametocytes that will be infectious to mosquitoes and the mosquitoes will produce infective sporozoites (Lensen, 1996). Hence the cultures can be used for studies on transmission blocking agents, either chemotherapeutics or immunological. Equally important, the cultures can be used for genetic crosses (Walliker et al., 1987; Wellems et al., 1987). In addition, the cultures can be used to produce mosquitoes infected with a well-defined strain or clone of parasites for the challenge of volunteers by sporozoite inoculation in vaccine trials (Davis et al., 1992).

Genetics: transfection

When P.falciparum parasites sensitive to pyrimethamine were transformed with plasmids containing the dihydrofolate reductase-thymidine synthetase gene (dhfr-ts) from clones resistant to pyrimethamine, the pyrimethamine resistance at first was maintained only under drug pressure with the transfected plasmids replicating as episomes. After a further prolonged period of drug pressure, however, homologous integration of the plasmids had occurred and the transformed parasites showed the stable level of pyrimethamine resistance characteristic of the source of the dhfr-ts gene (Wu et al.1996). The principle of transfection has been used to transfer resistivity to malaria infection in erstwhile susceptible strain and this provides ideas of the mechanisms of resistance to various antimalarial compounds.

Nutritional requirements

Malaria parasites ingest and digest haemoglobin of their host erythrocyte and probably other constituents of the red cell cytosol. With the use of extensively dialysed serum, it was possible to show that the intracellular parasites required an external source of hypoxanthine, panthothenate, cysteine, glutamate, glutamine, isoleucine, methionine, proline and tyrosine (Divo and Jensen, 1982b; Divo et al., 1985). A good substitute is Albumax I, which needs only to be supplemented with hypoxanthine. Vitamins such as ribloflavin, pyridoxamine and thiamine are also required.

The ability to have continuous axenic cultures greatly facilitates any type of investigation on P. falciparum. The laboratory protocols for the cultivation in vitro of malaria parasites are almost without exception minor modifications of the classic candle jar method first described by Trager and Jensen in 1976. The simpliest and most widely used is the petri dish candle jar method, or a variation of it using multiwell culture plates in place of the petri dishes. This method is excellent for experimental work for which numerous replicas are needed and small volumes are not a problem, such as parasite inhibition assays using antimalarial compounds or immunological factors, or in studies on parasite biology, ultrastructure and physiology (Jensen, 1981). Progresses in the various areas of malarial immunology and physiology has put increasing demands on in vitro cultures system to produce large quantities of the parasites (Palmer et al., 1982).

Though it is generally accepted that optimum growth of the malaria parasite Plasmodium falciparum in vitro is dependent on the presence of human serum in the culture medium, yet nummerous disadvantages associated with human serum, including variation in its growth promoting effectiveness between different batches, which necessitates batch testing, limited availability, relatively high cost, requirement for frozen storage, necessity for red cell/serum ABO group compatibility, and the obvious biohazard considerations, continue to provide an impetus toward the development of serum substitutes (Crammer et al., 1997).

In furtherance to these, the culture of P. falciparum, the most pernicious species of Plasmodium based on the Trager and Jensen candle jar system is very expensive when sub-tropical Africa is put in perspective. It would be an advantage to standardise the in vitro culture system by excluding the complex mixture of compounds found in serum (Zolg et al., 1982). Some investigators suggested that serum provides micronutrients and nonspecific growth-promoting factors to parasites while others ascribe it to nonspecific detoxifying properties (Barnes and Sato, 1980). There is a need to modify the existing methods of malaria culture system so that it becomes inexpensive and readily available in the regions where the most severe effects of the disease is felt and globally. The potential value of a suitable replacement for human serum in the in vitro cultivation of P. falciparum for chemotherapeutic and biochemical research is of particular importance in countries and areas of the world where malaria is endemic and non-immune human plasma is generally not available (Oduola et al., 1985). The present study provides a cheap alternative to the well established protocols by making use of plant extract which has been previously shown to support the growth of Trypanosoma cruzi in vitro (Fagbenro-Beyioku and Jagun, 1994 unpublished) rather than the commercial culture growth media. The formulation of a new, cheap and readily available culture system for P. falciparum would facilitate malaria research in respect of the exigencies of the developing countries and worldwide.

Recently the malaria situation has deteriorated and mortality from malaria is probably increasing in the whole of sub-saharan Africa. Some of the reasons for this are: drug resistance to most antimalarial drugs, insecticide resistance in mosquitoes, war and civil disturbances, environmental changes, climatic changes, travel and population increase. The main problem for malaria control, at present, is the antimalarial drug resistance, especially of Plasmodium falciparum, the most deadly malaria parasite (Krettli, 2001). Another important reason for the persistence of malaria in Africa is the presence of the vector, Anopheles gambiae, although social and economic factors are also worth mentioning. The female A. gambiae feeds preferentially on humans and is long-lived, making it particularly effective at transmitting malaria from one person to another. This makes the task of interrupting transmission daunting.

A major problem with the demise of inexpensive drugs such as chloroquine and sulphadoxine/pyrimethamine is that the newer drugs are unaffrodable by the poor people living in malaria endemic countries. According to Okochi and others (1999), one of the factors that draws back the rate of development of the health care system in Nigeria is the prohibitive high cost of importing drugs and producing new ones. Meanwhile as the levels of resistance to chloroquine and mefloquine continue to rise, the future for antimalarial treatment with existing drugs look increasingly bleak (Foley and Tilley, 1998). Artemisinin and related drugs are being used successfully in Southeast Asia and parts of Africa, but recrudescence after artemisinin treatment is a major problem and resistance to this drug is also appearing (Meshnick et al., 1996). With this increasing level of chloroquine resistance and fears of toxicity and decreased efficacy of sulphadoxine-pyrimethamine, there is an urgent need for an affordable, effective and safe alternative to chloroquine.

Medicinal plants, since times immemorial, have been used in virtually all cultures as a source of medicine (Hoareau and Dasilva, 1999). Traditional plants still play an important role in medical system in Nigeria and plant materials remain an important resource to combat serious diseases in the world. Pharmacognostic investigations of plants are carried out to find novel drugs or templates for the development of new therapeutic agents. Since many drugs, e.g quinine and artemisinin were isolated from plants and because of the increased resistance of many pathogens, e.g malaria parasites, towards established drugs, investigation of the chemical compounds within traditional plants is necessary, (Phillipson, 1991). New antimalarial drugs and approaches to overcome parasite resistance are needed to deal with the expanding problem of drug resistance which continues to challenge malaria control efforts based on early diagnosis and treatments. Only a limited number of antimalarial drugs are currently at an advanced stage of clinical development. In line with this, there is a renewed interest in plant products since the identification of sesquiterpene lactone artemisinin (quighaosu). An attractive option for Nigeria is the exploitation of the possible therapeutic effects of our local herbs. (Okochi et al., 1999)

Interestingly, many countries in the developing world are now regaining interest in making use of their indigenous resources that involve the use of herbal drugs and remedies and also considering the possibilities of combining synthetic medicine with traditional medicinal elements particularly at the primary health care level (Akerele, 1984; Hoareau and Dasilva, 1999). However, despite the collective efforts of scientists working on malaria around the world, the number of compounds tested against malaria remains very small compared with other diseases attracting more attention in the developed world. Relatively few compounds then prove active in inhibiting parasite growth in vitro and only a fraction of them is also effective in experimental animals (Olliaro and Yuthavong, 1999).

Majority of the West African people live in the rural areas where modern facilities are absent. Hence most people in such areas depend on plant extracts for the treatment of many diseases including malaria (Kimbi et al.,1998). In the traditional set up, the feverish patients usually consume either the aqueous or the alcoholic decoctions of the medicinal plants (Agomo, 1991). There is a need to increase the data that are available regarding the assessment of these plants and also the extent to which they are used in remedies in health care system. In Nigerian folk medicine several medicinal plants are highly valued to treat fever. Many of them have been previously investigated as antimalarial agents with suggestions for further research based on different forms of the extract, parts of the plants used and the determination of the active ingredients or substances. Since medicinal plants used in traditional medicine are an important source of biologically active compounds and have potential for the development of novel antiparasitic drugs (Phillipson, 1991; Wright et al., 1994), it is necessary to initiate screening program to evaluate the antiparasitic activity of the commonly used Nigerian medicinal plants and to isolate the active constutuents for therapeutic exploitation.

Background and Rationale for research on antimalarial plants

The current option for reducing the morbidity and mortality of malaria are still chemoprophylaxis and chemotherapy (Pradines et al., 2002). Failure of antimalarial prophylaxis with chloroquine and the combination of chloroquine with other antimalarials have been observed in Africa and other parts of the world where malaria is endemic (Sutherland, et al., 2003). The advent and spread of malaria parasites with a high level of resistance to most drugs recently available has created the need for the identification of new antimalarial drugs. Traditionally, medicinal plants have already provided valuable leads to potential compounds including Naphthoquinones terpenoids and alkaloids.

That antimalarial drugs are becoming less effective as the P. falciparum develop resistance to affordable drugs poses a serious further threat to clinical management and treatment of malaria. There has been a growing awareness by governments and the scientific and medical communities of the importance of medicinal plants in health-care systems in many developing countries. Greater importance is now being attached to the use of locally available medicines as a means of reducing reliance on expensive imported drugs. Recently, there have been some initiatives to promote research into the utilisation of traditional plant-based medicines (Bodeker and Willcox, 2000). Such plants are selected and screened on the basis of traditional reputation for efficacy in the treatment of malaria (Bhat and Surolia, 2001). Natural products have made an important contribution to antiparasitic drug research and despite all problems, there is every indication that they will continue to make contributions to the efforts to develop new and urgently needed drugs for now and the future.

In most African countries, chloroquine remains the only drug recommended by health authorities for the first-line treatment of uncomplicated malaria. It is also the only antimalarial frequently kept at home and used for self-treatment (Baird et al., 2002; Kofoed et al., 2002; Thomas et al., 2002; Trape et al., 2002). The continued use of chloroquine is understandable since the drug is cheap, always available and only associated with rather minor side effects (Kofoed et al., 2002). In addition, patients treated with this drug improve clinically, even though they remain parasitaemic after treatment (Trape et al., 2002). Meanwhile the spread of chloroquine-resistant P. falciparum through sub-saharan Africa has become a major obstacle for malaria control (Dorsey et al., 2000; Kofoed et al., 2002) and chloroquine resistance has been linked to malaria-specific mortality (Trape et al., 1998).

For historical and operational reasons, organised antimalarial vector-control campaigns are absent in most sub-saharan African countries. Malaria transmission intensities, therefore, are typically one or two orders of magnitude greater than those that occur in most other malaria-endemic regions of the world. (Alles et al., 1998). An understanding of the epidemiology of parasite diversity is thus important both in relation to the ability of parasites to escape antimalarial drugs as well as the immune system, including future vaccines (Farnett et al., 2002). The WHO (1993) had stated that the mainstay of malaria control remains early, effective treatment of clinical cases. Even though the effectiveness of this policy is likely to decrease as resistance to many available and affordable antimalarial increases (Schellenberg et al., 2002), it is almost certain that chemotherapy will remain the hallmark of malaria control especially in the absence of a malaria vaccine.

One of the approaches that have been proposed to limit this emergence and spreading of resistance to currently used antimalarials is the need to discover and develop novel compounds (Olliaro and Worth, 1997). In addition, there is a considerable interest in the use of multiple drugs, with different mechanisms of actions for the treatment of malaria cases (White et al., 2000). The rationale for combining drugs with independent modes of action and different resistance mechanisms is to improve therapeutic efficacy and to prevent or delay the emergence or development of resistance (Peters, 1990; White, 1998). The long-term benefit of using combination therapy in African settings may be realised if selective transmission of resistance is prevented (Sutherland et al., 2003). This may help to avoid the long wait for antimalarial drugs. The practice of combination therapy has long been utilised in the traditional treatment of fever and malaria In view of this, it will seem reasonable to suggest that the approach of drug combination is taken into consideration in the pre-clinical trials involving identification of active principles.

The unprecedented spread of multi-drug resistant P. flaciparum has highlighted the irrepressible need to develop new antimalarial drugs from natural products. Such drugs need be not only available but also affordable for developing countries where malaria undoubtedly remains one of the worst scourges contributing to mortality. The antimalarial potentials of compound derived from plants are proven by examples such as quinine, obtained from Cinchona species and artemisinin, obtained from Artemisia annua.

Apart from chemotherapy, the use of insecticides nets (ITNs) and curtains to prevent malaria has been emphasised as a possible effective means that could be very useful for both the individuals and the community. ITNs and curtains are a promising preventive measure because they have been documented to reduce malaria morbidity and mortality. Randomised controlled trials in different malaria transmission setting have shown ITNS, nets and curtains, to be effective in reducing mortality in children less than five years old (D’Alessandro et al., 1995; Binka et al., 1996; Nevill et al., 1996; Habluetzel et al., 1997). The implementation of ITN program is in progress in a number of countries in sub-Saharan Africa and more attention is been devoted to children in the first 2-18 months of life because high transmission pressure places the bulk of malaria-associated mortality in this age-group (Aidoo et al., 2002). Older children who survive this period have some degree of immunity that protects them from severe malaria (Molineaux 1997; Smith et al., 2001).

The mechanisms involved in the development of naturally acquired immunity against malaria are still not clear (Zhou et al., 2002) and protection from disease symptoms observed in older parasitaemic children, known as clinical immunity, is usually never reached in regions of very low or seasonal exposure to malaria transmission (Braga et al., 2002). Hence, Concerns have been raised, however, about the use of ITNs in high transmission areas, since ITNs reduce malaria exposure, a prerequisite for the development and maintenance of malaria-specific acquired immunity. In certain epidemiologic settings, this may in the long run be of detriment to the community because of the possible delay in the acquisition of effective immunity to malaria (Snow and Marsh, 1995; Snow et al., 1996). This view is supported by the epidemiologic studies in malarious areas showing that parasite densities and prevalence decrease with age and that severe disease manifestations and mortality are restricted to early childhood, a stage that is critical in the development of acquired immunity (Karuiki et al., 2003). The use of insecticides treated curtains, (ITCs) effectively provides a partial reduction in transmission intensity; children sleeping under ITCs probably still receive infectious mosquito bites but less frequently than those not using treated curtain and the rate at which curtain ITC users acquire effective immunity to malaria may therefore become reduced.

The hypothesis in this particular study is that the use of insecticides treated curtains (ITC) effectively provides a partial reduction in transmission intensity; children sleeping under ITC probably still receive infectious mosquito bites but less frequently than those not using treated curtain and the rate at which curtain ITC users acquire effective immunity to malaria may therefore become reduced. This hypothesis is supported, in part, by some studies in children that have shown that the use of ITNs was associated with a reduction in antibody response to malarial antigens (Genton et al., 1994; Snow et al., 1996, Askjaer et al., 2001). In this study therefore, investigation was carried out on the effect of the use of ITC on antibody immune responses in children living in villages with ITC and a control group of same age group living in another village without ITC. Also, the two allelic families of the msp-2 antigen represented by the parasite strain FCQ27 and ICI3D7 were analysed by PCR method.

Aims and Objectives of this research

□ To use readily available substitute for serum and RPMI 1640 in the medium used for the continuous cultivation of P. falciparum.

□ To formulate and develop a new and inexpensive in vitro medium for the cultivation of P. falciparum.

□ To assess the effect of variability of human serum on the cultivation of Plasmodium falciparum

□ To investigate the in vitro antimalarial activity of the crude extracts derived from four commonly used plants in Nigerian and West African folk medicine

□ To assess the potency of combination of antimalarial interaction between Enantia chlorantha and the three other aqueous extracts obtained from Azadirachta indica, Cympobogon giganteus and Morinda lucida.

□ To determine the mode of antimalarial activity of the (aqueous) boiled water extracts of the antimalarial herbs

□ To assess the role these extracts as used in traditional antimalarial treatments

□ Assessing the effects of insecticides treated curtains in preventing malaria

□ Determining and comparing the seroprevalences and the levels of malaria protective antibodies in children living in villages with and without ITCs

□ Studying the effect of ITC use on complexity of parasite population using PCR-based genotyping of parasites.

CHAPTER 2

MATERIALS AND METHODS

2.1 Study on malaria culture system

2.1.1 Preparation of Glucose solution

20g of glucose was dissolved in 100ml water (0.2g/ml). The solution was mixed until all the glucose dissolve to give a homogenous solution. The solution was autoclaved for 20 minutes and stored at 4 °C.

2. Preparation of saline solution

6.67g of sodium chloride was dissolved in 50ml water (0.13g/ml). The solution was made homogenous, autoclaved and stored at 4°C.

3. Preparation of (Phosphate buffer) PBS

Na2HPO412H2O (40g), KH2PO4 (5 g) and NaCI (81g) were dissolved in 1000 ml water and I part of the mixture was added to 100 parts of water to make PBX 1X with pH 7.3.

4. Preparation of liver extract

Approximately10g mice liver was crushed and 40ml PBS (1X) added. The mixture was centrifuged at 3000rpm for 20 minutes to remove the debris and suspended mass. Centrifugation was repeated until debris cleared. The resulting extract was autoclaved for 20 minutes, coagulant was removed by filteration and the final extract stored at 4°C.

2.1.5 Preparation of sap (plant exudate) solution

1ml of sap (Jatropha curcas) was dissolved in 99.0ml water. The resulting solution was filtered, autoclaved and kept as stock. The sap has been previously collected from the plant growing near the Library of the College of Medicine of the University of Lagos in January 2002. This was transported under cold condition to Stockholm.

2.1.6 Preparation of hypoxanthine solution

50mg hypoxanthine powder was dissolved in 1000ml water (0.05mg/ml).

2.1.7 Effects of water dilution of stock sap solution on uninfected erythrocytes

The objectives of this preliminary aspect of the research was to determine, prior to actual experiments, the appropriate dilutions of water and water plus glucose solution that would be necessary to :

• ensure that the infected and uninfected erythrocytes would not agglutinate in the new media.

• avoid the rapid discolouration of the media in the incubator due to excess and/or abnormal concentration of the sap solution (1% v/v).

• prevent the red cells in losing their form and function due to the concentration of the various solutions (conceptualisation)

Sequel to the above, 0.2ml washed uninfected blood was added to 1ml sap solution and diluted by water and water plus glucose solution in various ratios.

2.1.8 The newly formulated medium

Based on the results of the investigations above, i.e the effect of water dilution (data not shown), the following combinations were made to give a single batch of the newly formulated medium:

10 ml stock

20 ml water

2 ml glucose solution

1 ml saline solution

1 ml PBS 1X

12ml liver extract

15µl Gentamycin

The pH of the new media is about 6.6 but usually adjusted to 7.4-7.6 using NAHCO3 solution (0.002g/ml). The complete new medium was stored at -210C.

2.1.9 Experiment 1. New Malaria culture experiment

The candle jar method of Trager and Jensen (1976) was adopted in these experiments. 96 flat bottom culture wells were used because they are excellent for experimental work for which replicas are needed and small volumes are not a problem (Jensen, 1981). The parasitaemia was adjusted to 4% using blood washed thrice with Tris Hank solution and suspended in PBS (1X). Five replicas were set up for each approach as shown in table 1.

The total volume of each well of five replicas was 200µl of which 100µl was infected erythrocytes in PBS (1X) solution. In all cases, the haematocrit is 4%. The culture wells were incubated at 37oC after ensuring low oxygen by burnt out candles. The gas phase in a candle jar has about 3% CO2 and 15-17% O2. (Trager, 1987). The microtiter plates remained stationary at 37oC during the culture period, thus allowing the cells to settle at the bottom. When the medium was changed, culture plate were carefully moved to the culture hood area where the supernatant fluids were removed aseptically with sterilised Pastuer pipettes. At the same time blood films were prepared for the evaluation of parasite growth and multiplication. Fresh media were then placed as appropriate, the cells resuspended, and the culture plates returned to the incubator. These approaches, as described by Siddiqui, (1979) are conventional.

2.2 Experiment 2. The influence of serum variability of the in vitro cultivation of Plasmodium falciparum

2.2.1 Materials and methodology

Human sera from 1997 (SH97), 2002 (HS02) and a combination of the two (HS9702) were used for this experiment at 5% and 10% HS. The multiwell culture plates were set up as used in the previous experiment but with haematocrit of 2% and start parasitaemia 1%. Infected blood was washed 4 times with sterile RPMI 1640 solution and then synchronised by the sorbitol synchronization method (Vanderberg, 1979).

2.2.3 Synchronization of parasite culture.

The parasites in culture (8ml) were spin down at 2500rpm for 90 seconds and the supernatant remove. Thereafter, 8ml of 5% sorbitol was added and incubated for 10 mins at room temperature. The mixture was shaken twice, centrifuge after 10 minutes and washed twice with RPMI 1640 solution. The haematocrit was adjusted to 2% and parasitaemia 1% after synchronisation.

2.3 Experiment 3: The use of plant extract and human serum for the cultivation of P. falciparum malaria parasites.

The new plant based medium was prepared as described earlier and a similar medium was prepared but without autoclaving the plant exudate. In place of liver extract, human serum (HS) was added to the new media to make 5% or 10% of the media. Microtiter plates were used and the start parasitaemia was approximately 1.5%. The complete media with autoclaved exudate has pH 7.6 while that of the non-autoclaved was 7.5. The culture plate was kept in a CO2 incubator. Waki et al., (1984) had suggested that malaria parasites may be cultivated readily using a conventional CO2 incubator which is a common laboratory item.

2.4 Study on antimalarial plants

2.4.1 Plants used

The medicinal plants used in this experiment are shown in table 2 and these are, Azadirachta indica, Enantia chlorantha, Cymbopogon giganteus and Morinda lucida. They were obtained in Lagos, Nigeria in January, 2002.

2.4.2 Preparation of extracts

About 10g of each dried powdered extract was dissolved in 50ml alcohol (95%) for 7 days. The alcohol was allowed to evaporate at room temperature. 10ml PBS (1X) was added to 10mg of each extract to make 1mg/ml. The aqueous extracts were sterilized by passing through Acrodisc ( Syringe filter (0.2µm) to obtain clear filterate used for the inhibitory assay in culture wells.

2.4.3 Culture technique

P. falciparum, F32, that have been cultured continuously according to the methods of Trager and Jensen (1976) were used for this investigation. The parasites were exposed to a range of different concentrations of the four extracts in cornings microtest (96) culture wells at 1-2% parasitaemia. Each extract concentration has a replicate and serial dilutions of 1:2 (500µg/ml), 1:4 (250µg/ml], 1:8 (125µg/ml), 1:16 (62.5µg/ml) and 1:32 ( 31.3µg/ml). The number of parasitized cells per

40 000 cells were counted by fluorescent microscopy after 24 hours in culture incubated at 37o C.

2.4.4 Fluorescent microscopy

At 24 hours, the cells were harvested and put in 4ml centrifuge tubes, washed thrice with Tris-Hank solution. Tris-hank solution was prepared by adding 2.11g Tris HCl, 0.2g Tris base and 7.88g NACl in 1000ml distilled water. This soluton (Tris) was mixed with same volume of Hanks solution, hence the name Tris-Hank solution (0.15M, pH 7.2). The harvested cells were resuspended in 400µl Tris-Hank solution and fixed by Glutardialdehyde, GDA (4%) unto multitest slides coated with buffer. In 8 wells, with approximately 200 cells/field, an estimated 40 000 cells was counted in 25 fields for each well using high-power fluorescent microscope lens with oil immersion, and the percentage growth inhibition with respect to the control was determined by simple arithmetic calculation. This approach is one of the in vitro antiplasmodial tests for detecting antiplasmodial activity of plant extracts in the erythrocytic stage of malaria parasites (Rasoanaivo et al., 2003[In Press])

2.5 Antimalarial crude extract combination

2.5.1 The plant extracts were combined as follows:

Enantia chlorantha + Azadirachta indica (E+A)

Enantia chlorantha + Morinda lucida (E+M)

Enantia chlorantha + Cympobogon giganteus (E+C)

2.5.2 Plant extracts

In this experiment, the aqueous boiled water extracts of the plants were used. For the extraction, about 10g of each dried powdered extract was boiled in 50ml water until all the water evaporated. The extracts were thereafter dried by heat application. 10ml PBS (1X) was added to 10mg of each extract making 1mg/ml. These extracts in their PBS solutions were sterilized by passing them through syringe filters (0.2µm]. Equal amounts of each organic extracts were mixed and diluted with RPMI 1640 solution, were necessary, to achieve the desired concentrations in the culture wells.

2.5.3 In vitro inhibition assay for extract combination.

Serial double dilutions of the extracts were made in quadruplicate in 96 well micro titer plates. The extract solutions were diluted with RPMI 1640 to achieve the required concentration to be tested in culture. Each well contained 100µl of the diluted extract and 100µl of parasitized red blood cells at 4% haematocrit in MCM (1-2% parasitaemia, non-synchronised). The final combined extract concentrations tested ranged from 31.3-500µg/ml. The micortiter culture plates were incubated at 37oC in the candle jar. At 24 hours, fresh media with extracts were added and the experiment was terminated at 48 hours. After the incubation period, parasitized red blood cells were collected in 4ml centrifuge tubes, washed thrice with Tris- Hank solution and resuspended in 400µL Tris-Hank. The harvested cells were fixed unto 8 well multitest slide using buffer coat using GDA . Approximately 40 000 cells were counted under fluorescent microscopy after staining with dilute acridine orange.

2.5.4 Stage specificty of activity of antimalarial extracts

The parasites in culture were synchronised twice at 4-h interval using sorbitol lysis described previously. At 24hours after first synchronised, the culture was diluted to a parasitaemia of (1% and immediately used in the set up (96 wells, flat bottom micro-titer plates) for the stage specificity experiment. Each of the extract (uncombined) was used at a single concentration of 500µg/ml in 6 replicas. This was achieved by adding 100µl of infected (synchronised) culture and 100µl of each extract. The control was untreated and with the same number of replicas. At specific intervals (18-42h) after treatment, the cells were harvested and fixed in multitest slides using buffer coating and GDA. At 24 h after first treatment (and after second fixation), the media were changed in the remaining replicas meaning that fresh MCM-HS was added in the control and fresh extracts in the tested wells. The first harvest and fixation was at 18 hours and the last one was at 42 hours after first treatment or 54 hours after first synchronisation.

2.6 Materials and methods for studies on insecticides treted curtains

The study receives ethical approval from the Ministry of Health of Burkina Faso and the Research Ethical Committee of the Karolinska Institute (Stockholm, Sweden)

2.6.1 Study population

Blood samples were taken from asymptomatic children aged 3-7 years living in two villages, Goundry and Noungou near Ouagandougou, Burkina Faso. The study area is a typical zone of Sudanese savannah, with marked seasonal malaria transmission associated with the rainy season (July-October). During this period, the inhabitants of the area receive on the average more than one infective Anopheles bite per night (Esposito et al., 1988). Insecticides treated curtains are routinely used in Noungou village while they are absent in Goundry village. The children were treated after the first bleeding and blood samples were taken from the same children 21days after treatment.

2.6.2 Enzyme-linked immunosorbent assay (ELISA)

Determination of the concentration ot total anti-malarial antibodies was performed using an extract of matured stages of cultured P. falciparum as antigen (Troye-Blomberg et al., 1983). Ninety-six well ELISA plates (Costar) were coated with 50µL/ well of parasite extract (10µg/mL) (Perlmann et al., 1994). The immunoglobulin fractions were diluted 1:1000 and incubated for one hour at 37oC and IgG was assayed with phosphate-conjugated goat-anti-human IgG (Fc-fragment specific) (MABTECH AB, Nacka, Sweden) with p-nitrophenyl phosphate disodium salt (Sigma, St Louis, MO) as substrate. The immunoglobulin fractions used for both IgG1 and IgG3 was 1:400. Anti-IgG1 and IgG3 biotinylated monoclonal antibodies, (Pharmingen, San Diego, CA) were used for the assaying of these sub-classes. The concentrations of anti-malarial antibodies were calculated from standard curves obtained in a sandwich ELISA with six concentration of highly purified IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and goat anti-human IgG both as capture antibody (10µg/mL) and detecting antibody. Cut off values were calculated as the mean optical density values at 405nm plus 2SD of the values obtained with sera from eight Swedish donors who had not been exposed to malaria.

2.6.3 Preparation of parasite DNA

The DNA was prepared by a method described by Snounou et al., (1993). Blood was lysed with saponin and after centrifugation, the parasite-containing pellet was resuspended in lysis buffer (40nM Tris, pH 8.0, 80nM EDTA, 2% sodium dodecyl sulfate) and incubated in proteinase K. the DNA was extracted with phenol, followed by extraction with phenol-chloroform and chloroform. The DNA was then precipitated in 3M sodium acetate (pH 5.0) and absolute ethanol and incubated at -20oC overnight. After centrifugation, washing with 70% ethanol, and drying, the DNA pellet was resuspended in PCR water.

2.6.4 Polymerase chain reaction (PCR)

The PCR approach has been used to analyze the genome diversity of malaria parasites in the 2 groups of children. P. falciparum populations were genotyped by amplification of genes for merozoite surface protein-2 (msp-2). Block 3 of the msp-2 gene was amplified in the first reaction using the oligonucleotide primer pair M2-OF and M2-OR followed by two separate second amplifications to detect the IC allelic family, ICF and ICR. FCF and FCR primers were used for the detection of FC27 allelic family.

2.6.5 Gel electrophoresis

10-12(l aliquot from each PCR was loaded onto 1.8 % agarose gel in the presence of Ethidium bromide, subjected to electrophoresis and visualised with UV-light

CHAPTER 3

RESULTS

3.1 New malaria culture

Several attempts were made to grow P. falciparum in this plant-based medium. The most successful outcome is presented here. The dilution of the stock sap solution (1% v/v) by twice volume of water was found to be best suitable for the erythrocytes in view of the objectives analysed earlier. Growth of the parasites that have been coincubated in 96 wells plates was monitored for 5 days. The media were changed daily but no subcultivation was done. There had been many previous attempts to culture the parasite at other parasitaemia. It was however possible to observe substantial increase in parasitaemia in the various cultures (E-I) with a start parasitaemia of 4%. Table 3 and figure 1 showed the results obtained. The mean growth rates of the parasites on day 5 are shown in figure 2. Growth rates in these experiments, positive or negative, are expressed as percentage increase or decrease in parasitaemia over a specified period of time (formula given below). For MCM-HS, the culture was discontinued after day 3 due to decreased in number of parasitised erythrocytes. Since no subcultivation was carried out, the probable accumulation of lactic acid and other metabolites known to inhibit growth cannot be ruled out in this particular medium.

Growth rate= percentage parasitaemia-start percentage parasitaemia

Day(s) in culture

Where 24 hours represents one day in culture and 120 hours is 5 days in culture.

The results showed that the highest parasitaemia in the new medium was 9% at 48h in culture with medium G. This medium contains hypoxanthine at 0.02µM. After day 5, a parasitaemia of 8% was still observed in this medium. It was obvious that the conventional malaria culture medium supplemented with human serum (MCM.-HS) was far better than this new attempt as far as this trials are concerned. The parasitaemia was already 15% at 48h and the medium was discontinued at 72h. The mean growth rate with the MCM-HS was 4.2 as against 1.2 in medium G. It was not possible to re-establish parasites in this new medium by subsultivation. Perhaps more studies will be needed to achieve that. To show further that the plant exudate provided a source of nutrient required for parasite growth, it was supplemented with human serum at 5 and 10% and the parasites were observed for 48 hours. Tables 5a and 5b and figures 4a and 4b showed the observations recorded. The observations revealed that the newly formulated medium is capable of sustaining the malaria parasites even when hypoxanthine is excluded as in media J, L, N and R (figure 4a and 4b).

3.1.1 Influence of serum variability

Differences in the growth rate of parasites under in vitro are a common experience in the laboratory. One of the reasons for this may be the influence of the variability of the sera or materials used. The influence of the variability of human serum was demonstrated out using 2 different human sera whose combination effect proved more effective than when either was used alone. These media were changed daily for up to 8 days. The results are shown in tables 4a and 4b and figures 3a and 3b. Pooling of 2 human sera led to more growth compared to when either serum was used singly. Likewise it seems that the serum of 2002 did better than that of 1997. The reason for this is not known.

3.2 On antimalarial plants

The organic extracts of 4 medicinal plants at 5 different concentrations were tested for 24hours only in unsynchronised cultures of MCM-HS at 1-2% parasitaemia. The untreated culture served as the negative control. The basic measurement of antimalarial activity used in this study was a reduction in the number of parasitised cells in the test cultures compared to the negative control at 24 hours of incubation. Table 6 shows the percentage inhibition ± SEM. E.chlorantha showed the greatest antimalarial activity of 68.9% inhibition at 500µg/ml. This was followed by M.lucida with inhibition of 65.5% at same concentration while A.indica and C. giganteus gave 41.3 and 58.6 % inhibitions respectively. With respects to the administered concentrations, dose-dependent antimalarial activity was evident for all the crude extracts. The inhibition shown by A. indica at 500µg/ml and 250µg/ml was the same and it is unclear what could be responsible for this observation. At other concentrations and with other extracts, the dose-dependent activity were marked. The inhibition are higher with increasing extract concentration. The synergistic properties of the aqueous extracts of the antimalarial plants have also been examined. Table 7 showed the outcome of the combinations effects of E.chlorantha and each of the other three antimalarial plants namely, Azadirachta indica, Cypbopogon giganteus and Morinda lucida at different combined concentrations. There were remarkable inhibitory activites when E. chlorantha was combined with each of the other three extracts. These extracts were all in aqueous form. After 48 hours, greatest activity was observed when E.chlorantha is added to C.giganteus with inhibition of 86.2±1.6%. This was closely followed by E.chlorantha added to A.indica with 85±3.1% (table 7). Chloroquine was used in this experiment to compare the efficacy of these extracts to a commonly used antimalarial. The stage specificity experiment was performed using only a single concentration of the aqueous extract (500µg|ml) in their uncombined forms. Table 8 showed the inhibition from 18 to 42 hours after first treatment. The experiment was conducted using six replicas; the second treatment was carried out after harvesting of the cells for 24h fixation. Figure 5(a)-(e) showed the chemosuppressive and antiplasmodial effects of the aqueous extracts of the various medicinal plants on the various asexual stages of the parasite life cycle. It was also possible to compare the efficacy of the different forms of the extracts at 500µg/ml, i.e organic and aqueous extracts as shown in figure 6. The extracts exhibit varying inhibitory activities in respect of whether they are aqueous or organic. Interestingly, A.indica which gave an inhibition of 41.3±5.7% as organic extract was seen to inhibit about 75% of parasite growth as aqueous extract. The other extracts were found to be more active as organic extracts in this study.

3.3 Insecticides treated curtain

P. falciparum specific IgG

Eighty four samples have been analysed for ELISA out of which 46 were from children living in village with ITC and the remainder 38 were from a control group of children in the same age group living in another village without ITC. The results are shown in table 1. In Goundry village without ITC, it was observed that the mean for total IgG was higher for the first bleeding. The value was 30.2±3.9µg/mL while that of Noungou village with ITC was 21.4±4.0µg/mL. However, for the second bleeding, it was observed that the mean for total IgG was lower in Goundou village with a value of 12.2±1.8µg/mL as against 13.1±3.3µg/mL in Noungou village (Figure 7a and 7b). All the differences seen were not statistically significant.

Parasite specific IgG1 and IgG3 antibodies

IgG1 levels in village with ITC was higher than the levels in village without curtains for first and second bleeding. A mean of 6.73±2.5 and 5.1±1.5(g/mL was found in Noungou village compared to 4.35±0.5 and 3.24±0.3(g/mL in Goundry village (figure 8a and 8b). Similarly all the observed differences were not statistically significant.

The mean concentrations of IgG3 were higher in the village without ITC. The values obtained are 0.59±0.1(g/ml and 0.53±0.1(g/ml while a mean of 0.48±0.1(g/ml and 0.39±0.1(g/ml was observed in Noungou village for first and second bleeding respectively, (figure 9a and 9b)

P.falciparum genotypes

Both msp-2 allelic families, 1C37 and FC27 were found in the study groups. A comparative analysis showed that FC27 and IC37 were equally distributed among the children in the village where ITC is used with a mean occurrence of 1.67±0.2 for each allele. Meanwhile in the village without ITC, a higher occurrence of FC27 was seen over IC37 alleles. In comparing both study groups, we found that while the occurrence of FC27 was higher in the village without ITC, the same cannot be said of IC37, (figure 10a and 10b). This genotype, i.e IC37 occured more in the village with ITC. However, there were no statistically significant differences between users and non-users in observed infection multiplicity. This may imply that there was no preferential carriage of either type of genotypes in both groups.

RESULTS.

The synergistic properties and stage specificity antimalarial activities of the aqueous extracts have been examined. Tables 1 and 2 and figures 1-5 showed the outcome of the synergistic effects of combining E.chlorantha and each of the other three antimalarial plants namely, Azadirachta indica, Cypbopogon giganteus and Morinda lucida at different combined concentrations. Chloroquine was used in this experiment to compare the efficacy of these extracts. The stage specificity experiment was performed using only a single concentration of the aqueous extract (500µg|ml) in their uncombined forms. Table 3 showed the inhibition from 18 to 42 hours after first treatment. The experiment was conducted using six replicas; the second treatment was carried out after harvesting of the cells for 24hour fixation. Hence it was possible, using this approach to compare the efficacy of the different forms of the extracts at 500µg/ml, i.e organic and aqueous extracts as shown in figure 6. Figure 7 shows the chemosuppressive and antiplasmodial effects of the antimalarial plants on the blood stages of the parasites while figure 8 shows their increase in inhibitory activity over a 42hour period.

Results

Total IgG.

Eighty four samples have been analysed for ELISA out of which 46 were from children living in village with ITC and the remainder 38 were from a control group of children in the same age group living in another village without ITC. The results are shown in table 1. In Goundry village without ITC, it was observed that the mean for total IgG was higher for the first bleeding. The value was 30.2±3.9µg/mL while that of Noungou village with ITC was 21.4±4.0µg/mL. However, for the second bleeding, it was observed that the mean for total IgG was lower in Goundou village with a value of 12.2±1.8µg/mL as against 13.1±3.3µg/mL in Noungou village (Figure 1a and 1b). All the differences seen were not statistically significant.

IgG1 and IgG3 subclsses.

IgG1 levels in village with ITC was higher than the levels in village without curtains for first and second bleeding. A mean of 6.73±2.5 and 5.1±1.5(g/mL was found in Noungou village compared to 4.35±0.5 and 3.24±0.3(g/mL in Goundry village (figure 2a and 2b). Similarly all the observed differences were not statistically significant.

The mean concentrations of IgG3 were higher in the village without ITC. The values obtained are 0.59±0.1(g/ml and 0.53±0.1(g/ml while a mean of 0.48±0.1(g/ml and 0.39±0.1(g/ml was observed in Noungou village for first and second bleeding respectively, (figure 3a and 3b)

P.falciparum genotypes

Both msp-2 allelic families, 1C37 and FC27 were found in the study groups. A comparative analysis showed that FC27 and IC37 were equally distributed among the children in the village where ITC is used with a mean occurrence of 1.67±0.2 for each allele. Meanwhile in the village without ITC, a higher occurrence of FC27 was seen over IC37 alleles. In comparing both study groups, we found that while the occurrence of FC27 was higher in the village without ITC, the same cannot be said of IC37, (figure 4a and 4b). This genotype, i.e IC37 occurred more in the village with ITC. However, there were no statistically significant differences between users and non-users in observed infection multiplicity. This may imply that there was no preferential carriage of either type of genotypes in both groups.

CHAPTER 4

DISCUSSION

4.1 Malaria culture system

Plasmodium falciparum malaria is responsible for millions of deaths worldwide annually (Gatton and Cheng, 2002). The availability of a method for the continuous cultivation of P.falciparum described by Trager and Jensen (1976) has stimulated multiple and varied laboratory investigations on the most virulent of the human parasites. However the ease with which P.falciparum parasites adapt to culture by this method of Trager-Jensen is variable (Chin and Collins, 1980). According to WHO (1977), there has always been variation in the ease with which isolates or cultured parasites can be maintained moreso in a new medium. This variability in the ease of adapting to culture conditions is a common experience shared by investigators using the Trager-Jensen method to establish strains of falciparum parasites. In one experiment carried out by Chin and Collins (1980), it was observed that only 2 out of 15 isolates grew well upon culturing while the remainder strains required an adaptive period of 1-2 months before growth rates greater than 10 fold per 96 hours were reached. During the period of no apparent growth, the multiplication rate was seldom more than 2-fold every 96 hours and parasites were difficult to detect even in thick smears. Domarle and others (1997) found 4 out of 19 field samples adapted to culture conditions. These investigators considered a strain as adapted when the multiplication rate of the parasites exceeds 10-fold every 96 hours.

In this new study, several preliminary trials were conducted until P. falciparum F32 strains was succesfully sustained in vitro for a limited number of days. It is not clear why the parasites are difficult to re-establish by sub-cultivation but the problem of adaptability may be a likely reason. These results are basis for ongoing further investigations and optimisations. No previous attempts have been made to use plants extracts for the in vitro cultivation of P.falciparum except one report in which coconut extract was used (Renapurkar and Sutar, 1989). However the sap of Jatropha curcas used here as a basal medium has been shown to support the growth of Trypanosoma cruzi in our laboratory in Lagos (unpublished data). It is imperative to state that, since it takes substantial time for malaria parasites to adapt to a new growth medium, it will seem almost unlikely that the parasites would survive long enough to proliferate at a start parasitaemia of ≤1%. This can explain why a higher start parasitaemia was later used in this study. Indeed at 48h growth was observed in the culture wells especially those containing hypoxanthine. Sax and Rieckmann, (1980) suggested that the cultivation of parasites in a new culture medium should be pursued if comparable growth is not achieved immediately after changing from one serum (medium) to another.

Lingnau and others (1994) reported that continuous cultivation of various P. falciparum strain was possible with serum-free medium in Nutridoma-SR supplemented RPMI medium. However, the growth in the serum control was better and the reason for this was unclear. The ability of one serum-free formulation to support parasite growth also varies from strain to strain (Flores et al., 1997). For example, Nutridoma SR (N-SR; Boehringer Mannheim) has been shown to substitute serum in culturing of geographically distinct strains of malaria. Two of the test strains grew comparatively as well as the serum-grown controls, whereas another reached half the growth rate of control cultures (Lingnau et al., 1994). Likewise, other groups have substituted serum with a serum-defined lipid cholesterol-rich mixture and bovine albumin, with differing effects on parasite development (Asahi and Kanazawa, 1994; Ofulla et al., 1993).

The beneficial effects of adding glucose and hypoxanthine to parasite culture have previously been described (Zolg et al.,1982; Ofulla et al., 1993). Optimal glucose concentration in serum free media seems to be beneficial to parasites considering the fact that a parasitised erythrocytes uses 26 times more glucose than an unparasitised red blood cell (Jensen et al., 1983). Divo and Jensen, (1982b) tested various supplements, individually and in various supplements, with dialysed human serum and found that only hypoxanthine contributed to increased parasite growth. Hypoxanthine which is important for the formation of nucleic acid, was used in the experiments reported here to investigate its role as a growth-promoting factor. It was found to increase the mean growth of the parasites in culture compared to the media in which hypoxanthine was absent. However, there was no statistical significant difference between the growth observed in the media when used alone and when various percentages of hypoxanthine was added. The success of the various media that have been used for the cultivation of P. falciparum may be hinged on the presence of hypoxanthine ranging from 30 to 375µM in the culture media (Divo and Jensen, 1982b; Asahi et al., 1996) although Ofulla and others (1993) described that RPMI 1640 supplemented with Bovine Albumin Serum (BSA) sustained growth of the parasites for a long-term without added hypoxanthine. Obviously, there are various other supplements other than hypoxanthine that can place developmental demands on the parasites (Asahi et al., 1996).

The plant 'milky exudate' used in this study is known to contain water, protein, fat, carbohydrate, fiber and ash, (Duke and Atchley, 1984). It has been reported that albumin may be one of the major factors in the growth-promoting influence of serum. The mammalian liver is the source of albumin, a water-soluble protein. Liver extracts also contain many vitamins and minerals including iron. If it could be possible to use fractions of the liver extracts without autoclaving, it may be possible to obtain better parasite growth. The results of this study will be of practical and research value and may also help to unravel the precise role of serum in malaria culture medium. F32 strain of P. falciparum is commonly used in laboratory-based malaria research and a serum-free medium compatible to their growth requirements, as well as other strains of Plamodium falciparum, will be advantageous for standardisation of experimental data. In addition, to verify that that cells have not lost their physiological and/or metabolic functions, it will be interesting to perform drug sensitivity and metabolic assays on them when the medium is better established.

In establishing the influence of serum variability on malaria parasite culture, sera from 1997 and 2002 were used. Obviously, during routine laboratory culture of parasites, it was common to observe that sometimes the parasites grew well in cultures while at other times their growth is poor. In a specific instance, it was observed that combined sera from 1997 and 2002, (HS9702) gave an outstanding turnover of parasites in a short time depite a low start parasitaemia (data not shown). To be sure, a study was conducted to assess the effects of each serum (HS97) and (HS02) and the effect of combining both of them (HS9702). The results are shown in table figures 4a and 4b. At 5%HS, it was only in the wells having combined sera, HS9702 that the parasitaemia increased substantially at 96 hours. It was not possible to obtain a substantial increase in parasitaemia when the cultures in the HS97 and HS02 wells were adjusted to 10%HS even after 3 days (figure 3b). The parasites were maintained but hardly grew. It cannot be over ruled that the 1997 serum may have become unsuitable for culture but in comparison with the serum from 2002, it is also possible that the parasites will also require additional time to adjust to the new concentration of HS, i.e 10%. It seems almost invariably that the parasite requires corressponding adapatibility period to any kind of change in vitro, no matter how little. Rojas and Wasserman (1993) showed that when P. falciparum is subjected to altered or unconducive culture conditions, it is not unusual for the number of parasites to remain approximately constant. Experiments such as these, in which nutrients are made sub-optimal or temperatures made febrile, are used to manipulate culture so that parasites at any stage of development can be forced to mature at a given time.

Furthermore, Siddiqui (1979) had reported that the concentration of serum could be reduced to 2.5% to keep the stock cultures going. However, it is apparent from the results shown here that a serum concentration of 5% would not only keep the stock going but also increase the growth of the parasites if the serum proportion is from pooled sera. Since a number of factors exist in human serum (HS) that placed developmental demands on P.falciparum, to ensure minimal variation in the outcome of experimenta data, some investigators had suggested the pooling of human sera. This may also reduce the proportion of HS needed in the total medium. Human serum remains scarce and pooling up to 18 serum samples (Divo and Jensen, 1982a) may not be very practicable. The usage of 2 human sera even at 5% as shown here may be adequate to obtain desirable or optimal parasite growth in vitro.

In one single but short trial using the plant extract formulation and human serum, the malaria parasites were successfully maintained in culture for 48 hours (Tables 6a and 6b). It was possible to observe that non-autoclaved extract would support growth as much as autoclaved exudate though the influence of added hypoxanthine was difficult to asceratain during the 48 hours period. By carefully defining a way to obtain the exudate, it should be possible to minimise contamination, hence eliminating the need for autoclaving. Filteration using filter tops (0.22µm) should keep germs away. This result sheds more prospects on the fact that it could be possible to reach the goal of culturing the parasites and obtaining high yields using the plant exudate and animal sera. Ultimately, an avenue to achieve the aim of providing a cheap and readily available means of cultivating P.falciparum malaria parasites would have been opened. It will be interesting to use such parasites as antigens in in vivo study in mice because of the complete absence of human host factor.

2. On antimalarial plants

In sub-Saharan Africa where malaria is endemic and in other parts of the world, plants are extensively used for treating periodic fevers and malaria. The spread of multidrug-resistant P. falciparum has highlighted the urgent need to develop new antimalarial drugs, preferably inexpensive drugs that are affordable for developing countries, where malaria is prevalent (Miller, 1992; Vial, 1996). About 75% of the population in Africa do not have direct access to chemical treatment, such as chloroquine, but they have access to traditional medicine for treating fevers. However, treatment with these remedies had suffered a number of deficiencies; diagnosis is often a problem, identification of plant extracts may be insecure and the chemical content of extracts may vary considerably (Azas et al., 2002).

In this study, four crude organic extracts obtained from medicinal plants used in Nigerian folk medicine for the treatment of fever and malaria were tested in vitro against P.falciparum. The most active extract was obtained from E. chlorantha that showed appreciable inhibition of the parasites at all the concentrations used in the study. Enantia chlorantha (bark and leaf) which is used for sore treatment, fevers, coughs, vulnerary ulcer, haemostatic and febrifuge by traditional healers contains alkaloids, lignin, saponins and tannins (Gill and Akinwunmi, 1986). For M. lucida, dose-dependent inhibitory outcomes were marked. Awe and Makinde, (1997) reported the dose-dependent and seasonal variation in the activity of M. lucida using both in vitro and in vivo techniques. M. lucida was reported to contain anthraquinones which showed in vitro activity against P.falciparum and also possess antifungal properties. Morinda lucida is used locally in the treatment of yellow fever and jaundice (Guido et al., 1995). The inhibition shown by C. giganteus can be said to be remarkable because the plants is usually boiled with a mixture of certain other plants in Nigeria for prophylaxis or traditional chemotherapy of malaria. Occasionally, a few people take it alone. The relatively lower inhibition observed for the organic extract of Azadirachta indica in this study may correlates earlier findings that A. indica functions more as an antipyretic than as a schizonticidal agent in malaria therapy (Okpaniyi and Ezeuku, 1981). It is not clear why the observed inhibition was the same at 250µg/ml and 500µg/ml (table 6) but since biological systems are complex in nature, no kind of test can be expected to function perfectly (Rasoanaivo et al., 2003). Also, the saturation effect of the extract activity might be a factor.

It is noteworthy that whenever they have been studied, antimalarials such as chloroquine and quinine have not been found to have useful antipyretic properties (Krishna et al., 1995) contrary to dogma. Reasons to treat fever (using antipyretics) in malaria include making the patients more comfortable, minimising metabolic stresses of infection and perhaps reducing the risk of convulsions and neurological sequelae in children (Newton and Krishna, 1998). Treatment with antipyretic agents such as A. indica would lead to early relief of fever and pyrexia to eliminate the parasite thereby helping the body's immune system. Fever is a host response associated with schizont rupture and is the most common clinical manifestation of malaria (Gatton and Cheng, 2002).

Kimbi and others (1998), reported the chemosuppressive and prophylactic activities in mice of the medicinal herbs used in this present study. Their results showed that the boiled water extracts of C.giganteus and E.chlorantha have good potentials against chloroquine-resistant P.yoeli nigeriensis both as schizonticidal and prophylactic agents when compared to artemether. Also, in their study, very little antimalarial activity was reported for A.indica and M. lucida in the mice. However, Makinde and Obih (1985) reported that the boiled water extract of A.indica showed schizonticidal activity against chloroquine-sensitive P.berghei. It is therefore possible that the strain of the parasites or the species accounted for the differences observed. In addition it is not uncommon that some plants which are popularly used to treat fever or malaria in some areas may be found to be inactive or toxic in mice (Kimbi et al.,1998; Krettli et al.,2001). One plausible explanation is the unsuitability of the in vivo rodent malaria models to demonstrate the expected activity. Truly, no in vitro drug sensitivity test can entirely mimic the in vivo situation, but these in vitro methods should ideally utilise both uniform drug exposure and a test medium that approximates the in vivo milieu (Sixsmith et al., 1984). Additional in vivo models may be needed to adequately evaluate these antimalarial plants (Dow et al., 1999).

The strategy of drugs combination couple with early detection and confirmed diagnosis represent one of the way forward in the chemotherapy of malaria (Nosten and Brasseur, 2002). This will prevent or delay the emergence and spread of drug resistance and also interrupt the transmission of P. falciparum. In this study, the combination effects of boiled water extracts of four medicinal plants was observed at 44 hours. Drugs with different mechanisms of actions may enhance their respective efficacies and extend their therapeutic life span. This is a major public health consideration for developing countries whose needs for effective (and new) drugs is pressing (Taylor et al., 2001). In traditional settings, healers normally use decoctions to cure patients. Currents trends in malaria chemotherapy suggest a drift towards drug combination therapy. Drug combination approach has been adopted in cancer chemotherapy and in the treatment of infection with human immunodeficiency virus, tuberculosis, leprosy and Pseudomonas infection (Tjitra et al., 2001). Currents trends in malaria chemotherapy suggest a drift towards drug combination therapy. Specifically, combination such as artesunate and mefloquine have been found to be effective against multi-drug resistant P. falciparum (Nosten et al., 2000).

In this study, the combination effects of boiled water extracts of four medicinal plants was observed at 48 hours. The antimalarial activity of the organic extracts of these plants has been previously described. Treatment was repeated at 24hours after first treatment and the medium in the control was also replenished. All the combinations used showed significant but dose dependent inhibitory activity (Figures ?). These data are interesting because the combination effects or synergism permits the quantity of each extract to be reduced with higher efficacy which is comparable to the use of chloroquine alone. However at 31.3-125µg/ml, chloroquine showed a higher inhibitory activity than all the combinations of extracts used. Perhaps, at suboptimal levels, the extracts failed to show synergism even though the final extracts concentrations equates that of chloroquine. Since, the extractions are crude, their activities might have be devoid of the enhance efficacy that their isolated active principle (as in chloroquine) would have probably exerted at same concentrations. Nevertheless, considering the spread of resistance to chloroquine, which is the cheapest available antimalarial drug to date, the combination of active principles in drug research approaches using plant extracts or natural products will go a long way in tackling issues related new and cheap drugs that will improve cure rates, delay emergence of resistance and reduce transmission.

Usually, the end point for assessing drug sensitivity is parasite reinvasion of erythrocytes (Trigg, 1985). Inhibition of parasite invasion is due either to inhibition of the release of progeny merozoites or to inhibition of merozoite invasion of erythrocytes. The level of parasitaemia after merozoite release from erythrocytes containing schizont-stage parasites and subsequent invasion of new erythrocytes was less in all test cultures (with extracts) than in the untreated (negative control) cultures. Using an untreated culture as the control, it was possible to show that the culture adapted parasites were sensitive to the plant extracts and chloroquine because of the inhibition of parasite re-invasion and appreciable degree of schizonticidal effects as evident in test cultures A. indica and E. chlorantha at 42hours. This stage specificity experiment provided an opportunity to make a comparative analysis of the efficacy of the plant extracts at 24h with respect to the organic extract used in the previous study. It was observed that all the medicinal plants have higher inhibitory activity as organic extracts at same concentration of 500µg/ml except A.indica that showed that showed a more remarkable inhibition as aqueous extract (figure ?). It is possible that the solubility of the active ingredients of these plants differ based on the mechanisms of extraction. Another possible factor that may have influenced this variability is that the culture was synchronised in the stage-specificity experiment.

Investigations have been carried out on A.indica with a view to finding some scientific evidence for its use in traditional medicine. This plant has been shown to possess a steam-volatile, oily constituent in trace amounts, which showed one component common to leaf, stem and root bark when examined chromatographically (Sofowora, 1993). Ekanem (1978), Aladesanmi and others, (1988) and Kimbi and others, (1998) have reported the antimalarial effect of this plants on both chloroquine sensitive and multidrug resistant strains of P.falciparum. In this study, even though all stages of the parasites were still found at 42 hours, the schizonticidal effect of A.indica was marked. Morinda lucida showed a seemingly comparative but higher inhibition as organic over aqueous extracts. This correlated with the earlier findings that organic extract of M.lucida demonstrated higher antimalarial (schizonticidal) activity than the aqueous extract (Awe and Makinde, 1997). This plant is popularly known for its use against fever within the West Africa communities because of its schizonticidal activity and a number of compounds have been isolated from it including anthraquinones (Adesogan, 1979). At 42hours in this experiment M.lucida showed parasite inhibition that was more chemosuppresive than schizonticidal (figure 5b). Awe and Makinde (1997) suggested that the organic fraction of M.lucida could be further studied in order to isolate the antimalarial active principle in it as a promising medicinal plant.

The leaves of C. giganteus contains volatile oil, hesperidin bitter principles, and is used as flavouring agent, stimulant and anti-pyretic (Tyler et al., 1988). This plant, generally regarded as a fever-reducing herb, is also antiplasmodic as shown here. It has schizonticidal effect and had substantial inhibition on chloroquine sensitive P.falciparum at 42 hours (figure 5c). This plant has also been shown to have good potentials against chloroquine-resistant P.yoelii nigeriensis both as schizonticidal and prophylactic agents even when compared to artemether (Kimbi et al., 1998). E.chlorantha showed a higher schizonticidal effect than both M.lucida and C.giganteus. This plant also contains alkaloid, which are antiprotozoal even at low concentrations. Like C.giganteus, it has also been reported to have good potentials against chloroquine-resistant rodent malaria parasite in vivo. In rural, periurban and urban settings of West Africa, it is not uncommon that E.chlorantha is usually administered as alcoholic tinctures. The finding here that it has a higher antimalarial activity as an organic extracts supports its common traditional form of usage. Also, most other medicinal plants have specific ways in which they are utilised in traditional prophylaxis and chemotherapy of malaria.

4.3 On insecticides treated curtains

This study investigates the possible influence of the use of treated curtains on IgG antibodies levels in the sera of children compared to a control group of similar age group in Burkina Faso. Only asymptomatic children, (i.e children wihout fever) were included in this study. The children in this study were treated after the first collection of blood samples and analyses of sera collected 21days after treatment reveal a reduction in the total IgG as well as in IgG1 and IgG3 levels.

In this study one can suggest that treatment of asymptomatic individuals could lead to a reduction in malarial antigens and possibly a tendency towards the waning of anti-malarial IgG antibodies in hitherto exposed or protected children. The differences seen in these antibody titres were not statistically significant except in the IgG1 levels in Goundry village (without treated curtains). Here there was a statistically significant difference in the antibody levels before and after treatment. It has been previously reported that treatment with the consequent loss of parasites may be reflected in a decline in antibody levels (Rzepczyk et al., 1997; Boutlis et al., 2002; 2003). Hence there have been caution against the treatment of asymptomatic parasitemia in children and adolescents in whom possible protective effects may be reduced by treatment (Charlwood 1999; Boutlis et al., 2002).

Interestingly, it was found here that mean IgG1 antibodies was higher in Noungou village were treated curtains are routinely used. It has been hypothesised that in high transmission areas (such as Burkina Faso), the immune system especially in young children is overwhelmed by high density parasitaemia (and more parasite density) encountered during erythrocytic development, which results in an inefficient mounting of immune response (Kariuki et al., 2003). However, this new study does not indicate that the diversity of Plasmodium falciparum was less in the village were ITN is used even though the parasite density was lower.

Multiplicity of Infection

While FC 27 and IC37 have equal occurrence in children living in village with ITC, the occurrence of IC37 was more frequent in the non-ITC group. However there was no preferential carriage of either FC27 or IC37 alleles in both groups because there was no statistically significant difference in observed infection multiplicity. This shows that in areas of high P. falciparum endemicity, the use of curtains to prevent malaria may not delay the acquisition of malaria immunity in children. It has been speculated that any intervention to control the disease such as long-term chemoprophylaxis, impregnated bed-nets (Smith et al., 1999) or malaria immunization might interfere with the multiplicity of infection in individuals as it might act on parasite density loads and the spectrum of the disease. In addition, an evaluation of the extent of parasite diversity and the elucidation of the role that such a polymorphism plays in the human immune responses to those parasites is required to understand the parameters governing the acquisition of protective immunity (Ntoumi et al., 1995).

How the amount of disease in childhood varies with transmission is of central importance to malaria control (Snow and Marsh, 2002). It is possible that the development of an efficient antimalarial immunity requires continuous exposure to a large number of parasite variants and malaria antigens (Perlmann et al., 1995). The main concern for ITNs use is that in areas of high malaria transmission the long term use of these nets and curtains, which partly reduce the force of infection for people who use them, might result in an overall increase in malaria mortality relative to preintervention levels (Coleman et al., 2001). However, at all levels of transmission the overall balance of benefits, including reduced load on families and health services from non-life threatening malaria, favours the widespread introduction of ITNs in endemic areas of Africa (Snow and Marsh, 2002).

There are other fears associated with the use of insecticides treated materials which includes the cost implications, infrequent and improper use, very low retreatment rate (outside trial situation) and pyrethroid resistance. While ITMs will undoubtedly save many lives from malaria, particularly in the short-term, their long-term use especially in areas of high transmission which could lead to mortality rebound in later childhood needs to be carefully monitored (Snow et al., 1996).

CHAPTER 5

SUMMARY

The continuous cultivation of P. falciparum, achieved by Trager and Jensen in 1976 revolutionised research on human malaria. Their methods made it possible for research workers all over the world to study the clinically most important malaria parasite and hence formed the basis of many of the recent advances in malaria biochemistry, parasitology, immunology and chemotherapy (Trigg, 1985). The availabilty of fresh human serum remains a major limiting factor in growing P. falciparum on a mass scale.

The use of coconut water seems to be the only previous attempt made to cultivate P. falciparum using plant medium as reported by Renapurkar and Sutar (1989). Even then, RPMI and human serum were employed because the red cells were lysed when coconut water was used alone. However, by more thoroughly defining the nutritional requirements of P. falciparum, it may be possible to eliminate the human serum (and RPMI 1640) required for the continuous cultivation or to use animal sera without any reduction in growth (Divo and Jensen, 1982b). One of the potential uses of such a medium is in the standardisation of antimalarial drug sensitivity tests (Ofulla et al., 1994). In this new attempt, P. falciparum has been successfully cultivated in vitro in a medium that is free of both human serum and RPMI 1640. The features of this newly developed serum free formulation require further optimisation and investigation into its ability and sustainability for continuous cultivation of P. falciparum. Approaches such as this are also aimed to eliminating host factors influence in parasite biochemical and immunological experiments. This may probably be the first successful cultivation of P. falciparum using a plant exudate at basal level of nutrients and animal extracts to sustain development. Arguably, it also represents the cheapest way to keep the parasite alive.

Despite the considerable progress in malaria control over the past decade, malaria remains a disease of priority, particularly in Africa where about 90% of clinical cases occur. One of the greatest challenges facing malaria control worldwide is the spread and intensification of parasite resistance to antimalarial drugs. The limited number of such drugs has led to increasing difficulties in the development of antimalarial drug policy and adequate disease management (WHO, 2000). The most worrisome aspect of the encroachment of endemic malaria is the lack of infrastructure and implements with which to gain control (Barcus et al., 2002). Medicine, in several developing countries, using local traditions and beliefs, is still the mainstay of health care (Hoareau and Dasilva, 1999). Africa is a rich source of medicinal plants yet a relatively small number of drugs against malaria are available today. Although new drugs have appeared in the last 20 years, including atovaquone, malarone, halofantrine, mefloquine, proguanil, artemisinin derivatives and co-artemether, new and affordable drugs as well as better formulations are needed (Persidis, 2000). Likewise, the relatively high cost of new drugs is a major obstacle to their use in resource-poor settings where the burden of malaria is greatest (Olliaro et al., 1996; Goodman et al., 2000)

One of the key challenges in the fight against malaria is not just to develop effective and safe treatments, but also to make sure they are available to local governments and people at a price that will allow widespread use. The challenge ahead lies in determining the best alternative therapies for use now, the best prospect for drug development, regulatory approval and use in short term and the establishment of mechanisms and projects to ensure that improved drugs are sustainably discovered and developed into the future. Continued and sustainable improvements in antimalarial medicines research and development are essential for the world's future ability to treat and control malaria (Ridley, 2002).

Natural products isolated from plants used in traditional medicine, which have potent antiplasmodial action in vitro, represesents potential sources of new antimalarial drugs (Wright and Phillipson, 1990; Gasquet et al., 1993). Since plant materials remain an important resource to combat serious diseases in the world (Tshibangu et al., 2002), pharmacognostic investigations of plants are carried out to find novel drugs or templates for development of new therapeutic agents (König, 1992). It had been advocated that direct crude drug formulation of the herbs following toxicological absolution may not only produce dosage forms faster but will also lead to cheaper and more affordable drugs for the communities that need them (Elujoba, 1998). In addition, there is a belief that these medicines are safe because they are natural (Sofowora, 1993; Willcox et al., 2003. [In press])

The results in this study lend more credence to the use of the active species in traditional medicine in the treatment of fever and malaria although the potencies of these active extracts would have to be tested and compared to those of other standard drug test. It is suggested that E. chlorantha and the other three medicinal plants used in this study which are very popular in Nigerian rural and urban centres are potential sources of antimalarial agents and should therefore be the subject of further research to study their active principles or consituents. These plants remain effective as antimalarial treatments for fever and malaria. Since the life span of currently used antimalarial drugs are been extended by combination therapy, it is crucial that this implementation is accompanied by close monitoring of the impact, including malaria-specific and all-cause mortality, side effects of drugs, treatment seeking behaviour and parasite sensitivity to drugs (Trape et al., 2002). The combination of two or more of the extract leads to higher efficiency in terms of antimalarial activity while at the same time reducing the dosage of each extract required to achieve such. With due consideration to dosage administration to minimise toxicity and other side effects, these plants are effective for combination therapy, especially in the forms for which they are used locally. Subsequent to the high population, a high number of deaths occur from malaria in Nigeria. The actions to be taken include prevention efforts and research; search for new drugs or drug discovery ought to be an integral part of the research and development especially as the clues for an effective and licensed vaccines remain elusive.

Herbal remedies for malaria are already in popular use in developing countries especially due to lack of access to effective, cheap, safe and user-friendly medicines and many (of these herbal medicines) have been shown to have antiplasmodial activities in experimental studies (Willcox et al., 2003). The plants used in this study remain effective as antimalarial treatments for fever and malaria. The combination of two or more of the extract leads to higher efficiency in terms of antimalarial activity while at the same time reducing the dosage of each extract required to achieve such. With due consideration to dosage administration to minimise toxicity and other side effects, these plants are effective for combination therapy, especially in the form for which they are used locally. Subsequent to the high population, a high number of deaths occur from malaria in Nigeria. The actions to be taken include prevention efforts and research; search for new drugs or drug discovery ought to be an integral part of the research and development especially as the clues for an effective and licensed vaccines remain elusive.

That Chloroquine is still the primary drug for treatment in most sub-Saharan countries (Zucker et al., 2003), even though there is increasing resistance shows the enormous limitations of existing alternative treatments. For instance, artemisinin (qinghaosu) is not entirely a new substance; it had been in use in China for many centuries before the Chinese scientists isolated the active principle from the plant, Artemisia annua, in 1972 (Ringwald et al., 1999b). There are numerous traditional antimalarial plants in Nigeria and West Africa as a whole. It will not be out of context to suggest that a few of these plants will provide alternatives and possible replacements for the current antimalarials in routine use. Therefore, in view of the problems of inappropriate administration of dosage and insecurity due to toxicity, it is important that more chemotherapeutic research against malaria parasites be investigated especially with the commonly used plants known for their antiparasitic activities.

In concluding with the study on insecticides treated curtains, one may suggest that the use of ITCs do not prevent but they may reduce transmission of P. falciparum and as a consequence, the high parasite densities associated with malaria morbidity and mortality. It may be also be reasoned that the acquisition of anti-malarial immunity in children also depend on a number of social factors affecting their daily lives which determine their relative exposure risk. Even then, more accurate information on the relationship between transmission and malaria as a potentially fatal disease is needed. These intervention methods could help in saving the lives of more Africa children. Finally, it seems that insecticides treated material including nets and curtains do not prevent the acquisition of immunity in young children based on the comparative levels of protective immunoglobulin levels observed in users and non-users. This study revealed that Immunoglobulin G responses were not affected by the use of ITC and that there was no preferential carriage of msp-2 genotypes in the two groups of children in this study.

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Table 1. The various formulations of the new media used in the cultivation of malaria parasites showing hypoxanthine content (proportion and molarity values).

Culture Hypoxanthine content in 500µl Proportion Molarity (µM)

E new medium only - -

F new medium 15µl 3% 0.01

G new medium 30µl 6% 0.02

H new medium 50µl 10% 0.04

I new medium 100µl 20% 0.07

*MCM-HS malaria culture medium - -

*Malaria culture medium with 10% human serum (MCM-HS

Table 2. The medicinal plants and the various parts used.

Plant Parts used

E. chlorantha bark

M. lucida bark and leaves

C giganteus leaves

A. indica stem and leaves

Table 2. The percentage parasitaemia obtained in an attempt to raise the parasites from 1% parasitaemia in new media and MCM.

Time in culture(hours) Percentage parasitaemia (%)

A B C D MCM

0 1.5 1.5 1.5 1.5 1.5

24 1.5 1.6 1.9 2.5 1.5

48 1.5 1.6 1.5 1.5 3.5

72 1.5 2.0 1.5 2.5 5.0

96 2.1 1.3 2.1 1.9 8.0*

120 1.0 1.0 2.0 1.0 3.0

Molariity of hypoxanthine content, B=0.02µM, C=0.04µM,D=0.07µM * no subcultivation

Table 3. The percentage parasitaemia obtained at 4% start parasitaemia in new media and MCM-HS.

Time Percentage parasitaemia

E F G H I MCM-HS

0 4.0 4.0 4.0 4.0 4.0 4.0

24 5.4 (+1.4) 4.8 (+0.8) 3.9 (-0.1) 7.5 (+3.5) 3.7 (-0.3) 8.3 (+4.3)

48 5.0 (+0.5) 4.0 (0.0) 9.0 (+2.5) 5.0 (+0.5) 6.0 (+1.0) 15.0 (+5.5)

72 6.0 (+0.6) 6.7 (+0.9) 8.5 (+1.5) 5.5 (+0.5) 3.8 (-0.1) 12.0(+2.7)

96 7.6 (+0.9) 5.9 (+0.5) 8.3 (+1.1) 6.1 (+0.5) 7.4 (+0.9) **

120 7.1 (+0.6) 6.2 (+0.4) 8.0 (+0.8) 6.5 (+0.5) 7.5 (+0.7) **

Molarity of hypoxanthine: F=0.01µM, G =O.O2µM, H=0.04µM, I=0.07µM. Growth rate wrt start parasitaemia in parenthesis.

** not determined.

Table 4. The mean growth rate of the parasites at day 5 in cultures E-I and day 3 for MCM

Culture E F G H I MCM-HS

Growth rate (%) 0.8 0.5 1.2 1.2 0.4 4.2

Table 5a. Differences in growth of P. falciparum in different human serum at 5%

Percentage parasitaemia

Time in culture (hours) HS97 HS02 HS9702

0 1 1 1

24 1.9 2.4 2.8

48 1.9 2.3 3.8

72 1.3 2.6 3.9

96 1.2 3.6 8.0

120 1.6 2.7 7.0

124 1.3 1.5 5.7

HS97=HUMAN SERUM 1997, HS02=HUMAN SERUM 2002, HS9702=COMBINED

Table 5b The parasitaemia after 3 days in 10% HS

Percentage parasitaemia

Time in culture (hours) HS97 HS02 HS9702

0* 1.3 2.6 3.9

24 1.9 2.7 4.8

48 1.7 2.2 6.0

72 1.5 2.8 5.3

* After 72hours in MCM 5% HS.

Table 6a.The maintenance of P.falciparum in new plant medium and human serum at 10% HS

Percentage parasitaemia

Time in culture(h)

J K L M

0 1.5 1.5 1.5 1.5

24 2.0 3.2 2.9 3.6

48 2.5 3.5 3.5 2.0

Keys,

J, autoclaved plant exudate and human serum only

K, autoclaved plant exudate and human serum plus added hypoxanthine,0.04µM

L, non-autoclaved plant exudate and human serum only

M, non-autoclaved plant exudate and human serum plus added hypoxanthine,0.04µM

Table 6b. The maintenance of P.falciparum in new plant medium and human serum at 5% HS

Percentage parasitaemia

Time in culture(h)

N P R S

0 1.5 1.5 1.5 1.5

24 1.5 3.0 2.2 2.5

48 2.5 3.5 1.3 2.5

Keys,

N, autoclaved plant exudate and human serum only

P, autoclaved plant exudate and human serum plus added hypoxanthine, 0.04µM

R, non-autoclaved plant exudate and human serum only

S, non-autoclaved plant exudate and human serum plus added hypoxanthine, 0.04µM

Table 2. The total number of parasitised cells in test cultures at 24hours

Extract conc. Parasitised cells/40 000 cells Percentage Parasiataemia (%)

(µg/ml)

AI

500 670 1.7

250 680 1.7

125 724 1.8

62.5 890 2.2

31.3 898 2.3

CG

500 485 1.2

250 504 1.3

125 570 1.4

62.5 669 1.7

31.3 723 1.8

ML

500 415 1.0

250 592 1.5

125 718 1.8

62.5 804 2.0

31.3 836 2.1

EC

500 372 0.9

250 412 1.0

125 438 1.1

62.5 502 1.3

31.3 583 1.3

AZ = Azadirachta indica, CG = Cymbopogon giganteus, ML= Morinda lucida, EC = Enantia chlorantha

Table 3. The percentage inhibition±SEM of the organic extracts of the medicinal plants

Extracts concentration (µg/ml) Percentage inhibition (%) ±SEM

AZ

500 41.3±5.7

250 41.3±4.7

125 37.9±2.1

62.5 24.1±7.4

31.3 20.7±3.6

CG

500 58.6±1.5

250 55.2±2.2

125 51.7±3.9

62.5 41.4±3.3

31.3 37.9±4.0

ML

500 65.5±1.4

250 48.3±4.7

125 37.9±4.2

62.5 31.0±5.0

31.3 27.6±2.5

EC

500 68.9±2.7

250 65.5±2.5

125 62.1±2.3

62.5 55.2±1.4

31.3 48.3±3.6

AZ = Azadirachta indica, CG = Cymbopogon giganteus, ML= Morinda lucida, EC = Enantia chlorantha

Table 1. Synergistic effect: The total number of parasitised cells at 48 hours

Final extract conc. Parasitised cells/40 000 cells Percentage Parasitaemia (%)

(µg/ml)

E+A

500 176 0.4

250 253 0.6

125 620 1.6

62.5 899 2.3

31.3 984 2.5

E+C

500 164 0.4

250 349 0.9

125 620 1.6

62.5 740 1.9

31.3 803 2.0

E+M

500 336 0.8

250 440 1.1

125 548 1.4

62.5 560 1.4

31.3 643 1.6

CQ*

500 236 0.6

250 312 0.8

125 374 0.9

62.5 440 1.1

31.3 523 1.3

E+A= E. chlorantha and A. indica: E+C= E.chlorantha and C.giganteus: E+M= E.chlorantha and M. lucida. CQ = chloroquine *not combined

Table 2. The final concentration and percentage inhibition±SEM of the various extract combination

Final extract concentration (µg/ml) Percentage inhibition ± SEM

E+A

500 85.2 ± 3.1

250 78.8 ± 3.6

125 47.8 ± 5.6

62.5 24.2 ± 6.9

31.3 17.2 ± 6.2

E+C

500 86.2 ± 1.6

250 70.2 ± 5.1

125 47.8 ± 8.6

62.5 37.7 ± 4.1

31.3 32.7 ± 7.8

E+M

500 71.7 ± 4.6

250 63.0 ± 8.2

125 53.9 ± 9.9

62.5 52.9 ±3.4

31.3 45.8 ± 7.3

CQ*

500 80.1 ± 6.7

250 73.7 ± 3.1

125 68.4 ± 6.1

62.5 63.0 ± 5.4

31.3 55.9±5.77

E+A= E. chlorantha and A. indica: E+C= E.chlorantha and C.giganteus: E+M= E.chlorantha and M. lucida. CQ = chloroquine *not combined

Table 3. Stage specificity: The inhibition of parasite growth by the aqueous extracts of the 4 medicinal plants

Time (hours)

After 1st Sync. After treatment Percentage inhibition (%)

EC ML CG A1

30 18 36.0 32.0 36.0 40.0

36 24* 54.0 60.0 52.1 75.4

42 30 75.4 67.7 73.8 79.6

48 36 78.4 79.6 83.8 77.4

54 42 91.7 84.2 92.5 95.0

EC=E.chlorantha, ML=M.lucida, CG=C.giganteus, AI=A.indica. *2nd treatment after fixation of second replicas.

TABLE 1

Mean concentrations ±SEM of Immunoglobulin (IgG) antibodies (µg/mL)

Total IgG IgG3 IgG1

First bleeding

ITC 21.4±4.0 0.53±0.1 6.73±2.5

No ITC 30.2±3.9 0.59±0.1 4.35±0.5

Second bleeding

ITC 13.1±3.3 0.39±0.1 5.10±1.5

No ITC 12.2±1.8 0.48±0.1 3.24±0.3

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