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

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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 2,298 Views PDF Format No. of Chapters: 1 to 5 Chapters

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 Î¼g/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 (Ï‡2 = 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
11 Background - - - - - - - - - 1
12 Justification/Rationale of the study - - - - - - 6
13 objectives of the study - - - - - - - - - 7
14 Scientific Hypothesis - - - - - - - - 7

CHAPTER TWO â€" LITERATURE REVIEW
21Disease incidence and trends - - - - - - - 8
211 Geographical distribution and populations at risk - - - - 8
22 Causative agents - - - - - - - - - 10
23 Transmission and biology of P falciparum - - - - - 10
24 Symptoms - - - - - - - - - - 15
25 Diagnosis - - - - - - - - - - 16
251 Microscopy - - - - - - - - 16
252 Clinical (presumptive) diagnosis - - - - - - 17
253 Antigen detection tests (rapid or â€˜dipstickâ€™ diagnostic tests) - 18
254 Molecular tests - - - - - - - 18
255Serology - - - - - - - - - 19
26 Antimalarial Drugs - - - - - - - - - 19
261 Quinine and related compounds - - - - - 19
262 Antifolate drugs - - - - - - - - 23
263 Antibiotics - - - - - - - - - - 25
264 Artemisinin compounds - - - - - - 26
27 Combination therapy with antimalarials - - - - - 28
271 Non-Artemisinin based combinations - - - - - 29
272 Artemisinin-based combinations - - - - - 29
273 Traditional Antimalarial Herbs - - - - - - 31
28 Antimalarial Drug Resistance - - - - - - - 33
2 81 Definition of antimalarial drug resistance - - - - - - 34
282 Malaria treatment failure - - - - - - 34
283 Mechanisms of antimalarial resistance - - - - - 35
2831 Chloroquine resistance - - - - - - - 35
2832 Antifolate combination drugs - - - - - - 36
29 Spread of resistance - - - - - - - - - 36
291 Biological influences on resistance - - - - - - 37
292 Programmatic influences on resistance - - - - - 40
210 Detection of resistance - - - - - - - - 42
2101 In vivotests - - - - - - - - - 42
2102 In vitro tests - - - - - - - - - 43
2103 Animal model studies - - - - - - - 45
2104 Molecular techniques - - - - - - - 45
2105 Case reports and passive detection of treatment failure - - 46
211 The future: prevention of drug resistance - - - - - - 46

CHAPTER THREE â€" MATERIALS AND METHODS
31 Study Area - - - - - - - - - - 49
32 Study Patients - - - - - - - - - 49
33 Sampling Procedure - - - - - - - - 49
34 Ethical Consideration - - - - - - - - 51
35 Sample Collection - - - - - - - - - 51
36 Cryopreservation - - - - - - - - - - 52
37 Processing of sample - - - - - - - - 52
371 Microscopic examination - - - - - - - - 52
38 Antimalarial sensitivity testing - - - - - - 52
381 Revival of cryopreserved parasites - - - - - - 52
382 In vitro microtest (Mark III Test) - - - - - - - 53
39 Antimalarial Activity Testing of Crude Organic Extracts of - - - 53
Medicinal Plants: Momordica charantia (Ejirin), Diospyros
monbuttensis (Eegun eja) andMorinda lucida (Oruwo)
391 Preparation of plant extract - - - - - - 53
392 In vitrotest - - - - - - - - 53
310 Molecular Studies - - - - - - - - 54
3101 DNA extraction - - - - - - - - 54
3102 PCR for detection of Pfcrtgene - - - - - - 54
3103 Nested PCR and RFLP for Pfcrtmutation-specific detection - - - 55
3104 PCR and RFLP for detection of Pfmdr1gene - - - - - 55
3105 PCR assays for the detection of Pfdhfr and Pfdhps genes - - 56
3106 PCR and RPLP assay for (SERCA) PfATPase6 - - - - 57
3107 Molecular Genotyping of isolates using MSP1&2 and Glurp - - - 57
3108 Questionnaire Administration - - - - - 60

CHAPTER FOUR â€" RESULTS
41 Incidence of Malaria in Ogun State, Southwestern Nigeria - - - - 61
411 Patients Characteristics - - - - - - - - 61
412 Incidence of Malaria - - - - - - - - - 61
42 In VitroDrug sensitivity Tests - - - - - - - - 61
43 Prevalence of drug resistant molecular markers - - - - - 62
44 In vitroantimalarial activity of herbal extracts - - - - - - 62
45 Genetic Diversity of P falciparum - - - - - - - - - - - - - - - - - - - - - - - - 63
46 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-NÂ´-(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 21 Life Cycle of PlasmodiumSpecies - - - - - - - 14
Fig 31 Map of Ogun State, South Western Nigeria - - - - - 50
Fig 41 Sample of HN-NonLinn Software Statistical Package - - - 75
Fig 42 Cross Resistance between Chloroquine and Amodiaquine, n=100 - - 76

LIST OF TABLES

Table 31 PCR Primers for MSP1, MSP2 and Glutamate rich protein - - 59
Table 41 Incidence of P falciparum infection in Ogun State - - - 65
Table 42 Zone wise Incidence of Malaria in Ogun State - - - - - 66
Table 43 In vitrosusceptibility of P falciparum isolates to Antimalarial Drugs - - 67
Table 44 Zonewise resistance pattern of P falciparum to antimalarial drugs - 68
Table 45 Zonewise Prevalence of molecular markers of resistance to
antimalarial drugs in Plasmodium falciparumfrom Ogun State,
South Western Nigeria - - - - - - - - 69
Table 46 In vitrosusceptibility of P falciparum isolates to Local
Antimalarial Herbs - - - - - - - - 70
Table 47 Genetic diversity of Plasmodium falciparum isolates from Ogun State,
South Western Nigeria - - - - - - - 71
Table 48 Zonewise Genetic Diversity of P falciparum from Ogun State,
Southwestern Nigeria - - - - - - - 72
Table 49 Occupation of respondents - - - - - - - - 73
Table 410 Knowledge on prevention and control of malaria among respondents - 74

LIST OF PLATES

Plate 41 DNA bands of wild type and
mutated P falciparum chloroquine resistance genes - - - - 77
Plate 42 P falciparumMultidrug Resistance Genes showing the wild
type and mutated genes - - - - - - - 78
Plate 43 DNA band of Dihydrofolate reductase gene (DHFR 108) - - - - 79
Plate 44 DNA band of Dihydropteroate synthase gene (DHPS 540) - - - 80
Plate 45 DNA band of wild type PfATPase6 - - - - - - 81
Plate 46 DNA bands of P falciparumMSP1 MAD20 on Gel - - - - 82
Plate 47 DNA bands of P falciparumMSP1 K1 on Gel - - - - 83
Plate 48 DNA bands of P falciparumMSP1 RO33 on Gel - - - 84
Plate 49 DNA bands of P falciparumMSP2 3D7 on gel - - - - 85
Plate 410 DNA bands of P falciparumMerozoite Surface Protein2 FC27 on gel - 86
Plate 411 DNA band of P falciparumGlutarmate rich protein - - - - 87

153 Pages 38,603 Words 2,298 Views PDF Format No. of Chapters: 1 to 5 Chapters

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