in-vitro and molecular studies on the resistance of p - Covenant ...

IN-VITRO AND MOLECULAR STUDIES ON THE RESISTANCE
OF P. falciparum TO ANTIMALARIAL DRUGS IN OGUN STATE,
SOUTHWESTERN NIGERIA
BY
OLASEHINDE GRACE IYABO
B.Sc (HONS) Unilor<strong>in</strong>; M.Sc (Unilag)
A THESIS SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL
SCIENCES, COLLEGE OF SCIENCE AND TECHNOLOGY
COVENANT UNIVERSITY, OTA,
NIGERIA
IN PARTIAL FULFILMENT OF THE REQUIREMENT
FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY
(Ph.D) IN MICROBIOLOGY
2010

CERTIFICATION
We certify that this is an orig<strong>in</strong>al research study by OLASEHINDE Grace Iyabo, for the award of
Ph.D <strong>in</strong> Microbiology <strong>in</strong> the Department of Biological Sciences, Covenant University, Ota,
Nigeria.
Dr. Olusola Ojurongbe Signature…………….. Date……………
(Supervisor)
Prof. L. O. Egwari Signature………………. Date……………
(Co-Supervisor)
Dr. E. O. Fagade Signature……………… Date……………..
(Co-Supervisor)
ii

DECLARATION
We declare that this thesis entitled “IN-VITRO AND MOLECULAR STUDIES ON THE
RESISTANCE OF P. falciparum TO ANTIMALARIAL DRUGS IN OGUN STATE,
SOUTHWESTERN NIGERIA” which is an orig<strong>in</strong>al work of Olaseh<strong>in</strong>de, Grace Iyabo
(Matriculation Number CUGP050156) has been exam<strong>in</strong>ed <strong>and</strong> found to have met the requirements
of the Covenant University for the award of The degree of Doctor of Philosophy (Ph.D). We
therefore recommend the work for the award of the Ph.D. degree <strong>in</strong> Microbiology.
Prof. A. O. Adeyeba Signature……………… Date…………..
(Chief Supervisor <strong>and</strong> Professor of Medical Parasitology)
Dr. Olusola Ojurongbe Signature…………….. Date……………
(Supervisor <strong>and</strong> Senior Lecturer, Medical Parasitology)
Prof. L. O. Egwari Signature………………. Date……………
(Co-Supervisor <strong>and</strong> Professor of Medical Microbiology)
Dr. E. O. Fagade Signature……………… Date……………..
(Co-Supervisor <strong>and</strong> Senior Lecturer, Environmental Microbiology <strong>and</strong> Biotechnology)
Dr. S. N. Ch<strong>in</strong>edu Signature………………. Date
(Head of Department)
Prof. S.O. Otokiti
(School of Postgraduate Studies Representative)
Prof. (Mrs) A.F. Fagbenro Beyioku
(External Exam<strong>in</strong>er)
iii

DEDICATION
This Ph.D work is dedicated to The Almighty God, The giver of all knowledge <strong>and</strong> wisdom.
iv

ACKNOWLEDGEMENTS
‘Except the Lord builds a house, the builders labour <strong>in</strong> va<strong>in</strong>’. I return all thanks to the Almighty
God for his mercies, faithfulness, grace, favour <strong>and</strong> <strong>in</strong>spiration that I enjoyed dur<strong>in</strong>g the course of
this Ph.D programme. To him alone is all glory for a successful completion.
I profoundly acknowledge the Chancellor of Covenant University for his spiritual motivation <strong>and</strong>
for provid<strong>in</strong>g such an enabl<strong>in</strong>g environment from which I have benefited. I wish to express my
profound gratitude to the management of Covenant University under the leadership of Prof. (Mrs)
Aize Obayan for the support, advice <strong>and</strong> encouragement while the programme lasted.
My s<strong>in</strong>cere appreciation goes to my chief supervisor, Professor A.O. Adeyeba for his awesome
contributions, fatherly advice, thorough supervision <strong>and</strong> mentorship roles while on the research
programme. I am privileged to have studied under his tutelage, most especially I want to
appreciate him for h<strong>and</strong><strong>in</strong>g me over to Dr. Olusola Ojurongbe to take on the supervision when he
was <strong>in</strong>disposed. I pray God for the speedy restoration of his health. I am s<strong>in</strong>cerely grateful to my
supervisor, Dr. Ojurongbe for his useful advice, guided supervision <strong>and</strong> timely encouragement. He
came to my rescue when I would have been str<strong>and</strong>ed. I am most grateful. I also want to appreciate
Prof. Louis O. Egwari <strong>and</strong> Dr. O.E. Fagade who jo<strong>in</strong>tly co-supervised this work. I am <strong>in</strong>debted to
their thoroughness <strong>and</strong> eagle eyes that shaped this work.
I profoundly appreciate the Third World Organisation for Women <strong>in</strong> Science-Third World
Academy of Science (TWOWS-TWAS) for grant<strong>in</strong>g me the 2007 TWAS-TWOWS postgraduate
research fellowship award which was utilized at the National Institute of Malaria Research, New
(NIMR) Delhi, India. I greatly appreciate the <strong>in</strong>tellectual, material <strong>and</strong> f<strong>in</strong>ancial contributions of
TWOWS, TWAS <strong>and</strong> NIMR. I will forever be grateful for the <strong>in</strong>valuable contributions of Dr.
Neena Valecha, my host supervisor <strong>in</strong> India, Dr. Anup Anvikar, the Head of malaria parasite bank
<strong>and</strong> cell culture laboratories, Dr. Ruchi S<strong>in</strong>gh of Molecular biology laboratories <strong>and</strong> Dr. Pillai for
useful advice care <strong>and</strong>. My special appreciation also goes to all the staff <strong>and</strong> students of
Microscopy laboratories, Parasite bank <strong>and</strong> <strong>molecular</strong> biology laboratories for all their love <strong>and</strong>
v

support while I was <strong>in</strong> India. Special thanks to Mallick Prashant <strong>and</strong> Joy for their k<strong>in</strong>d assistance
while I was <strong>in</strong> the <strong>molecular</strong> biology laboratories of NIMR, <strong>in</strong> India.
My s<strong>in</strong>cere appreciation goes to the Dean of College of Science <strong>and</strong> Technology, Prof. James
Katende for his fatherly advice. Special thanks to the entire members of staff of Biological
Sciences Department, my HOD, Dr. S.N. Ch<strong>in</strong>edu for his words of encouragement, Dr. (Mrs)
A.A. Ajayi, my senior colleague, mentor <strong>and</strong> friend for her underst<strong>and</strong><strong>in</strong>g <strong>and</strong> for fill<strong>in</strong>g <strong>in</strong> the gap
for me while I was away on this research work, Dr. O.O Obembe for his encouragement <strong>and</strong>
advice, Mr O. Taiwo, Mr R.O. Ishola, Dr <strong>and</strong> Mrs Jide Adekeye <strong>and</strong> Miss Opeyemi Abegunde for
their technical support <strong>and</strong> assistance dur<strong>in</strong>g the course of this work. Special thanks to Mr. Enoch
Alabi for his professional advice on the arrangement <strong>and</strong> pr<strong>in</strong>t<strong>in</strong>g of this work. May God bless you
all.
My profound gratitude goes to my brothers <strong>and</strong> sisters, Dr. <strong>and</strong> Mrs Henry Owolabi, Mr. <strong>and</strong> Mrs
Victor Owolabi, Mr <strong>and</strong> Mrs J.J. Dada, Engr. & Mrs Femi Owolabi <strong>and</strong> Mr. <strong>and</strong> Mrs A. Adebiyi
for their love, prayers <strong>and</strong> words of encouragement to make sure that this Ph.D work is an
accomplished mission. Many thanks to my late dad, Chief John A. Owolabi whose wish has now
be<strong>in</strong>g accomplished. I shall forever be grateful to my sweet mummy, Mrs Alice E. Owolabi for her
constant prayers love <strong>and</strong> support <strong>in</strong> mak<strong>in</strong>g sure that we get the best out of life. I s<strong>in</strong>cerely
appreciate my uncle’s wife <strong>and</strong> mentor, Dr. (Mrs) A.O. Adedayo for her encouragement, love <strong>and</strong>
prayers. I deeply appreciate my <strong>in</strong>-laws, the entire Olaseh<strong>in</strong>des for their underst<strong>and</strong><strong>in</strong>g, moral
support, prayers <strong>and</strong> encouragement. I s<strong>in</strong>cerely appreciate my darl<strong>in</strong>g husb<strong>and</strong>, Pastor Lawrence
Olaseh<strong>in</strong>de, for his deep underst<strong>and</strong><strong>in</strong>g, patience, love, spiritual <strong>and</strong> f<strong>in</strong>ancial back<strong>in</strong>g even when I
had to leave my family to ensure the completion of this work. Many thanks to our dear children:
Esther, Joseph <strong>and</strong> Daniel for their love, underst<strong>and</strong><strong>in</strong>g, for be<strong>in</strong>g peaceful children, for their
prayers <strong>and</strong> support. I deeply appreciate everyone who rendered assistance to me <strong>in</strong> the course of
this work who for time <strong>and</strong> space could not be mentioned <strong>in</strong> this text. May God bless <strong>and</strong> reward
you all <strong>in</strong> Jesus name. I ascribe all glory <strong>and</strong> honor to the Almighty God for the success of this
research.
vi
Grace

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 – 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 – LITERATURE REVIEW
2.1.Disease <strong>in</strong>cidence <strong>and</strong> trends………………………………………………….8
2.1.1 Geographical distribution <strong>and</strong> populations at risk…………………..8
2.2. Causative agents……………………………………………………………10
2.3 Transmission <strong>and</strong> biology of P. falciparum ………………………………..10
2.4 Symptoms…………………………………………………………………..15
2.5 Diagnosis……………………………………………………………………16
2.5.1 Microscopy…………………………………………………………16
2.5.2 Cl<strong>in</strong>ical (presumptive) diagnosis……………………………………17
2.5.3 Antigen detection tests (rapid or ‘dipstick’ diagnostic tests)……….18
2.5.4 Molecular tests………………………………………………………18
vii

2.5.5Serology………………………………………………………………….19
2.6 Antimalarial Drugs…………………………………………………………….19
2.6.1 Qu<strong>in</strong><strong>in</strong>e <strong>and</strong> related compounds……………………………………….19
2.6.2 Antifolate drugs………………………………………………………..23
2.6.3 Antibiotics……………………………………………………………..25
2.6.4 Artemis<strong>in</strong><strong>in</strong> compounds……………………………………………….26
2.7 Comb<strong>in</strong>ation therapy with antimalarials………………………………………28
2.7.1 Non-Artemis<strong>in</strong><strong>in</strong> based comb<strong>in</strong>ations…………………………………29
2.7.2 Artemis<strong>in</strong><strong>in</strong>-based comb<strong>in</strong>ations………………………………………29
2.7.3 Traditional Antimalarial Herbs………………………………………...31
2.8 Antimalarial Drug Resistance …………………………………………………33
2.8.1 Def<strong>in</strong>ition of antimalarial drug resistance……………………………..34
2.8.2 Malaria treatment failure………………………………………………34
2.8.3 Mechanisms of antimalarial resistance………………………………..35
2.8.3.1 Chloroqu<strong>in</strong>e resistance………………………………………………...35
2.8.3.2 Antifolate comb<strong>in</strong>ation drugs………………………………………….36
2.9 Spread of resistance…………………………………………………………....36
2.9.1 Biological <strong>in</strong>fluences on resistance…………………………………....37
2.9.2 Programmatic <strong>in</strong>fluences on resistance………………………………..40
2.10 Detection of resistance………………………………………………………...42
2.10.1 In vivo tests…………………………………………………………....42
2.10.2 In <strong>vitro</strong> tests…………………………………………………………...43
2.10.3 Animal model <strong>studies</strong>………………………………………………...45
2.10.4 Molecular techniques…………………………………………………45
2.10.5 Case reports <strong>and</strong> passive detection of treatment failure………………46
2.11 The future: prevention of drug resistance…………………………………….46
CHAPTER THREE – MATERIALS AND METHODS
3.1 Study Area…………………………………………………………………..49
3.2 Study Patients………………………………………………………………..49
3.3 Sampl<strong>in</strong>g Procedure………………………………………………………….49
3.4 Ethical Consideration………………………………………………………...51
viii

3.5 Sample Collection……………………………………………………………51
3.6 Cryopreservation……………………………………………………………..52
3.7 Process<strong>in</strong>g of sample………………………………………………………....52
3.7.1 Microscopic exam<strong>in</strong>ation……………………………………………...52
3.8 Antimalarial sensitivity test<strong>in</strong>g……………………………………………….52
3.8.1 Revival of cryopreserved parasites…………………………………….52
3.8.2 In <strong>vitro</strong> microtest (Mark III Test)……………………………………...53
3.9 Antimalarial Activity Test<strong>in</strong>g of Crude Organic Extracts of …………………53
Medic<strong>in</strong>al Plants: Momordica charantia (Ejir<strong>in</strong>), Diospyros
monbuttensis (Eegun eja) <strong>and</strong> Mor<strong>in</strong>da lucida (Oruwo)
3.9.1 Preparation of plant extract…………………………………………53
3.9.2 In <strong>vitro</strong> test…………………………………………………………53
3.10 Molecular Studies………………………………………………………….54
3.10.1 DNA extraction…………………………………………………….54
3.10.2 PCR for detection of Pfcrt gene……………………………………54
3.10.3 Nested PCR <strong>and</strong> RFLP for Pfcrt mutation-specific detection……...55
3.10.4 PCR <strong>and</strong> RFLP for detection of Pfmdr1 gene……………………...55
3.10.5 PCR assays for the detection of Pfdhfr <strong>and</strong> Pfdhps genes………….56
3.10.6 PCR <strong>and</strong> RPLP assay for (SERCA) PfATPase6……………………57
3.10.7 Molecular Genotyp<strong>in</strong>g of isolates us<strong>in</strong>g MSP1&2 <strong>and</strong> Glurp……...57
3.10.8 Questionnaire Adm<strong>in</strong>istration………………………………………60
CHAPTER FOUR – RESULTS
4.1. Incidence of Malaria <strong>in</strong> Ogun State, Southwestern Nigeria…………………….61
4.1.1 Patients Characteristics…………………………………………………..61
4.1.2 Incidence of Malaria……………………………………………………..61
4.2. In Vitro Drug sensitivity Tests…………………………………………………...61
4.3 Prevalence of drug resistant <strong>molecular</strong> markers…………………………………62
4.4 In <strong>vitro</strong> antimalarial activity of herbal extracts…………………………………..62
4.5 Genetic Diversity of P. falciparum........................................................................63
4.6 Knowledge <strong>and</strong> practice on the use of antimalarial drugs……………………….64
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CHAPTER FIVE –
DISCUSSION ……………………………………………………………………..88
CONCLUSION…………………………………………………………………....101
CONTRIBUTION TO KNOWLEDGE…………………………………………....102
REFERENCES……………………………………………………………………..103
APPENDICES……………………………………………………………………...128
x

ABBREVIATIONS
ADP Adenos<strong>in</strong>e diphosphate
ATP Adenos<strong>in</strong>e triphosphate
pfATPase P. falciparum Adenos<strong>in</strong>e Triphosphatase 6 genes
SERCA Sarco/endoplasmic reticulum calcium-dependent
DELI Double-site Enzyme-l<strong>in</strong>ked Lactate dehydrogenase
Immunodetection
DHFR Dihydrofolate reductase
DHPS Dihydropteroate synthase
DNA Deoxyribonucleic acid
EDTA Ethylenediam<strong>in</strong>etetraacetic acid
ELISA Enzyme-l<strong>in</strong>ked immunosorbent assay
HEPES N-(2-hydroxyethyl)piperaz<strong>in</strong>e-N´-(2-ethanesulfonic acid)
HPLC High-performance liquid chromatography
HRP II Histid<strong>in</strong>e-rich prote<strong>in</strong> II
IC50 50% <strong>in</strong>hibitory concentration
LDH Lactate dehydrogenase
MIC M<strong>in</strong>imal <strong>in</strong>hibitory concentration
NAD Nicot<strong>in</strong>amide aden<strong>in</strong>e d<strong>in</strong>ucleotide
PABA Para-am<strong>in</strong>obenzoic acid
PCR Polymerase cha<strong>in</strong> reaction
Pfcrt P. falciparum Chloroqu<strong>in</strong>e resistance transporter gene
PCR Polymerase cha<strong>in</strong> reaction
pfmdr1 P. falciparum multidrug resistance gene 1
RPMI Roswell Park Memorial Institute
TDR Special Programme for Research <strong>and</strong> Tra<strong>in</strong><strong>in</strong>g <strong>in</strong> Tropical
Diseases
Tween 80 polyoxyethylenesorbitan monooleate
vs versus
WHO World Health Organization
DMSO Dimethyl sulphoxide
MSP1 Merozoite Surface Prote<strong>in</strong> 1
MSP2 Merozoite Surface Prote<strong>in</strong> 2
GLURP Glutarmate Rich Prote<strong>in</strong>
QT-NASBA Quantitative Nucleic Acid Sequence Based Amplification
BSA Bov<strong>in</strong>e Serum Album<strong>in</strong>
WBC White blood cell(s)
TCM Tissue Culture Medium
xi

LIST OF FIGURES
Fig Title Page
Fig 2.1 Life Cycle of Plasmodium Species……………………………………..14
Fig 3.1 Map of Ogun State, South Western Nigeria…………………………….50
Fig 4.1 Sample of HN-NonL<strong>in</strong>n Software Statistical Package…………………75
Fig 4.2 Cross Resistance between Chloroqu<strong>in</strong>e <strong>and</strong> Amodiaqu<strong>in</strong>e, n=100…….76
xii

LIST OF TABLES
Table Title Page
Table 3.1 PCR Primers for MSP1, MSP2 <strong>and</strong> Glutamate rich prote<strong>in</strong>……………….59
Table 4.1 Incidence of P. falciparum <strong>in</strong>fection <strong>in</strong> Ogun State. ………………………65
Table 4.2 Zone wise Incidence of Malaria <strong>in</strong> Ogun State…………………………….66
Table 4.3 In <strong>vitro</strong> susceptibility 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 <strong>molecular</strong> markers of resistance to
antimalarial drugs <strong>in</strong> Plasmodium falciparum from Ogun State,
South Western Nigeria. …………………………………………………….69
Table 4.6 In <strong>vitro</strong> susceptibility 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 <strong>and</strong> control of malaria among respondents………74
xiii

LIST OF PLATES
Plate Title Page
Plate 4.1 DNA b<strong>and</strong>s of wild type <strong>and</strong>
mutated P. falciparum chloroqu<strong>in</strong>e resistance genes……………….....77
Plate 4.2 P. falciparum Multidrug Resistance Genes show<strong>in</strong>g the wild
type <strong>and</strong> mutated genes………………………………………………...78
Plate 4.3 DNA b<strong>and</strong> of Dihydrofolate reductase gene (DHFR 108)……………..79
Plate 4.4 DNA b<strong>and</strong> of Dihydropteroate synthase gene (DHPS 540)…………….80
Plate 4.5 DNA b<strong>and</strong> of wild type PfATPase6 …………………………………….81
Plate 4.6 DNA b<strong>and</strong>s of P. falciparum MSP1 MAD20 on Gel……………………82
Plate 4.7 DNA b<strong>and</strong>s of P. falciparum MSP1 K1 on Gel…………………………83
Plate 4.8 DNA b<strong>and</strong>s of P. falciparum MSP1 RO33 on Gel………………………84
Plate 4.9 DNA b<strong>and</strong>s of P. falciparum MSP2 3D7 on gel…………………………85
Plate 4.10 DNA b<strong>and</strong>s of P. falciparum Merozoite Surface Prote<strong>in</strong>2 FC27 on gel…86
Plate 4.11 DNA b<strong>and</strong> of P. falciparum Glutarmate rich prote<strong>in</strong> ……………………87
xiv

ABSTRACT
The widespread of drug resistant Plasmodium falciparum has led to a rise <strong>in</strong> malaria-
associated mortality most especially <strong>in</strong> sub-Saharan Africa. In-<strong>vitro</strong> <strong>and</strong> <strong>molecular</strong> <strong>studies</strong>
were carried out <strong>in</strong> order to determ<strong>in</strong>e the resistant pattern of P. falciparum to antimalarial
drugs <strong>and</strong> some local antimalarial herbs <strong>in</strong> Ogun State, Southwestern Nigeria. Prevalence of
falciparum malaria was determ<strong>in</strong>ed by microscopic exam<strong>in</strong>ation of Giemsa-sta<strong>in</strong>ed blood
samples of patients who presented with fever <strong>in</strong> selected State Hospitals <strong>in</strong> Ogun State.
Antimalarial drug sensitivity of one hundred (100) P. falciparum isolates to chloroqu<strong>in</strong>e,
amodiaqu<strong>in</strong>e, mefloqu<strong>in</strong>e, qu<strong>in</strong><strong>in</strong>e, sulphadox<strong>in</strong>e/pyrimetham<strong>in</strong>e, artesunate <strong>and</strong> three local
antimalarial herbs: Momordica charantia (Ejir<strong>in</strong>,) Diospyros monbuttensis (Eegun eja) <strong>and</strong>
Mor<strong>in</strong>da lucida (Oruwo) was determ<strong>in</strong>ed us<strong>in</strong>g the <strong>in</strong>-<strong>vitro</strong> microtest (Mark III) technique.
For <strong>molecular</strong> <strong>studies</strong> <strong>and</strong> genotyp<strong>in</strong>g, DNA was extracted from patient blood us<strong>in</strong>g the
QiaAmp DNA Blood M<strong>in</strong>ikit extraction method. Nested Polymerase Cha<strong>in</strong> Reaction followed
by Restriction Fragment Length Polymorphisms (PCR/RFLP) were used for the detection of
P. falciparum chloroqu<strong>in</strong>e resistance transporter (Pfcrt), P. falciparum multidrug resistance 1
(pfmdr1), P. falciparum dihydrofolate reductase (Pfdhfr), P. falciparum dihydropteroate
synthase (Pfdhps) <strong>and</strong> P. falciparum sarco/endoplasmic reticulum calcium-dependent ATPase
(SERCA) PfATPase6 genes. Genetic diversity of the isolates was determ<strong>in</strong>ed us<strong>in</strong>g merozoite
surface prote<strong>in</strong>s 1 <strong>and</strong> 2 (msp1 <strong>and</strong> msp2) <strong>and</strong> Glutamate rich Prote<strong>in</strong> (Glurp). Structured
Questionnaires were adm<strong>in</strong>istered to patients or/<strong>and</strong> parents of <strong>in</strong>fants to determ<strong>in</strong>e the factors
that could lead to the development of drug resistance by the parasite <strong>in</strong> the study population.
Out of 4066 subjects screened dur<strong>in</strong>g the period of study, 2550 (61.1%) were positive. Highest
prevalence (72%) was recorded <strong>in</strong> children 1-5 years while the same group also had the
highest parasitaemia of 1080. All the isolates tested were sensitive to Qu<strong>in</strong><strong>in</strong>e, Mefloqu<strong>in</strong>e <strong>and</strong>
Artesunate. Only 51% of the isolates were resistant to chloroqu<strong>in</strong>e, 13% to amodiaqu<strong>in</strong>e <strong>and</strong>
5% to sulphadox<strong>in</strong>e pyrimetham<strong>in</strong>e respectively. Highest resistance to chloroqu<strong>in</strong>e (68.9%)
was recorded among isolates from Yewa zone while highest resistance to amodiaqu<strong>in</strong>e (30%)
was observed <strong>in</strong> Ijebu zone. Highest resistance to sulphadox<strong>in</strong>e <strong>and</strong> pyrimetham<strong>in</strong>e was
recorded <strong>in</strong> Yewa <strong>and</strong> Egba zones respectively. A significant positive correlation was observed
between the responses to artemis<strong>in</strong><strong>in</strong> <strong>and</strong> mefloqu<strong>in</strong>e (P=0.001), artemis<strong>in</strong><strong>in</strong> <strong>and</strong> qu<strong>in</strong><strong>in</strong>e
(P=0.05), Qu<strong>in</strong><strong>in</strong>e <strong>and</strong> mefloqu<strong>in</strong>e (P= 0.01). A significant negative correlation was observed
between the responses to chloroqu<strong>in</strong>e <strong>and</strong> mefloqu<strong>in</strong>e (P=0.05). For the local herbs highest
xv

antiplasmodial activity was obta<strong>in</strong>ed with the ethanolic extract of Diospyros monbuttensis (IC50
= 32 µg/ml). P. falciparum isolates analyzed dur<strong>in</strong>g this study have
demonstrated highly diverse nature of field isolates <strong>in</strong> respect of msp-1
(block 2) <strong>and</strong> msp-2 (central repeat region, block3). All the three reported
families of msp-1(K1, MAD20 <strong>and</strong> RO33) <strong>and</strong> two of msp-2 (FC27 <strong>and</strong> 3D7)
were observed among the isolates. Proportion of isolates with K1 family
was 68% with 4 alleles <strong>in</strong> the range of 100 to 300 basepairs (bp).
Proportion of isolates with MAD20 family was 40% <strong>and</strong> a total of 3 alleles
were observed with<strong>in</strong> 100 to 300 bp. RO33 proportion was 20% <strong>and</strong> the
family was observed to be monomorphic with an allele size of 200 bp. In
msp-2 the proportion of FC27 family was 76% <strong>and</strong> that of 3D7 was 56%.
Proportional Prevalence of FC27 <strong>and</strong> 3D7 families was significantly different
(χ2 = 16.5, P = 0.002). Eighty percent of the isolates harbor the genes that code for
Glutamate rich prote<strong>in</strong> with size rang<strong>in</strong>g between 700 <strong>and</strong> 900bp. Pfcrt (K76T ) Pfmdr1 (mdr
1 ) Pfdhfr (S108N), <strong>and</strong> Pfdhps (K540E ) resistant genes were detected among the isolates
while resistant SERCAPfATPase6 gene which codes for artemis<strong>in</strong><strong>in</strong> resistance was not
detected <strong>in</strong> 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 <strong>in</strong> their management of
malaria. Those who comb<strong>in</strong>ed antimalarial drugs with traditional medic<strong>in</strong>e from local healers
were found to be 17.4%. Only 18% of the sample population used Insecticide treated mosquito
nets, 42.3% use w<strong>in</strong>dow <strong>and</strong> door nets while 13% do not employ any mosquito preventive
method. Cont<strong>in</strong>uous use of the current antimalarial drugs <strong>in</strong>creases the chance of resistance
develop<strong>in</strong>g to those drugs. Control of drug use <strong>and</strong> reduc<strong>in</strong>g exposure of parasites to the drugs
are most effective where the parasite is still sensitive to the drug. Molecular methods are most
effective for monitor<strong>in</strong>g the spread of resistant stra<strong>in</strong>s of P. falciparum.
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