IN-VITRO AND MOLECULAR STUDIES ON THE RESISTANCE OF P. falciparum TO ANTIMALARIAL DRUGS IN OGUN STATE,SOUTHWESTERN NIGERIA

By

OLASEHINDE GRACE IYABO

Presented To

Department of Biological Science

153 Pages 38,603 Words 156 Views PDF Format No. of Chapters: 1 to 5 Chapters
Reference/Bibliography: YES 4,500 NGN 4,000 NGN PROJECT
ABSTRACTS

The widespread of drug resistant Plasmodium falciparum has led to a rise in malariaassociated mortality most especially in sub-Saharan Africa. In-vitro and molecular studies were carried out in order to determine the resistant pattern of P. falciparum to antimalarial drugs and some local antimalarial herbs in Ogun State, Southwestern Nigeria. Prevalence of falciparum malaria was determined by microscopic examination of Giemsa-stained blood samples of patients who presented with fever in selected State Hospitals in Ogun State. Antimalarial drug sensitivity of one hundred (100) P. falciparum isolates to chloroquine, amodiaquine, mefloquine, quinine, sulphadoxine/pyrimethamine, artesunate and three local antimalarial herbs: Momordica charantia (Ejirin,) Diospyros monbuttensis (Eegun eja) and Morinda lucida (Oruwo) was determined using the in-vitro microtest (Mark III) technique. For molecular studies and genotyping, DNA was extracted from patient blood using the QiaAmp DNA Blood Minikit extraction method. Nested Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphisms (PCR/RFLP) were used for the detection of P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum multidrug resistance 1 (pfmdr1), P. falciparum dihydrofolate reductase (Pfdhfr), P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum sarco/endoplasmic reticulum calcium-dependent ATPase (SERCA) PfATPase6 genes. Genetic diversity of the isolates was determined using merozoite surface proteins 1 and 2 (msp1 and msp2) and Glutamate rich Protein (Glurp). Structured Questionnaires were administered to patients or/and parents of infants to determine the factors that could lead to the development of drug resistance by the parasite in the study population. Out of 4066 subjects screened during the period of study, 2550 (61.1%) were positive. Highest prevalence (72%) was recorded in children 1-5 years while the same group also had the highest parasitaemia of 1080. All the isolates tested were sensitive to Quinine, Mefloquine and Artesunate. Only 51% of the isolates were resistant to chloroquine, 13% to amodiaquine and 5% to sulphadoxine pyrimethamine respectively. Highest resistance to chloroquine (68.9%) was recorded among isolates from Yewa zone while highest resistance to amodiaquine (30%) was observed in Ijebu zone. Highest resistance to sulphadoxine and pyrimethamine was recorded in Yewa and Egba zones respectively. A significant positive correlation was observed between the responses to artemisinin and mefloquine (P=0.001), artemisinin and quinine (P=0.05), Quinine and mefloquine (P= 0.01). A significant negative correlation was observed between the responses to chloroquine and mefloquine (P=0.05). For the local herbs highest xv antiplasmodial activity was obtained with the ethanolic extract of Diospyros monbuttensis (IC50 = 32 μg/ml). P. falciparum isolates analyzed during this study have demonstrated highly diverse nature of field isolates in respect of msp-1 (block 2) and msp-2 (central repeat region, block3). All the three reported families of msp-1(K1, MAD20 and RO33) and two of msp-2 (FC27 and 3D7) were observed among the isolates. Proportion of isolates with K1 family was 68% with 4 alleles in the range of 100 to 300 basepairs (bp). Proportion of isolates with MAD20 family was 40% and a total of 3 alleles were observed within 100 to 300 bp. RO33 proportion was 20% and the family was observed to be monomorphic with an allele size of 200 bp. In msp-2 the proportion of FC27 family was 76% and that of 3D7 was 56%. Proportional Prevalence of FC27 and 3D7 families was significantly different (χ2 = 16.5, P = 0.002). Eighty percent of the isolates harbor the genes that code for Glutamate rich protein with size ranging between 700 and 900bp. Pfcrt (K76T ) Pfmdr1 (mdr 1 ) Pfdhfr (S108N), and Pfdhps (K540E ) resistant genes were detected among the isolates while resistant SERCAPfATPase6 gene which codes for artemisinin resistance was not detected in the population. The questionnaire study showed that 24.6% of the patient visit hospitals for treatment, 12.0% use local healers while 25.0% buy antimalarial drugs without prescription. It was also observed that some use more than one method in their management of malaria. Those who combined antimalarial drugs with traditional medicine from local healers were found to be 17.4%. Only 18% of the sample population used Insecticide treated mosquito nets, 42.3% use window and door nets while 13% do not employ any mosquito preventive method. Continuous use of the current antimalarial drugs increases the chance of resistance developing to those drugs. Control of drug use and reducing exposure of parasites to the drugs are most effective where the parasite is still sensitive to the drug. Molecular methods are most effective for monitoring the spread of resistant strains of P. falciparum
CONTENT


Title  Page
Title Page -  -  -  -  -  -  -  -  - .i
Certification -  -  -  -  -  -  -  -  -  -  - ii
Declaration -  -  -  -  -  -  -  -  -  -  - iii
Dedication -  -  -  -  -  -  -  -  - iv
Acknowledgements -  -  -  -  -  -  -  -  -  - v
Content Page -  -  -  -  -  -  -  -  -  - vii
Abbreviations -  -  -  -  -  -  -  -  -  - xi
List of Figures -  -  -  -  -  -  -  -  -  - xii
List of Tables -  -  -  -  -  -  -  -  -  - xiii
List of Plates -  -  -  -  -  -  -  -  -  - .xiv
Abstract -  -  -  -  -  -  -  -  -  - .xv

CHAPTER ONE â€" INTRODUCTION
1.1 Background  -  -  -  -  -  -  -  -  - 1
1.2 Justification/Rationale of the study -  -  -  -  -  - 6
1.3 objectives of the study  -  -  -  -  -  -  -  -  - 7
1.4 Scientific Hypothesis -  -  -  -  -  -  -  - 7

CHAPTER TWO â€" LITERATURE REVIEW
2.1.Disease incidence and trends -  -  -  -  -  -  - .8
2.1.1 Geographical distribution and populations at risk -  -  -  - 8
2.2.  Causative agents -  -  -  -  -  -  -  -  - 10
2.3  Transmission and biology of P. falciparum -  -  -  -  - 10
2.4 Symptoms -  -  -  -  -  -  -  -  -  - 15
2.5 Diagnosis -  -  -  -  -  -  -  -  -  - 16
2.5.1 Microscopy -  -  -  -  -  -  -  - 16
2.5.2 Clinical (presumptive) diagnosis -  -  -  -  -  - 17
2.5.3 Antigen detection tests (rapid or ‘dipstick’ diagnostic tests) - .18
2.5.4 Molecular tests -  -  -  -  -  -  - 18
2.5.5Serology -  -  -  -  -  -  -  -  - .19
2.6 Antimalarial Drugs -  -  -  -  -  -  -  -  - .19
2.6.1 Quinine and related compounds -  -  -  -  - .19
2.6.2 Antifolate drugs -  -  -  -  -  -  -  - 23
2.6.3 Antibiotics -  -  -  -  -  -  -  -  -  - 25
2.6.4 Artemisinin compounds -  -  -  -  -  - .26
2.7 Combination therapy with antimalarials -  -  -  -  - 28
2.7.1 Non-Artemisinin based combinations -  -  -  -  - 29
2.7.2 Artemisinin-based combinations -  -  -  -  - 29
2.7.3 Traditional Antimalarial Herbs -  -  -  -  -  - 31
2.8 Antimalarial Drug Resistance  -  -  -  -  -  -  - 33
2.8.1 Definition of antimalarial drug resistance -  -  -  -  -  - 34
2.8.2 Malaria treatment failure -  -  -  -  -  - 34
2.8.3 Mechanisms of antimalarial resistance -  -  -  -  - 35
2.8.3.1 Chloroquine resistance -  -  -  -  -  -  - 35
2.8.3.2 Antifolate combination drugs -  -  -  -  -  - .36
2.9 Spread of resistance -  -  -  -  -  -  -  -  - .36
2.9.1 Biological influences on resistance -  -  -  -  -  - .37
2.9.2 Programmatic influences on resistance -  -  -  -  - 40
2.10 Detection of resistance -  -  -  -  -  -  -  - 42
2.10.1 In vivotests -  -  -  -  -  -  -  -  - .42
2.10.2 In vitro tests -  -  -  -  -  -  -  -  - 43
2.10.3 Animal model studies -  -  -  -  -  -  - 45
2.10.4 Molecular techniques -  -  -  -  -  -  - 45
2.10.5 Case reports and passive detection of treatment failure -  - 46
2.11 The future: prevention of drug resistance -  -  -  -  -  - .46

CHAPTER THREE â€" MATERIALS AND METHODS
3.1 Study Area -  -  -  -  -  -  -  -  -  - 49
3.2 Study Patients -  -  -  -  -  -  -  -  - 49
3.3 Sampling Procedure -  -  -  -  -  -  -  - .49
3.4 Ethical Consideration -  -  -  -  -  -  -  - 51
3.5 Sample Collection -  -  -  -  -  -  -  -  - 51
3.6 Cryopreservation -  -  -  -  -  -  -  -  -  - 52
3.7 Processing of sample -  -  -  -  -  -  -  - .52
3.7.1 Microscopic examination -  -  -  -  -  -  -  - 52
3.8 Antimalarial sensitivity testing -  -  -  -  -  - .52
3.8.1 Revival of cryopreserved parasites -  -  -  -  -  - .52
3.8.2 In vitro microtest (Mark III Test) -  -  -  -  -  -  - 53
3.9 Antimalarial Activity Testing of Crude Organic Extracts of  -  -  - 53
Medicinal Plants: Momordica charantia (Ejirin), Diospyros
monbuttensis (Eegun eja) andMorinda lucida (Oruwo)
3.9.1 Preparation of plant extract -  -  -  -  -  - 53
3.9.2 In vitrotest -  -  -  -  -  -  -  - 53
3.10 Molecular Studies -  -  -  -  -  -  -  - .54
3.10.1 DNA extraction -  -  -  -  -  -  -  - .54
3.10.2  PCR for detection of Pfcrtgene -  -  -  -  -  - 54
3.10.3 Nested PCR and RFLP for Pfcrtmutation-specific detection -  -  - 55
3.10.4 PCR and RFLP for detection of Pfmdr1gene -  -  -  -  - 55
3.10.5 PCR assays for the detection of Pfdhfr and Pfdhps genes -  - .56
3.10.6 PCR and RPLP assay for (SERCA) PfATPase6 -  -  -  - 57
3.10.7 Molecular Genotyping of isolates using MSP1&2 and Glurp -  -  - 57
3.10.8 Questionnaire Administration -  -  -  -  - 60

CHAPTER FOUR â€" RESULTS
4.1. Incidence of Malaria in Ogun State, Southwestern Nigeria -  -  -  - .61
4.1.1 Patients Characteristics -  -  -  -  -  -  -  - 61
4.1.2 Incidence of Malaria -  -  -  -  -  -  -  -  - 61
4.2.  In VitroDrug sensitivity Tests -  -  -  -  -  -  -  - 61
4.3 Prevalence of drug resistant molecular markers -  -  -  -  - 62
4.4 In vitroantimalarial activity of herbal extracts -  -  -  -  -  - 62
4.5 Genetic Diversity of P. falciparum -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  - 63
4.6 Knowledge and practice on the use of antimalarial drugs -  -  - .64

CHAPTER FIVE â€" DISCUSSION  -  -  -  -  -  -  - 88
CONCLUSION -  -  -  -  -  -  -  -  -  - .101
CONTRIBUTION TO KNOWLEDGE -  -  -  -  -  -  - .102
REFERENCES -  -  -  -  -  -  -  -  -  -  - 103
APPENDICES -  -  -  -  -  -  -  -  -  -  - 128


ABBREVIATIONS

ADP Adenosine diphosphate
ATP  Adenosine triphosphate
pfATPase P. falciparumAdenosine Triphosphatase 6 genes
SERCA Sarco/endoplasmic reticulum calcium-dependent
DELI  Double-site Enzyme-linked Lactate dehydrogenase Immunodetection
DHFR  Dihydrofolate reductase
DHPS  Dihydropteroate synthase
DNA  Deoxyribonucleic acid
EDTA  Ethylenediaminetetraacetic acid
ELISA  Enzyme-linked immunosorbent assay
HEPES  N-(2-hydroxyethyl)piperazine-N´-(2-ethanesulfonic acid)
HPLC  High-performance liquid chromatography
HRP II  Histidine-rich protein II
IC50  50% inhibitory concentration
LDH  Lactate dehydrogenase
MIC  Minimal inhibitory concentration
NAD  Nicotinamide adenine dinucleotide
PABA  Para-aminobenzoic acid
PCR  Polymerase chain reaction
Pfcrt P. falciparum  Chloroquine resistance transporter gene
PCR  Polymerase chain reaction
pfmdr1 P. falciparum multidrug resistance gene 1
RPMI  Roswell Park Memorial Institute
TDR  Special Programme for Research and Training in Tropical Diseases Tween 80 polyoxyethylenesorbitan monooleate vs  versus
WHO  World Health Organization
DMSO Dimethyl sulphoxide
MSP1 Merozoite Surface Protein 1
MSP2 Merozoite Surface Protein 2
GLURP Glutarmate Rich Protein
QT-NASBA Quantitative Nucleic Acid Sequence Based Amplification
BSA Bovine Serum Albumin
WBC White blood cell(s)
TCM Tissue Culture Medium


LIST OF FIGURES

Fig 2.1  Life Cycle of PlasmodiumSpecies -  -  -  -  -  -  - 14
Fig 3.1  Map of Ogun State, South Western Nigeria -  -  -  -  - .50
Fig 4.1   Sample of HN-NonLinn Software Statistical Package -  -  - 75
Fig 4.2  Cross Resistance between Chloroquine and Amodiaquine, n=100 -  - .76

LIST OF TABLES

Table 3.1  PCR Primers for MSP1, MSP2 and Glutamate rich protein -  - .59
Table 4.1  Incidence of P. falciparum infection in Ogun State.  -  -  - 65
Table 4.2  Zone wise Incidence of Malaria in Ogun State -  -  -  -  - .66
Table 4.3  In vitrosusceptibility of P. falciparum isolates to Antimalarial Drugs -  - 67
Table 4.4  Zonewise resistance pattern of P. falciparum to antimalarial drugs - .68
Table 4.5  Zonewise Prevalence of molecular markers of resistance to
antimalarial drugs in Plasmodium falciparumfrom Ogun State,
South Western Nigeria.  -  -  -  -  -  -  -  - .69
Table 4.6  In vitrosusceptibility of P. falciparum isolates to Local
Antimalarial Herbs -  -  -  -  -  -  -  - .70
Table 4.7 Genetic diversity of Plasmodium falciparum isolates from Ogun State,
South Western Nigeria -  -  -  -  -  -  - 71
Table 4.8  Zonewise Genetic Diversity of P. falciparum from Ogun State,
Southwestern Nigeria -  -  -  -  -  -  - .72
Table 4.9  Occupation of respondents -  -  -  -  -  -  -  - 73
Table 4.10  Knowledge on prevention and control of malaria among respondents - 74

LIST OF PLATES

Plate 4.1 DNA bands of wild type and
mutated P. falciparum chloroquine resistance genes -  -  -  - 77
Plate 4.2 P. falciparumMultidrug Resistance Genes showing the wild
type and mutated genes -  -  -  -  -  -  - 78
Plate 4.3  DNA band of Dihydrofolate reductase gene (DHFR 108) -  -  -  - 79
Plate 4.4  DNA band of Dihydropteroate synthase gene (DHPS 540) -  -  - .80
Plate 4.5 DNA band of wild type PfATPase6  -  -  -  -  -  - .81
Plate 4.6 DNA bands of P. falciparumMSP1 MAD20 on Gel -  -  -  - 82
Plate 4.7 DNA bands of P. falciparumMSP1 K1 on Gel -  -  -  - 83
Plate 4.8 DNA bands of P. falciparumMSP1 RO33 on Gel -  -  - 84
Plate 4.9 DNA bands of P. falciparumMSP2 3D7 on gel -  -  -  - 85
Plate 4.10 DNA bands of P. falciparumMerozoite Surface Protein2 FC27 on gel - 86
Plate 4.11 DNA band of P. falciparumGlutarmate rich protein  -  -  -  - 87.
153 Pages 38,603 Words 156 Views PDF Format No. of Chapters: 1 to 5 Chapters
Reference/Bibliography: YES 4,500.00 NGN 4,000 NGN PROJECT

About e-Project Material Centre


e-Project Material Centre is a web service aimed at successfully assisting final year students with quality, well-researched, reliable, and ready-made project work. Our materials are recent, complete (chapter 1 to Minimum of Chapter 5, with references), and well-written. INSTANT ACCESS! INSTANT DOWNLOAD. Simply select your department, choose from our list of topics available, and explore your data.

Why Students Love to Use e-Project Material?


Guaranteed Delivery: Getting your project delivered on time is essential. You cannot afford to turn in your project past the deadline. That is why you must get your project online from a company that guarantees to meet your deadline. e-Project Topics Material Centre is happy to offer instant delivery of projects listed on our website. We can handle just about any deadline you send our way. Satisfaction Guaranteed: We always do whatever is necessary to ensure every customer's satisfaction.

Disclaimer


e-Project Topics Material Centre will only provide projects as a reference for your research. The projects ordered and produced should be used as a guide or framework for your own project. The contents of the projects should help you generate new ideas and thoughts for your own project. It is the aim of e-Project Topics Centre to only provide guidance by which the projects should be pursued. We are neither encouraging any form of plagiarism nor are we advocating the use of the projects produced herein for cheating.

Terms and Conditions


Using our service is LEGAL and IS NOT prohibited by any university/college policies. You are allowed to use the original model papers you will receive in the following ways:
  • As a source for additional understanding of the subject
  • As a source for ideas for your own research (if properly referenced)
  • For PROPER paraphrasing (see your university definition of plagiarism and acceptable paraphrase) Direct citing (if referenced properly)
Thank you so much for your respect to the author's copyright.

Refund and Privacy Policy


  • Refunds: All sales are final. However, if you encounter any issues with accessing your purchased material, kindly contact our support team for immediate resolution.
  • Privacy Policy: Your personal information is protected and will not be shared with third parties. We ensure secure payment processing and data confidentiality.

Contact Information


e-projecttopics.com © copyright 2025 || Nigeria
Designed By: Powersoft Technology
X

Need Help Finding or Downloading Your Project Material?

If you don't see the topic you're looking for or You need urgent/express attention, click the WhatsApp Icon/link below to contact ADMIN and get the material you need instantly. We are always available online to attend to your needs. Thanks