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

By

OLASEHINDE GRACE IYABO

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Biological Science Department

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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 ÎÂ�g/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
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 â€˜dipstickâ€ 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-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 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.

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