TABLE OF CONTENTS
Title page…………………………………………………………………………………………………………………… i
Certification……………………………………………………………………………………………………………… ii
Dedication……………………………………………………………………………………………………………….. iii
Acknowledgement…………………………………………………………………………………………………….. iv
Table of Contents………………………………………………………………………………………………………. v
List of Tables…………………………………………………………………………………………………………. viii
List of Figures………………………………………………………………………………………………………….. ix
List of Abbreviations………………………………………………………………………………………………….. x
Abstract…………………………………………………………………………………………………………………… xi
CHAPTER ONE:………………………………………………………………………………………………………. 1
- Introduction and Literature Review……………………………………………………………………. 1
- Chemotherapy as a Major Malaria Control Method………………………………………….. 10
- Population/Genetic Diversity of Plasmodium falciparum…………………………………… 22
CHAPTER TWO:……………………………………………………………………………………………………. 28
- Materials and Methods…………………………………………………………………………………… 28
- Detection of pfmdr1 Polymorphisms……………………………………………………………….. 30
- Parasite Population Genotyping Using Antigenic Markers –
MSP-1, MSP-2 and GLURP……………………………………………………………………………. 35
CHAPTER THREE:…………………………………………………………………………………………………. 38
- Results………………………………………………………………………………………………………… 38
- Plasmodium falciparum population structure profile and
Complexity of Infection………………………………………………………………………………… 39
- Correlation of the pfmdr1 polymorphisms with the parasite
population structure of the isolates…………………………………………………………………. 39
CHAPTER FOUR……………………………………………………………………………………………………. 47
- Discussion…………………………………………………………………………………………………… 47
- Conclusion and Recommendations…………………………………………………………………. 49
REFERENCES………………………………………………………………………………………………………… 51
LIST OF TABLES
Table 1: Primer sequence and reaction conditions of pfmdr1
N86Y/Y184F and pfmdr1 D1246Y Genes……………………………………………………… 34
Table 2: Primer sequence and reaction conditions for MSP-1, MSP-2 and GLURP………….. 37
Table 3: Demographic characteristics of children at enrolment………………………………………. 40
LIST OF FIGURES
Figure 1: Map showing Global Malarial Mortality…………………………………………………. 5
Figure 2: Microscopy view of trophozoites and gametocytes of P. falciparium
on thin film of blood after Geimsa staining……………………………………………… 9
Figure 3: Chemical structures of Artemisinn and derivatives…………………………………. 14
Figure 4: RFLP analysis of pfmdr1 N86Ypolymorphism………………………………………. 41
Figure 5: Prevalence of N86Y, Y184F and D1246Y pfmdr1 polymorphisms in
P. falciparum isolates obtained from the children…………………………………… 42
Figure 6: Prevalence of pfmdr1 N86Y/Y184F/D1246Y haplotypes………………………… 43
Figure 7: Parasite clones using MSP-2………………………………………………………………… 44
Figure 8: Prevalence of monoclonal and polyclonal infection by
MSP-1, MSP-2 and GLURP………………………………………………………………… 45
Figure 9: Correlation of pfmdr1 polymorphism with the parasite
population structure in the isolates………………………………………………………… 46
LIST OF ABBREVIATIONS
AA: Artesunate-Amodiaquine
ACT: Artemisinin-based Combination Therapy AL: Artemether-Lumefantrine
DBS: Dry Blood Spot
DPC: Delayed Parasite Clearance COI: Complexity of Infections CQ: Chloroquine
DNA: De-oxyriboNucleic Acid DPC: Delay Parasite Clearance GLURP: Glutamate-rich Protein GMS: Greater Mekong Sub-region
H-VNB: Hemozoin –Generated Vapour Nanobubble MSP-1: Merozoites Surface Protein 1
MSP-2: Merozoites Surface Protein 2 PCR: Polymerase Chain Reaction
Pfmdr-1: Plasmodium falciparum Multidrug Resistance 1 Pfcrt: Plasmodium falciparum Chloroquine Resistance Transporter RFLP: Restriction Fragment Length Polymorphism
SERCA: Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase SNP: Single Nucleotides Polymorphisms
SP: Sulfadoxine-Pyrimethamine WHO: World Health Organization
ABSTRACT
The artemisinin-based combination therapies (ACTs) have been the first line treatment for uncomplicated malaria in most sub-Saharan African countries following the widespread of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP). Mutations on pfmdr1 gene have been implicated in drug resistance to CQ and the partner drugs in the ACTs. There is therefore a need to evaluate the impact of the combination therapies since its adoption in 2005 in Nigeria on polymorphisms on pfmdr1 gene and the parasite diversity. A total of 98 dried blood spot (DBS) samples were collected from children aged 6 months to 5 years with microscopically confirmed P. falciparum infection in Ibadan in 2010. Parasite genomic DNA was isolated using the QIAamp extraction kits. Nested PCR followed by restriction fragment length polymorphism (RFLP) was used to detect polymorphisms on Pfmdr1gene while nested PCR was used to evaluate parasite diversity. The pfmdr1 Y86, F184 and Y1246 mutant alleles were present in 27%, 56% and 48% of the isolates respectively. Based on the polymorphic regions of MSP-1, MSP-2 and GLURP genes, monoclonal infections were observed in 81.6%, 51.6% and 5.6% respectively. The multiplicity of infection in all the isolates analysed using the polymorphic region of MSP-1, MSP-2 and GLURP were 1.8, 2.0 and 2.4 respectively. Results from this study showed that there was a relative decline in the prevalence of Y86, F184 and Y1246 mutant alleles of pfmdr1 gene in P. falciparum obtained from children in Ibadan South- west Nigeria five years after the adoption of the ACTs. Also there was no significant change in the parasite population diversity. This study showed that the change in antimalarial treatment policy in Nigeria does not have any effect on polymorphisms on pfmdr1 gene and parasite diversity. There is however a need to carry out more studies and also includes other drug resistance markers.