t i r y  O f  OM AHA  UIBARV
QL368. H33 D85 
bite C.l 
G369105
3 0 6 9 2  1 0 02  1 153  9
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STUDIES ON GENETIC MUTATIONS IN PLASMODIUM 
FALCIPARUM STRAINS ASSOCIATED WITH 4- 
AMINOQUINOLINES (CHLOROQUINE) AND 
PYRIMETHAMINE-SULPHADOXINE (FANSIDAR) RESISTANCE 
IN GHANAIAN MALARIA PATIENTS.
A THESIS PRESENTED TO THE DEPARTMENT OF ZOOLOGY,' 
UNIVERSITY OF GHANA
BY
NANCY ODUROWAH DUAH
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF MASTER OF PHILOSOPHY IN 
ZOOLOGY (PARASITOLOGY)
SEPTEMBER, 2001
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This thesis is the result o f research work undertaken by Nancy O. Duah in the 
Department o f Zoology, University o f  Ghana, under the supervisions o f  Dr Michael 
D. Wilson, Dr. Kwadwo A. Koram and Dr. Dominic Edoh.
DECLARATION
MISS NANCY ODUROWAH DUAH 
(Student)
Date..........................................................
(Supervisor) 
Date....................................
DR. KWADWO ANSAH KORAM 
(Supervisor)
Date..........................................
(Supervisor)
Date..........
11
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DEDICATION
TO THE MEMBERS OF THE DU AH FAMILY
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I wish to express my sincere appreciation to my supervisors Dr. M. D. Wilson, Dr. K. 
A. Koram and Dr. D. Edoh for the technical advice and guidance, which contributed 
towards the success o f  this project.
I would like to acknowledge the tremendous help and goodwill received from 
Noguchi Memorial Institute for Medical Research (N.M.I.M.R.). In particular, Prof. 
D. Ofori-Adjei, Director, for allowing the work to be carried out in the Institute. My 
sincere gratitude also goes to the staff o f Epidemiology Unit (Mr. Abuaku, Mr. 
Fenteng, Mr. Attiogbe, Mr. Osei, Mr Boafo and Miss Ampomah) for their assistance 
throughout the fieldwork. Thanks are due the following people for their valuable 
assistance in the laboratory; Dr. D. A. Boakye, Mr. Charles Brown, Mr. Jim 
Brandful, Mrs. Anita Ghansah, Mr Neils Quashie, Mrs. Bridget-Marian Ogoe, Mr. 
Boafo Afrim, Miss Janet Midega, Miss Helena Baidoo, M iss Naiki Puplampu, Miss 
Olivia Tetteh, Miss Marian Opoku-Agyakwah, Mr. Bismark Sarfo, Mr. Evans Glah 
and all others who variously helped me.
My heartfelt thanks and gratitude go to all the members o f  my family, especially to 
my Mum and Dad for their support and encouragement, my sisters, Louisa, Julia, 
Joana, Naa Merley and my brother Sammie; my cousins, Mavis, Cynthia, Abigail, 
and Susana; my auntie Paulina; my grannies; my brother-in-law, Steve; and my two 
sweet nieces, Stephanie and Shawna, for their prayers, support and love. To my 
friends, Sarah, Cynthia, Akosua, Neils, Derek, Martin, Michael, Andrew, Charles, 
Emmanuel and Benard, I extend my sincere appreciation for their prayers and love.
To my mates, Godfred Futagbe and Samuel Kwapong, thanks for the encouragement.
ACKNOWLEDGEMENTS
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An outstanding appreciation goes to Almighty God for all the grace that has 
sustained me till now and all that will keep me growing stronger in the future.
The Multilateral Initiative on Malaria (MIM) grant from WHO/TDR to Dr. Kwadwo 
A. Koram (N.M.I.M.R) funded this research work.
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DECLARATION..............................................................................................................11
DEDICATION.................................................................................................................
ACKNOWLEDGEMENTS......................................................................................... iv
TABLE OF CONTENTS.............................................................................................. vi
LIST OF ILLUSTRATION..........................................................................................ix
LIST OF TABLES.......................................................................................................... x
LIST OF APPENDICES..............................................................................................xii
LIST OF ABBREVIATIONS....................................................................................xiii
ABSTRACT....................................................................   xiv
CHAPTER 1 ....................................................................................................................... 1
GENERAL INTRODUCTION..................................................................................1
1.1 Introduction.............................................................................................................1
1.2 R ationale .................................................................................................................. 6
1.3 General O bjective.................................................................................................. 8
1.3.1 Specific ob jectives.......................................................................................... 8
CHAPTER 2 ...................................................................................................................... 10
LITERATURE REVIEW ........................................................................................... 10
2.1 Malaria: The Disease............................................................................................. 10
2.1.1 Disease symptom s..........................................................................................11
2.2 The Global Distribution o f  M alaria ...................................................................13
2.3 The Socio-economic Impact o f M alaria ........................................................... 15
2.4 The Life Cycle and Transmission o f Human P lasm odium ..........................17
2.5 The Life Cycle o f the Anopheline Vectors o f  M a la ria ................................. 20
2.6. Control o f M alaria ............................................................................................24
2.6.1 Historical overv iew ........................................................................................24
2.6.2 Vector con tro l................................................................................................. 26
2.6.3. Chemotherapy...............................................................................................29
2.7 Genetic Mutations Associated with Chloroquine and Fansidar Resistance 
in Plasmodium fa lc ipa rum ................................................................
TABLE OF CONTENTS
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2.7.1 Chloroquine resistance ................................................................................41
2.7.1.1 Mutations in the Pfcrt g e n e ................................................................. 41
2.7.1.2 Mutation in the Pfmdrl g e n e .............................................................. 42
2.7.2 Fansidar resistance.......................................................................................42
2.7.2.1 Mutations in the Dhfr g e n e ..................................................................42
2.12.2  Mutations in the Dhps g e n e ................................................................ 43
2.8 Methods for Detection o f Plasmodium fa lc ip a rum .................................. 44
2.8.1 M icroscopy....................................................................................................44
2.8.2 In-vitro test o f  parasite sensitivity to ch loroquine.................................4 4
2.8.3 Polymerase Chain Reaction (PC R ).......................................................... 46
2.8.3.1 Standard PCR amplification p ro toco l............................................... 48
2.8.3.2 Nested PCR amplification p ro toco l...................................................48
CHAPTER 3 ....................................................................................................................49
MATERIALS AND M ETHOD S ........................................................................... 4 9
3.1 Study A re a ........................................................................................................... 49
3.1.1 Hohoe D istric t.............................................................................................. 49
3.1.2. Navrongo D istric t.................................................................................... 4 9
3.2 Field Sample Collection ....................................................................................52
3.3 Chemicals and R eagen ts................................................................................... 53
3.4 Laboratory S tud ies............................................................................................. 54
3.4.1 Preparation and examination o f blood s lid e s .........................................54
3.4.2 In-vitro susceptibility test o f  P. falciparum  to C h loroquine ...............54
3.4.3 The identification o f Plasmodium  spec ies..............................................56
3.4.3.1 Isolation o f Plasmodium  DNA from filter paper blood b lo t  56
a) Chelex extraction m ethod .........................................................................56
b) Methanol fixation m ethod ........................................................................57
3.4.3.2 Nested PCR method for Plasmodium  species identification  57
3.4.3.3 Analysis o f PCR p roducts...................................................................58
3.4.4 Detection o f Plasmodium falciparum  genetic mutations associated 
with antimalarial drugs resistance......................................................................60
3.4.4.1 Chloroquine resistance associated genetic m u ta tions...................60
a) Pfcrt 7 6 ........................................................................................................ 60
b) Pfmdrl 8 6 ....................................................................................................62
3.4.4.2 Fansidar resistance associated genetic m u ta tions.......................... 64
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a) Dhfr 51, 59 and 1 0 8 .....................................................................................04
b) Dhps 437 and 540 ........................................................................................ 65
3.5 Statistical analysis................................................................................................ 68
CHAPTER 4 ......................................................................................................................68
RESU LTS ....................................................................................................................... 68
4.1 Study Population...................................................................................................68
4.2 Parasitaemic Profile o f  the Study Popu lation .................................................70
4.3 In vitro Chloroquine Sensitivity S tud ies .........................................................74
4.4 PCR Detection o f Plasmodium  S pec ies ...........................................................76
4.5 Distribution of P. falciparum  Genotypes Associated with Chloroquine 
Resistance......................................................................................................................77
4.5.1 Pfcrt 7 6 .............................................................................................................77
4.5.2 Pfmdrl 8 6 ........................................................................................................ 78
4.5.3 Double mutations (both pfcrt T76 and p fm drl Y 8 6 ) ...............................79
4.6 Distribution o f P. falciparum  Genotypes Associated with Fansidar 
R esistance......................................................................................................................87
4.6.1 Dhfr 51 ............................................................................................................ 87
4.6.2 Dhfr 5 9 ............................................................................................................ 87
4.6.3 Dhfr 1 0 8 .......................................................................................................... 88
4.6.4 Triple mutation (dhfr 51, 59 and 108 ) ...................................................... 89
4.6.5 Dhps 437 ......................................................................................................... 89
4.6.6 Dhps 540 .........................................................................................................90
4.6.7 Double mutations (dhps 437 and 5 4 0 ) ...................................................... 90
4.6.8 Quintuple mutations (dhfr 51, 59, 108, dhps 437 and 5 4 0 ) ................... 90
4.7 Comparison between Molecular Analysis, In vivo and In vitro 
O utcom es..................................................................................................................... 96
CHAPTER 5 .....................................................................................................................103
DISCUSSION AND CONCLUSION .................................................................... 103
REFERENCES ................................................................................................................ H I
A PPEND ICES ................................................................................................................. 125
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LIST OF ILLUSTRATIONS 
Figure 2.1 Global distribution o f  malaria
Figure 2.2 Schematic illustration o f  the life cycle o f  P. falciparum
Figure 2.3 Schematic illustration o f  the life cycle o f  Anopheles vectors o f  malaria
Figure 3.1 Map showing the location o f the study sites, Hohoe and Navrongo in 
Ghana
Figure 4.1 Age distribution o f  patients recruited for the study at Hohoe
Figure 4.2 Age distribution o f  patients recruited for the study at Navrongo
Figure 4.3 Allelic frequencies o f pfcrt 76 o f  non-respondents from Hohoe at pre 
and post-treatment
Figure 4.4 Allelic frequencies o f  pfcrt 76 o f  non-respondents from Navrongo at
pre and post-treatment
Figure 4.5 Prevalence o f  p fm drl 86 mutation in non-respondents from Hohoe at
pre and post-treatment
Figure 4.6 Prevalence o f pfm drl 86 mutation in non-respondents from Navrongo
at pre and post-treatment
Figure 4.7 Agarose gel electrophoresis o f nested PCR products from the
amplification o f alleles o f pfcrt codon 76.
Figure 4.8 Agarose gel electrophoresis o f  nested PCR products from the
amplification o f  alleles o f p fm drl codon 86.
Figure 4.9 Agarose gel electrophoresis o f  nested PCR products from the
amplification o f alleles o f  dhfr codon 108.
Figure 4.10 Agarose gel electrophoresis o f nested PCR products from the
amplification o f alleles o f dhfr codons 51 and 59.
Figure 4.11 Agarose gel electrophoresis o f nested PCR products from the
amplification o f alleles o f dhps codons 437 and 540.
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Table 3.2
Table 3.3 
Table 3.4
Table 4.1 
Table 4.2 
Table 4.3
Table 4.4
Table 4.5
Table 4.6
Table 4.7
Table 4.8
Table 4.9
Table 3.1 DNA sequences o f  the synthetic oligonucleotide primers for PCR 
identification o f Plasmodium  species
DNA sequences o f  the synthetic oligonucleotide primers for the 
detection o f  mutations associated with Chloroquine resistance 
Recognition sites o f  restriction enzyme and product sizes for p fcrt 76 
DNA sequences o f the synthetic oligonucleotide primers for the 
detection o f mutations associated with Fansidar resistance.
Distribution o f sexes and treatment outcome o f  the two study sites 
Parasitaemic profile o f  patients before treatment w ith chloroquine 
Chloroquine inhibition levels o f Plasmodium falciparum  isolates from 
Hohoe and Navrongo
Prevalence o f  pfcrt codon 76 alleles in patients at pre-treatment from 
Hohoe and Navrongo
Prevalence o f  pfm drl codon 86 alleles in patients at pre-treatment 
from Hohoe and Navrongo
Prevalence o f  double mutations, pfcrtT76  and p fm drlYS6 , at pre and 
post-treatment in patients from Hohoe and Navrongo 
Baseline prevalence o f alleles o f the dhfr codon 51 in patients from 
Hohoe and Navrongo
Baseline prevalence o f alleles o f the dhfr codon 59 in patients from 
Hohoe and Navrongo
Baseline prevalence o f  alleles o f the dhfr codon 108 in patients from 
Hohoe and Navrongo
LIST OF TABLES
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Table 4.10
Table 4.11
Table 4.12
Table 4.13
Table 4.14
Table 4.15
Baseline prevalence o f alleles o f the dhps codon 437 in patients from 
Hohoe and Navrongo
Baseline prevalence o f  alleles o f  the dhps codon 540 in patients from 
Hohoe and Navrongo
Baseline prevalence o f double mutations o f  dhps (437 + 540) in 
patients from Hohoe and Navrongo
Baseline prevalence o f  quintuple mutations (dhfr 51 + 59 + 108 + 
dhps 437 + 540) in patients from Hohoe and Navrongo 
Outcomes o f  in-vivo and in-vitro analysis for selected samples from 
Hohoe and Navrongo
Baseline prevalence o f  triple mutations o f  dhfr (51 + 59 + 108) in
patients from Hohoe and Navrongo
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Appendix
Appendix
Appendix
Appendix
Appendix
I Preparation o f standard solutions
II Chemicals, reagents and equipments
III Recruitment and Case Record form
IV  An example o f  plot o f  log molecular weight against mobility 
to determine sizes o f  PCR products
V Data from field and laboratory work
LIST OF APPENDICES
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bp base pair
dATP deoxyadenosine triphosphate
dCTP deoxycytidine triphosphate
dGTP deoxyguanosine triphosphate
dTTP deoxythymidine triphosphate
ddw double-distilled water
DNA deoxyribonucleic acid
EDTA Disodium ethylene diamine tetraacetate. 2 H2O
EtBr Ethidium bromide
EtOH Ethanol
M Molar
Mw molecular weight
PCR Polymerase chain reaction
pfcrt Plasmodium falciparum  transporter gene
PfCRT Plasmodium falciparum  transmembrane protein
pfmdrl Plasmodium falciparum  multi-drug resistant gene
dhfr dihydrofolate reductase gene
dhps dihydropteroate synthase gene
rpm revolution per minute
sddw sterile double distilled water
TAE Tris-acetate-EDTA
Tm melting temperature
Hi microlitre
HM micromolar
LIST OF ABBREVIATIONS
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ABSTRACT
The malaria drug policy for Ghana is chloroquine, Fansidar and quinine as the first 
line, second line and third line drugs respectively. However, the burden o f  malaria 
has been complicated by the emergence o f resistance especially to chloroquine, 
which is a cheap and effective drug. It has therefore become imperative that the 
levels o f  resistance o f  Plasmodium falciparum  to anti-malarial drugs (chloroquine 
and Fansidar) in Ghana be established and the information used to develop an 
appropriate drug policy for effective case management. This present study therefore 
used molecular techniques, mostly, polymerase chain reaction (PCR) to detect and 
characterise mutations in the putative P. falciparum  transporter gene (pfcrt) and P. 
falciparum  multi-drug resistance gene (pfmdrl) that are known from previous studies 
to be associated with chloroquine resistance. Mutations in the dihydrofolate 
reductase g ene ( dhfr) and dihydropteroate synthase gene (dhps) that are associated 
with pyrimethamine-sulphadoxine (Fansidar) resistance were also studied. Children 
aged 5 years and below diagnosed as having uncomplicated malaria were recruited at 
two sentinel hospitals in Ghana (Hohoe and Navrongo) for the study with an 
informed consent from parents or guardians. Blood films obtained from the patients 
were examined fo r the  presence o f  malaria parasites before treatment (Day 0) and 
then on Days 7 and 14 after treatment. Filter paper blood blots were also obtained at 
the same time, for use in PCR to detect the mutations. In addition to these, in-vitro 
chloroquine sensitivity test was performed on P. falciparum  isolates from 26 
patients. The in vivo studies revealed that 62% and 31 % o f the patients from Hohoe 
and Navrongo respectively were resistant to chloroquine. The classification o f 
resistance according to parasitological clearance at Hohoe was 55%, 33% and 13% 
for RI, RII and RIII levels respectively; it was 43%, 33% and 23% respectively at
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Navrongo. T he b aseline p revalence o f  t he p fc r t  76 and p fm drl  86 mutations were 
82.5% and 82.0% in Hohoe and 43.8% and 61.5% in Navrongo. An association 
between pfcrt and  p fm d r l  mutations and  clinical outcome w as observed a t  Hohoe 
(odds ratio = 12.40, p = 0.0001) but not at Navrongo (odds ratio = 1.16, p = 0.75). 
The baseline prevalence o f the quintuple mutations o f  dhfr and dhps were 31.1% and 
1.04% for Hohoe and Navrongo respectively. The in-vitro  results showed that 7 o f  
the 26 isolates were resistant to chloroquine with an IC50 value o f  1 .5x l0 '6 mol/litre. 
The results from this study suggest that mutations in p fcrt and p fm drl can be used to 
predict the outcome o f chloroquine treatment at Hohoe but not at Navrongo. The 
observed differences in the pfcrt and pfm drl prevalence rates and in the association 
between genetic mutations and treatment outcome, is thought to be due to differences 
in drug pressure at the two areas. The relatively high prevalence o f  the quintuple 
mutations o f  dhfr and dhps observed at Hohoe gives an idea o f  the use o f  Fansidar 
whilst is the contrary for Navrongo.
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CHAPTER 1
GENERAL INTRODUCTION
1.1 Introduction
Malaria is a serious and a major public health problem facing humanity in most 
tropical countries particularly sub-Saharan Africa. An estimated 300 to 500 million 
individuals are infected each year worldwide and between 1.5 and 2.7 million people 
die from it in Africa annually. Over 90% o f the deaths occur in  children under 5 
years o f  age (WHO, 1999). According to the WHO (1998), malaria represents 2.3% 
of the overall global disease burden and 9% in Africa, ranking third among major 
infectious disease threats after pneumococcal acute respiratory infections (3.5%) and 
tuberculosis (2.8%).
The disease is caused by protozoan parasites belonging to the family Plasmodiidae 
and genus Plasmodium. There are four species o f Plasmodium  known to cause the 
disease in its various forms in man, namely; P. falciparum, P. malariae, P. vivax and 
P. ovale. O f these four Plasmodium  species, P. falciparum  is the most widespread 
and causes by far the most morbidity and mortality as well as presenting therapeutic 
challenge o f drug resistance (WHO, 1999). In sub-Saharan Africa, the disease is 
transmitted from person to person by the female anopheline mosquito, mostly 
members o f  the An. gambiae species complex (Lindsay et al., 1991).
The disease symptoms appear 10-16 days after an infectious bite w ith the bursting o f 
infected red blood cells that releases merozoites into blood circulation. It is 
characterised by recurrent attacks or paroxysms with three stages involving chills, 
followed by fever and then sweating.
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There a re p rincipally t wo a pproaches t o t he c ontrol o f  m alaria, v ector control and 
chemotherapy. Vector control involves the use o f  insecticides and environmental 
management whilst chemotherapy involves mainly the use o f  antimalarial drugs. 
Both approaches have limitations, which have hindered the effective control o f  the 
disease. The development o f  insecticide resistance in the vectors and o f  drug 
resistance in the parasite are the two major limitations.
The use o f  chemotherapy to control malaria was known in the 15th century with the 
use o f quinine extracted from the bark o f  cinchona tree by the Peruvian Indians 
(WHO, 1999). Chloroquine was introduced in the 1940s and since then chloroquine 
has been a drug o f  choice for most control programmes because it is cheap and 
effective in curing the disease. Other types o f  drugs, which are basically antifolates, 
pyrimethamine and sulphones, were also developed later and used.
However, the effective use o f  the different groups o f  drugs in disease management 
has been hampered by the development o f  resistance to one or more in different parts 
o f  the world. Chloroquine-resistant P. falciparum  was first reported in Thailand in 
1961 (Kain et al., 1994) and this rapidly spread worldwide such that there is virtually 
no endemic area that has not reported it. The problem o f  drug resistance can be 
attributed primarily to increased selection pressures on P. falciparum  in particular, 
due to indiscriminate and incomplete drug use for self-treatment (WHO, 1995).
The Plasmodium  parasites are known to have complex genomes and P. falciparum  
resistance to antimalarials has been attributed to mutations in the genome o f the
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parasite (Wellems et al., 1997). The authors discovered the cg2 gene on chromosome 
7 and provided evidence that chloroquine resistance in Indochina arose with a 
particular s et o f  m utations in the cg2 allele, which spread across Asia and Africa. 
Polymorphisms in this gene then were thought to be associated with chloroquine 
resistance but allelic modification experiments have ruled out a role for this gene in 
chloroquine resistance (Su et al., 1997; Basco and Ringwald, 1999; Adagu and 
Warhurst, 1999; Fidock et al., 2000).
Another genetic mechanism o f chloroquine resistance that has been suggested is the 
single base substitutions in the P .falciparum  multi-drug-resistance gene (pfmdrl) on 
chromosome 5 w hich i s a ssociated w ith e nhance e fflux o f  t he d rug from  r esistant 
parasites (Foote et al., 1990). The mutation results in the substitution o f  asparagine 
for tyrosine at position 86. Association o f  chloroquine resistance w ith  p fm d r l  has 
been reported in genetic studies (Wellems et al., 1990). Work done in Sudan, 
Tanzania and Kenya revealed an association between the mutation and chloroquine 
resistance (IAEA, 2001; Duraisingh et al., 1997). However, in some field studies 
done in Mali, Cameroon and Southern Africa, no association was found between the 
presence o f the mutation and chloroquine resistance (Basco and Ringwald, 1998; 
Djimde et al., 2001, McCutcheon et al, 1999).
Chloroquine resistance has also been linked to mutant alleles o f  the P. falciparum  
transporter gene (pfcrt). This gene has 13 exons and was identified near cg2 on 
chromosome 7 (Fidock et al., 2000). It encodes for the digestive-vacuole 
transmembrane protein known as PfCRT. Sets o f  point mutations in pfcrt were 
associated with chloroquine resistance in laboratory lines o f  P. falciparum  from
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Southeast Asia, Africa and South America (Plowe and Wellems, 1999; Djimde et al., 
2001). One mutation that results in the substitution o f threonine for lysine at position 
76 (K76T) o f  the gene’s DNA sequence was present in all resistant isolates and 
absent from all sensitive isolates tested in vitro (Djimde et al., 2001). Also, genetic 
transformation experiments with plasmids expressing mutant forms o f  pfcrt 
conferred chloroquine resistance on three different chloroquine sensitive clones 
(Djimde et al., 2001).
Fansidar which is a combination o f folate antagonists and sulphonamides, has also 
become important because it is almost as cheap as chloroquine (Sudre et al., 1992) 
and because it is often effective against chloroquine resistant P. falciparum  
(Mharakurwa and Mugochi, 1994; Soto et al., 1995). However, treatment failure has 
become so common that, it is no longer a reliable choice for the treatment o f  P. 
falciparum  infection in many parts o f  the world (Peters, 1987). The resistance o f  P. 
falciparum  to pyrimethamine-sulphadoxine has been attributed to point mutations in 
the dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) as well as 
dihydropteroate synthase gene (dhps) [Peterson et al., 1990; Wang et al., 1997], The 
genotypes defined by the dhfr mutations are the Asn-108, lleu-51, Arg-59 and Leu- 
164 whilst those for dhps are Gly-437, Glu-540 and Gly-581.
Isolates from areas where in vivo Fansidar resistance is common often have one or 
more o f  the dhfr point mutations associated with pyrimethamine resistance in-vitro 
(Peterson et al., 1988). Fansidar contains two drugs therefore it is possible that 
combination o f point mutations, each o f  which has minimal or undetectable effect 
but may have profound effect together (synergistic). Both sets o f  mutations tend to
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occur in a progressive, step-wise fashion, with higher levels o f  in vitro resistance 
occurring in the presence o f  multiple mutations, leading to the suggestion that 
different levels o f  in vivo resistance may be determined by specific sets o f  dhfr and 
dhps mutations (Plowe et al., 1997; Wang et al., 1997). Recently, in vitro 
pharmacokinetic studies o f  the synergistic action o f  pyrimethamine and sulphadoxine 
carried out under physiologic folate and para-aminobenzoic acid conditions suggest 
that the in vivo response to Fansidar may be determined primarily by parasite 
sensitivity to pyrimethamine (Watkins et al., 1997). This therefore suggests that 
mutations in the dhfr are responsible for Fansidar resistance in P. falciparum.
The fact that there is no third antimalarial drug suitable for widespread use to replace 
chloroquine and Fansidar, the ability to map resistant malaria quickly and accurately 
on an epidemiological scale will be important in efforts to control the spread o f  
resistance (Plowe et al., 1999). Mapping o f  the mutations associated with resistance 
is one such means for monitoring drug resistance. Even though there are new drugs 
such as Malarone, Halfan, Mefloquine and others, these are very expensive and 
therefore cannot be used by the poor malaria endemic countries.
Methods for detection o f such mutations using the polymerase chain reaction (PCR), 
which is an in vitro method for DNA amplification already exist (Plowe et al., 1995). 
The nested PCR, which involves two rounds o f  amplification, primary and secondary 
(Snounou, 1993a) increases the sensitivity o f detecting even point mutations in DNA 
sequences.
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Ghana is a malaria endemic country and distribution o f  the disease follows distinct 
climatic and ecological zones, with more cases occurring in the middle forest 
ecological zone, followed by coastal zone and then the northern savannah area 
(Ahmed, 1989). The disease accounts for about 40-42% o f all outpatient visits in  the 
country (MOH, 1 987). The transmission o f  the disease occurs throughout the year 
but more especially just before and after rains (Ahmed, 1989). The commonest 
malaria parasite in the country is P. falciparum.
Chloroquine is the most widely used antimalarial and it is also the first line drug for 
disease management (Ofori-Adjei, 1989). Fansidar is used as a second line drug with 
quinine as the third line drug. The occurrence o f  chloroquine resistant parasite was 
first reported in 1986 in Accra (Neequaye, 1986) and since then other reports o f  .P. 
falciparum  resistance in patients have also been made (Ofori-Adjei et al., 1988; 
Neequaye et al., 1988). Afari et al. (1992) in a field study assessed sensitivity to 
chloroquine in three ecological zones in Ghana and confirmed the occurrence o f 
chloroquine resistant P. falciparum  parasites. Recent studies carried out in some 
parts o f  the country revealed that about 40% o f patients treated with chloroquine did 
not respond to treatment in some parts o f  the country (Ofori-Adjei, 2001).
A study o f schoolchildren at Madina, a suburb o f Accra revealed a high incidence o f 
Fansidar treatment failure (Landgraf et al., 1994). It is however likely that this may 
be the situation in the other parts o f  the country because increasing drug treatment 
failures have been observed with chloroquine and Fansidar at most hospitals (Koram, 
2001 ).
1.2 Rationale
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Probably because o f  it’s effectiveness, low cost and low rate o f  side effects, 
chloroquine still remains the drug o f  choice in most malaria endemic countries but 
appearance and spread o f chloroquine resistant parasites in Africa poses a major 
challenge to malaria control. With the spread o f  drug-resistant parasites the efficacy 
o f chloroquine may deteriorate beyond a level at which it will cease to be effective as 
first line drug for the treatment o f  malaria.
However, when there is little or no evidence to support a switch in first line drugs, 
the use o f  chloroquine may be retained long beyond the point at which it retains its 
comparative advantage with adverse effects on malaria morbidity and mortality 
(Koram, 2001). The experience from Kenya and Malawi suggests that this might 
have b een t he c ase a nd c hloroquine w as r etained 1 ong a fter i ts efficacy had fallen 
below desirable levels (Bloland e ta  I ,  1 993). A lthough translation o f  results from  
simplified in vivo tests into treatment policy may be problematic, the absence o f 
reliable data to base such decisions on do not augur well for a control strategy based 
on chemotherapy. Therefore the following questions need to be answered
1. Is the use o f  chloroquine as a first line drug in Ghana still justified?
2. What is the level o f P. falciparum  resistance to registered antimalaria drugs in 
the country?
This study will seek to partially answer these questions by determining the level o f 
infection with resistant parasites at two sentinel sites so that drug resistance can be 
monitored to enable the effective use o f the front line drugs in disease management.
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This will be achieved by establishing the levels o f  resistance and the resistance- 
associated mutations in the parasite to the drugs used to manage malaria in the 
country.
1.3 General Objective
The overall aim o f this study is to determine chloroquine and Fansidar resistance 
levels and resistance-associated genetic mutations in P. falciparum  populations at 
two district hospitals in Ghana. The following mutations in P. falciparum-, putative 
transporter gene {pfcrt) and P. falciparum  multi-drug resistance gene {pfmdrl) both 
associated with chloroquine resistance, and dihydrofolate reductase gene (dhfr) and 
dihydropteroate synthetase gene (dhps) which are associated w ith Fansidar 
(pyrimethamine-sulphadoxine) resistance in patients will be investigated.
1.3.1 Specific objectives
1. To recruit and obtain demographic data on age and sex o f  a cohort o f children 
aged 5 years and below.
2. To collect filter paper blood blot samples from malaria patients on days 0, 3, 7, 
14 and 21 and also venous blood from all recruited patients on day 0 for 
molecular analysis and parasite culture respectively.
3. To conduct in vitro chloroquine sensitivity tests on parasites in venous blood 
samples from patients.
4. To detect and identify the species o f Plasmodium  present in the blood blot 
samples using PCR
5. To amplify target P. falciparum  DNA sequences which have the mutations o f 
pfcrt, pfmdr, dhfr and dhps genes using the polymerase chain reaction (PCR).
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6. To determine the distribution and frequencies o f  both resistant and wildtype 
alleles o f parasite in the sampled populations.
7. To analyse the data to reveal any associations between Chloroquine and Fansidar 
in vivo resistance and the mutations.
8. To confirm the association between these genetic mutations and chloroquine 
resistance using the results from in-vitro drug sensitivity test.
9
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CHAPTER 2
LITERATURE REVIEW
2.1 Malaria: The Disease
Malaria and its symptoms have been known since time immemorial in m an’s 
recorded history with the occurrence o f  mosquito in amber suggesting its prevalence 
in pre-historic times (Smith, 1996). The symptoms o f the disease were first described 
by Hippocrates, who related them to the time o f the year and to where the patients 
lived (Bradley, 1996). A variety o f  names were given in describing the disease such 
as shakes, intermittent fever, ague and chills. It was realised then that there was an 
aetiological relationship between the disease and swamps. This led the Romans to 
begin drainage programmes because o f  the bad air that was associated with fever- 
producing areas and hence the term mala aria, written mal'aria  (Smith, 1996). W ith 
time the apostrophe was removed to get the present day term malaria.
The causal agent was discovered by Alphonse Laveran in 1880. Ronald Ross later 
worked out the transmission o f  p arasite from  o ne p erson t o another b y  A nopheles 
mosquitoes in 1898. The first known intervention against the disease was by native 
Peruvian Indians (before the discovery o f the parasite and vector) in 1600, who used 
the bitter bark o f cinchona tree (Bradley, 1996). By 1649, the bark was available in 
England as “Jesuits powder” (Bruce-Chwatt, 1980) to cure agues as it was called 
then. Vector control through insectides began in 1942 with the discovery o f  DDT, 
which was very effective but there was rapid insectide resistance. Other methods 
were implemented such as coating marshes with paraffin, draining stagnant water 
and t he u se o f  n ets. C hemotherapy h as b een t he m ain m ethod fo r p arasite c ontrol
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with the use o f  drugs like quinine, chloroquine, pyrimethamine-sulphadoxine and 
mefloquine.
2.1.1 Disease symptoms
Clinical manifestations o f  the disease are dependent on the immune status o f the host 
and also the species o f Plasmodium  an individual is harbouring. The first symptoms 
o f malaria are non-specific and resemble influenza. These symptoms are similar for 
all four species o f Plasmodium. They include headache, muscular ache, vague 
abdominal discomfort, lethargy, lassitude and dysphoria. These precede fever up to 2 
days. Then there is fever with temperature rising intermittently (>37.5°C), shivering, 
mild chills, worsening headache, malaise and loss o f  appetite. The periodicity o f  
fever depends on the type o f parasite species a patient harbours. I f  the infection is left 
untreated, the fever in P. vivax and P. ovale infections regularises to a 2-day cycle 
(tertian) and P. malariae fever occurs every 3 days. For P. falciparum , fever remains 
erratic and  m ay no t regularise to  tertian  pattern  (White, 1996). There is paroxysm 
characterised by teeth-chattering rigors, cold intense headache and muscular ache, 
which can last up to 30 minutes, and then followed by profuse sweats. As the 
infection continues there is splenomegaly and hepatomegaly and development o f 
anaemia.
Severe malaria, which is the acute form o f falciparum malaria, as defined by WHO 
(1990) includes features such as severe anaemia, renal failure, pulmonary oedema, 
hypoglycaemia, circulatory collapse, bleeding, convulsions, haemoglobinuria, coma 
hyperparasitaemia, jaundice and hyperpyrexia. Cerebral malaria is the most 
prominent feature o f  severe malaria and is defined strictly as unarousable coma. This
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is caused by the sequestration o f infected erythrocytes in the microvasculature o f  the 
brain. Most deaths due to malaria are caused by severe malaria.
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2.2 The Global Distribution of Malaria
The distribution o f the disease was previously widespread but over the past 50 years, 
the geographical area affected by malaria has shrunk considerably and the disease is 
mainly confined to poorer tropical areas o f  Africa, Latin America, and Asia (Fig 2.1) 
where there are problems o f controlling the disease because o f  inadequate health 
structures and poor socio-economic conditions (WHO, 1998). Malaria is endemic in 
a total o f  101 countries and territories which have been divided into WHO’s malaria 
regions. There are 45 countries in WHO's African Region, 21 in WHO's Americas 
Region, 4 in WHO's European region, 14 in WHO's Eastern Mediterranean Region, 8 
in WHO's S outh-East A sia  Region, and 9 in WHO's Western Pacific Region. The 
disease is now a public health problem in these countries, which are inhabited by an 
estimated total o f 2400 million people representing 40% o f the world's population 
(WHO, 1998). It was further estimated that worldwide prevalence o f  the disease is 
300-500 million clinical cases each year o f which more than 90% are in sub-Saharan 
Africa. Furthermore, an estimated mortality due to malaria is over 1 million deaths 
each year with the vast majority o f  deaths occurring among young children in Africa, 
especially those in remote rural areas with poor h ealth s ervices. P regnant w omen, 
non-immune travellers, refugees, displaced persons and labourers entering endemic 
areas are also at risk o f  death due to the disease. According to WHO, distribution o f 
the disease varies from one country to another and even within countries because o f 
the flight range o f the vector, which is about 2 miles and is irrespective o f  the 
prevailing wind.
13
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2.3 The Socio-economic Impact of Malaria
Malaria has been a major obstacle to development in disease endemic areaS'espgcially in 
Africa, which is the worst affected malaria region. More than any other disease, malaria 
hits the poor and the disease endemic countries are some of the world's poorest (WHO, 
1998). The disease causes 1 0.6% o f  lost disability adjusted life years (DALYs) being 
second only to HIV/AIDS (WHO, 1999).
In Africa, malaria accounts for up to a third o f all hospital admissions and up to a quarter 
of all deaths o f children under the age of 5 (WHO, 1998). There are up to 800,000 
infantile mortalities and a substantial number of miscarriages and very low birth weight 
(VLBW) babies per year due to the disease (Bradley, 1996). The cost o f malaria in 
economic terms is also high; treatment ranges in cost between $US 0.80 and SUS 5.30 
depending on local drug resistance, and the total cost in Africa is estimated at about 
SUS 1.8 billion per year (NIAID, 2000). A bout of malaria typically costs 10 working 
days, adding to the economic burden. It is also estimated that, costs to countries include 
costs for control and lost workdays to be 1-5% o f GPD in Africa. For the individual, 
costs include the price of treatment and prevention and lost income (WHO, 1998).
The rural communities are the most affected because the rainy season is often a time of 
intense agricultural activity and time for poor families to earn most o f their annual 
income. The disease can make these families even poorer due to the lost o f labour. In 
children, malaria leads to chronic school absenteeism and there can be impairment of 
learning ability. Urban malaria is increasing due to unplanned development around large 
cities, particularly in Africa and South Asia.
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Malaria epidemics related to political/civil upheavals, economic difficulties and 
environmental problems also contribute in the most dramatic way to death tolls and 
human suffering (WHO, 1998). Increased risk o f the disease is linked with changes in 
land use activities like road construction, mining, logging and agricultural and irrigation 
projects, particularly in "frontier" areas like the Amazon and in South-East Asia. Other 
causes of its spread include global climatic change, disintegration o f health services, 
armed conflicts and mass movements of refugees. The emergence o f multi-drug resistant 
strains o f parasite is also exacerbating the situation. New drugs developed are expensive, 
further straining the economies of the rural communities and endemic countries.
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2.4 The Life Cycle and Transmission of Human Plasmodium
The life cycle o f the parasite is split between a vertebrate host and an insect vector. 
Parasites are transmitted from one human to  another b y  female Anopheline mosquito 
(Figure 2.2). There are three phases o f development in the life cycle o f the parasite 
namely, exo-erythrocytic stages in liver, erythrocytic schizogony in erythrocytes and 
sexual process in the mosquito (Smith, 1996). A human infection begins with a bite from 
an infected Anopheles mosquito when it takes a blood meal. During the process 
numerous threadlike sporozoites, the infective stage o f the parasite, may be injected as 
the mosquito feels about with its proboscis before it strikes a small capillary. The 
sporozoites are directly injected into the blood stream. Some die but the survivors after 
30 minutes migrate to  the  liver (Smith, 1996). A ll sporozoites leave peripheral blood 
circulation within 45 minutes of injection into the host (WHO, 1987).
In the hepatocytes, the sporozoites multiply asexually (schizogony) to become a schizont 
that contains tiny rounded merozoites. When schizogony is completed usually after 9-16 
days, merozoites are released and invade the erythrocytes. The invasion into the 
erythrocytes is achieved through endocytosis (Aikawa, 1980). Within the erthrocytes 
they fuel their activities by consuming haemoglobin and develop into ring-shaped 
trophozoites. There is another round o f asexual reproduction within the erythrocytes, 
which takes 48 hours and this time when the merozoites are released some invade other 
erythrocytes whilst others change into sexual forms (micro and macrogametocytes) 
within 10-12 days (Mons, 1985). Normally, a variable number o f  asexual cycles occur 
before any gametocytes are produced (Carter & Gwadz, 1980).
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The gametocytes have no further activity in the human host. These circulate in the blood 
stream until they are picked up by another mosquito as it takes blood from the infected 
human. In the gut of the mosquito, there is exflagellation o f the microgametocytes and 
subsequent fertilization of the macrogametocytes. The zygote, which is called ookinete, 
penetrates the wall o f the midgut and develops into an oocyst. Sporogony within the 
oocyst produces many threadlike sporozoites, which are released as the oocyst ruptures. 
The sporozoites develop and become up to 1000 times more infective than when in the 
oocyst (WHO, 1987). They then migrate to the salivary glands where they reside until 
injection into another human host.
18
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in the Gut of a female 
Anopheles
Schizonts
Merozoites
Schizonts
Merozoites
Traphozoite
Ring Stages
Blood Vessels
iPOROZOIftS
?o mosquito
Fig. 2.2 Schematic illustration of the life cycle of P. falciparum  (from Malaria 
Foundation International Website, after Shobhona Sharma, 2001)
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2.5 The Life Cycle of the Anopheline Vectors of Malaria
There are four distinct stages in the life cycle o f the mosquito, namely, egg, larva, pupa, 
and adult stages (Fig. 2.3). The immature stages are aquatic whilst the adult is terrestrial.
After mating, the female mosquito feeds on blood, which helps in the development of 
the eggs. Each blood meal provides enough nutrition for a female mosquito to lay a 
batch of eggs. Anophelines show the most regular cycle o f blood feeding and egg laying. 
The females will also feed on nectar between blood meals. Blood feeding can take place 
before or after mating; this depends on the species and local circumstances (White,
1998).
Depending on the type of mosquito species, there are specific breeding sites where the 
female mosquito lays her eggs. Eggs are deposited on damp soil or vegetation, in moist 
tree-holes or containers, and sometimes on to water. It is the choice of specific 
oviposition sites by female mosquitoes that determines the breeding places o f each 
species (White, 1998). The female mosquito lays 30-500 or more eggs at one 
oviposition. The number o f eggs deposited depends much on the species although all of 
them are not deposited at one site (Service, 1993). The number o f eggs laid depends also 
on nutrition and size of adults. Mosquitoes of the genus Anopheles lay single eggs that 
float on the water's surface. Aedes mosquitoes for example, lay their eggs singly on 
damp soil and the eggs hatch only when flooded that is, they undergo diapause. In the 
case of Culex and Culiseta species, the eggs are stuck together in groups or rafts of 200 
or more on the surface of the water. Eggs hatch soon after completion o f the embryonic
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development, in as little as two or three days in tropical species but a week or more in 
temperate and subartic species. Temperature plays a role in the success o f eggs hatching 
into larvae. Eggs o f tropical Anopheles, stephensi and An. culicifacies die when the 
temperatures fall below about 10°C (Service, 1993).
All mosquito larvae are aquatic and metapneustic and pass through four larval instars 
(Service, 1993). They breathe at the water's surface through a specialized siphon tube. 
Mosquito larvae are very active and move almost continuously as they shuttle to the 
surface to obtain oxygen and dive to the bottom to find food. There are five functional 
feeding types among larvae namely, scavengers that ingest mainly dead food by scraping 
and nibbling at animal carcasses and detritus; bottom-feeders that browse over bottom 
debris, feeding on living and nonliving material; filter-feeders that strain phytoplankton 
and zooplankton from the water; filter-feeders that feed from the surface film and 
predators that consume other living organisms.
Rates of larval growth are influenced by environmental factors such as water 
temperature, photoperiodicity, food supply, degree of overcrowding and the species 
(Service, 1993; White, 1998). No known mosquito has larvae that can withstand 
desiccation although larvae may be able to survive short periods among wet mud etc. 
Larvae shed (moult) their skins four times, growing larger after each moulting.
Within a few days to several months, depending on the species and environmental
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factors, the larvae develop into pupae. Pupation occurs at the water surface and the 
whole process takes about three to five minutes. Pupae are non-feeding and normally 
spend most o f their time at the water surface breathing through the paired trumpets. 
When pupae are disturbed by shadows or vibrations, they descend in a zigzagging 
fashion. Mosquito pupae are the most active of any insect pupae (White, 1998).
Pupal duration is mainly determined by temperature. In hot tropical countries it is 
usually two or three days, but can be as short as 26 hours at temperature o f about 30°C 
(Service, 1993). Duration also varies according to species. When this process is 
complete, the fully formed adult emerges from the pupal case. Emergence usually takes 
12-15 minutes and within minutes afterwards the newly emerged adult can make very 
short flights. Male larvae generally pupate before females and in most species male 
pupal life is shorter than female pupal life. Males usually emerge a day  o r  so  before 
females (Service, 1993). The entire life cycle from egg to adult can be completed in less 
than 10 days during periods of favourable temperatures.
After emergence from the pupa the teneral adult usually seeks a shelter amongst 
vegetation until ready for mating, which in the case of the female usually occurs a few 
hours after emergence, but sometimes much sooner (Service, 1993). In some species one 
or two days may elapse before emergence and mating. M ales do no t mate until their 
genitalia have rotated through 180°C, a process that takes 20-24 hours but in some 
species as little as 6-12 hours (Service, 1963; White, 1998). Mating usually occurs in 
flight and commonly around dusk. Adult mosquitoes can live for several weeks to 
several months, depending upon numerous environmental conditions.
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MOSQUITO LIFE CYCLE
Figure 2.3: Schematic illustration of the life cycle o f Anopheles vectors of malaria 
(After Service, 1980).
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2.6. Control o f Malaria
2.6.1 Historical overview
Systematic control of malaria could only begin after the discovery o f the causative 
parasite by Alphonse Laveran in 1880 and the demonstration by Ronald Ross in 1897 
that mosquito was the vector.
The first control mounted against the disease was in the 1950s and 1960 (NIAID, 2000). 
With the discovery of dichlorodiphenyltrichloroethane (DDT) during World War II, it 
was thought eradication of malaria was possible (WHO, 1998). The WHO Global 
Malaria Eradication Programme was then created with the aim o f spraying homes with 
residual insecticide in this case DDT and manipulation o f the environment to reduce 
mosquito breeding. This strategy proved successful in large areas o f North America, 
Southern Europe, the former Soviet Union and some territories of Asia and South 
America by halting transmission and thereby eradicating malaria. The disease however 
persisted in most countries in Latin America, Asia and Africa. Large-scale eradication 
was never attempted in Africa because of the problems and logistics associated with 
malaria control, which were beyond the scope o f the vast majority of the countries then.
Whilst malaria has remained endemic in most African countries, the disease is re- 
emerging in areas where it was previously under control or eradicated, for example, in 
the Central Asian Republics of Tajikistan and Azerbaijan, and in Korea (WHO, 1999). 
Easy international travel has enhanced the importation o f cases of malaria and therefore 
it is now more frequently registered in developed countries
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Malaria has been a priority for WHO since its inception in 1948. The WHO’s 
Programme on Communicable Diseases (CDS) coordinated control activities worldwide. 
The four basic technical elements o f the current WHO's Global Malaria Control Strategy 
are the provision o f early diagnosis and prompt treatment for the disease, the planning 
and i mplementation o f  s elective and sustainable preventive measures including vector 
control, early detection for the prevention or containment of epidemics and the 
strengthening of local research capacities to promote regular assessment of countries' 
malaria situations, in particular the ecological, social and economic determinants o f the 
disease (WHO, 1998).
The UNDP, UNICEF, WHO and World Bank in 1998 launched the Roll Back Malaria 
programme (RBM) with the aim o f obtaining deeper commitment to win the fight 
against malaria (WHO, 1998). This was going to require not only the commitment of the 
health sector, but also other governmental sectors, the private sector where activities 
may d irectly or indirectly affect the malaria situation, nongovernmental organizations, 
and affected communities themselves. The achievements o f the RBM so far, have been 
political commitment to malaria control, a progressive strengthening of national and 
local capacities for assessing malaria situations and selecting appropriate measures 
aimed at reducing or preventing the disease (WHO, 1998). National plans o f action have 
also been developed in more than 80% of malaria endemic countries.
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The main approach to controlling adult mosquitoes has been the spraying o f  inside 
surfaces o f walls and ceiling or roof of homes with residual insecticide. The rationale 
being that mosquitoes will rest on the walls before or after biting and remain long 
enough to pick up lethal dose of the insecticide.
The main insecticide used during the 1950s and 1960s malaria control programmes was 
DDT. It was w idely used because of its  cheapness per unit weight and its  durability, 
which enabled spraying to be carried out twice a year, or only once in areas with a short 
annual malaria mosquito season (Curtis, 1996). Earlier studies demonstrated that DDT 
was also a repellent, irritant and has toxic effects on malaria vectors (WHO, 1998). 
When DDT was sprayed on house walls at doses o f 2 g/m2, it exerted powerful control 
over indoor transmission of malaria (Cavalie and Mouchet, 1962). There were claims in 
the 1960s and 70s about supposed effects o f DDT on human health such as DDT 
residues in human breast milk which was usually attributed to contaminated food 
(Curtis, 1994). Also resistance to DDT developed in the insects, which rendered it 
ineffective after a while. DDT was then replaced w ith organophosphate o r  carbamate 
insecticides such as malathion or bendiocarb in Sri Lanka, parts o f India, Pakistan, 
Turkey and Central America. However, these insecticides proved to be considerably 
more expensive to use than DDT and malathion does not persist well on mud walls.
2.6.2 Vector control
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Pyrethroids such as deltamethrin and lambdacyhalothrin are used presently and are 
effective at far lower doses than DDT (0.25 mg/sq metre compared with 2 gm/sq metre). 
Although more expensive per unit weight, these pyrethroids are not much more 
expensive per house protected per year (Curtis, 1994). They are also much more 
acceptable to householders because they leave no visible deposit on walls and also 
because they kill nuisance insects such as cockroaches. Like DDT, pyrethroids tend to 
irritate mosquitoes so that they do not rest on deposits for long. However, the 
pyrethroids paralyse the nervous system so fast that contact for a few minutes is enough 
to kill, whereas much longer contact is required with DDT and mosquitoes may escape it 
without picking up a lethal dose. For these reasons, better malaria control has generally 
been achieved with pyrethroids rather than with DDT and other chemical insecticides.
Large scale use o f insecticide impregnated treated bednets (IIBNS) in endemic countries 
is being promoted as a means to  control malaria. Studies conducted in rural northern 
Ghana showed a reduction in mortality in users o f the IIBNS and it also revealed that 
protection extends about 100m to non-user neighbours (Binka et al., 1998). Other 
studies conducted in rural Tanzania, Ifakara, over a three-year period reported of 
reduction in mortality such that IIBNS prevented 1 in 20 child deaths in the community 
(Schellenberg et al, 2001). To assess the impact o f  distributing IIBNS on malaria 
parasitaemia and anaemia in young children following the Ifakara studies, it was 
revealed that IIBNS had a protective efficacy against parasitaemia and anaemia in 
children and therefore a substantial effect on morbidity (Abdulla et al, 2001). Nets have 
long been appreciated as a protection against night biting mosquitoes including malaria
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vectors. However, nets are often tom or hung in such a way that mosquitoes can enter or 
bite through them. The initial motive for impregnating them with an insecticide, which 
was safe for close human contact, was to add a chemical barrier to the imperfect 
physical barrier presented by the net. This apparently arises because a treated net either 
kills or irritates and drives away mosquitoes before they have found a hole in the net to 
enter. An additional argument for treating bednets is that they are a rational place in  
which to deploy a residual insecticide because mosquitoes are attracted to them by the 
carbon dioxide and body odour emitted by the sleeper. Thus the net acts like a baited 
trap. In comparison with spraying a family's house, the amount o f insecticide needed to 
treat their nets is much less and synthetic netting is a more favourable substrate for a 
residual insecticide than is a mud wall. The concern has been raised that large scale use 
of pyrethroid impregnated nets may select for pyrethroid resistance o f a physiological 
kind or may change mosquito behaviour so that they bite out o f doors instead (WHO,
1999).
Anopheles breeding is sufficiently limited in extent and definable therefore, larval 
control also makes a significant contribution to malaria control. Thus draining or filling 
breeding sites, screening of tanks, spraying of larvicide such as Temephos even into 
potable water, stocking breeding sites with larvivorous fish such as Ctenopharyngodon 
idella, spraying breeding sites with bacterial toxin from Bacillus thuringiensis 
israelensis have all been used to control larval populations (Curtis, 1991).
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There is considerable interest in the idea o f rendering mosquito populations genetically 
harmless by introducing genes, which make them non-susceptible to Plasmodium  
(Collins and Paskewitz, 1995) and also to divert them from being strongly attracted to 
biting humans and animals. Thus, for the desirable genes to be spread in Anopheles 
populations, genetic systems such as transposable elements or the intracellular symbiont 
Wolbachia have been used (Curtis, 1996). However, this approach is still being 
researched and may be a long time yet before it becomes operational.
2.6.3. Chemotherapy
Chemotherapy has been the main method for parasite control and clinical management 
with the use of antimalarial drugs such as quinine, chloroquine and others.
Antimalarials can be grouped under four categories according to the stage in the life 
cycle of the parasite that they attack. The four categories are tissue schizonticides, blood 
schizonticides, gametocytocidals and sporonticidals. The causal prophylactics appear to 
kill the early but not the late liver stages and prevent erythrocytic infections. Drugs that 
destroy all exo-erythrocytic forms are referred to as tissue schizonticides. Blood 
schizonticides act on the asexual erythrocytic parasites. The gametocytocidal drugs act 
on gametocytes, particularly the immature forms. The antimalarials which when taken 
up in a blood meal inhibit the development o f oocyst and thereby prevent the production 
of sporozoites in the mosquito are referred to as sporonticidals.
29
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Artemisinin
The infusion of qinghao Artemisia annua has been used in China for at least the last 
2000 years (WHO, 1999) and its active ingredient “qinghaosu” also known as 
artemisinin have recently been identified. The two most widely used derivatives of 
artemisinin are artesunate and artemether and both are blood schizonticides. Artesunate 
is given orally in the form o f tablets at a dose of 4mg/kg for 3 days and sodium 
artesunate is in the form of powder that is reconstituted for intravenous injection. 
Artemether is given through intramuscular injection (anterior thigh) at a dose of 
3.2mg/kg and then 1.6kmg/kg (Fernandez, 2001). While they are widely used in 
Southeast Asia, they are not licensed in much o f the "Western World" including 
Australia (Davis, 2001). A high rate o f treatment failures has been reported and it is now 
being combined with mefloquine for the treatment o f P. falciparum  malaria (WHO, 
1998).
Halofantrin
Halofantrin belongs to a class of compound called the phenanthrene-methanols and it is 
not related to quinine. It was first introduced in the 1980s as a blood schizonticidal 
against the erthrocytic stages of chloroquine resistant P. falciparum  and also the 
erythrocytic stages of P.vivax but not the hypnozoites. The drug is used to treat acute 
forms o f  uncomplicated and multi-resistant P. falciparum  malaria and as a ‘stand by’ 
drug if  chemoprophylaxis fails and there is no medical aid available. It is given at 
8mg/kg in 3 doses. It is slowly and irregularly absorbed if  taken orally (but aided by a
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fatty meal), with a peak plasma concentration 4-6 hours later. Its half-life is 1-2 days and 
it is eliminated through the faeces. But due to its short half life o f 1 to 2 days, i t  is  
therefore not suitable as a prophylactic (Davis, 2001). Halofantrin has been associated 
with neuropsychiatric disturbances and also it is contra-indicated during pregnancy and 
not advised for women who are breastfeeding (WHO, 1998). Abdominal pain, diarrhoea, 
prurities and skin rash have also been reported as side effects by WHO.
Malar one
Malarone was first introduced in 1998 in Australia. It is a combination of proguanil and 
atovaquone. Atovaquone was introduced in 1992 and it was used with success for the 
treatment of Pneumocystis carrinii. It is a synthetic derivative of 
hydroxynaphthoquinone, and may exert its effect by selectively inhibiting electron 
transport in the mitochondria. When combined with proguanil there is a synergistic 
effect and the combination is at the present time a very effective antimalarial treatment 
(Davis, 2001). I t is  given a t lOOOmg/day for 3 days. Malarone has undergone several 
large scale clinical trials and has been found to  be 95% effective in drug resistant P. 
falciparum  malaria (WHO, 1998). It has been claimed to be largely free from 
undesirable side effects even though proguanil is an antifolate. The common side effects 
listed in order o f occurrence are rash, nausea, diarrhoea, headache, fever and vomiting. 
However, it is a very expensive drug.
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Mefloquine
Mefloquine was first introduced in  1 971. It is  a quinoline methanol derivative that is 
related structurally to quinine. Mefloquine is a blood schizonticide and it is also 
effective in killing hypnozoites if  given as a combination treatment with primaquine. 
The compound was found to be effective against malaria that was resistant to other 
forms of treatment and because o f its long half life is also a good prophylactic.
The drug interferes w ith transportation of haemoglobin products and other substances 
from the host cell to the parasite’s food vacuole. (Hellgren et al., 1997; Weidekamm et 
al., 1998). Because o f its relationship to quinine, the two drugs are not recommended to 
be used together. Mefloquine is taken orally at 25mg/kg once and is rapidly absorbed. 
Though it has a slow onset of action, it is very long acting with a plasma half-life of 30 
days. There have been reports of various undesirable side effects including several cases 
of acute brain syndrome, which is estimated to occur in 1 in 10,000 to 1 in 20,000 of the 
people taking this drug (WHO, 1998). This side effect usually develops about two weeks 
after starting mefloquine and generally resolves after a few days. Widespread resistance 
has now developed to mefloquine and this together with undesirable side effects has 
resulted in a decline in its use (Fernandez, 2001).
Mepacrine
Mepacrine was introduced in 1935 but is now considered obsolete. This drug, which is a 
9-amino-acridine, is an effective blood schizonticide but among its disadvantages is the 
fact that it is laid down in the skin and the recipient turns bright yellow. It was used as a 
prophylactic on a large scale during the World War II (WHO, 1998). It had a major
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influence in reducing the incidence of malaria in troops serving in South East Asia 
(Davis, 2001). It is given intravenously at 20mg/kg over 4 hours followed by lOmg/kg 
over 8 hours (Fernandez, 2001) It is now considered to have too many undesirable side 
effects and is no longer used.
Primaquine
Primaquine was first used in 1951, replacing the related pamaquine, which was one of 
the first synthetic antimalarials. Both are 8-aminiquinolines which destroy pre- 
erythrocytic stages (sporontocides). The important role o f primaquine is in the treatment 
of P. vivax infection which has persistent liver stages. It is taken orally and is rapidly 
absorbed and metabolised. Primaquine is given as its base, 15 mg/day for 14 days. The 
half-life o f the drug is 3-6 hours. Its main unwanted effect is due to an inherited genetic 
metabolic condition, a deficiency o f glucose-6-phosphate dehydrogenase in the red 
blood cell. Large doses of primaquine usually result in methaemaglobinaemia with 
cyanosis haemolysis (Fernandez, 2001).
Proguanil
Proguanil was first synthesised in 1946 and introduced in 1948 as a Biguanide (Davis, 
2001). It has a biguanide chain attached at one end to a chlorophenyl ring and its 
structure is very close to pyrimethamine. It destroys the early tissue stages, especially in 
P. falciparum  and hence is used as causal prophylactics. It also acts as a blood 
schizonticide working more slowly than chloroquine and as a sporontocide. It is given at 
200mg for 3 to 7 days (Navy Medical Dept, 2001). It is free from unpleasant side effects 
and because of its mode of action, it is among the compounds known as folate
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antagonists o r  antifolates and destroys the malarial parasite by binding to the enzyme 
dihydrofolate reductase. It is still used as a prophylactic in some countries.
Pyrimethamine
Pyrimethamine was introduced in 1952 as a diaminopyrimidine with similar activities as 
proguanil. It is a drug that affects the synthesis and utilisation o f  folate. Pyrimethamine 
(2,4, diaminopyrimidine) acts by inhibiting the dihydrofolate reductase necessary for 
synthesis of tetrahydrofolate, a precursor in the parasite DNA synthesis. Pyrimethamine 
is given at 25mg weekly has a half life o f 4 days (Navy Medical Dept, 2001). Some of 
the side effects are skin rashes and higher doses will affect human dihydrofolate releases 
leading to megaloblastic anaemia. Folate supplement is usually given in pregnancy, but 
this reduces the efficacy of the drug (Nwanynwu et al., 1996; Basco and Ringwald, 
1998).
Quinine
Quinine is an alkaloid originally extracted from the bark o f Cinchona ledgeriana 
(Cinchona tree). It is one of the four main alkaloids found in the bark and is the only 
drug, which over a long period o f time has remained largely effective for treating the 
disease (Davis, 2001). The antifebrile properties of the bitter bark o f the cinchona tree 
were known by local populations well before the 15th century (WHO, 1999). History 
has it that the Spanish learned about quinine from the Peruvian Indians in the 1600s. 
Export o f quinine to Europe and to United States became a lucrative business until 
World War II cut off access to the world supply of cinchona bark. Quinine is a bitter
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tasting blood schizonticide and in case o f P. vivax and P. malariae also acts as a 
gametocytocide. It was isolated in 1820 by pharmacist Pelletier and Caventou ( Haas, 
1994). It is now used only for emergency treatment o f P. falciparum  malaria, when 
response to chloroquine and most antimalarials have failed. Its mechanism o f action is 
similar to Chloroquine, it causes cytotoxicity o f the parasite by inhibiting the plasmodial 
haem polymerisation with the subsequent built up o f toxic haem. It also intercalates with 
parasite DNA. It is given orally at lOmg.salt/kg as a 7-day course (Fernandez, 2001). It 
is well absorbed in the gut but 80% of it may be bound to plasma protein. Its half-life is 
10 hours and is metabolised in the liver and excreted in  urine within 24 hours. Side 
effects are numerous and could be fatal. In the 1930's and 40's it was common for people 
to take quinine when they thought they had "a touch o f malaria” in Africa. The 
association of repeated infections with falciparum malaria and inadequate treatment with 
quinine, resulted in the development in some o f acute massive intravascular haemolysis 
and haemoglobinuria (black water fever). In the 1940s, an intensive research program to 
find alternatives to quinine resulted in the manufacture o f chloroquine and other 
chemical compounds that are more effective and less toxic (NIAID, 2000).
Sulphones
Sulphones such as Dapsone and Sulphonamides have antimalarial activity as blood 
schizonticides and are usually used in conjunction with other blood schizonticides 
usually pyrimethamine. They are also folate antagonists and have antibacterial activity 
whilst Dapsone is an antileprosy agent. A combination o f Dapsone and pyrimethamine is
35
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marketed as Maloprim. Sulphonamide (e.g. sulphadoxine) and sulphones (Dapsone) act 
by competing for the enzyme dihydropteroate synthetase with para-aminobenzoic acid 
and therefore inhibit folate synthesis. They act on  erythrocytic P . falciparum , but no t 
sporozoites or hypnozoites. It is a top up treatment after a 7-day course o f quinine in 
acute attack of chloroquine resistant falciparum  malaria. It is also a prophylaxis for 
chloroquine resistant strain P. falciparum  or P. vivax. Proguanil or pyrimethamine- 
dapsone (Fansidar) is combined with chloroquine to prevent transmission by killing 
gametocytes. It is orally administered and slow but well absorbed.
Chloroquine
Chloroquine is one of the most successful antimalarial drug ever synthesised, because of 
its safety, affordability, ease o f use and its great efficacy (Ginsburg et al., 1999). 
Chloroquine and its close relative Amodiaquine are all 4-aminoquinolines. It was 
introduced in 1945 and is an excellent blood schizonticide. It is also a gametocytocide 
against P. vivax, P. ovale and P. malariae. For the rapid control of acute malaria, 
chloroquine is usually the drug o f choice. It is a weak base and concentrates in the 
parasites’ lysosome, possibly by  a p arasite-specific drug-concentrating m echanism. I ts 
beneficial effects on public health have been very enormous but after nearly a half- 
century o f use, how it works at the molecular level remains unknown (Wellems, 1992).
36
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The drug is administered orally, unless where not feasible or in severe attack, then it is 
given by continuous intravenous infusion and frequent intramuscular o r s ubcutaneous 
injection. It is rapidly and completely absorbed and extensively distributed throughout 
the tissues. There is a slow release from the tissue thus resulting in long term protection. 
It is metabolised in the liver to be excreted in urine. The half-life o f the drug is 50 hours, 
though 70% comes out as intact drug. It is also used for prophylactic purposes. It is 
considered safe for pregnant and lactating women and also for children. However, it is a 
recommendation to discourage pregnant women from non-endemic areas to travel to 
endemic areas because of the difficulties associated with treatment and the risk to the 
foetus should they contract malaria.
Chloroquine now no longer offers protection against South East Asian P. falciparum  and 
increasingly in other regions because o f resistance (Navy Med. Dept, 2001). While 
effective in  suppressing P. falciparum  in  some parts o f Africa and most strains o f P. 
vivax, resistant forms of P. vivax are appearing and have been reported in Papua New 
Guinea, Indonesia, Thailand and India (WHO, 1998).
Mode o f  action o f  chloroquine
Although, the mechanism of action of chloroquine is not known with certainty, many 
studies are under way to discover the cause o f resistance in the parasite. Also, the
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biochemical and molecular mechanisms o f drug resistance in P. falciparum  remains 
unknown (Wellems et. al., 1997; Martiney et al., 1999).
Slater and Cerami (1992) first reported that chloroquine interferes with a haem 
detoxification enzyme that is essential to the survival o f the parasites in red blood cells. 
The rapid propagation of the parasites in part is met through the digestion o f the host cell 
haemoglobin in the parasites’ acid food vacuoles (Wellems, 1992). The digestion of 
haemoglobin results in the release of large amounts o f a toxic haem moiety called 
ferriprotoporphyrin IX (FPIX), which cannot be degraded by the parasite. As such FPIX 
is sequestered within the food vacuoles as innocuous dark brown granules called the 
malaria pigment or haemozoin. Chloroquine and other related quinoline ring groups of 
antimalarials attack the intra-erythrocytic stages o f pigment-producing parasites by 
inhibiting pigment production. Chloroquine is thought to bind tightly to soluble FPIX to 
form a complex in the food vacuole (Fitch, 1983). The toxic FPIX-drug complexes thus 
would poison the acid food vacuole and thereby kill the parasite (Wellems, 1992).
It is thought that chloroquine being a weak base accumulates in the acidic food vacuole 
of the intra-erythrocytic parasite (Ginsburg et al., 1999). FPIX is toxic to the parasite, it 
increases membrane permeability, which if not detoxified may end up in lysis o f the cell. 
The p olymerisation o f  the toxic F PIX i nto h armless h aemozoin ( HZ) i s a mechanism 
used by the parasite to protect itself from the toxicity. Chloroquine inhibits the 
polymerisation of the FPIX because o f its ability to form a complex which results in an 
increase of the FPIX concentration in the parasite. Chloroquine also causes 
fragmentation of the parasite's RNA and intercalates with its DNA (Ward, 1988).
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Fansidar
Fansidar, a combination of pyrimethamine and sulphadoxine was pressed into service as 
the first line of defence against chloroquine-resistant P. falciparum  malaria in South 
America and Southeast Asia (Plowe et al., 1997). It is used for uncomplicated malaria 
that cannot be cured by chloroquine, or as first-line treatment where chloroquine has 
been dropped from use (Krogstad, 1996). Fansidar is cheap, practically, only one dose is 
needed and is highly effective in much of Africa and South America. As a combination 
drug, each tablet contains 500mg o f sulphadoxine and 25mg o f pyrimethamine. It is 
given as: Adults: 3 tablets in a single dose; Children: 1 tab in children 4-8 years old, 2 
tabs in adolescents 9-14 years old, 3 tabs in those >14 years old.
The effects o f Fansidar are confined to late trophozoites. As a result, it is said to have a 
slow onset o f action, and is not generally recommended for severe malaria. The rate of 
parasite clearance achieved by Fansidar is about the same as that o f quinine (Winstanley, 
1996). Drug-sensitive P. falciparum  malaria is generally treated with a single oral dose 
of Fansidar, which makes the treatment of out-patients very practicable. An 
intramuscular (IM) formulation is available, and is used for patients with protracted 
vomiting and its efficacy in severe forms of childhood malaria has been compared 
favourably with that of quinine (Simao et al., 1991). Oral pyrimethamine and 
sulphadoxine are extensively and rapidly absorbed at about 12 and 4 hours respectively 
(Winstanley et al., 1992a). Pyrimethamine, but not Sulphadoxine, is absorbed more 
slowly after IM injection, possibly because of its poor aqueous solubility (Winstanley, 
1996). The elimination half life of Pyrimethamine varies from 40 to 100 hours, and that
39
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of Sulphadoxine is about 200 hours. It is because o f this slow elimination rate that 
Fansidar need only be given once.
In Southeast Asia, where parasites are often resistant to both pyrimethamine and 
sulphadoxine, Fansidar is clinically useless (Winstanley, 1996). Resistance to 
pyrimethamine has been reported in Africa (WHO, 1990) and also resistance to Fansidar 
is now widespread and serious side effects have been reported (WHO, 1998).
Mode o f  action o f  Fansidar
Plasmodium parasites synthesise their folic acid, which is needed to make the nucleotide 
building blocks o f DNA de novo. Folic acid has three major components namely, 
glutamic acid, para-aminobenzoic acid and pteridine. Fansidar has two main modes of 
action. In the presence of para-aminobenzoic acid, folate can be synthesised but 
sulphadoxine in Fansidar inhibits the incorporation o f para-aminobenzoic acid [PABA] 
into dihydropteroate, a precursor of dihydrofolate, by competitive inhibition of 
dihydropteroate synthetase (dhps).
In the presence of folate, dihydropteroate is reduced to dihydrofolate by an enzyme 
known as dihydrofolate synthase and then to tetrahydrofolate by dihydrofolate reductase 
(dhfr). Pyrimethamine is a competitive inhibitor of dihydrofolate reductase (dhfr) and it 
binds and inhibits dihydrofolate reductase (dhfr). The sulphadoxine and pyrimethamine 
both act synergistically against the parasite (Chulay et a/., 1984).
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2.7 Genetic Mutations Associated with Chloroquine and Fansidar Resistance in 
Plasmodium falciparum
2.7.1 Chloroquine resistance
2.7.1.1 Mutations in the Pfcrt gene
The effectiveness o f chloroquine depends on its concentration in the parasite’s digestive 
vacuole and in resistant parasites the accumulation of chloroquine inside the vacuole is 
diminished (Marsh, 1998). This is attributed to the mutation in the transporter gene such 
that chloroquine entry into the digestive vacuole is hindered because o f mutant 
transporter proteins.
Most chloroquine resistance studies to find point mutations in the parasite genome have 
been conducted in vitro. Mutations have been reported to occur in P. falciparum  
digestive-vacuole transmembrane proteins referred to as PfCRT. The gene that encodes 
for the protein is known as the pfcrt gene which is located on chromosome 7 with 13 
exons (Plowe and Wellems, 1999; Fidock et al., 2000; Djimde et al., 2001).
A single substitution o f Threonine for Lysine at position 76 (pfcrt 76) was found to be 
present in chloroquine-resistant isolates and absent in chloroquine-sensitive isolates 
(Plowe and Wellems, 1999). In addition to pfcrt 76, multiple mutations have also been 
identified in various arrangements at positions 72, 74, 75, 97, 220, 271, 326, 356 and 
371 (Plowe and Wellems, 1999). The roles of these other mutations are still being 
investigated. But studies conducted in Mali by Djimde et al. (2001) suggest the mutation 
in pfcrt 76 putatively confers chloroquine resistance in P. falciparum.
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2.7.1.2 Mutation in the Pfmdrl gene
Foote et al. (1990) have suggested that single base mutations in the P. falciparum  
multidrug resistance gene {pfmdr 1) on chromosome 5 are associated with enhanced 
efflux of chloroquine from resistant parasites. The pfmdr 1 gene encodes for P- 
glycoproteins in the digestive vacuole referred to as Pgh 1 (Foote et al., 1989). The 
mutation is as a result of a single base substitution of Asparagine to Tyrosine at position 
86 (pfmdr 1 86). Some field works done have found an association between pfmdrl and 
chloroquine resistance (Foote et al., 1990) whilst others have not (Basco and Ringwald, 
1998; McCutcheon et al, 1999).
2.7.2 Fansidar resistance
2.7.2.1 Mutations in the Dhfr gene
Point mutations in the dhfr gene which have been reported to be associated with 
Fansidar resistance occur in codons 108, 51, 59 and 164 (Zolg et al., 1989). The 
mutations are as a result of substitution o f single bases in the gene sequence, which 
results in altering the shape of the proteins active site cavity (Plowe et al., 1997). There 
are two point mutations at codon 108, one is a Serine to Asparagine change and the other 
is a Serine to Threonine change. Asparagine to Isoleucine change occurs at codon 51 
whilst Cysteine to Arginine change occurs at 59. The Isoleucine to Leucine change at 
164 together with Asn-108, Ile-51 and Arg-59 have been reported to confer high-level 
resistance to the drug (Peterson et al., 1988).
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2.7.2.2 Mutations in the Dhps gene
The mutations in the dhps gene that are associated with Fansidar resistance are found at 
codons 437 and 540. The substitutions result in Alanine to Glycine change and Lysine to 
Glutamic acid change respectively (Brooks et ah, 1994). These mutations have been 
associated with decreased susceptibility to sulphadoxine (Plowe et ah, 1997). In addition 
to these two mutations, others that have been reported include codons 436-Serine to 
Alanine and Serine to Phenylalanine, 581-Alanine to Glycine and 613-Alanine to 
Threonine/ Serine (Wang et al, 1997).
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2.8 Methods for Detection of Plasmodium falciparum
2.8.1 Microscopy
This method o f diagnosing malaria is efficient in confirming an infection with 
Plasmodium. It involves the preparation o f blood films for malaria parasites from 
capillary blood or venous blood. There are two types o f blood films, thin and thick, 
which are both made on glass slides. The thin blood film is used for the identification of 
malarial parasite species and consists o f one layer o f evenly distributed blood cells 
whilst the thick blood film is made by concentrating the blood spot on the glass slide. 
After staining in Giemsa stain (10%), the parasites appear as pinkish rings among white 
cells against a background of lightly stained red cell debris under the microscope.
In P. falciparum  infection, schizonts are rarely seen because erythrocytic schizogony 
takes place in the internal organs. Therefore only trophozoites (rings) and gametocytes 
are seen in blood films. Absolute numbers o f parasites (number/pl) can be estimated in 
thick blood films by counting the parasite against white cells and using fixed white 
blood cell (WBC) count value to calculate the number o f parasite per jil o f blood by the 
equation:
Parasite number/ul blood = number of parasites x 8000/number o f WBC
2.8.2 In vitro test of parasite sensitivity to chloroquine
The culturing of malaria parasites was first developed by Trager and Jensen (1976). This 
involves the culturing of parasites in various concentrations o f the drug. The principle
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underlying the culture is maintaining human erythrocytes under conditions that support 
intracellular development of the parasites.
The in vitro drug sensitivity test has been developed to include the measurement of 
extent of growth of parasites using the radio labelled metabolite. It involves the 
incorporation of the radioactively labelled precursor 3H-hypoxantin as a marker for 
parasite growth (Desjardins et al., 1979). The principle involved here is that since the 
malaria parasite cannot synthesise purines de novo, they rely on the host for their supply 
of bases using a salvage pathway. The parasites have different degrees of preference for 
obtaining host-derive purines, the preference being hypoxanthin followed by adenosine 
and adenine. Hence on the addition of 3H-hypoxanthin to a culture media, the parasite 
rapidly picks up the radioactively labelled hypoxanthin. The use of 3H-hypoxanthin as 
an i ndex o f  p arasite g rowth i s u sually p referred over m icroscopic m ethods b ecause i t 
yields results more rapidly and more reliably. However it must be stressed that the 
microscopic method is still important in studies in which the effect o f drugs on the 
various group stages is being conducted. Its disadvantages however are that it is time 
consuming, strenuous and has a higher degree of error.
The major problem in  assessing drug sensitivity o f  parasites in  in  vitro  culture is  the 
presence o f different stages of development of the parasite. With the use of radio 
labelled hypoxanthin (3H-hypoxantin), the problem seems a bit solved. This nucleic acid 
precursor appears to be efficient because it is preferentially incoiporated into the more 
matured parasites of P. falciparum.
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A culture medium consists o f RPMI-1640 (composition shown in appendix II) 
developed by Moore et al. (1967), supplemented with Herpes buffer and normal human 
serum or complex mixtures o f protein, lipid and other growth factors. It also contains 
erythrocyte suspension giving a typical 5% hematocrit and 1% or less initial malaria 
parasitaemia (Trager and Jensen, 1978). The dish or flask containing the culture is 
incubated at 37-38°C in an atmosphere o f 3-5% CO2 and 17% or less of O2 under 
aseptic conditions. In the simplest set up, this mixture of gases is obtained using a 
“candle jar”. The candle jar is basically a dessicator with a stop cork in the lid with a 
plain white candle inside. After the dish or flask has been set in, the candle is lit and the 
cover put on with the stop-cork opened. The moment the  candle goes ou t due to  the  
accumulation o f CO2, the stop cork is closed and the dessicator is set in an incubator at 
37°C. At least once a day the dish is removed from the dessicator and provided with 
fresh medium by following the appropriate procedure. This simple candle jar method is 
ideal for many kinds of work and lends itself to screening for new antimalarial agents 
(Trager, 1978), and for drug resistance experiments (Nguyen-Dinh and Trager, 1978).
2.8.3 Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) is an in vitro method o f nucleic acid synthesis by 
which a target DNA fragment is exponentially replicated (Erlich, 1989). It uses a thermo 
stable DNA polymerase isolated from Thermus aquaticus and two oligonucleotide 
primers that flank the target DNA to be amplified. The reaction involves repeated cycles 
of heat denaturing of DNA, annealing of the primers to their complementary sequences 
at a lower temperature, and extension of the annealed primers with the DNA 
polymerase. The primers hybridise to the opposite strands o f target DNA and are
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oriented (3’ ends pointing towards each other) so that DNA synthesis by the polymerase 
enzyme proceeds across the region between the primers. The extension products are 
complimentary to and capable of binding primers, therefore successive cycles of 
amplification result in the doubling o f the target DNA synthesised in the previous cycle.
The primers are usually designed such that the annealing temperatures in the PCR are as 
high as possible to ensure specificity during amplification. Under s tandard c onditions 
the annealing temperature in a reaction should be 5°C lower than the melting 
temperatures (Tm) of the primers, which can be determined using the following formula 
(after Thein and Wallace, 1986):
Tm = 4(G + C) + 2(A + T)
Where G, C, A and T are guanine, cytosine, adenine and thymine respectively.
Ideally, primers with sequences containing runs o f nucleotides that might promote 
annealing with strands outside the target sequences and also with significant secondary 
structures are avoided.
In designing primers to detect point mutations with PCR, the mutant base is located at 
most, three bases from the 3’ end of the primer. At the 3’ end of the primer, annealing is 
more specific than the 5’ end and also initiation of extension occurs at the 3’ end. 
Therefore complementarity of bases is preserved with no altering of bases with the use 
of a polymerase with high proofreading ability.
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2.8.3.1 Standard PCR amplification protocol
The standard PCR amplification protocol (Innis and Gelfand, 1986) will amplify most 
target DNA, however, optimal performance is sought by varying most parameters and 
conditions for each new application.
The standard PCR reaction mix comprises of the following:
- 1 x PCR reaction buffer
- 200|iM each o f dATP, dCTP, dGTP, dTTP (deoxyribonucleotides)
- 2.5units/ 1 OOjj.1 Taq DNA polymerase
- 0.5|iM each of forward and reverse primers (synthetic oligonucleotides)
- lng- l|xl o f template DNA
The standard PCR thermal cycling conditions as follows:
Denaturation 94°C for 30 seconds
Primer annealing (Tm - 5°C) for 30 seconds 
Primer extension 72°C for 2 minutes 
The reaction is usually concluded with a final extension step o f 72°C for 5 minutes to
enable completion of all strands.
2.8.3.2 Nested PCR amplification protocol
This is a form of PCR amplification protocol that involves two rounds of amplification 
with the product or amplicon of the first amplification (described as ‘outer PCR’) used 
as template for the second amplification (described as ‘inner PCR’). This nested 
approach has been shown to have an increased sensitivity of between 10-100 fold for
detecting blood parasites (Snounou et al., 1993). Several studies have demonstrated that
this approach reveals genetic polymorphisms (Cortese and Plowe, 1999; Djimde et al., 
2001).
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CHAPTER 3
MATERIALS AND METHODS
3.1 Study Area
The studies were carried out at Hohoe and Navrongo (Fig. 3.1). These were selected on 
the basis that they are located in two different ecological zones and also had the facilities 
to handle a minimum daily attendance of about 200 patients at the out patient 
department (OPD). The facilities were also to have reliable supply o f water, electricity 
and adequate laboratory space for the study.
3.1.1 Hohoe District
The Hohoe district lies in the middle belt o f the country with semi deciduous forest 
vegetation. The population of this district is estimated to be 110,000 (National Census, 
1984). The district is divided into six sub-districts for health administration purposes. 
There is one government hospital in the district capital, one health centre, 17 health 
posts and a Roman Catholic Church clinic. Disease transmission is perennial with peaks 
occurring after the major rains in June to October. The major vector for disease 
transmission is An. gambiae s. I. but An. funestus predominates in the dry season. More 
than 95% of all infections are caused by P. falciparum  whilst mixed infections with P. 
malariae are also seen (Afari et al., 1992).
3.1.2. Navrongo District
The Kassena Nankana district lies in the northern part of the Guinea savannah belt and 
Navrongo is the district capital. The population is estimated to be about 140,000 living
49
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in dispersed compounds (Binka et al., 1994). The Navrongo War Memorial Hospital 
serves the whole d istrict. M alaria transmission i s i ntense and h ighly s easonal w ith P. 
falciparum  being the predominant parasite. The main transmission vectors are An. 
gambiae s.s. and An. funestus.
50
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BURKINA FASO
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' (— WESTERN
’ CENTRALU \
y_
-  -- GREATER ACCRA
GULF OF GUINEA
Figure 3.1 Map showing the location of the study sites Hohoe and Navrongo 
Ghana.
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3.2 Field Sample Collection
Children aged 5 years and below reporting with symptoms o f malaria or history o f fever 
(with temperatures of >37.5°C in the past 48-72hours) and parasite count between 
2000/fj.l and 100,000/p.l were recruited for the study. The consents o f parents or 
guardians o f  the children were sought before recruiting them  into the  study. Children 
who were unable to drink or breast-feed, vomiting intermittently, convulsing and 
unconscious were excluded from the study. The recruitment was carried out over a 
period of one year at both sites.
Blood films were prepared before treatment, stained with Giemsa and parasites counted. 
Venous blood was collected into heparinised tubes for in-vitro drug sensitivity testing. 
Blood blots were made on 110mm Whatman filter paper, dried and stored individually 
in plastic bags a t room  temperature fo r PCR analysis. The children were then treated 
with chloroquine at a standard dose of 25 mg/kg over 3 days (Appendix El). They were 
followed up daily to day 3 then on days 7 and 14. Blood film for parasite count and filter 
paper blood blot for PCR were collected on each follow-up day. Children with 
parasitaemia above 25% of pre-treatment level on day 3 were treated with Fansidar at 
25mg/kg in one dose and were asked to report on day 7. Filter paper blood blots were 
kept at room temperature until ready for use.
Non-respondents were classified as RI, RII, Rill according to the classic parasitological 
definition by WHO (1996). According to the criteria, patients were classified as RI if 
there was initial clearance of parasites but parasitaemia recurred by day 14. Those who
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had persistent parasitaemia with reduction to less than 25% o f the initial level by day 3 
were classified as RII whilst those who had persistent parasitaemia with no reduction in 
the level of parasitaemia or with a reduction to 25% or more of the initial level by day 3 
were classified as RIII. A patient was however considered to be chloroquine sensitive if 
there was clearance of parasites initially and no parasitaemia was observed by day 14.
3.3 Chemicals and Reagents
The chemicals and reagents used for the present study are listed in Appendix I. The 
various buffers and solutions were prepared as described in Appendix II.
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3.4.1 Preparation and examination of blood slides
A trained technician did microscopic preparation and the examination o f the blood slides 
using standard examination protocol.
3.4.2 In vitro susceptibility test of P. falciparum  to Chloroquine
This test was performed following the modification done by Rieckmann (1978).
About 5ml o f patient b lood w as c ollected i nto h eparinised t ubes a nd w ashed 4 t imes 
with neutral RPMI 1640 to remove WBCs leaving behind RBCs, some o f which will be 
infected with the trophozoites stage o f the parasite. Then 5(il o f the washed parasitized 
RBC were dispensed into the wells o f the microtitre plate containing 45j_l1 o f sterile 
parasite growth medium (Appendix II). The plate was tapped gently to ensure that the 
contents were mixed well. Then chloroquine at different concentrations o f 1, 2, 4, 8, 16, 
32 and 64 pmol were added. Some wells with no chloroquine served as controls. The 
microtitre plate was then covered with its lid and placed in a candle jar which has 
already been warmed in an oven. A candle was lit and placed in the jar with the exhaust 
cork opened. Thereafter, the jar was tightly closed when the candlelight was almost 
extinguished. The jar and contents were then placed in an incubator at 37.5°C for 24 
hours. After that, the jar was removed and opened in a sterile hood,the microtitre plate 
was removed and 1 Ojal o f radioactively labelled hypoxanthin (H3) was added to each 
well and mixed gently. The plate was placed back in the candle jar and was put into the 
incubator for another 24 hours.
3.4 Laboratory Studies
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The cells were harvested after incubation using the Filtermate 196 harvester (Canbetta 
Comp). The levels o f radioactivity were determined using a Matrix Direct Beta Counter 
96 (Canbetta Comp).
The percentage inhibition of parasite growth was then calculated as follows:
Inhibition = CPMn -  CPMr  x 100%
CPM0
where
CPM = counts per minute 
CPMo =CPM in wells without chloroquine 
CPMc = CPM in wells with concentration o f chloroquine 
The inhibition concentration (IC50), which is the effective concentration at which 50% of 
the parasites are inhibited, was estimated from a plot o f inhibition levels against 
chloroquine concentration.
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3.4.3 The identification of Plasmodium  species
3.4.3.1 Isolation of Plasmodium DNA from filter paper blood blot
Two methods for isolating of DNA from filter paper blood blots were used. The first 
which is the Chelex method (Wooden et al., 1993) was used initially, but was later 
abandoned for the methanol fixation method (Cortese and Plowe, 1999) because the 
latter was relatively faster.
a) Chelex extraction method
About 3mm filter paper blood blots were individually cut out and transferred into a 
1.5ml microfuge tube. Then 1ml of PBS (pH 7.4) and 50^1 o f 10% saponin were added, 
inverted three times and stored at 4°C overnight. Each tube was centrifuged at 
14,000rpm for 5secs and the reddish PBS/saponin supernatant discarded. Another 1ml of 
PBS was then added to the filter paper in the tube, inverted several times and incubated 
at 4°C for 15mins after which the tube was centrifuged under the same conditions as 
above and the supernatant also discarded. Then 100|il o f sterile water and 50|il o f 20% 
Chelex were then added to the tube and vortexed. Parasite DNA was extracted by 
incubating the tube and its content at 95°C for lOmins, vortexing at 2 minutes intervals. 
After incubation, the tube was centrifuged at 14,000rpm for 5mins and as much solution 
as possible was transferred into a 0.5ml tube, centrifuged again and the supernatant 
transferred into a fresh tube making sure the Chelex was not carried over. The content of 
the tube was stored at -20 °C until ready for use.
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b) Methanol fixation method
An approximately 3mm-square piece of blood-impregnated filter paper was cut out 
using a sterile razor (wiping off the razor with tissue paper soaked in 75% ethanol 
between cuttings). Then each piece o f the filter paper blood blot was transferred into a 
0.5ml PCR microfuge tube and 50|ul o f methanol was added making sure the paper was 
totally submerged. The tube was incubated for 15mins at room temp. After incubation, 
the methanol was pipetted out with care taken to retain the paper in the tube. The tube 
was left opened on the bench on its side to allow remaining methanol to evaporate. This 
usually took 15mins for the paper blot to be fully dried. 50|il o f water was then added 
and the tube heated for 15mins at 95°C with occasional vortexing at 2 minutes intervals 
during the incubation to improve DNA yield. It was then stored at -20°C  until ready for 
use.
3.4.3.2 Nested PCR method for Plasmodium  species identification
The nested PCR method for the identification of human Plasmodium  (Snounou et al., 
1993) was used. The initial amplification reaction involved the use o f genus-specific 
oligonucleotide primer pair, rPLU5 and rPLU6 to amplify DNA targets from the four 
malaria species. The PCR product obtained was used as the template for the nested PCR 
using species-specific oligonucleotide primer pairs for Plasmodium  species. The primer 
pairs were rFALl and rFAL2 for P. falciparum, rMALl and rMAL2 for P. malariae, 
rOVAl and rOVA2 for P. ovale and rVIVl and rVIV2 for P. vivax (Table 1).
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All the reactions were carried out in  a final volume o f  20 |il which contained 1 xPCR 
buffer (50mM KC1, 20mM Tris-HCl, pH 8.3, 0.1 mg/ml o f gelatin), 2mM MgCh, 
200|iM dNTP mix, 125nM of each primer, 0.5U Taq polymerase and 3pl of DNA 
template for primary amplification. This was thoroughly mixed and overlaid with 20\A 
o f mineral oil to avoid evaporation and refluxing of the reaction mixture. For the nested 
reaction, the mix remained the same except that 2\x\ of the PCR product was used as 
DNA template.
For both the primary and secondary amplifications, PCR was carried out using a PTC- 
1001 hernial c ycler (MJ Research, U SA) w ith c ycling p arameters o f  a n i nitial melt at 
94°C for 2mins followed by 30cycles o f 94°C for 30secs (denaturation), 58°C for 
30secs (annealing), 72°C for 2mins (extension) and a final cycle o f 72 °C for 5mins.
3.4.3.3 Analysis of PCR products
Following the PCR, lOpl of the PCR products were electrophoresed on a 2% agarose gel 
stained with 5|_ig/ml ethidium bromide. The electrophoresis was run in lx  TAE buffer at 
80 volts for 1 hour. The gel was visualised under a Dual Intensity UV transilluminator 
(UVP) after which a photograph of the gel was taken using a Polaroid camera with 
Polaroid type 667 films (Sigma). The sizes of the PCR products were estimated by 
comparison with the mobility o f a standard o f known band sizes. The diagnostic sizes 
expected o f the PCR amplified fragments are 205bp for P. falciparum, 144bp for P. 
malariae, 800bp for P. ovale and 120bp for P. vivax.
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Table 3.1
DNA sequences of the synthetic oligonucleotide primers for PCR identification of 
Plasmodium species
Melting
Name of temp.
Primer* DNA Sequence (5’ -  3 ’) (Tm°C)
rPLU5 (f) CCT GTT GTT GCC TTA AAC TTC 58
rPLU6 (r) TTA AAA TTG TTG CAG TTA AAA CG 58
rFALl (f) TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT 76
rFAL2 (r) ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC 86
rMALl (f) ATA ACA TAG TTG TAC GTT AAG AAT AAC CGC 82
rMAL2 (r) AAA ATT CCC ATG CAT AAA AAA TTA TAC AAA 72
rOVAl (f) ATC TCT TTT GCT ATT TTT TAG TAT TGG AGA 76
rOVA2 (r) GGA AAA GGA CAC ATT AAT TGT ATC CTA GTG 74
rVIVl (f) CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC 82
rVIV2 (r) ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA 86
*Where f  is forward and r is reverse
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3.4.4 Detection of Plasmodium falciparum  genetic mutations associated with 
antimalarial drugs resistance.
3.4.4.1 Chloroquine resistance associated genetic mutations
The same methods as described in 3.4.3.1.1 and 3.4.3.1.2 above were used to isolate P. 
falciparum  DNA from filter paper blood blot.
a) Pfcrt 76
The approach used by Cortese and Plowe (1999) for amplification of the DNA 
sequences of interest was used. With this approach, an initial PCR to amplify the region 
with the mutation, then two more PCRs, one to detect either mutant or wildtype and the 
second for the restriction analysis with Apol to confirm the presence or absence of 
mutation. For the details of primers sequences see Table 3.2.
The initial amplification reaction involved the use o f  the oligonucleotide primer pair, 
CRTP1 and CRTP2 to amplify a 537bp region around the mutation K.76T. The reaction 
volume of 25|il contained lx  PCR buffer (50mM KC1, 20mM Tris-HCl, pH 8.3, 
O.lmg/ml o f gelatin), 2.5mM MgC^, 200)^M of dNTP mix, 0.1 jiM o f each primer, 
0.625U o f Taq polymerase and 5|il of DNA template. It was overlaid with 20ul of 
mineral oil. PCR assay were carried out using a Hybaid thermal cycler (Hybaid, UK) 
with cycling parameters of an initial melt at 94°C for 3mins followed by 30 cycles of 
94°C for 30secs (denaturation), 56°C for 30secs (annealing), 72°C for lm in (extension) 
followed by a final extension o f72°C  for 3mins.
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The oligonucleotide primers pairs CRTP3 and CRTP4m were used to amplify mutant 
alleles and CRTP3 and CRTP4w for wildtype alleles, both with an expected size of 
366bp. The reaction volume of 25jal contained lx  PCR buffer (50mM KC1, 20mM Tris- 
HC1, pH 8.3, 0.1 mg/ml of gelatin), 1.5mM MgCh, 200pM of dNTP mix, 0.1 |jM o f each 
primer, 0.625U o f  Taq polymerase and 0.5jul of amplicon DNA. It was overlaid with 
20(j.l o f mineral oil. The cycling conditions were an initial melt at 94°C for 3mins 
followed by 25 cycles of 94°C for 30secs (denaturation), 50°C for 30secs (annealing), 
64°C for lm in (extension) and a final extension o f 64 °C for 3mins.
The confirmatory PCR used primers CRTD1 and CRTD2 to obtain a 134bp fragment for 
restriction digestion analysis. The reaction volume of 25p.l contained lx  PCR buffer 
(50mM KC1, 20mM Tris-HCl, pH 8.3, 0.1 mg/ml of gelatin), 2.5mM MgCl2, 200|iM of 
dNTP m ix, 0.1 pM o f  e ach p rimer, 0.625U o f  Taq polymerase and 0.5pl of amplicon 
DNA. The cycling c onditions w ere a n i nitial m elt a 19 5°C f  or 5 mins f  oil owed b y 2 5 
cycles of 92°C for 30secs, 48°C for 30secs, 65°C for 30secs and a final extension of 65 
°C for 3mins.
The restriction enzyme Apol was used and the digestion was performed in a 20pl 
volume, which contained 5|al of the PCR product, 2.5pl of 4U/|il enzyme (0.5U) and 
2.5pl of buffer. The mix was incubated at 50°C for 6 hours. Then the restriction 
products were run on 2% ethidium bromide agarose gel. The restriction site and the 
expected product sizes after digestion are shown in Table 3.3.
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b) Pfmdrl 86
The primary amplification reaction involved the use o f oligonucleotide primer pair, 
MDR-1 and MDR-2 to amplify a 603bp fragment which had the mutation at codon 86. 
The reaction volume of 25jul contained lx  PCR buffer (50mM KC1, 20mM Tris-HCl, pH 
8.3, O.lmg/ml of gelatin), 2.5mM MgCh, 200pM o f dNTP mix, l.OpM o f each primer, 
0.625U of Taq polymerase and 5pl of DNA template. The cycling parameters were an 
initial melt at 95°C for 3mins followed by 30cycles of 92°C for 30secs (denaturation), 
48°C for 45secs (annealing), 65°C for lmin (extension) and a final extension of 65°C 
for 5mins.
The secondary amplification reaction involved primer pair MDR-3 and MDR-4, which 
amplified a 521 bp, fragment if the mutation was present. A reaction volume o f2  5pl 
contained lx  PCR buffer (50mM KC1, 20mM Tris-HCl, pH 8.3, O.lmg/ml of gelatin), 
2.5mM MgCh, 200pM of dNTP mix, l.OpM of each primer, 0.625U of Taq polymerase 
and 0.5(j.l o f amplicon DNA. The cycling parameters were an initial melt at 95°C for 
3mins followed by 15 cycles of 92°C for 30secs (denaturation), 48°C for 30secs 
(annealing), 65°C for 45secs (extension) and a final extension o f 65°C for 5mins.
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Table 3.2
DNA sequences o f synthetic oligonucleotide primers for the detection of mutations 
associated with chloroquine resistance.
Gene loci Primer DNA Sequence (5’ -  3 ’)
Melting 
temp. 
(Tm C)
pfcrt 76 CRTP1 (0 CCG TTA ATA ATA AAT ACA CGC AG 62
CRTP2 (r) CGG ATG TTA CAA AAC TAT AGT TAC C 68
CRTP3 (f) TGA CGA GCG TTA TAG AG 50
CRTP4m (r) GTT CTT TTA GCA AAA ATT G 48
CRTP4w (r) GTT CTT TTA GCA AAA ATC T 48
CRTD1 (f) TGT GCT CAT GTG TTT AAA CTT 56
CRTD2 (r) CAA AAC TAT AGT TAC CAA TTT TG 58
pfmdrl 86 MDR-1 (f) ATG GGG TAA AGA GCA GAA AGA 60
MDR-2 (r) AAC GCA AGT AAT ACA TAA AGT CA 60
MDR-3 (f) TGG TAA CCT CAG TAT CAA AGA A 60
MDR-4 (r) ATA AAC CTA AAA AGG AAC TGG 56
Where f  is forward and r is reverse
Table 3.3
Recognition sites of restriction enzyme and product sizes for pfcrt 76
Product sizes (bp)
Restriction Before After
enzyme Recognition site digestion digestion Allele cut
Apol 5’ PujAATTPy 
3’ PyTTAAtPu
134 100 + 34 wildtype
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3.4.4.2 Fansidar resistance associated genetic mutations
a) Dhfr 51,59 and 108
Primary amplification reaction involved the use of oligonucleotide primer pair, AMP1 
and AMP2 to amplify a 720bp o f the dhfr coding region. The reaction volume of 25(j.l 
contained lx  PCR buffer (50mM KC1, 20mM Tris-HCl, pH 8.3, O.lmg/ml o f gelatin), 
3.5mM MgCh, 200^M of dNTP mix, l.Oj^M o f each primer, 0.625U of Taq polymerase 
and 5(il of DNA template. The cycling parameters were an initial melt at 95°C for 3mins 
followed by 30cycles of 92°C for 30secs, 45°C for 45secs, 72°C for 45secs and a final 
extension of 72°C for 3mins.
The PCR product obtained was used as the DNA template in two separate reactions 
using primer pair MUM-D and FR-51MB1 for mutant allele and MUM-D and FR- 
51WB1 for wildtype allele. Both have an expected size of 238bp. Each reaction volume 
of 25|il contained lx  PCR buffer (50mM KC1, 20mM Tris-HCl, pH 8.3, O.lmg/ml of 
gelatin), 2.5mM MgC^, 200(iM of dNTP mix, 0.5jaM of each primer, 0.625U o f Taq 
polymerase and 0.5|al of amplicon DNA. The cycling parameters were an initial melt at 
95°C for 3mins followed by 15 cycles of 92°C for 30secs, 54°C for 30secs, 72°C for 
lmin and a final extension of 72°C for 3min.
The second nested PCR involved using primer pairs SP1 and FR59m for the codon 59 
mutant allele and S PI and FR59w for wildtype allele in two separate reactions. Both 
have an expected size of 190bp. The reaction volume of 25|al contained lx  PCR buffer 
(50mM KC1, 20mM Tris-HCl, pH 8.3, O.lmg/ml of gelatin), 1.5mM MgCl2, 200|jM of
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dNTP m ix, 0 ,5jiM o f  each primer, 0 .625U o f  Taq polymerase and 0.5pl o f amplicon 
DNA. The cycling parameters were an initial melt at 95°C for 3mins followed by 15 
cycles o f 92°C for 30secs, 54°C for 30secs, 72°C for 30secs and a final extension of 
72°C for 3min.
The third secondary amplification reaction involved primer pairs SPI and DIA-12 and 
SPI and D1A-9 for the two codon 108 mutant alleles and SPI and DIA-3 for wildtype 
allele in three separate reactions. All three have an expected size o f  337bp. Each reaction 
volume of 25p.l contained lx  PCR buffer (50mM KC1, 20mM Tris-HCl, pH 8.3, 
O.lmg/ml of gelatin), 1.5mM MgCl2, 200pM o f dNTP mix, 0.5jaM o f each primer, 
0.625U of Taq polymerase and 0.5|il o f amplicon DNA. The cycling parameters were an 
initial melt at 95°C for 3mins followed by 15 cycles o f 92°C for 30secs, 55°C for 
45secs, 72°C for 45secs and a final extension of 72°C for 3min.
b) Dhps 437 and 540
The primary amplification reaction involved the use of oligonucleotide primer pair, 
DHPS-1 and DHPS-2 to amplify a 1328bp of the dhps coding region. The reaction 
volume o f 25pl contained lx  PCR buffer (50mM KC1, 20mM Tris-HCl, pH 8.3, 
O.lmg/ml of gelatin), 3.5mM MgCl2, 200|iM of dNTP mix, l.OpM of each primer, 
0.625U o f Taq polymerase and 5pl o f DNA template. The cycling parameters were an 
initial melt at 95°C for 3mins followed by 30cycles of 92°C for 30secs, 45°C for 
45secs, 65°C for lmin and a final extension of 65°C for 3mins.
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The first nested PCR involved primer pairs 185S and 437M-A for the codon 437 mutant 
allele and 185S and 437W-2C for wildtype in two separate reactions. Both have an 
expected size o f 333bp. Each reaction volume of 25^ x1 contained lx  PCR buffer (50mM 
KC1, 20mM Tris-HCl, pH 8.3, O.lmg/ml o f gelatin), 2.5mM MgCb, 200|iM o f dNTP 
mix, 0.5(^M o f  each primer, 0.625U o f  Taq polymerase and 0.5jj.1 o f amplicon DNA. 
The cycling parameters were an initial melt at 95°C for 3mins followed by 30 cycles of 
92°C for 30secs, 48°C for 45secs, 65°C for lm in and a final extension of 65°C fo r 
3min.
The second nested PCR involved primer pairs 185S and 540M for the codon 540 mutant 
allele and 540W and 218A for the wildtype in two separate reactions. Expected sizes for 
the wildtype and the mutant are 636bp and 561bp respectively. Each reaction volume of 
25ju.l contains lx  PCR buffer (50mM KC1, 20mM Tris-HCl, pH 8.3, O.lmg/ml of 
gelatin), 2.5mM MgCU, 200|aM of dNTP mix, 0.5|uM of each primer, 0.625U of Taq 
polymerase and 0.5|_il o f amplicon DNA. The cycling parameters were an initial melt at 
95°C for 3mins followed by 30 cycles of 92°C for 30secs, 52°C for 45secs, 72°C for 
lmin and a final extension of 72°C for 3min.
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DNA sequences o f the synthetic oligonucleotide primers for the detection of mutations
Table 3.4
associated with Fansidar resistance.
Gene
locus Primer DNA Sequence (5’ -  3’)
Melting 
temp. 
(Tm C)
dhfr AMP1 (f) TTT ATA TTT TCT CCT TTT TA 40
AMP2 (r) CAT TTT ATT ATT CGT TTT CT 48
51 MUM-D (f) TTT ATC CTA TTG CTT AAA GGT TTA 60
FR-51MB1 (r) GGA GTA TTA CCA TGG AAA TGT CT 64
FR-51WB1 (r) GGA GTA TTA CCA TGG AAA TGT CA 64
59 SPI (f) ATG ATG GAA CAA GTC TGC GAC 62
FR59M (r) ATG TTG TAA CTG CAC G 46
FR59W (r) ATG TTG TAA CTG CAC A 44
108 SPI (f) ATG ATG GAA CAA GTC TGC GAC 62
DIA-12 (r) GAA TGC TTT CCC AGT 44
DIA-9 (r) GAA TGC TTT CCC AGG 46
DIA-3 (r) GAA TGC TTT CCC AGC 46
dhps DHPS-1 (f) TTT TAG AGA TCC ACA AGA 48
DHPS-2 (r) TTA AAA CAT CCA AAA CC 44
437 185S (f) TGA TAC CCG AAT ATA AGC ATA ATG 64
437M-A (r) TTT GGA TTA TGG TAT AAC AAA AGT CC 68
437W-2C (r) TTT GGA TTA TGG TAT AAC AAA AGT CG 68
540 185S (f) TGA TAC CCG AAT ATA AGC ATA ATG 64
540M (r) CTA GAT TAT CAT AAT TTG TTA GTA C 62
540W (f) GGA AAT CCA CAT ACA ATG GAA A 60
218 A (r) ATA ATA GCT GTA GGA AGC AAT TG 62
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CHAPTER 4
RESULTS
4.1 Study Population
A total of 199 patients comprising 103 from Hohoe and 96 from Navrongo were selected 
for this study. The details o f the demographic information, parasitaemia levels during 
pre and post treatment, and PCR data for each patient are given in Appendix V.
The 103 patients studied at Hohoe comprised of 48 (46.6%) males and 55 (53.3%) 
females. The mean age of the study population was 27.5 months with a standard 
deviation of 14.8 (range 6-59 months) and figure 4.1 shows the age distribution. Thirty- 
nine patients (37.8%) responded adequately to treatment with chloroquine whilst the 
non-respondents were 64 (62.1%). On the basis of parasitologic clearance, out of the 64 
non-respondents 35 (55%) were class I (RI) resistant, 21 (33%) were RII and the rest 8 
(13%) were RIII (Table 4.1).
O f the 96 patients studied at Navrongo, 52 (54%) were males and 44 (46%) were 
females. The mean age o f the study population was 28.7 months with a standard 
deviation o f 15.0 (range: 6-59 months) and figure 4.2 shows the age distribution. Sixty- 
nine (69%) patients responded adequately to chloroquine whilst treatment failed in 30 
(31%) of them. O f these 30 non-respondents, 13 (43%) were RI, 10 (33%) were RII and 
7 (23%) were RIII (Table 4.1).
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4.2 Parasitaemic Profile of the Study Population
The pre-treatment parasitaemic profile o f  respondents and non-respondents at the two 
sites are shown in Table 4.2. The geometric mean parasitaemia o f the Hohoe patients 
was 15,348parasites/|al o f blood. The majority (56%) o f the patients with adequate 
treatment response had pre-treatment parasitaemia below 10,000 parasites/^1 whilst 
majority (64%) of the non-respondents had levels higher than 10,000 parasites/pl. There 
was no significant difference in the mean parasitaemia in the respondents and non­
respondents groups (p = 0.44).
For Navrongo, the geometric mean parasitaemia was 22,089 parasites/pl with the 
majority (70%) of respondents having levels above 10,000 parasites/pl. Eighty percent 
of the non-respondents had pre-treatment parasitaemia above 10,000 parasites/|il. There 
was no significant difference between the mean parasitaemia in the respondents and 
non-respondents groups (p = 0.52).
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Table 4.1
Distribution of sexes and treatment outcome of the study sites
Hohoe Navrongo
No. of males 48 52
No. of females 55 44
No. of patients sensitive to chloroquine 39 66
No. of patients with the classes of resistance
RI 35 13
RII 21 10
Rm 8 7
Table 4.2
Parasitaemic profiles of patients before treatment with chloroquine
Number of patients
Parasitaemia Hohoe (n = 103) Navrongo(n = 96)
(parasite/^!) Respondents Non-respondents Respondents Non-respondents
<2000 0(0) 1 (0.97) 2(2.1) 0(0)
2001-5000 16(15.5) 17(16.5) 10(10.4) 3(3.1)
5001-10,000 6 (5.8) 6(5.8) 8(8.3) 3(3.1)
10,001-20,000 7 (6.7) 7 (6.7) 10(10.4) 7(7.3)
20,001-50,000 4(3.8) 11 (10.4) 19 (20.0) 7 (7.3)
50,001-100,000 4(3.8) 11 (10.4) 13 13.5) 6(6.3)
>100,001 2(1.9) 12(11.6) 4 (4.2) 4 (4.2)
Percentages are in brackets.
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6-11. 12-23. 24-35 
Age (months)
36-47 48-60
Figure 4.1 Age distribution of patients recruited for the study
at Hohoe
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Pr
op
or
tio
n 
of 
pa
tie
nt
s 
(%
)
35
30 -
25 -
20  -
15 -
10 -
M m .
K v : .
6-11. 12-23. 24-35 36-47
Age (months)
48-60
Figure 4.2 Age distribution of patients recruited for the 
study at Navrongo
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4.3 In vitro Chloroquine Sensitivity Studies
A total o f 26 isolates were successfully tested at the two sites (Appendix V). Eight o f 
these were from Hohoe and 18 from Navrongo. Mean percentage inhibition o f growth of 
P. falciparum  isolates at chloroquine concentrations of 1, 2, 4, 8, 16, 32 and 64 pmol 
respectively for the two sites are shown in Table 4.3. The IC50 value determined was 
1.5xl0"6 mol/litre for the 26 isolates.
Out o f the 8 isolates from Hohoe, 7 (86%) were sensitive to chloroquine and one showed 
resistance even at concentrations above 8.0 pmol. Overall, the mean percentage 
inhibition at 8pmol was 89.1%, which indicate a high level of sensitivity of P. 
falciparum  to chloroquine in the area.
For Navrongo, 12 (67%) o f the 18 isolates tested were sensitive to chloroquine whilst 6 
(33%) were observed to be resistant. The mean inhibition at 8pmol was 67.8%.
74
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Table 4.3.
Chloroquine inhibition levels of Plasmodium falciparum isolates from Hohoe and 
Navrongo.
No. of
Mean percentage inhibition at concentrations of 
chloroquine (pmol)
Site patients 1 2 4 8 16 32 64
Hohoe 8 15 29.6 36.9 89.1 100 100 100
Navrongo 18 7.6 12.6 24.6 67.8 72.9 87.4 100
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4.4 PCR Detection of Plasmodium Species
The results obtained demonstrated that PCR quality grade DNA was obtained using the 
two rapid DNA extraction methods. In all, parasite DNA was extracted from 385 filter 
paper blood blot samples. The nested PCR amplifications for detection and identification 
were all successful. The diagnostic sizes of the PCR products were 205bp for P. 
falciparum  and 144bp for P. malariae.
At Hohoe, P. falciparum  was identified as single infections in 98 (95.2%) o f the patients 
and as mixed infection with P. malariae in 5 (4.8%). Neither of the other two human 
parasites, P. ovale and P. vivax was found.
At Navrongo, P. falciparum  was identified as single infections in 86 (89.6%) o f the 
patients and as mixed infection with P. malariae in 10 (10.4%). No P. ovale and P. vivax 
were found.
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4.5 Distribution of P. falciparum  Genotypes Associated with Chloroquine 
Resistance
The PCR based assay o f P. falciparum  genotypes, pfcrt and pfmdrl were all successful. 
A total of 2865 individual PCR assays were performed.
4.5.1 Pfcrt 76
At Hohoe, the baseline prevalence (Day 0) o f the pfcrt T76 within the patients was 
82.5% (85/103). The details of how the mutation was distributed within respondents and 
non-respondents are shown in Table 4.4. O f the 39 respondents, 5 had the mutant allele 
only, 12 had the wildtype only and 22 had both alleles, whilst for the 64 non­
respondents, 31 had the mutant only, 6 had the wildtype only and 27 had both alleles. 
The distribution of the mutation in the Day 0 specimens o f the different non-respondent 
categories RI, RII and RIII were 86% (30/35), 95% (20/21) and 100% (8/8) respectively. 
The prevalence o f the mutation in the 46 non-respondents whose parasitaemia had not 
cleared from Day 7 post-treatment was 100% (46/46); 43 had the mutant only and 3 had 
mixed alleles. The allelic frequencies of pfcrt 76 at Day 0, 7 and 14 samples o f the non­
respondents are shown in Figure 4.3. The sensitivity and specificity of the detection of 
resistance were 71% and 84% respectively. The presence o f pfcrt T76 was found to be 
strongly associated with the development of chloroquine resistance (odds ratio = 12.40,
p = 0.0001).
At Navrongo, the baseline prevalence of the pfcrt T76 mutation within the patients was 
43.8% (46/96). The distribution of the mutation within respondents and non-respondents 
are shown in Table 4.4. Of the 66 respondents, 24 had the mutant only, 37 had the
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wildtype only and 4 had both alleles, whilst for the 30 non-respondents 12 had the 
mutant only, 16 had the wildtype only and 2 had both alleles. The distribution o f the 
mutation among the different categories of the non-respondents RI, RII and RIII at Day 
0 were 69% (9/13), 33% (3/10) and 29% (2/7) respectively. The prevalence of the 
mutation in the 28 non-respondents whose parasitaemia had not cleared from Day 7 
post-treatment was 75% (21/28); 20 had the mutant only and one had mixed alleles. 
None of the alleles was detected in the remaining 7 (i.e. PCR negative). The allelic 
frequencies of pfcrt 76 in Day 0, 7 and 14 specimens o f the non-respondents are shown 
in Figure 4.4. The sensitivity and specificity o f detection o f resistance was 61% and 57% 
respectively. There was no association between the presence o f the mutation and 
treatment outcome (odds ratio = 1.16, p = 0.75).
4.5.2 Pfmdrl 86
At Hohoe, the baseline prevalence of the pfmdrl Y86 among patients was 821fl®/o^) 
(84/103). Details on the distribution of the mutation within respondents and non­
respondents are shown in Table 4.5. O f the 39 respondents, 30 had the mutant and 9 had 
the wildtype, whilst for the 64 non-respondents, 57 had the mutant allele and 7 had the 
wildtype allele. The distribution of the mutation among the different categories o f the 
non-respondents RI, RII and RIII at Day 0 were 83% (29/35), 95% (20/21) and 100 (8/8) 
respectively. The prevalence of the mutation in the 46 non-respondents whose 
parasitaemia had not cleared from Day 7 post-treatment was 100% (46/46). The allelic 
frequencies o f pfmdrl 86 in Days 0, 7 and 14 samples of the non-respondents are shown 
in Figure 4.5. The sensitivity and specificity of detection o f resistance was 89.1% and
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At Navrongo, the baseline prevalence o f  pfmdrl Y86 was 61.5% (59/96). O f the 66 
respondents, 3 8 had  the mutant only and 28 had the wildtype allele whilst for the 30 
non-respondents, 21 had the mutant and 9 had the wildtype. The distribution of the 
mutation among the three non-respondent categories, RI, R II and RHI at Day 0 were 
69% (9/13), 60% (6/10) and 86% (6/7) respectively. The prevalence of the mutation in 
the 28 non-respondents whose parasitaemia had not cleared from Day 7 post-treatment 
was 68% (19/28). None of the alleles was detected in the remaining 9 (i.e. PCR 
negative). The allelic frequencies of pfmdrl 86 in Days 0, 7 and 14 samples of the non­
respondents are shown in Figure 4.6. The sensitivity and specificity o f detection of the 
mutation were 70.0% and 42.4% respectively. The association between the presence of 
mutation and treatment outcome was not significant (odds ratio = 1.72, p = 0.25).
4.5.3 Double mutations (both pfcrt T76 and pfmdrl Y86)
At Hohoe, the baseline prevalence o f the double mutations within the patients was 
73.8% (76/103). The details of the distribution of the mutation are shown in Table 4.6. 
O f the 39 respondents, 56% (22/39) had both mutations whilst for the 64 non­
respondents 84% (54/64) had both mutations. The distribution o f both mutations in the 
Day 0 specimen of the different non-respondent categories RI, RII and RIII were 83% 
(29/35), 95% (20/21) and 100% (8/8) respectively. The prevalence of both mutations in 
the 46 non-respondents whose parasitaemia had not cleared from Day 7 post-treatment 
was 61% (28/46). The remaining 18 had either pfcrt T76 or pfmdrl Y86 but not both.
33.0% respectively. The association between the mutation and treatment outcome was
significant (odds ratio = 3.62, p = 0.01).
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At Navrongo, the baseline prevalence o f the double mutations within the patients was 
36.5% (35/96). O f the 66 respondents, 36% (24/66) had both mutations whilst for the 30 
non-respondents, 37% (11/30) had both mutations. The distribution o f both mutations in 
the Day 0 specimen among the different non-respondent categories RI, RH and RHI 
were 69% (9/13), 33% (3/10) and 29% (2/7) respectively The prevalence o f both 
mutations in the 2 8 non-respondents w hose p arasitaemia h ad n ot c leared f  rom D ay 7 
post-treatment was 39.3% (11/28). O f the remaining 17, 10 had either pfcrt T76 or 
pfmdrl Y86 and none of the mutations was detected in 7 samples (i.e. PCR negative). 
There was no association between clinical outcome and the presence o f both mutations 
(odds ratio = 1.01, p = 0.98).
There was a strong association between treatment outcome and the presence of both
mutations (odds ratio = 4.17, p = 0.002).
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Table 4.4
Prevalence of pfcrt codon 76 alleles in patients at pre-treatment from Hohoe and 
Navrongo
Prevalence of pfcrt 76 alleles (%)
Hohoe Navrongo
Alleles
Respondents 
(n = 39)
Non-respondents 
(n = 64)
Respondents Non-respondents 
(n = 66) (n = 30)
Mutant only 12.8 48.4 36.4 40.0
Wildtype only 30.8 9.4 56.1 53.3
Mixed 56.4 42.2 6.1 6.7
Table 4.5
Prevalence of pfmdrl codon 86 alleles in patients at pre-treatment from Hohoe and
Navrongo
Prevalence of pfmdrl 86 alleles (%)
Hohoe Navrongo
Alleles
Respondents Non-respondents 
(n = 39) (n = 64)
Respondents Non-respondents 
(n = 66) (30)
Mutant 69.2 89.1 57.6 70.0
Wildtype 30.8 10.9 42.4 30.0
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Table 4.6
Prevalence of double mutations, pfcrtT16 and pfmdr 1Y86, at pre and post-treatment in
patients from Hohoe and Navrongo.
Prevalence of double mutations {pfcrt T76 + pfmdrl Y86)/%
Respondents Non-respondents
Site Day 0 Day 0 Day 7 Day 14
Hohoe 54.0 84.0 9.0 61.0
Navrongo 36.0 37.0 39.3 32.0
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Fr
eq
ue
nc
y 
(%
)
90
80 -
70
60
50 -
40 -
30
20  -
10
mutant wildtype
Alleles
mixed
Figure 4.3 Allelic frequencies of pfcrt 76 of non-respondents 
from Hohoe at pre and post-treatment
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Fr
eq
ue
nc
y 
(%
)
60 -|
50 -I
mutant wildtype mixed
Alleles
Figure 4.4 Allelic frequencies of pfcrt 76 of non-respondents 
from Navrongo at pre and post-treatment
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Pr
ev
al
en
ce
 
(%
)
100
Figure 4.5 Prevalence of pfmdrl 86 mutation of non­
respondents from Hohoe at pre and post-treatment
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Day
0 7 14
Figure 4.6 Prevalence of pfmdrl 86 mutation of non­
respondents from Navrongo at pre and post-treatment
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4.6 Distribution of P. falciparum  Genotypes Associated with Fansidar Resistance
The PCR based assay o f P. falciparum  genotypes, dhfr and dhps were all successful. In 
all, 4450 individual PCR assays were performed. It is worth noting that all chloroquine 
non-respondents who were given Fansidar showed adequate treatment response.
4.6.1 Dhfr 51
At Hohoe, the baseline prevalence of the mutations at codon 51 within the patients was 
44.6% (46/103). The details o f the distribution o f alleles of this codon within the malaria 
patients are shown in Table 4.7. In all, 40% (41/103) of the samples analysed were PCR 
negative (no allele detected by PCR). O f the 14 patients given Fansidar, 8 had mixed 
alleles, 1 had the mutant allele and 5 were PCR negative at Day 0. At post-treatment, 2 
of them had mixed alleles, 4 had the mutant only and 2 had the wildtype only.
At Navrongo, the baseline prevalence o f the mutation within the patients was 6.3% 
(6/96). The details o f the distribution of the alleles of codon 51 are shown in Table 4.7. 
Sixty-five percent (62/96) of the samples analysed were PCR negative. Three patients 
were given Fansidar and at pre-treatment, 1 had the wildtype only and the 2 were PCR 
negative. At post-treatment, it was as above.
4.6.2 Dhfr 59
The baseline prevalence of the mutation at codon 59 within the patients at Hohoe was 
57.3% (59/103). The details of the distribution of the alleles of this codon are shown in 
Table 4.8. In all 35% (36/103) were PCR negative. O f the 14 patients given Fansidar, at 
pre-treatment, 8 had mixed alleles, 4 had the mutant allele and 2 were PCR negative. At
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post-treatment, 1 had mixed alleles, 2 had the mutant only and 2 had the wildtype.
At Navrongo, the baseline prevalence of the mutation at codon 59 was 28% (27/96). The 
details o f the distribution of the alleles o f this codon are shown in Table 4.8. Sixty-nine 
percent (66/96) o f the samples were PCR negative. O f the 3 patients given Fansidar, at 
pre-treatment, 1 had the mutant only and the 2 were PCR negative. At post-treatment, it 
was the same.
4.6.3 Dhfr 108
At Hohoe, the baseline prevalence of the mutation at codon 108 within the patients was 
66% (68/103). The details o f the distribution of the alleles of the codon 108 are shown in 
Table 4.9. O f the 103 samples analysed, 28.2% (29/103) were PCR negative. O f the 14 
patients given Fansidar, at pre-treatment, 9 had mixed alleles, 2 had the mutant allele 
and 3 were PCR negative. At post-treatment, 1 had mixed alleles and 4 had the mutant 
only and 2 had the wildtype only.
At Navrongo, the baseline prevalence of the mutation was 31.3% (30/96). Details of the 
distribution of alleles are shown in Table 4.9. O f the 96 samples analysed, 59.4% 
(57/96) were PCR negative. O f the 3 patients given Fansidar, at pre-treatment, 1 had the 
mutant only and the 2 were PCR negative. At post-treatment, 1 had the wildtype and 1 
had the mutant only.
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4.6.4 Triple mutation (dhfr 51, 59 and 108)
At Hohoe, the baseline prevalence o f the presence of all three mutations in the dhfr 
region within the patients was 33% (34/103) [Table 4.10]. O f the 14 patients given 
Fansidar, at pre-treatment, 12 had the triple mutations. At post-treatment, 3 had the triple 
mutations.
At Navrongo, the baseline prevalence was 5.2% (5/96) within the patients. O f the 3 
patients given Fansidar, at pre-treatment, none had the triple mutations.
4.6.5 Dhps 437
The baseline prevalence of the mutation at codon 437 within the patients at Hohoe was 
76% (78/103). The details o f the distribution of the alleles are shown in Table 4.11. O f 
the 103 samples analysed 18.5% (19/103) were PCR negative. O f the 14 patients given 
Fansidar, at pre-treatment, 4 had mixed alleles, 6 had the mutant allele, 1 had the 
wildtype and 3 were PCR negative. At post-treatment, 1 had mixed alleles, 2 had the 
mutant only, 7 had the wildtype only.
For Navrongo, the baseline prevalence of the mutation was 9.4% (9/96). Details o f the 
distribution o f alleles are shown I Table 4.11. About 72% o f the samples were PCR 
negative. O f the 3 patients given Fansidar, at pre-treatment, 1 had the wildtype only and 
the 2 were PCR negative. At post-treatment, none of them had any o f the alleles.
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4.6.6 Dhps 540
At Hohoe, the baseline prevalence of the mutation at codon 540 within the patients was 
53.4% (55/103). The details o f the distribution of the alleles are shown in Table 4.12. 
Twenty-two percent (23/103) were PCR negative. O f the 14 patients given Fansidar, at 
pre-treatment, 9 had mixed alleles, 1 had the wildtype allele and 4 were PCR negative. 
At post-treatment, 5 had mixed alleles, 3 had the mutant only, 2 had the wildtype only.
At Navrongo, the baseline prevalence of the mutation within the patients was 
6.3%(6/96). Details o f the distribution of alleles are shown in Table 4.12. Seventy-two 
percent (69/96) were PCR negative. O f the 3 patients who were given Fansidar, none of 
them had any of the codon 540 alleles at both pre and post-treatment.
4.6.7 Double mutations (dhps 437 and 540)
At Hohoe, the baseline prevalence of patients with both codons 437 and 540 mutations 
was 51.5% (53/103) [Table 4.13], O f the 14 patients given Fansidar, at pre-treatment, 8 
had the double mutations. At post-treatment, 3 o f them had the double mutations.
For Navrongo the baseline prevalence was 3.1% (3/96). All the 3 patients who were 
given Fansidar did not have both mutations at both pre and post-treatment.
4.6.8 Quintuple mutations (dhfr 51, 59, 108, dhps 437 and 540)
At Hohoe, the baseline prevalence o f  patients w ith all five mutations a ssociated w ith 
Fansidar resistance was 31.1% (32/103)[Table 4.14], O f the 14 patients given Fansidar, 
at pre-treatment, 8 had the quintuple mutations. At post-treatment, 1 had the quintuple
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mutations
For Navrongo, the baseline prevalence was 1.04% (1/96) [Table 4.14]. None of the 3 
patients given Fansidar had the quintuple mutations either at pre or post-treatment.
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Table 4.7
Baseline prevalence of alleles of the dhfr codon 51 in patients at Hohoe and Navrongo
Alleles
Prevalence of alleles of dhfr  51/%
Hohoe (103) Navrongo (96)
Mutant only 12.0 2.0
Wildtype only 15.5 29.0
Mixed alleles 33.0 4.0
Table 4.8
Baseline prevalence of alleles of the dhfr codon 59 in patients at Hohoe and Navrongo
Prevalence of alleles of dhfr  59/%
Alleles Hohoe Navrongo
Mutant only 24.3 19.8
Wildtype only 7.7 3.0
Mixed alleles 33.0 8.3
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Table 4.9
Baseline prevalence of alleles of the dhfr codon 108 in patients at Hohoe and Navrongo
Prevalence of alleles of dhfr 108/%
Alleles Hohoe Navrongo
Mutant only 18.4 7.3
Wildtype only 5.8 9.4
Mixed alleles 47.6 24.0
Table 4.10
Baseline prevalence of triple mutations of dhfr (51 + 59 + 108) in patients at Hohoe and
Navrongo.
Site Prevalence of triple mutations (dhfr 59, 59 and 108)/%
Hohoe 33.0
Navrongo 5.2
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Table 4.11
Baseline prevalence of alleles of the dhps codon 437 in patients at Hohoe and Navrongo
Prevalence of alleles of dhps 437
Alleles Hohoe Navrongo
Mutant only 38.8 0
Wildtype only 5.8 18.8
Mixed alleles 36.9 9.4
Table 4.12
Baseline prevalence of alleles of the dhps codon 540 ini patients at Hohoe and Navrongo
Prevalence of alleles of dhps 540
Alleles Hohoe Navrongo
Mutant only 1.0 0
Wildtype only 24.3 21.0
Mixed alleles 52.4 6.3
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Table 4.13
Baseline prevalence of double mutations of dhps (437 + 540) in patients at Hohoe and
Navrongo
Site Prevalence of double mutations (dhps 437 and 540)
Hohoe 51.5
Navrongo 3.1
Table 4.14
Baseline prevalence of quintuple mutations (dhfr 51 + 59 + 108 + dhps 437 + 540) in 
patients at Hohoe and Navrongo
Prevalence of quintuple mutations (dhfr  59, 59,108,
Site dhps 437 and 540)
Hohoe 31.1
Navrongo 1.04
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4.7 Comparison between Molecular Analysis, In vivo and In  vitro Outcomes
Generally, the results of the in-vivo studies correlated with that o f the in vitro at Hohoe 
(Table 4.15). One o f the isolates, which was sensitive in-vivo showed resistance in the in 
vitro testing. The presence o f pfcrt T76 and pfmdrl Y86 were observed in pre-treatment 
samples o f both in vivo sensitive and resistant infections and also in the post-treatment 
samples of the resistant infections.
For Navrongo, the outcomes of both in-vivo and in vitro analysis were generally similar 
(Table 4.15). However, all RII infections were sensitive in-vitro and 3 o f the sensitive 
infections in vivo were resistant in vitro. For all RIII infections, there was growth of the 
parasite in vitro even at concentrations of 8-32pmol but were totally inhibited at 64pmol. 
Pfcrt T76 was not detected in any o f the pre-treatment samples analysed but was 
detected in p ost-treatment samples of the in-vivo c hloroquine r esistant infections. F or 
pfmdrl Y86, there was detection in 15 of the 18 samples and in all resistant infections.
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Table 4.15
Outcomes of in-vivo and in-vitro analysis for the selected samples from Hohoe and 
Navrongo
In-vitro  outcome
lit vivo Hohoe Navrongo
outcome S R s R
Sensitive 6 1 9 3
Resistant 1 0 3 3
Where S and R are sensitive and resistant respectively
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1 2  3 M 4 5 6 7 M 8 9 10 11 M 12 13 14
Fig 4.7 Agarose gel electrophoresis of PCR products obtained from the nested 
amplification o f alleles of pfcrt codon 76. Lanes 1-3 = primary amplification using 
primers CRTP1 and CRTP2, Lanes M = lOObp molecular weight marker, Lanes 4-6 = 
secondary amplification using primers CRTP3 and CRTP4m, Lanes 8-10 = secondary 
amplification using CRTP3 and CRTP4w, Lanes 12-14 = secondary amplification with 
primers CRTD1 and CRTD2, Lanes 7 and 11 are negative controls.
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1 2 M 3 4 5 6
603bp —► -4—521bp
Fig 4.8 Agarose gel electrophoresis of PCR products obtained from the nested 
amplification of alleles of pfmdrl codon 86. Lanes 1-2 = primary amplification using 
primers MDR-1 and MDR-2, Lane M = lOObp molecular weight marker, Lanes 3-6 = 
secondary amplification with primers MDR-3 and MDR-4.
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1 2 m  3 4 5 6 7 8 9 10 11 12 13 14 M
Fig 4.9 Agarose gel electrophoresis o f PCR products obtained from the nested 
amplification of alleles of dhfr codon 108. Lanes 1-2 = primary amplification using 
primers AMP1 and AMP2, Lanes M = lOObp molecular weight marker, Lanes 3-5 = 
secondary amplification with primers SP1 and DIA-12, Lanes 7-9 = secondary 
amplification with primers SP1 and DIA-9, Lanes 11-13 = secondary amplification with 
primers SP1 and DIA-3, Lanes 6,10 and 14 are negative controls.
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1 2 3 4  5 6 7 8 M 9 10 11 12
Fig 4.10 Agarose gel electrophoresis o f PCR products obtained from the nested 
amplification o f alleles of dhfr codons 51 and 59. Lanes 1-3 = secondary amplification 
for codon 51 using primers MUM-D and FR-51MB1, Lanes 5-8 = secondary 
amplification for codon 51 using primers MUM-D and FR-51WB1, Lane M = lOObp 
molecular weight marker, Lanes 9-10 = secondary amplification for codon 59 with 
primers SP1 and FR59m, Lanes 11-12 = secondary amplification for codon 59 with 
primers SP1 and FR59w, Lane 4 is a negative control.
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1 2 3 4 5 6 7 M 8 9 10 11 M 12 13 14
Fig 4.11 Agarose gel electrophoresis o f PCR products from the nested amplification of 
alleles of dhps codons 437 and 540. Lanes 1-3 = secondary amplification for codon 437 
using primers 185S and 437M-A, Lanes 4-6 secondary amplification for codon 437 
using primers 185S and 437W-2C, Lanes M = lOObp molecular weight marker. Lanes 8- 
10 = secondary amplification for codon 540 using primers 218A and 540W-S, Lanes 12- 
14 = secondary amplification for codon 540 with primers 185S and 540M-A, Lanes 7 
and 11 are negative controls.
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CHAPTER 5
DISCUSSION AND CONCLUSION
This study investigated the species o f  Plasmodium  responsible fo r malaria in patients 
and compared the in-vivo and in-vitro classification of resistance in 26 isolates at two 
selected sentinel sites in Ghana. Also, the relationship between responses to the 
antimalarial drugs and the presence of mutations at several gene loci, pfcrt and pfmdrl 
for chloroquine as well as pfdhfr and pfdhps for Fansidar in P. falciparum  were 
investigated.
The results obtained revealed that the majority of the malaria cases (94.2% and 89.6% 
for Hohoe and Navrongo respectively) were caused by P. falciparum. Mixed infections 
of P. falciparum  and P. malariae were also recorded, occurring in 4.8% and 10.4% at 
Hohoe and Navrongo respectively. The findings are similar to those o f previous studies 
in Ghana which found that about 95% of all infections in the country is caused by P. 
falciparum  and the rest by P. malariae and P. ovale in that order o f importance (Ahmed, 
1989; Binka et al., 1994). Surprisingly, no significant differences were found between 
the parasitaemia of respondents and non-respondents at the study sites (p = 0.44 and 
0.51 for Hohoe and Navrongo respectively).
Results from this study suggest that the presence of P. falciparum pfcrt 76 genotypes can 
be used to predict chloroquine resistance levels at some (Hohoe) but not all sites 
(Navrongo) in the country. A high frequency of the pfcrt mutant gene (82%) was 
observed in Hohoe, which was reflected in a high rate (62.1%) o f chloroquine treatment
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failure. The relatively higher frequency of the mutation as compared to that observed in 
the in vivo outcome of treatment supports the findings of Djimde el al. (2001) that 
clinical failure to chloroquine occurs at rates lower than the frequency o f pfcrt T76 
allele.
A significant association was found between treatment outcome and the presence of 
pfcrt T76 (odds ratio 12.40, p-value 0.0001) in Hohoe and moreover, all the non­
respondents were found to be infected with pfcrt T76 parasites at post-treatment. The 
observations made in Hohoe are consistent with findings in Mali, Mauritania, Sudan and 
Cameroon, where the high prevalence o f the resistant genotypes was associated with 
treatment failures (Djimde et al., 2001; Jelinek et al., 2001; Babiker et al., 2001; Basco 
and Ringwald, 2001), which lends support to the belief that T76 plays an essential role 
in chloroquine resistance.
However, 27 patients carrying mutant parasites responded satisfactorily to chloroquine. 
Of these, 5 patients had the mutant only and 22 had mixed alleles of pfcrt 76, which is 
almost 69% (27/39) of the total number of respondents. A similar observation was also 
made in the Mali study where Djimde et al. (2001) found 37% (40/107) of the 
respondents having the mutant allele. The possible explanations for this observation 
could be that, mutant parasites as compared to the wildtype parasites were few in 
numbers in these patients and therefore were taken care of by the host’s enhanced 
immunity. However, it is difficult to explain away the 5 cases which had only the mutant 
parasites at pre-treatment, though it is likely that other factors including low 
parasitaemia and host immunity played a role in their elimination. Another interesting
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observation was that pfcrt T76 was not detected in 5 (4.8%) cases at pre-treatment at 
Hohoe but was found in post-treatment samples. Here also, three possible explanations 
could have accounted for this. Firstly, the resistant parasites could have been in the liver 
or sequestrated in deep tissues during pre-treatment and have then emerged into the 
blood stream after treatment. Secondly, it could have been due to reinfection. However, 
reinfection is unlikely by day 14 because the incubation period o f the parasite and the 
time taken to appear in the blood after an infective bite is averagely about 12 days 
(White, 1996). Thirdly, it could have been due to mixed infections consisting mainly of 
sensitive p arasites with a very small population of resistant parasites, which were not 
detected by PCR since this method is known to be poor at detecting minority alleles (at 
<0.1%) [Ranford Cartwright, 2001]. With the administration o f chloroquine, the 
sensitive parasites were cleared whilst the resistant parasite population persisted 
resulting in treatment failure and subsequent detection of the mutant parasites in the 
post-treatment samples. The detection of low levels mutant parasites can only be 
achieved by employing quantitative PCR, which is very expensive to undertake and 
therefore was not done.
With regards to the other chloroquine resistant gene pfmdr\, the prevalence of the 
mutation among the Hohoe patients was 82% and a positive association was found 
between the pfmdr\ Y86 and chloroquine treatment outcome (odds ratio 3.62; p-value 
0.01). Moreover, the mutant parasites were persistent in all the non-respondents at post­
treatment. Similar findings have been made by researchers in Mauritania, Sudan and 
Kenya (Jelinek et al., 2001; IAEA, 2001).
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The presence of both mutant forms o f the pfcrt 76 and pfmdrl 86 were observed in 74% 
of the patients. Both mutations were found to be strongly associated with treatment 
failure in Hohoe (odds ratio 4.17, p-value 0.001) and similar findings have been reported 
by Jelinek et al. (2001) in Mauritania. Since pfmdrl and pfcrt are on different 
chromosomes, their selection cannot be attributed to physical linkage. Rather it could be 
that pfmdrl confers some advantage to the parasite in the presence o f chloroquine by 
augmenting the level o f resistance due to pfcrt mutation (additive effect).
At Navrongo, a different scenario was observed. The prevalence o f pfcrt T76 in the 
patients was low (44%) and no association was found between the mutation and clinical 
outcome (odds ratio 1.16, p-value 0.75). Also, pfcrt T76 was not detected in 10 (10.4%) 
non-respondents at pre-treatment but was however detected in them at post-treatment. 
Although there was no association between the pfcrt mutation and treatment outcome, 
the mutant alleles were found after treatment in all the non-respondents. Djimde et al. 
(2001) had suggested that other mutations at different codons o f the pfcrt gene such as 
174, E75, S220 and T356 may be important for chloroquine resistance therefore it is 
likely that these or other mutations may be responsible for resistance in the parasites at 
Navrongo. Further studies are required to investigate the presence of these other 
mutations and to determine their association to chloroquine resistance in Navrongo.
The prevalence of the pfmdrl mutation was 62% and similarly no association was found 
between pfmdrl mutation and clinical response in Navrongo (odds ratio 1.72, p-value 
0.24). This finding has also been reported in Mali and Cameroon (Djimde et al, 2001; 
Basco and Ringwald, 1998). Moreover, no association was found between the presence
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of both mutations, pfcrt T76 and pfmdrl Y86 with clinical response (odds ratio 1.01, p- 
value 0.97).
There were marked differences in treatment responses at the study sites with 
significantly more non-respondents in Hohoe (62%) than at Navrongo (31%). The 
differences in the observed resistance between the urban (Hohoe) and the rural 
community (Navrongo) could be attributed to self-medication due to easy accessibility 
to chloroquine in the former. Most often patients resort to buying from chemical sellers 
to avoid spending too much time and money at health centres and hospitals. Failure to 
adhere to prescription for treatment (non-compliance) by patients could also be largely 
responsible for the occurrence of drug resistance. The consequences o f inappropriate use 
of the drug lead to an increase in drug pressure, resulting in the selection for resistant 
strains of the parasites. Hohoe is urban in its settings and access to drugs is easier. This 
is most likely the reason for the selection of mutant pfcrt T76 parasites and therefore an 
association between this genotype and clinical outcome but not at Navrongo, which is 
relatively rural in comparison.
Parasite antimalarial drug resistance is known to occur naturally. Genetically diverse 
wildtype populations of P. falciparum  have heterogenous sensitivity to antimalarial 
drugs (Thaithong, 1983). Additionally, resistance may arise from spontaneous 
chromosomal p oint mutations, w hich a re i ndependent o f  drug pressure. Once formed, 
these more resistant mutants have a survival advantage in the presence o f antimalarial 
drugs (Curtis and Otoo, 1986; Cross and Singer, 1991). Resistant parasites may also be 
selected when parasites are exposed to subtherapeutic drug concentrations which are
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below the concentrations that kill the parasites. This occurs if  there is widespread use of 
the drug at inadequate doses. Resistance is also very likely if  the drug is eliminated 
slowly from the body because new parasites introduced to low drug concentrations are 
not eliminated (White, 1992). There is an assertion that high transmission rates favour 
the spread of drug resistance when combined with heavy drug pressure which is itself 
the most important factor in the survival o f drug resistant mutants (Mackinnon, 1997). 
This was observed at the two study sites. Hohoe is characterised by perennial malaria 
transmission and this could be the reason for the spread o f resistant parasites but not at 
Navrongo with an intense but seasonal transmission.
The alleles o f dhfr and dhps associated with response to pyrimethamine and 
sulphadoxine were also characterised in the present study.. The presence of mutations in 
codons 51, 59 and 108 of the dhfr gene was detected in pre-treatment P. falciparum  
isolates as well as in some post-treatment samples o f non-respondents to chloroquine. 
Out of the total number of isolates analysed in both sites (103 from Hohoe and 96 from 
Navrongo), about 40% were PCR negative for dhfr codons 51, 59 and 108 at both sites. 
Similarly, about 30% were PCR negative in the case of dhps codons 437 and 540. The 
reason may be that the regions amplified in this study, which has been found to be 
associated with Fansidar sensitivity in other countries like Mali, Tanzania, Cambodia, 
Vietnam and others (Plowe et al., 1999) may differ in sequence from what is present at 
the two sites, hence the primers used may be inappropriate for the detection.
The results from this study showed that the presence of dhfr and dhps mutations does not 
accurately predict Fansidar sensitivity or resistance in vivo in Hohoe and Navrongo. It is
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possible that these mutations may become more useful in the future with increasing 
treatment failure. However, it is worth noting that all chloroquine treatment failures 
responded adequately to Fansidar treatment. This gives credence to the usefulness of 
Fansidar as the second line antimalarial drug in Ghana The observed prevalence o f the 
mutations in these settings, in the face o f very low Fansidar resistance, suggests that in- 
vivo Fansidar resistance may evolve more quickly than predicted should the drug 
become the first line antimalarial to replace o f chloroquine in the country.
This study has demonstrated that the method of sequential processing of whole blood 
spots and performance of PCR is valuable in detecting mutations associated with drug 
resistance but very expensive. Samples collected from patients using minimally invasive 
technique (blood spots) can therefore be used for extensive hospital or field surveys of 
the relationship between mutations in P. falciparum  and antimalarial drug resistance. 
Although, the molecular technique described is relatively easy and rapid to perform, the 
relevance of genetic mutation as reliable genetic markers of in vivo antimalarial drugs 
resistance in Ghana needs further detailed investigation.
The results o f the in-vitro chloroquine sensitivity test correlated very well with that of 
the in-vivo observation thus, supporting the idea that in-vitro sensitivity is a useful 
epidemiological tool. However, the in-vitro drug sensitivity testing does not predict 
clinical response to treatment since it does not take into account individual differences in 
antimalarial pharmacokinetics, immunity or stage of disease.
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The estimated IC50 for chloroquine was 1.5xlO'6M indicating that isolates encountered 
in this study were fairly sensitive to the drug. All resistant cases at RIII level in-vivo 
showed isolate in-vitro inhibition at concentrations higher than the cut off 8pmol 
concentration indicative o f chloroquine resistance.
To summarise, the present study has shown that the pfcrt and pfmdrl genotyping could 
be useful in predicting the level of chloroquine resistance at Hohoe but not at Navrongo. 
The likely reason being that the level of drug pressure is higher at urbanised Hohoe than 
in the relatively rural Navrongo, which was reflected in the observed low prevalences of 
both the pfcrt and pfmdrl mutations at the latter study site. Other pfcrt mutations in the 
P. falciparum genome also need to be investigated in isolates at Navrongo to find out if  
they could predict the level of chloroquine resistance. However at both sites, all the non­
respondents harboured pfcrt T76 parasites, providing evidence and thus confirming the 
results o f other studies that this mutation is associated with chloroquine resistance. For 
Fansidar, the low prevalences of dhfr and dhps mutations observed revealed that there is 
low-level use o f  the drug especially in Navrongo whilst for Hohoe with a relatively high 
prevalence o f the quintuple mutations is indicative of a rapid emergence and spread of 
Fansidar resistance in the event o f drug policy change.
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chloroquine-resistant malaria in African children: a cost-effectiveness 
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THAITHONG, S. (1983). Clones of different sensitivities in drug-resistant isolates 
of Plasmodium falciparum. Bull. World Health Organ., 61 (4): 709-712.
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hybridization probes in the diagnoses o f genetic disorders. In Human 
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TRAGER, W. & JENSEN, J. B. (1976). Human malaria parasite in continuous 
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273: 627-628.
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in the human parasite Plasmodium falciparum  is determined by mutations 
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folate utilization. Molec. Microbiol., 23: 979-986.
WARD, S. A. (1988). Mechanisms of chloroquine resistance in malarial 
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WATKINS, W. M., MBERU, E. K., WINSTANLEY, P. A. & PLOWE, C. V.
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C. (1998). Lack o f bioequivalence o f a generic mefloquine tablet with the 
standard product. Eur. J. Clin. Pharmacol., 54 (8): 615-619.
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WELLEMS, T. E„ PANTON, L. J., GLUZMAN, I. Y., DO ROSARIO, V. E., 
GWADZ, R. W., WALKER-JONAH, A. & KROGSTAD, D. J. (1990). 
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Malaria Network. URL: http://www.who.ch/
ZOLG, J. W., PLITT, J. R., CHEN, G. X. & PALMER, S. (1989). Point mutations in 
the dihydrofolate reductase-thymidylate synthase gene as a molecular 
basis for pyrimethamine resistance in Plasmodium falciparum. Mol. 
Biochem Parasitol., 36: 253-262.
124
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APPENDICES
Appendix I
PREPARATION OF STANDARD SOLUTIONS
The following standard solutions were prepared using sterilised double distilled water.
Where appropriate, the solutions were autoclaved at 121 lb/sq for 15minutes in an Eyela 
Autoclave (Rikikkaki, Tokyo).
DNA Extraction
10% saponin
lOg of saponin was dissolved in 100ml o f sterilised doubled distilled water 
lx  PBS (Phosphate buffered saline)
One tablet dissolved in 200ml of water to obtain 0.01M phosphate buffer, 
0.0027M potassium chloride, pH 7.4 at 25°C.
20% Chelex
20g o f Chelex-100 was dissolved in 100ml of double distilled water and 
autoclaved.
Gel Electrophoresis (agarose)
Ethidium bromide (lOmg/ml)
lg  o f ethidium bromide was completely dissolved in 100ml o f sterilised double 
distilled water and stored in the dark at 4°C.
50x TAE buffer
242g Tris base, 57.1ml glacial acetic acid, 100ml 0.5M EDTA, pH adjusted to
7.7 and the volume made to 100ml with doubled distilled water.
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0.5M EDTA (pH 8.0)
186g of EDTA, dissolved in 800ml double distilled water, pH adjusted with 
NaOH pellets and stored at room temperature.
Gel Loading Buffers
6x Bromophenol blue
0.25% bromophenol blue, 40% sucrose in water and stored at 4°C.
5x Orange G
20% (w/v) Ficoll, 25mM EDTA, 2.5% (w/v) Orange G stored at room 
temperature.
DNA Molecular Weight Marker
1 OObp ladder
The first band is lOObp, the subsequent bands measure 200, 300, 400, ..., 
lOOObp.
126
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Appendix II 
CHEMICALS AND REAGENTS
DNA Extraction and PCR
lOx PCR buffer (Sigma)
Magnesium chloride (Sigma)
Taq DNA polymerase (Sigma)
Deoxyribonucleotide triphosphates, dATP, dCTP, dGTP and dTTP (Amersham 
Pharmacia Biotech).
Oligonucleotide primers (Oswel)
Saponin (Sigma)
Silica gel (Sigma)
Absolute ethanol (Sigma)
Mineral oil 
Chelex-100 (Sigma)
Bromophenol blue (Sigma)
Agarose (molecular biology grade, Sigma)
Methanol (Sigma)
Tris(hydroxymethyl)aminomethane (Trizmabase, Sigma)
Ethylene diamine tetra acetate disodium salt (EDTA, Sigma)
Ethidium bromide (Sigma)
1 OObp molecular weight markers (Gibco-BRL)
Ficoll (Sigma)
127
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In vitro Drug Sensitivity Test
Neutral RPMI 1640 (without glutamine but with bicarbonates, Sigma)
Glutamine
Glucose
HEPES
AB sera
Gentamycine
Chloroquine phosphate powder 
Radiolabelled Hypoxanthin mono-hydrochloride
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COMPOSITION OF RPMI 1640
From Gibco catalogue___________
Component_______________________________________ Amount (mg/1)
Inorganic salts
Ca(N03)2.4H20 100.0
KCI 400.0
Mg2S 0 4 48.84
NaCl 6000.0
NaHCOj 2000.0 (added before use)
Na2HP04 800.0
Amino acids
L-Arginine (free base) 200.0
L-Asparagine 50.0
L-Aspartic acid 20.0
L-Cysteine 65.0 (2HC1)
L-Glutamic acid 20.0
L-Glutamine Glycine 300.0
L-Histidine (free base) 10.0
L-Hydroxyproline 15.0
L-Isoleucine (allo-free)( 20.0
L-Leucine (methionine free) 50.0
L-Lycine-HCl 40.0
L-Methionine 15.0
L-Phenylalanine 15.0
L-Proline (hydroxy-proline free) 20.0
L-Seme 30.0
L-Threonine (allo-free) 20.0
L-Tryptophan 5.0
L-Tyrosine 28.94 (sodium salt)
L-Valine 20.0
Vitamins
Calcium pantothenate 0.20
Choline chloride 3.00
Folic acid 1.0
Isoinositol 35.0
Nicotinamide 1.0
P-Aminobenzoic 1.0
Pyridoxine-HCl 1.0
Riboflavin 0.20
Thiamine-HCl 1.0
Vitamin B12 0.005
Others
Glucose 2000.0
Glutathione 1.0
Phenol red 5.0
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Appendix III
RECRUITMENT AND CASE RECORD FORM
DISTRICT:...................................................................DATE:....................
NAME:...........................................................................STUDY NO.:........
AGE (MTHS):...........................SEX:..........................WEIGHT(KG):..
HOME ADDRESS:.....................................................LANDMARK:...
GUARDIAN’S NAME:..............................................OCCUPATION:,
FAMILY HEAD:...........................................................................................
ANTI-MALARIAL INTAKE DURING THE PAST WEEK:...........
DAY
0
DAY
1
DAY
2
DAY
3
DAY
7
DAY
14
DAY
21
DATE
FEVER (Yes/No)
VOMITING (Yes/No)
DIARRHOEA (Yes/No)
CONVULSION (Yes/No)
* UNABLE TO STAND/SIT UP. 
(Yes/No)
*UNABLE TO DRINK/ 
BREASTFEED/EAT (Yes/No)
♦LETHARGIC/UNCONSCIOUS
(Yes/No)
AXILLARY TEMP. (°C)
RESPIRATORY RATE
PARASIT COUNT/WBC
DRUG
DOSE
HB. (g/dl)
* Danger signs-: Do not recruit if  they are present on Day 0, and remove from study and refer if  present 
after Day 0.
+ For those given alternative drug on Day 14
Comments:.............................................................................
130
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CONSENT FORM: MAPPING THE RESPONSE OF PLASMODIUM FALCIPARUM  
TO CHLOROQUINE AND OTHER ANTIMALARIAL DRUGS 
IN GHANA.
I___________________________________ being the mother/father/legal guardian of
__________________________________ , am being asked to a llow _____________________
to participate in a medical study called “Mapping the Response of Plasmodium 
falciparum  to Chloroquine and other anti-malarial drugs in Ghana”.
It has been explained to me that the study is being conducted in 6 districts across Ghana 
and that it is a collaborative study between Ministry o f Health, Accra, the Noguchi 
Memorial Institute for Medical Research (NMIMR), Legon, the University o f Ghana 
Medical School (UGMS), Korle-Bu and the Institute de Medicine Tropicale du Service 
de Sante des Armees (IMTSSA), Marseille, France. The information gathered would be 
used to determine how useful chloroquine is for treatment o f malaria in Ghana.
It has been explained to me that my child/ward will be one o f several children in the 
country (about 300 in this district) being asked to volunteer for the study. If  I agree for 
my child/ward to join the study s/he will be asked to give a small blood sample (less 
than 1 ml) before treatment is given for malaria. The treatment given will be chloroquine 
and I should not give any other medication to my child/ward without making the 
investigators aware.
I understand that I will bring my child/ward to the hospital for review by the 
investigators two days, a week and two weeks from today. At each o f these visits, my 
child/ward will be examined and a small volume o f blood taken from my child/ward by 
finger prick to determine whether treatment given him or her is effective for treating the 
malaria infection. Finger pricks may cause mild pain during the short time it takes to 
insert the lancet and withdraw the blood. There is a very small risk of infection with 
finger pricks but the investigators have informed me that this is very unlikely to occur. 
The study investigators will take all available precautions such as using sterile 
equipment only once, and alcohol swabs to protect my childAvard. If I choose not to let 
my child/ward participate in the study s/he will be treated in the usual way and the 
decision not to let my child/ward participate will in no w'ay affect the care that s/he will 
be given in this hospital. If my child/ward is less than 6 months and more than 5 years or 
very ill or anaemic, s/he will not be allowed to participate in the study. These restrictions 
are necessary for the safety of the children.
During the study if my child/ward is found not be responding to the chloroquine 
treatment, other drugs will be given that will cure the child o f the infection. These will 
include but not limited to Fansidar and Quinine. In the event that my childAvard is given 
another drug besides chloroquine s/he will be required to come to the clinic to be 
examined to ensure that the infection is completely cured.
I have been informed that the risks of participating in the study are those o f finger pricks 
as mentioned before. The benefits of participating in the study include monitoring of the 
child/ward to ensure that the malaria infection is completely cured and the treatment of
131
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any other illness while the child/ward is under supervision. If I have any question or 
concerns about the study I can contact any of the following persons,
1 The Medical Officer in charge at the district hospital
2 The Regional Director of Health Services
3 Dr. Kwadwo A. Koram at the Noguchi Memorial Institute for Medical Research, 
University of Ghana, Legon (tel no. 021 500374) or in person at the district hospital
4 Dr. Richard Y. Osei, Malaria Control Programme, Ministry of Health, Accra or in 
person at the district hospital.
5 Prof. David Ofori-Adjei, University of Ghana Medical School, Korle-Bu, Accra
I understand that joining the study is voluntary (I do not have to let my child/ward join). 
If I allow my child/ward to join, I will be expected to bring the child for all the follow up 
visits but I may withdraw my child/ward’s participation at any point without any penalty 
or loss o f benefits to which I’m otherwise entitled. The doctors may also withdraw my 
child/ward without my consent if they fel this is necessary for the child’s safety. The 
information in my child/ward’s records is confidential, but may be examined by 
representatives of the Ghanaian Ministry of Health, the NMIMR, UGMS or by doctors 
providing care for the child.
The study described above has been explained to me and I voluntarily agree to let my 
child/ward participate in the study. I have had the opportunity to ask questions and 
understand that future questions I have will be answered by one o f the study 
investigators.
NAME:_______________________
(MOTHER/FATHER/GUARDIAN) 
SIGNATURE/RT. THUMPRINT
DATE:
WITNESS NAME:_______________________________ DATE:_____
WITNESS SIGNATURE:______________________________________
INVESTIGATOR’S NAME:_____________________________DATE:
INVESTIGATOR’S SIGNATURE: _______________
132
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Guidelines for the oral administration of malaria drugs 
Chloroquine doses in mg/kg body weight using 500mg salt tablets
WEIGHT RANGE/kg DAY 1 DAY 2 DAY 3
3 .4 -7 .4  ~2 ~2 ~
7 .5 -9 .9  % 3/4 '/2
10 .0 -14 .4  1 1 Zi
14 .5 -18 .4  l'A l'A  3A
18 .5 -34 .9  2Vi 2A  1
Sulphadoxine/Pyrimethamine (Fansidar) doses in mg/kg body weight using 500mg S  
+ 25mg P  tablets.
WEIGHT RANGE/kg FANSIDAR
5 .0 -6 .0 %
7 .0 -1 1 .0 Z2
12 .0 -17 .0 3A
18 .0 -22 .0 1
23 .0 -25 .0 1 %
133
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Lo
g 
DN
A 
si
ze
Appendix IV
AN EXAMPLE PLOT OF LOG MOLECULAR WEIGHT AGAINST MOBILITY 
TO DETERMINE SIZES OF PCR PRODUCTS
3.50
Mobility (mm)
The size of a DNA fragment that has a mobility x is estimated by extrapolating 
on to the Y-axis. The anti-log ot the figure where it transects the Y-axis is the 
molecular weight of the fragment
134
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Appendix V
DATA FROM FIELD AND LABORATORY WORK
I) Demographic data and parasitaemia for Hohoe and Navrongo
Definition o f codes and numbers in the data 
Site code:
1 -  Hohoe 
3 -  Navrongo 
Age in months 
Sex:
1 -  Male
2 -  Female
Trt resp. -  Treatment response
1 -  Sensitive
2 -  Resistant
Class -  Classification o f resistance 
0 -  Sensitive
1 - R I
2 -R I I
3 - RIII 
d-0 -  Day 0 
d-3 -  Day 3 
d-7 -  Day 7 
d-14 -  Day 14 
d-21 -  Day 21
135
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Site
code idno. age sex trtresp class
Parasite count/|J 
d-0 d-3 d-7 d-14 d-21
3 21 1 2 1 3480 40 0 1400 0
4 9 2 2 3 2680 1360 0 10680 2840
10 14 2 2 3 2000 9960 0 0 0
12 11 2 2 1 12680 120 40 20160 0
23 36 2 2 3 19520 2200 7440 13000 1680
33 58 2 2 1 96800 0 0 2560 0
59 14 1 2 1 32000 0 280 920 0
60 8 2 2 1 14400 40 800 40800 0
130 28 2 2 3 44800 15200 0 0 0
132 48 1 2 1 56800 0 0 9880 0
141 20 1 2 2 25600 5600 0 0 0
149 48 1 2 1 4800 0 0 2320 0
153 18 2 2 1 79200 280 0 1160 0
257 28 2 2 1 24080 0 0 3560 120
290 16 1 2 1 3400 0 0 120 46720
303 7 1 2 2 50400 160 0 40600 0
305 13 2 2 2 29600 320 1400 3440 160
307 23 1 2 1 46800 0 0 0 2680
313 56 2 2 2 122240 440 160 1200 0
318 48 2 2 1 138960 0 40 9920 0
324 17 1 2 2 72080 520 640 4880 0
329 36 2 2 2 116080 18600 440 4960 0
353 30 2 2 2 151200 1480 0 2000 0
357 59 1 2 1 127720 200 0 9800 0
362 30 2 2 2 3920 360 600 0 40
364 15 1 2 3 125520 52000 0 0 61200
371 23 2 2 2 2280 360 1040 560 0
374 28 2 2 1 22720 0 0 160 0
376 20 2 2 1 5920 0 4400 0 0
379 9 2 2 1 120000 0 0 240 1880
383 54 2 2 3 32840 8760 48800 0 0
397 13 1 2 2 272000 160 0 360 0
455 8 2 2 2 92600 3480 0 1000 400
468 20 2 2 1 34160 0 0 0 5200
479 26 1 2 1 56800 0 0 10640 0
482 42 1 2 1 72000 0 0 0 69240
552 24 2 2 2 124600 4080 0 91200 0
558 12 1 2 1 576040 0 0 5360 0
584 52 2 2 1 8160 0 0 0 130000
585 25 2 2 1 25120 0 480 320 0
586 28 2 2 1 93040 0 0 800 26200
610 8 2 2 3 9680 15400 0 1120 8440
620 36 2 2 1 15880 0 0 0 560
684 18 2 2 2 4040 120 0 200 40
136
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Sit.
coc
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Parasite count/(^l
idno. aue sex trtresD class d-0 d-3 d-7 d-14 d-21
685 31 1 2 1 120000 0 0 17280 0
690 34 2 2 1 3440 0 0 1400 0
700 9 1 2 1 6960 0 0 0 440
703 20 1 2 1 3360 0 0 0 1160
707 14 1 2 2 2440 120 240 120 0
709 6 2 2 2 2480 0 0 40 120
710 9 2 2 2 2480 80 40 600 0
713 13 2 2 2 22000 840 720 0 40
730 33 1 2 2 3400 1080 120 41600 9600
731 28 2 2 1 4880 0 0 0 19840
733 17 1 2 1 15360 0 0 0 160
735 15 2 2 3 14000 1920 2320 0 6200
741 16 1 2 1 6320 40 0 0 1520
742 29 2 2 1 8960 0 0 200 1160
744 27 2 2 2 2360 200 0 0 4800
745 24 1 2 2 3720 240 0 0 24200
747 12 2 2 1 80960 0 40 80 0
945 12 1 2 1 128000 26520 0 0 0
949 21 2 2 1 4400 3040 0 0 0
956 59 2 2 2 10600 0 0 1280 0
14 26 2 2 1 6480 0 0 7480 0
36 19 2 2 2 87200 240 0 5520 0
66 25 2 2 1 12080 0 0 360 0
154 9 1 2 1 8840 0 0 14880 0
155 24 1 2 1 2560 0 0 13920 0
157 19 2 2 2 17120 160 0 21040 0
162 11 2 2 3 72800 16280 0 14440 0
170 45 1 2 2 12720 120 0 2720 0
182 27 1 2 1 13320 0 0 0 720
237 37 2 2 1 16520 0 0 5120 0
240 30 2 2 1 28920 0 3360 1120 0
248 9 1 2 2 20840 840 0 2240 0
255 21 1 2 1 55280 0 0 2520 0
258 22 1 2 2 75360 8240 0 1920 0
263 40 1 2 1 20240 0 0 7320 0
269 24 2 2 1 35360 0 0 1040 0
279 33 2 2 1 39440 0 8240 0 0
313 19 1 2 1 2080 0 0 8600 0
315 34 1 2 2 380800 4800 0 2320 0
319 2 2 1 3440 0 0 9280 0
337 25 1 2 2 108800 28320 0 0 0
341 15 2 2 3 34640 5080 8240 8360 0
343 16 1 2 3 174400 1520 0 39440 0
403 14 2 2 3 8240 640 0 16800 0
137
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Site Parasite count/^il
code idno. aee sex trtresp class d-0 d-3 d-7 d-14 d-21
3 411 23 2 2 3 127680 0 80240 159680 0
3 412 23 2 2 3 87320 0 11280 12960 0
3 420 15 1 2 2 17280 0 22400 0 0
3 445 33 2 2 2 16960 4800 5840 0 0
3 447 49 1 2 2 62800 120 12640 0 0
3 450 43 2 2 3 28400 0 0 3280 0
1 13 36 1 1 0 28000 0 0 0 0
1 14 24 1 1 0 4760 0 0 240 0
1 16 59 1 1 0 3240 0 0 0 0
1 46 30 1 1 0 5000 0 0 0 0
1 47 22 2 1 0 16800 0 0 0 0
1 54 42 1 1 0 34000 0 0 0 0
1 550 16 1 1 0 96800 0 0 0 0
1 551 19 1 1 0 4080 0 0 0 0
1 567 56 2 1 0 95200 800 2680 0 0
1 572 48 2 1 0 15200 80 0 0 0
1 599 24 2 1 0 2560 0 0 0 0
1 605 36 1 1 0 130000 4880 0 0 0
1 609 6 2 1 0 33440 0 0 0 0
1 663 25 2 1 0 2760 0 0 0 0
1 695 20 2 1 0 4400 0 0 40 0
1 697 29 2 1 0 8800 0 0 0 0
1 698 38 1 1 0 16600 0 120 0 0
1 699 27 1 1 0 2120 0 0 0 0
1 708 36 1 1 0 10360 160 0 0 0
1 712 8 2 1 0 22520 80 0 0 0
1 714 40 2 1 0 3760 0 0 0 0
1 729 56 1 1 0 2040 360 40 0 0
1 732 43 2 1 0 2240 0 0 0 0
1 738 38 1 1 0 5960 0 0 0 0
1 739 51 1 1 0 9680 0 0 40 0
1 740 13 2 1 0 7520 0 0 0 0
1 743 30 2 1 0 11800 80 0 0 0
1 944 25 1 1 0 3600 0 0 0 0
1 946 46 1 1 0 91400 0 0 0 0
1 947 35 1 1 0 10400 600 0 0 0
1 948 15 2 1 0 2320 680 0 0 0
1 950 49 1 1 0 5040 0 0 0 0
1 951 49 2 1 0 5040 0 0 0 0
1 952 25 1 1 0 3320 0 0 0 0
1 954 10 1 1 0 3000 0 0 0 0
1 955 10 1 1 0 17600 0 0 0 0
1 957 42 1 1 0 51120 0 0 0 0
1 958 37 1 1 0 2440 0 0 0 0
138
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Siti
coc
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Parasite count/p.1
ldno. ace sex trtresp class d-0 d-3 d-7 d -b
959 35 1 1 0 140000 0 0 0
3 51 1 1 0 27320 120 0 0
5 58 1 1 0 19560 0 0 0
6 33 2 1 0 23880 0 0 0
17 36 1 1 0 2480 0 0 0
45 24 2 1 0 83560 80 0 0
61 51 1 1 0 86720 0 0 0
67 6 1 1 0 3320 0 0 0
77 32 1 1 0 37120 280 0 0
83 17 2 1 0 23560 0 0 0
145 59 2 1 0 30720 0 0 0
159 13 1 1 0 8240 0 0 0
164 58 2 1 0 9160 0 0 0
166 14 2 1 0 73040 1120 0 0
171 20 1 1 0 3320 0 0 0
173 27 1 1 0 1600 0 0 0
176 48 2 1 0 35240 0 0 0
177 12 1 1 0 24680 0 0 0
183 21 2 1 0 16040 0 0 0
185 51 1 1 0 8120 0 0 0
188 32 1 1 0 74720 0 0 0
189 49 2 1 0 3080 19200 0 0
197 14 2 1 0 76920 6520 0 0
198 16 2 1 0 24080 0 0 0
202 10 1 1 0 13080 0 0 0
205 21 2 1 0 4920 0 720 0
207 9 1 1 0 73040 720 0 0
234 42 1 1 0 8240 0 0 0
238 59 1 1 0 24960 0 0 0
243 39 2 1 0 12760 0 0 0
247 50 2 1 0 25120 0 0 0
251 52 1 1 0 16600 0 0 0
254 52 2 1 0 32080 0 0 0
256 32 1 1 0 12360 0 0 0
260 50 2 1 0 2840 0 0 0
264 40 1 1 0 18720 0 0 0
268 27 1 1 0 37040 1120 0 0
273 10 1 1 0 690440 8640 0 0
276 7 2 1 0 4960 0 0 0
304 15 2 1 0 95200 0 0 0
323 35 2 1 0 42880 0 0 0
329 12 2 1 0 2560 0 0 0
332 36 2 1 0 113600 0 0 0
339 14 1 1 0 78720 0 0 0
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3
d-21
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Parasite count/|il
ldno. aee sex trtresp class d-0 d-3 d-7 d-14
342 56 1 1 0 29040 0 0 0
404 6 2 1 0 6160 0 0 0
405 17 2 1 0 20240 0 0 0
406 25 1 1 0 16080 1120 0 0
407 24 1 1 0 7480 0 0 0
409 31 1 1 0 80240 0 0 0
413 19 1 1 0 3520 0 0 0
414 50 1 0 49120 0 0 0
416 42 1 1 0 31840 0 0 0
423 37 1 0 9280 0 440 0
424 10 1 1 0 32800 0 0 0
426 41 1 1 0 2480 0 0 0
428 8 1 1 0 1560 0 0 0
431 24 1 0 71200 0 0 0
436 51 1 1 0 72800 0 0 0
437 20 1 1 0 110720 1520 0 0
438 46 1 1 0 76480 0 0 0
441 38 1 1 0 8400 0 0 0
442 34 1 0 71600 0 0 0
443 15 1 1 0 18800 160 0 0
444 11 1 0 10200 80 0 0
448 14 1 1 0 32320 0 0 0
451 20 1 1 0 242400 0 0 0
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Definitions of codes and numbers in the data.
Site code
1 -  Hohoe 
3 -  Navrongo 
Id no. -  Identification number 
Trt. Resp. -  Treatment response
1 -  Sensitive
2 -  Resistant 
pfcrt -  pfcrt 76 
pfmdrl -p fm d rl 86 
dr-51 -  dhfr 51 
dr-59 -  dhfr 59
dr-108 -  dhfr 108 
ds-437 -  dhps 437 
ds-540 -  dhps 540 
For all the gene codons
0 -  no alleles
1 -  mutant allele only
2 -  wildtype allele only
3 -  mixed alleles
II) Data from PCR assays at pre-treatment for Hohoe and Navrongo
141
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Site Mutation specific PCR for the four gene loci at day 0
code id no. trt resp.pfcrt pfmdrl dr-51 dr-59 dr-108 ds-437 ds-540
1 3 2 1 1 0 0 0 0 0
1 4 2 1 1 0 0 0 0 0
1 10 2 3 1 0 0 0 0 0
1 12 2 1 1 3 3 3 3 2
1 23 2 1 1 3 3 3 1 3
1 33 2 3 1 3 3 3 3 3
1 59 2 3 1 2 1 3 1 3
1 60 2 1 1 2 3 3 1 3
1 130 2 1 1 0 1 3 1 3
1 132 2 1 1 1 3 3 1 3
1 141 2 3 1 3 3 3 3
1 149 2 3 1 1 3 3 1 3
1 153 2 1 1 3 3 3 1 3
1 257 2 1 1 3 3 1 1 3
1 290 2 1 0 3 2 3 1 3
1 303 2 1 1 3 3 3 3
1 305 2 1 1 3 3 3 1 3
1 307 2 1 1 3 2 3 1 3
1 313 2 1 1 3 3 3 1 3
1 318 2 1 1 3 3 3 3
1 324 2 1 1 3 3 3 1 3
1 329 2 3 1 2 3 3 1 2
1 353 2 3 1 3 3 3 1 3
1 357 2 3 1 2 3 3 3
1 362 2 1 1 3 3 3 3
1 364 2 3 1 3 3 3 1 3
1 371 2 1 1 3 1 3 1 2
1 374 2 1 1 1 1 1 1 3
1 376 2 1 1 3 1 3 1 3
1 379 2 1 1 3 3 3 1 3
1 383 2 1 1 1 1 1 1 3
1 397 2 2 1 3 1 3 1 3
1 455 2 1 1 0 0 0 0
1 468 2 1 1 1 1 2 1 3
1 479 2 2 0 1 0 0 2
1 482 2 2 0 2 0 2 1 2
1 552 2 3 1 1 1 3 1 3
1 558 2 3 1 3 0 3 1 3
1 584 2 3 1 3 3 3 1 3
1 585 2 1 1 3 3 3 3 3
1 586 2 3 1 3 3 3 1 3
1 610 2 1 1 0 0 0 0 0
142
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Mutation specific PCR for the four gene loci at day 0
id no. trt resp.pfcrt pfmdrl dr-51 dr-59 dr-108 ds-437 ds-540
620 2 3 0 3 0 0 0 0
684 2 1 1 3 1 1 3 2
685 2 2 1 0 0 0 0 2
690 2 2 1 0 0 0 3 2
700 2 3 1 0 0 0 0 0
703 2 3 1 1 3 1 1 3
707 2 1 1 2 1 1 1 1
709 2 3 1 0 0 0 0 0
710 2 3 1 3 1 1 1 3
713 2 1 1 3 3 3 2 3
730 2 1 1 0 2 0 3 2
731 2 3 1 2 2 3 0 2
733 2 3 1 1 3 3 3 3
735 2 3 1 3 3 3 3 3
741 2 3 1 2 0 3 3 3
742 2 3 0 0 0 0 3 2
744 2 3 1 2 3 2 3 2
745 2 3 1 1 2 1 1 3
747 2 3 1 1 3 3 2 2
945 2 2 0 0 1 2 3 0
949 2 1 1 0 3 1 0 0
956 2 3 0 2 1 3 1 0
14 2 1 1 2 1 3 0 0
36 2 3 0 2 3 3 0 2
66 2 1 1 2 3 2 0 0
154 2 1 0 0 0 0 0 0
155 2 2 1 0 0 0 0 0
157 2 2 0 0 0 0 0 0
162 2 1 1 0 0 0 0 0
170 2 2 0 0 0 0 0 0
182 2 2 0 0 0 0 0 0
237 2 1 1 0 1 3 0 0
240 2 1 1 0 1 1 0 0
248 2 1 1 1 1 1 0 0
255 2 1 1 0 1 0 0 2
258 2 3 1 2 3 3 0 0
263 2 1 1 0 0 0 0 0
269 2 1 0 0 0 0 0 0
279 2 1 1 0 0 0 0 0
313 2 2 1 0 0 0 0 0
315 2 2 1 0 0 0 0 0
319 2 2 0 0 0 0 0 0
143
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Site Mutation specific PCR for the four gene loci at day 0
code id no. trt resn.pfcrt pfmdrl dr-51 dr-59 dr-108 ds-437 ds-540
3 337 2 2 0
3 341 2 1 1
3 343 2 2 0
3 403 2 2 1
3 411 2 2 1
3 412 2 2 1
3 420 2 2 1
3 445 2 2 1
3 447 2 2 1
3 450 2 2 1
1 13 1 1
1 14 1 2 1
1 16 1 2
1 46 1 3 1
1 47 1 3 1
1 54 1 0
1 550 1 3 1
1 551 1 3 1
1 567 1 3 1
1 572 1 3 1
1 599 1 2 0
1 605 1 2 0
1 609 1 1 0
1 663 1 2 0
1 695 1 1 1
1 697 1 1 1
1 698 1 3 1
1 699 1 3 1
1 708 1 3 0
1 712 1 3 1
1 714 1 3 1
1 729 1 3 1
1 732 1 2 0
1 738 1 1 0
1 739 1 2 1
1 740 1 3 1
1 743 1 3 1
1 944 1 2 0
1 946 1 3 1
1 947 1 2 1
1 948 1 2 1
1 950 1 2 0
0 0 0 0 0
2 1 3 0 2
2 3 2 0 0
2 0 2 0 2
3 1 1 2 2
3 0 3 2 3
2 1 1 2 0
2 0 2 3 2
2 1 3 3 2
0 0 0 2 0
0 0 0 3 3
0 1 3 1 3
0 0 0 3 0
2 2 1 1 3
3 2 3 3 3
0 0 0 0 0
2 3 1 3 3
2 3 3 3 3
3 0 1 3 2
1 1 3 1 3
0 2 1 3 0
0 1 0 3 2
0 0 0 3 0
0 0 0 3 3
3 1 1 3 3
0 1 0 3 0
0 0 0 3 2
0 0 0 3 2
0 0 0 3 0
0 0 0 3 3
0 0 0 3 2
3 0 1 1 3
0 0 0 0 0
0 0 2 0 2
0 0 0 1 2
3 3 3 3 3
0 0 0 3 3
0 1 3 2 0
2 0 3 3 3
0 1 3 0 3
0 3 1 0 2
2 0 0 0 0
144
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3
3
3
3
3
3
3
3
3
3
3
3
3
3
Mutation specific PCR for the four gene loci at day 0
id no. trt resp.pfcrt pfmdrl dr-51 dr-59 dr-108 ds-437 ds-540
951 1 3 1 2 0 2 0 0
952 1 3 1 0 0 3 2 0
954 1 3 1 0 1 1 2 2
955 1 2 1 0 0 1 1 2
957 1 3 1 0 1 3 1 2
958 1 3 1 0 1 1 2 0
959 1 3 1 0 1 3 3 2
3 1 1 1 0  0 0 0 0
5 1 2 0 0 0 0 0 0  
6 1 2 0 0 0 0 0 0  
17 1 1 1 0 0 0 0 0
45 1 1 1 0 0 0 0 0
61 1 2 0 0 0 0 0 0
67 1 1 0 0 0 0 0 0
77 1 2 0 0 0 0 0 0
83 1 1 1 0 0 0 0 0
145 1 2 0 2 0 2 0 0
159 1 1 1 0 0 0 0 0
164 1 2 0 0 0 0 0 0
166 1 1 1 0 0 0 0 0
171 1 1 1 0  0 0 0 0
173 1 2 0 0 0 0 0 0
176 1 2 0 0 0 0 0 0
177 1 2 1 0 0 0 0 0
183 1 2 0 0 0 0 0 0
185 1 2 0 0 0 0 0 0
188 1 2 0 0 0 0 0 0
189 1 2 0 0 0 0 0 0
197 1 2 0 0 0 0 0 0
198 1 2 0 0 0 0 0 0
202 1 2 0 0 0 0 0 0
205 1 1 1 0  0 0 0 0
207 1 0 0 0 0 0 0 0
234 1 3 1 0 0 0 0 0
238 1 3 1 0 0 0 0 0
243 1 1 l o 0 0 0 0
247 1 1 l o 0 0 0 0
251 1 3 1 0 0 0 0 0
254 1 2 0 0 0 0 0 0
256 1 2 0 0 0 0 0 0
260 1 1 o 0 0 0 0 0
264 1 1 1 0 0 0 0 0
145
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3
Mutation specific PCR for the four gene loci at day 0
id no. trt resp.pfcrt
268 1 1
273 1 1
276 1 1
304 1 3
323 1 2
329 1 1
332 1 1
339 1 1
342 1 1
404 1 2
405 1 2
406 1 2
407 1 2
409 1 2
413 1 1
414 1 2
416 1 2
423 1 1
424 1 2
426 1 2
428 1 2
431 1 2
436 1 2
437 1 2
438 1 2
441 1 1
442 1 1
443 1 2
444 1 2
448 1 2
451 1 2
pfmdrl dr-51 dr-59
1 0 0
1 0 0
1 0 0
1 2 2
0 2 3
0 0 3
1 2 2
0 2 1
1 0 0
1 2 1
1 2 1
1 3 1
1 0 0
1 2 1
1 0 0
0 0 0
0 2 0
1 3 1
1 2 1
1 2 0
1 0 3
0 2 3
1 2 0
1 2 1
0 0 0
1 2 0
1 1 1
1 2 0
0 0 2
1 2 0
1 2 1
dr-108 ds-437 ds-540
0 0 0
0 0 0
0 0 0
2 0 2
3 0 2
3 0 2
2 0 0
0 0 0
0 0 0
3 2 2
3 2 2
3 3 3
1 2 0
1 2 2
1 2 3
0 2 0
2 2 0
3 2 2
0 3 3
3 0 0
3 2 3
3 2 2
3 3 3
3 3 2
2 2 0
3 2 2
3 2 2
3 2 2
0 3 0
3 3 2
3 3 2
146
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Ill) Data from in-vitro chloroquine sensitivity test on P. falciparum  isolates from
Hohoe and Navrongo
Definitions o f codes and numbers in the data 
Site code
1 -  Hohoe
3 -  Navrongo
Id no. -  Identification number
Trt. Resp. -  In-vivo treatment response
1 -  Sensitive
2 -  Resistant 
Concentration o f chloroquine pmol
1 pmol
2 pmol
4 pmol 
8 pmol 
16 pmol 
32 pmol 
64 pmol
In-vitro response
5 -  Sensitive 
R - Resistant
147
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Percentage inhibition at concentrations of 
chloroquine pmol
id no. trt resD 1 2 4 8 16 32 64
946 1 32 37.6 46 100 100 100 100
947 1 18 12.5 16.7 100 100 100 100
949 2 11 9.1 14.4 100 100 100 100
951 1 25.2 22.7 18 100 100 100 100
952 1 0 0 0 100 100 100 100
954 1 2.9 0 12.5 100 100 100
958 1 3.8 25.3 100 100 100 100 100
959 1 27.2 100 100 100 100 100 100
403 2 0 0 0 0 0 0 100
404 1 11 7 0 100 100 100 100
405 1 0 1 7 100 100 100 100
406 1 0 8 1 2 4 21 100
407 1 41 43 100 100 100 100 100
409 1 3 2 18 100 100 100 100
411 2 0 11 26 9 100 100 100
412 2 0 0 0 0 0 100 100
414 1 0 0 100 100 100 100 100
418 0 0 0 0 0 53
420 2 36 31 64 100 100 100 100
424 1 0 0 0 3 9 100 100
437 1 0 0 4 100 100 100 100
440 0 7 14 100 100 100 100
443 1 0 3 0 100 100 100 100
445 2 9 7 7 100 100 100 100
447 2 32 100 100 100 100 100 100
448 1 5 8 2 100 100 100 100
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