IN VITRO AND GENETIC ASPECTS OF TREATMENTS FOR FALCIPARUM MALARIA: STUDIES OF CONVENTIONAL AND NOVEL DRUGS WITH A PARTICULAR FOCUS ON PAPUA NEW GUINEA Rina Pok-Man Fu nee Wong B. Sc. (First Hons) School of Medicine & Pharmacology This thesis is submitted to the University of Western Australia for the degree of DOCTOR OF PHILOSOPHY OF MEDICINE 2011
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IN VITRO AND GENETIC ASPECTS OF
TREATMENTS FOR FALCIPARUM MALARIA:
STUDIES OF CONVENTIONAL AND NOVEL DRUGS
WITH A PARTICULAR FOCUS ON
PAPUA NEW GUINEA
Rina Pok-Man Fu nee Wong
B. Sc. (First Hons)
School of Medicine & Pharmacology
This thesis is submitted to the University of Western Australia
for the degree of DOCTOR OF PHILOSOPHY OF MEDICINE
2011
DECLARATION
The research presented in this thesis is my own work unless otherwise stated. The majority of this work was undertaken in the School of Medicine and Pharmacology (Fremantle Unit), the University of Western Australia. Field components were carried out at collaborating institutions, specifically the Papua New Guinea Institute of Medical Research (PNGIMR), Madang, Papua New Guinea and Case Western Reserve University, Cleveland, Ohio, United States of America. This thesis has not been submitted for any other degree at this or any other tertiary institution.
• Patient recruitment, blood collection and P. falciparum screening were carried out by the nursing team at Alexishafen Health Centre, Madang, Papua New Guinea as part of an antimalarial treatment trial.
• Restriction fragment length polymorphism assays of P. falciparum field isolates were performed by laboratory staff at the PNGIMR as part of the same treatment trial.
Rina Pok-Man Fu nee Wong Perth, Australia 2011
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DECLARATION FOR THESIS CONTAINING PUBLISHED WORK AN D/OR WORK PREPARED FOR PUBLICATION
This thesis contains published work and/or work prepared for publication, some of which has been co-authored. The bibliographical details and where it appears in the thesis are outlined below. The candidate must attach to this declaration a statement for each publication detailing the percentage contribution by the candidate. This statement must be signed by all authors. If signatures from all the authors cannot be obtained, the statement detailing the candidate’s contribution to the published work must be signed by the coordinating supervisor.
• Harin, A. Karunajeewa, Ivo Mueller, Michele Senn, Enmoore Lin, Irwin Law, Servina P. Gomorrai, Olive Oa, Suzanne Griffin, Kaye Kotab, Penias Suano, Nandao Tarongka, Alice Ura, Dulcie Lautu, Madhu Page-Sharp, Rina P. M. Wong, Sam Salman, Peter Siba, Kenneth F. Ilett and Timothy M. E. Davis. (2008) A trial of combination antimalarial therapies in children from Papua New Guinea. N Engl J Med 359 (24) 2545-57. Precise Contributions: performed in vitro drug sensitivity testing and analysis, assisted with patient recruitment and sample processing. Overall Contribution: 5%.
• Rina P. M. Wong and Timothy M. E. Davis. (2009) Statins as potential antimalarial drugs: Low relative potency and lack of synergy with conventional antimalarial drugs. Antimicrob Agents Chemother 53 (5) 2212-2214. Precise Contributions: designed and performed all in vitro experiments, data analysis and interpretation, drafting of manuscript. Overall Contribution: 90%.
• Stephan Karl and Rina P.M. Wong (equal-first author), Tim St. Pierre, Timothy M.E. Davis. (2009) A comparative study of a flow-cytometry-based assessment of in vitro Plasmodium falciparum drug sensitivity. Malaria Journal 8, 294. Precise Contributions: designed and performed drug sensitivity assays suitable for three methods of growth response assessment, data collection for the reference isotopic and enzyme methods, data analysis and drafting of manuscript. Overall Contribution: 40%.
• Rina P. M. Wong, Dulcie Lautu, Livingstone Tavul, Sarah L. Hackett, Peter Siba, Harin A. Karunjeewa, Kenneth F Ilett, Ivo Mueller, Timothy M. E. Davis. (2010) In vitro sensitivity of Plasmodium falciparum to conventional and novel antimalarial drugs in Papua New Guinea. Trop Med Int Health 15(2) 342-349.
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Precise Contributions: designed and performed drug sensitivity assays, method validation and optimisation, assisted with sample collection and processing, data analysis and interpretation, drafting of manuscript. Overall Contribution: 80%.
• Rina P. M. Wong, Harin Karunajeewa, Ivo Mueller, Peter Siba, Peter A. Zimmerman, Timothy M. E. Davis. (2011) Molecular assessment of Plasmodium falciparum resistance to antimalarial drugs in Papua New Guinea using an extended ligase detection reaction-fluorescent microsphere assay. Antimicrob Agents Chemother 55(2) 798-805. Precise Contributions: performed molecular screening of Plasmodium species, and drug resistant genes, optimisation and extension of the LDR-FMA technique to include the screening of 10 additional SNPs in the pfmdr1 gene, data analysis and interpretation, determination of positive threshold parameters, drafting of manuscript. Overall Contribution: 85%.
• Rina P. M. Wong, Sam Salman, Kenneth F Ilett, Ivo Mueller, Timothy M. E. Davis. (2011) Desbutyl-lumefantrine is a metabolite of lumefantrine with potent in vitro antimalarial activity that may influence artemether-lumefantrine treatment outcome. Antimicrob Agents Chemother 55(3) 1194-1198. Precise Contributions: designed and performed in vitro antimalarial assays, drug interaction asssays, data analysis and interpretation, drafting of manuscript. Overall Contribution: 75%.
• Louise R. Whittell, Kevin T. Batty, Rina P. M. Wong, Erin Bolitho, Simon A. Fox, Timothy M. E. Davis, and Paul E. Murray. (2011) Synthesis and antimalarial evaluation of novel isocryptolepine derivatives. Bioorg Med Chem 19, 7519-7525. Precise Contributions: designed and performed in vitro antimalarial assays, data analysis and interpretation, manuscript preparation. Overall Contribution: 25%.
• Rina P. M. Wong and Timothy M. E. Davis. (2011) In vitro antimalarial efficacy and drug interactions of fenofibric acid. Antimicrob Agents Chemother (Manuscript submitted # AAC05076-11) Precise Contributions: designed and performed in vitro antimalarial assays, drug interaction asssays, data analysis and interpretation, drafting of manuscript. Overall Contribution: 90%.
• Rina P. M. Wong, Gavin R. Flematti and Timothy M. E. Davis. (2011) Detection of volatile organic compounds produced by Plasmodium falciparum in culture. Manuscript in preparation. Precise Contributions: designed culture-volatile compounds capture apparatus, performed experiments, GCMS and data analysis, and drafting of manuscript. Overall Contribution: 70%.
• Harin, A. Karunajeewa, Ivo Mueller, Michele Senn, Enmoore Lin, Irwin Law, Servina P. Gomorrai, Olive Oa, Suzanne Griffin, Kaye Kotab, Penias Suano, Nandao Tarongka, Alice Ura, Dulcie Lautu, Madhu Page-Sharp, Rina P. M. Wong, Sam Salman, Peter Siba, Kenneth F. Ilett and Timothy M. E. Davis. (2008) A trial of combination antimalarial therapies in children from Papua New Guinea. N Engl J Med 359 (24) 2545-57.
• Rina P. M. Wong & Timothy M. E. Davis. (2009) Statins as potential antimalarial drugs: Low relative potency and lack of synergy with conventional antimalarial drugs. Antimicrob Agents Chemother 53 (5) 2212-2214.
• Stephan Karl and Rina P.M. Wong (equal-first author), Tim St. Pierre, Timothy M.E. Davis. (2009) A comparative study of a flow-cytometry-based assessment of in vitro Plasmodium falciparum drug sensitivity. Malaria Journal 8, 294-305.
• Rina P. M. Wong, Dulcie Lautu, Livingstone Tavul, Sarah L. Hackett, Peter Siba, Harin A. Karunjeewa, Kenneth F Ilett, Ivo Mueller, Timothy M. E. Davis. (2010) In vitro sensitivity of Plasmodium falciparum to conventional and novel antimalarial drugs in Papua New Guinea. Trop Med Int Health 15(2) 342-349.
• Rina P. M. Wong, Harin Karunajeewa, Ivo Mueller, Peter Siba, Peter A. Zimmerman, Timothy M. E. Davis. (2011) Molecular assessment of Plasmodium falciparum resistance to antimalarial drugs in Papua New Guinea using an extended ligase detection reaction-fluorescent microsphere assay. Antimicrob Agents Chemother 55(2) 798-805.
• Rina P. M. Wong, S. Salman, Kenneth F Ilett, Ivo Mueller, Timothy M. E. Davis. (2011) Desbutyl-lumefantrine is a metabolite of lumefantrine with potent in vitro antimalarial activity that may influence artemether-lumefantrine treatment outcome. Antimicrob Agents Chemother 55(3) 1194-1198.
• Louise R. Whittell, Kevin T. Batty, Rina P. M. Wong, Erin Bolitho, Simon A. Fox, Timothy M. E. Davis, and Paul E. Murray. (2011) Synthesis and antimalarial evaluation of novel isocryptolepine derivatives. Bioorg Med Chem 19, 7519-7525.
• Rina P. M. Wong and Timothy M. E. Davis. (2011) In vitro antimalarial efficacy and drug interactions of fenofibric acid. Antimicrob Agents Chemother (Manuscript submitted # AAC05076-11)
• Rina P. M. Wong, Gavin R. Flematti and Timothy M. E. Davis. (2011) Detection of volatile organic compounds produced by Plasmodium falciparum in culture. Manuscript in preparation.
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CONFERENCE PRESENTATIONS
• Rina P. M. Wong & Timothy M. E. Davis. In vitro susceptibility and inter-relationships of nine standard and new antimalarials against Plasmodium falciparum isolates from Papua New Guinean children. (2008) Research Showcase: School of Medicine & Pharmacology, Perth, Australia. (Oral)
• Rina P. M. Wong & Timothy M. E. Davis. Malaria and statins. (2008) Annual Research Showcase: School of Medicine & Pharmacology, Perth, Australia. (Oral: Best Student Oral Award)
• Rina P. M. Wong & Timothy M. E. Davis. Statins and fibrates as potential antimalarial agents. (2009) The Australian Society for Medical Research, Medical Research Week Scientific Symposium, Perth, Australia. (Oral)
• Rina P. M. Wong, Harin Karunajeewa, Ivo Mueller, Peter Siba, Eric P. Carnevale, Peter A. Zimmerman and Timothy M. E. Davis. Drug Resistance Polymorphisms in Plasmodium falciparum from children in Papua New Guinea by a recently developed LDR-FMA technique. (2009) Combined Biological Sciences Meeting, Perth, Western Australia. (Oral: New Investigator Award)
• Rina P. M. Wong, Harin Karunajeewa, Ivo Mueller, Peter Siba, Eric P. Carnevale, Peter, A. Zimmerman and Timothy M.E. Davis. Novel molecular detection of drug resistance markers in Plasmodium falciparum from paediatric uncomplicated malaria in Papua New Guinea. (2010) 14th International Congress on Infectious Diseases, Miami, Florida, United States of America. (Oral)
• Rina P. M. Wong, S. Salman, Kenneth F. Ilett, Ivo Mueller and Timothy M. E. Davis. In vitro and in vivo evaluation of desbutyl-benflumetol, a promising antimalarial drug. (2010) XII International Congress on Parasitology, Melbourne, Australia. (Oral)
• Rina P. M. Wong, S. Salman, Kenneth F. Ilett, Ivo Mueller and Timothy M. E. Davis. Desbutyl-lumefantrine, a promising antimalarial drug. (2010) Combined Biological Sciences Meeting, Perth, Western Australia. (Poster: Best Postgraduate Poster Award)
• Rina. P. M. Wong – Three Minute Thesis Oration, Resistance of Plasmodium falciparum to antimalarials in Papua New Guinea. (2010) The Australian Society for Medical Research, Medical Research Week, Scientific Symposium, Perth, Australia (Song).
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• Rina. P. M. Wong – Three Minute Thesis Oration, Multi-resistant malaria: drugs, genes and sick babies. (2010) Three Minute Thesis Competition: The University of Western Australia, Perth, Australia (Finalist).
• Rina. P. M. Wong and Timothy. M. E. Davis. Fenofibric acid, metabolite of fenofibrate is a promising, novel antimalarial drug. (2011) Australian Society for Parasitology Annual Conference, Carins, Queensland, Australia. (Oral: Best Student Oral Prize).
• Rina. P. M. Wong, Gavin. R. Flematti and Timothy M. E. Davis. Detection of volatile organic compounds produced by Plasmodium falciparum in culture. (2011) The Australian Society for Medical Research, Medical Research Week, Scientific Symposium, Perth, Australia. (Oral).
• Rina. P. M. Wong and Timothy M. E. Davis. Lipid-modifying drugs as novel antimalarial therapy. (2011) BIT’s 1st Annual World Congress of Microbes: 1st Annual Symposium of Antiparasites, Beijing, China. (Oral, submitted by Invitation).
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ABSTRACT
Malaria remains a significant global health problem. Plasmodium falciparum, the
predominant and most virulent infecting species, has developed resistance to most
antimalarial drugs. Drug sensitivity is monitored by i) in vivo (clinical) outcome, ii) in
vitro response of cultured parasites to a range of drug concentrations, and iii) presence
of resistance-associated molecular markers. Few studies have integrated these
approaches which can all contribute to the development of treatment regimens that
improve clinical outcome and delay spread of resistance.
Recent clinical studies have shown high rates of treatment failure in Papua New Guinea
(PNG), necessitating a proposed change from chloroquine (CQ) or amodiaquine (AQ)
plus sulfadoxine-pyrimethamine (SP) to artemisinin combination therapy (ACT). The in
vitro sensitivity of 64 P. falciparum isolates from Madang Province to CQ, AQ,
4.3.1 Pfmdr1 LDR-FMA Development ..................................................................................... 113 4.3.1.1 PCR Optimisation...........................................................................................................................113 4.3.1.2 LDR Optimisation ..........................................................................................................................118 4.3.1.3 Optimised LDR-FMA for pfmdr1 and Multiplexed Detection of SNPs in pfdhfr, pfdhps and pfcrt
4.3.2.1 Comparison between LDR-FMA and RFLP speciation .................................................................124 4.3.2.2 Inter-assay concordance .................................................................................................................124 4.3.2.3 Identification of drug resistance alleles ..........................................................................................125
4.3.3 Field Application of the LDR-FMA ................................................................................. 126 4.3.3.1 Speciation and drug resistance genes in PNG field isolates............................................................126 4.3.3.2 Prevalence of polymorphic alleles in pfcrt, pfmdr1, pfdhfr and pfdhps .........................................127 4.3.3.3 Parasite drug resistance mutations and treatment outcome.............................................................130
Drug Half-life Resistance to chlorproguanil-dapsone (short half-life) develops more slowly than that to SP (long
half-life)^.
Dosing Use of subtherapeutic doses in self-treatment such as with antifolate drugs in Thailand in the 1970s; poor drug compliance; mass drug administration with subtherapeutic doses; use of chloroquinised
salt** .
Non-target drug pressure
Presumptive use of antimalarial drugs without laboratory diagnosis or for indications other than
malaria#,҂.
Pharmacokinetics Use of drug formulations with reduced bioavailability*.
Cross-resistance SP and sulfamethoxazole-trimethoprim*.
Quality Poor manufacturing practices with substandard content of active ingredients, intentional
counterfeiting, deterioration due to storage and handling^^.
Human Host immunity Non-immune, migrant gem-miners and resistance to mefloquine on the Thai-Cambodian border*.
Health Malnourished and HIV infected individuals have significantly poorer parasitological response٭.
Maintenance of resistant parasite
reservoir
Non-detection of drug failure*.
Parasite Genetic mutations Polymorphisms in the genes: pfcrt, pfmdr1, pfdhps, pfdhfr, pfserca*.
Transmission level
Whether low or high transmission has more influence on drug resistance is debatable;
prevalence of drug resistance is higher in regions of low transmission, whereas a model suggests the
benefits of transmission control in delaying resistance development§.
Vector and environment
Mosquito affinity of parasites
Increased infectivity and productivity of CQ-resistant parasites in Anopheles dirus and the
propagation of CQ resistance in South-East Asia and Western Oceania*.
Chapter 1 General Introduction
42
The interactions between parasite genetic polymorphisms have been identified to confer
or modulate antimalarial drug resistance. These involve genes that encode membrane
transporter proteins such as pfcrt and P-glycoprotein homologue 1 (pfmdr1) associated
with 4-aminoquinoline resistance. Mutations in the enzymes dihydrofolate reductase
and dihydropteroate synthase involved in folate synthesis, decreases sensitivity to
pyrimethamine and sulfadoxine respectively. The involvement of parasite genetics and
drug resistance are discussed in Section 1.18.
P. falciparum strains that already demonstrate resistance to a number of antimalarials
display a level of genetic plasticity that enables them to rapidly adapt to a new drug not
chemically related (Rathod et al. 1997; Nzila et al. 2010). Early reports using rodent
malaria have shown a strain resistant to one drug is more prone to give rise to resistant
lines to another drug, compared to strains that are fully susceptible (Powers et al. 1969;
Peters et al. 1976). Similar observations are evident during in vitro induction of
resistance, where the chance of generating parasite resistant lines increases with the
number of drug-resistant phenotypes. For instance, the ease of generating parasite
resistant line against atovaquone was greatest using the culture-adapted P. falciparum
strains W2 (cycloguanil, pyrimethamine and sulfadoxine resistant), followed by FCR3
(pyrimethamine and cycloguanil resistant), then 3D7 (sulfadoxine resistant) and the
least in the fully drug-susceptible D6 strain which failed to generate any drug resistant
parasite line (Rathod et al. 1997). This phenomenon is termed ‘accelerated resistance to
multidrug (ARMD)’, which is different to the known multidrug-resistant phenotype
(Rathod et al. 1997). The occurrence of ARMD may be due to low efficiency of DNA
repair mechanisms (Trotta et al. 2004), which can be attributed to the high mutation rate
during parasite multiplication. Hence, under drug pressure, parasites with the ARMD
phenotype have higher ability to produce a drug-resistant clone.
1.14.5 Mechanism of Resistance to 4-Aminoquinolines and Arylaminoalcohols
Much evidence attributes the activity of CQ to its capacity to concentrate itself from nM
concentrations in the extra-cellular environment to mM levels within the parasite food-
vacuole (Bray et al. 1998; Le Bras et al. 2003). Resistant parasites are found to
accumulate CQ less efficiently (Saliba et al. 1998). Verapamil is a modulator of P-
Chapter 1 General Introduction
43
glycoprotein in mammalian cells expressing multidrug resistance. The observation that
CQ resistance is reversible by verapamil has led to the discovery of an analogous efflux
protein in the food vacuole of P. falciparum. Mutations in the corresponding gene, the
pfmdr1 gene has been associated with in vivo CQ resistance which modulates in vitro
resistance (Foote et al. 1990; Reed et al. 2000; Nagesha et al. 2001). It also plays a
significant role in the parasite’s sensitivity to structurally related compounds such as
quinine and AQ. Genetic and field studies have linked parasite possession of the wild
type pfmdr1 gene and its amplification is associated with increased resistance to MQ,
lumefantrine, halofantrine and artemisinin (Price et al. 2004; Sidhu et al. 2006;
Chavchich et al. 2010). Interestingly, there is an inverse relationship between parasite
sensitivity to CQ and MQ (Duraisingh et al. 2005). Mutations in the pfcrt gene which
encodes for another transmembrane protein in the parasite digestive vacuole also confer
CQ resistance. Specific polymorphisms encoding for resistance in the pfmdr1 and pfcrt
genes are discussed in Section 1.18.
1.14.6 Mechanism of Resistance to Antifolates
Antifolate combination drugs such as SP act via sequential and synergistic inhibition of
two key enzymes involved in parasite folate synthesis. Dihydrofolate reductase and
dihydropteroate synthetase are encoded by the P. falciparum dihydrofolate reductase
(pfdhfr) and P. falciparum dihydropteroate synthase (pfdhps) genes respectively.
Mutation in pfdhfr reduces its affinity to pyrimethamine or related compounds, whereby
inhibition is attenuated (Le Bras et al. 2003). Similarly, mutation in the pfdhps is
associated with sulfadoxine resistance (Wang et al. 1997). Specific combinations of
mutations in both pfdhfr and pfdhps have been associated with varying degrees of
antifolate resistance; these are discussed in more detail in Section 1.18.
1.14.7 Mechanism of Resistance to Artemisinin and Derivatives
Although a number of putative targets have been proposed, the exact mechanisms of
action of artemisinin remain uncertain (O'Neill et al. 2010). Chavchich et al have
selected parasite lines that are resistant to artemisinin and artelinic acid under
Chapter 1 General Introduction
44
continuous drug pressure (Chavchich et al. 2010). The changes in parasite susceptibility
were accompanied by concomitant increase in pfmdr1 gene copy number and protein
expression. In additional to these molecular changes, the authors reported a reduction in
parasite sensitivity to MQ, quinine, lumefantrine and halofantrine, in concordance with
field observations (Sidhu et al. 2006). Besides pfmdr1 gene copy number, the
acquisition of artemisinin tolerance has been associated with parasite developmental
arrest and changes in transcriptomic modifications as a result of drug pressure
(Witkowski et al. 2010).
Studies of the cytotoxic effects of artemisinin suggested its mechanism of action may
be via interactions between the artemisinin endoperoxide bridge and haem-iron
(Kannan et al. 2005). Subsequent production of alkylated haem derivatives of
artemisinin (i.e. haemarts) has been proposed to cause parasite death due to its
interference with haemazoin formation as well as harmful effects due to free radicals
(Kannan et al. 2005). This hypothesis is consistent with studies demonstrating that
artemisinin activity can be enhanced by oxidising agents and attenuated by reducing
agents (Krungkrai et al. 1987). However, this theory has been challenged since
artemisinin is active against ring-stage parasites that do not harbour high concentrations
of haem (Olliaro et al. 2001). Wu et al proposed that artemisinin is activated on the
reductive cleavage of the peroxide bond by iron-sulfur redox centres common to
Plasmodium enzymes. As a result, alkylation of these enzymes may be responsible for
parasite death (Wu 2002a). This hypothesis is supported by the interactions between
radiolabelled artemisinin and various parasite proteins, highlighting the possibility that
parasite death may be due to endogenous protein alkylation and inactivation
(Bhisutthibhan et al. 2001). Some of the proposed target proteins for artemisinin
include those involved in the electron transport chain, cysteine protease, translationally
controlled tumour protein, and pfATP6 (Eckstein-Ludwig 2003; Li 2005). The latter is a
SERCA-type calcium ATPase where field and in vitro evidence show parasites
expressing the L263E mutation in pfATP6 have decreased sensitivity to artemisinin and
its derivatives (Uhlemann 2005; Krishna et al. 2006; Fidock et al. 2008).
Chapter 1 General Introduction
45
1.15 IN VITRO DETECTION OF RESISTANCE IN P.
FALCIPARUM
Vigilant detection and monitoring of antimalarial resistance is prudent for ensuring the
best choice of treatment within a given locality. In vitro testing of parasite susceptibility
is a valuable tool for resistance surveillance to complement clinical trials which are
much more time and resource-consuming. In vitro susceptibility testing involves the
short term culture of parasites isolated from an infected individual, and determining the
level of growth inhibition after exposing them to various drug concentrations.
Sensitivity is usually measured in terms of the concentration of drug required to inhibit
growth by 50% (IC50) (Rieckmann et al. 1978; WHO 2001; Noedl et al. 2007). The IC50
is subsequently compared against a threshold value for in vitro resistance, which is
uniquely determined for each drug. In the example of CQ for which the in vitro
resistance threshold is 100 nM, the value was determined by comparing IC50 values
obtained from 11 geographically distinct culture-adapted and field isolates (Cremer et
al. 1995). Most in vitro drug resistance thresholds were selected without information on
clinical outcome hence may not directly predict in vivo resistance (Ekland et al. 2008).
Nevertheless, IC50s have been used extensively as an international currency in the
assessment of drug susceptibility from different geographic regions.
The first in vitro drug sensitivity test was published in the late 1960s where infected
blood samples were treated with CQ, QN and cycloguanil and the extent of parasite
maturation in the presence of these drugs was assessed (Rieckmann et al. 1968). This
marked the beginning of short-term in vitro culture of malaria parasites and continuous
culture methods were described shortly after (Trager et al. 1976). Based on the new
milestones in malaria culture techniques, assessments of drug inhibition against all
developmental stages of parasites using 48 or 96 hr assays were developed (Trager
1978; Richards et al. 1979). These methods involved longer test periods which enabled
the testing of slower-acting antimalarials. Further development over the next few
decades produced diverse approaches including visual examination and counting of
mature parasite stages, determination of parasite enzyme activity, and sophisticated
assays that quantify the amount of newly synthesised DNA during parasite development
Chapter 1 General Introduction
46
by radio-isotope labelling (Rieckmann et al. 1978; Desjardin 1979; Makler et al. 1993a;
Radfar et al. 2009). The following sections provide an overview of the main types of in
vitro assays used for the assessment of P. falciparum growth inhibition in response to
different drug doses.
1.15.1 Schizont Maturation
1.15.1.1 Macro test
The WHO macro in vitro test is based on maturation of trophozoites and formation of
schizonts in the presence of different concentrations of drugs (WHO 1978). This assay
is supplied in a kit, primarily developed for the field assessment of P. falciparum
susceptibility to CQ. Briefly, 8 mL of venous blood are collected and transferred into a
sterile Erlenmeyer flask (25 mL) containing glass beads and stoppered. The sample is
defibrinated by physical rotation for 5 min. Into each test vial, 1 mL of blood is
aliquoted in the specified sequence for areas with suspected resistance: 2 control vials,
CQ vials containing 0.5, 1, 1.5, 2, 3, 0.25, 0.75, 1.25 nM and, for areas with no prior
indication of resistance 2 control vials, CQ vials containing 0.5, 1.0, 0.75, 0.25, 1.5, 2,
1.25, 3 nM. This sequence of testing increases the chance of parasite sensitivity being
assessed in the optimal range of drug concentrations in case of inadequate sample
volume. After gentle mixing the vials are closed and incubated in water at 38.5°C for 24
- 28 hr. Thick and thin blood smears are prepared after incubation, and the percentage
of schizonts in test wells relative to those of control wells are determined (Dulay et al.
1987).
The macro in vitro method is simple to use with little specialised equipment required. In
view of the fact that there are limited resources in malaria endemic areas, it has been
useful in field settings (Cattani et al. 1986). One limitation however, is the need for a
large-volume blood sample and waterbath space. Many large scale studies employ a
micro technique as a result (Rieckmann et al. 1978).
Chapter 1 General Introduction
47
1.15.1.2 Micro test
A scaled down modification of the macro test commonly known as the Rieckmann
microtechnique enables the assessment of parasite drug sensitivity using a small amount
of blood (Rieckmann et al. 1978). Briefly, finger-prick blood samples (100 µL) are
diluted 1:10 with complete culture medium and dispensed (50 µL) into 96-well plates
provided by the WHO that are pre-dosed with drugs at final concentrations from 0.25 to
16 pmol/well. Test plates are incubated in a candle jar to achieve a microaerophilic
atmosphere at 37°C for 24 to 36 hr depending on the rate of maturation in control wells.
Thick smears are subsequently prepared and the number of schizonts, which is defined
as intra-erythrocytic parasites with 3 or more nuclei, per 200 asexual parasites is
counted. Sensitivity is reported as the highest concentration of drug in which
schizogony occurred.
The in vitro microtechnique has been widely employed in field studies due to its
simplicity and requirement for minimal specialised equipment and technical personnel
(Kouznetsov et al. 1980; Trenholme et al. 1993; Noedl et al. 2001; Menezes et al.
2002). With further refinement, the microtechnique (Mark III) is now the WHO
standard assessment of P. falciparum antimalarial drug susceptibility (WHO 2001).
Despite these advantages, a number of limiting factors should be considered. Since the
sample is used directly in the test system, the parasite density in the inoculum
influences the susceptibility outcome (Kouznetsov et al. 1980; Ponnudurai et al. 1981).
Thaithong et al evaluated the effect of initial parasitaemia on parasite survival in the
presence of 5 antimalarials. The authors found that the actions of CQ, AQ, MQ and
quinine were significantly reduced in the presence of parasitaemia >1% (Thaithong et
al. 1983). In addition, the WHO in vitro micro kit only caters for a single test per
sample and not duplicates or triplicates as in the isotope incorporation assay and
Plasmodium lactate dehydrogenase measurement (Desjardin 1979; Makler et al.
1993b). In practical terms this is advantageous as schizont counting is labour intensive
and time consuming, especially when the method already requires the counting of 8
thick films per sample to determine sensitivity outcome. Another consideration
important for data interpretation is that schizont enumeration by microscopy tends to
Chapter 1 General Introduction
48
produce IC50 values that are two to three times higher than by the isotopic method
(Wernsdorfer et al. 1988).
1.15.2 3H-Hypoxanthine Incorporation Assay
The isotopic assay utilises the parasite’s requirement for hypoxanthine as a nucleic acid
precursor during its development (Desjardin 1979). The assay is applicable for both
field isolates and culture-adapted samples as detailed in Section 2.2.5. The isotopic
assay provides a semi-automated technique to assess drug susceptibility. It is
reproducible and sensitive, and is considered the reference method for drug sensitivity
assays (Makler et al. 1993b; Druilhe et al. 2001). Numerous in vitro studies have used
this method, and have circumvented the need for specialised equipment in the field by
transporting samples to a centralised laboratory for testing (Basco et al. 1998; Pradines
et al. 2006). This approach has proven successful in various African countries where
the high throughput assay enables more effective monitoring of resistance epidemiology
(Nzila-Mounda et al. 1998; Pradines et al. 1999a; Basco et al. 2003a; Jambou et al.
2005). However, the use of radioisotope involves high costs for consumables and
laboratory infrastructure, requiring supporting facilities such as safe disposal of
radioactive waste and reliable couriers. Technical training of research personnel to
perform meticulous handling of unsealed radio-isotopes is usually unavailable in
developing countries such as PNG. The high costs and technical constraints have
hindered the implementation of the isotopic method in many malaria-endemic
countries. Nonetheless, strategic collaboration between developing and developed
countries should enable the use of this sensitive method for the prudent ‘gold standard’
Plasmodium lactate dehydrogenase (pLDH) is the terminal enzyme of the anaerobic
Embden-Meyerhoff glycolytic pathway and is essential for energy production in
malaria parasites (Sherman 1979). Early interest in using pLDH as a marker for
parasitaemia and parasite growth stem from the favourable characteristics of the
enzyme. PLDH can be distinguished from human LDH based on its ability to rapidly
Chapter 1 General Introduction
49
utilise 3-acetyl pyridine adenine dinucleotide (APAD) as a coenzyme to convert lactate
to pyruvate at a rate 200-fold more effectively than the human isoforms (Sherman 1961;
Gomez et al. 1997). In addition, the clearance of pLDH from plasma is rapid (3 - 5
days) and correlates with parasitaemia in vitro and in vivo (Makler et al. 1993a). The
pLDH level declines rapidly when parasites are no longer metabolically viable (Piper et
al. 1999). These biochemical features constitute a sensitive and specific marker for the
determination of parasite growth. It has been applied to various assay formats for
assessing in vitro drug susceptibility as described in Section 2.2.4.
1.15.3.1 Colourimetric pLDH microtests
The original drug sensitivity assay using pLDH was described by Makler et al (Makler
et al. 1993a). The assay follows a similar set up for parasite drug exposure in the
isotopic assay, as described in Section 2.2.4. The reaction is allowed to develop at room
temperature (RT) and the consequential change in colour can be monitored visually or
measured spectrophotometrically at 650 nm (Figure 2.6). PLDH activity can also be
determined kinetically at 30 sec intervals for 30 min by the formation of reduced
APAD. The enzymatic approach has been successfully used in field studies (Makler et
al. 1993a; Basco et al. 1995; Wong et al. 2010). Inhibition profiles and IC50s obtained
by pLDH are comparable to those determined by the isotopic and microscopic assay
(Makler et al. 1993; Basco et al. 1995). For optimal sensitivity, however, the enzymatic
assay requires an initial parasitaemia between 1 and 2% at 1.5% haematocrit (hct), with
a detection limit of 0.4% parasitaemia. This range of initial parasitaemia is often too
high for most field isolates from sub-Saharan Africa where patients with acute
uncomplicated falciparum malaria usually present with parasitaemia <1% (Basco et al.
1995). The method is therefore not sensitive enough as a diagnostic method.
Nonetheless, it has been employed in monitoring therapeutic efficacy of drug treatments
in Chinese patients infected with P. falciparum and P. vivax (Wu et al. 2002b). A
modification to the colourimetric assay is the use of sodium-2,3-bis-[2-methoxy-4-
nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilid (XTT) in place of NBT and the
reaction is followed by optical density (OD) measurement at 450 nm (Delhaes 1999).
However, this method requires even higher initial hct and parasitaemia, therefore, offers
Chapter 1 General Introduction
50
no advantage over the unmodified version.
1.15.3.2 Immunocapture of pLDH
Other variations based on immunocapture of pLDH have been developed with the use
of monoclonal antibodies (Makler et al. 1998; Kaddouri et al. 2006; Mayxay et al.
2007). This approach significantly improves assay sensitivity, as it is less prone to non-
specific reduction of NBT in the haemolysate and in the reagent mixture (Knobloch et
al. 1995; Oduola et al. 1997). In a Nigerian clinical study, Oduola et al (1997)
developed an enzyme-linked immunosorbent assay (ELISA) that combines the use of
antibody capture technique with APAD to enhance sensitivity and specificity of pLDH
detection. The immunocapture pLDH (IcpLDH) assay uses 96-well plate coated with
mouse monoclonal antibody specific to pLDH, to which the blood sample is added.
During incubation, pLDH is captured on the plate by antibodies, whilst non-specific
contents are washed off. NBT is subsequently added and end point absorbance is
measured. The authors observed a specific and immediate relationship between
dissipation of pLDH enzyme activities and resolution of infection in vivo (Oduola et al.
1997; Piper et al. 1999). Further refinement of this technique led to the development of
a double-site enzyme-linked pLDH immunodetection (DELI) assay (Moreno et al.
2001). Field trials in Thailand, Laos and Senegal demonstrated the DELI microtest to
be highly sensitive, allowing for the inclusion of isolates with parasitaemia as low as
0.005% (Moreno et al. 2001; Brockman et al. 2004; Mayxay et al. 2007). The IC50s
obtained using the DELI approach correlated well with the isotopic test, showing
similar proportions of drug resistant and sensitive isolates. It is also easier and faster to
implement than the isotopic test, and does not require sophisticated equipment (Moreno
et al. 2001; Kaddouri et al. 2008). A current drawback to its implementation is that
monoclonal antibodies towards pLDH are not commercially available, with its use
limited to collaborating laboratories. Overall, immunocapture pLDH colourimetric
assays are cost-effective and straight forward to perform, with great potential for world-
wide implementation for epidemiological monitoring of drug resistance.
Chapter 1 General Introduction
51
1.15.4 Histidine-Rich Protein II (HRP2) Assay
Histidine-rich protein II is a naturally occurring protein found in several cellular
compartments in the parasite including the cytoplasm. It has been implicated as an
important factor in the detoxification of haem and is readily recovered from plasma,
infected RBC membrane and culture supernatants (Howard et al. 1986; Sullivan et al.
1996; Lynn et al. 1999; Papalexis et al. 2001). The level of HRP2 in malaria cultures
increases with parasite development and multiplication (Desakorn et al. 1997), hence
making it an excellent indicator of parasite growth in response to antimalarial drugs. In
2002, a novel approach was described to assess drug sensitivity in Thai isolates by
measuring the level of HRP2 in an ELISA (Noedl et al. 2002). Briefly, parasitised RBC
samples were standardised (0.05% parasitaemia, 1.5% hct) and incubated with various
drug concentrations in 96-well plates similar to that in the pLDH assay. These were
subsequently frozen-thawed and the haemolysed samples transferred to commercial
ELISA plates pre-coated with mononclonal antibodies against HRP2. The plates were
incubated at RT for one hr and washed repeatedly to remove unbound content. Diluted
antibody conjugate was added to each well and incubated as previously. After several
washes, diluted chromogen was added for colour development in the dark for 15 min.
At the end of the reaction, a stop solution was added and OD measured at 450 nm for
IC50 determination (Noedl et al. 2002).
A number of field studies have implemented the new HRP2 microtest and found it
highly sensitive and simple to perform with results closely aligned to those obtained by 3H-hypoxanthine incorporation and the WHO schizont maturation test (Noedl et al.
2003; Attlmayr et al. 2005; Noedl et al. 2005). HRP2 assay is expensive to perform for
the purpose of in vitro drug susceptibility as three columns of the ELISA plates are
required for one drug to be tested in triplicate per sample. For multiple drug testing
required in large scale studies, the use of commercial kits (~AUD $100/plate) is costly
(Cellabs, Australia). More recently, an in-house HRP2 ELISA has been described using
commercially available monoclonal antibodies and is a cheaper alternative to using test
kits (~AUD $20/plate) (Noedl et al. 2005). The method requires additional coating of
test plates which is labour intensive and potentially introduces a bias if antibodies are
Chapter 1 General Introduction
52
not coated evenly across the wells. This in-house version produces similar results to
those by commercial kits (Noedl et al. 2005), and the availability of monoclonal
antibodies facilitates a more rapid implementation of a cost effective and sensitive in
vitro assay.
1.15.5 Dual Detection of HRP2 and PLDH
It is presently accepted that HRP2 levels reflect both past and current infections due to
its slow clearance (10 - 14 days) while pLDH levels reflect the current infection. The
concurrent measurement of these two bio-markers by means of a unified ELISA
approach has been recently described (Martin et al. 2009). The unified protocol enables
the direct comparison of both HRP2 and pLDH results and provide a more enhanced
assessment of parasite burden to include sequestered parasites, which would be
clinically relevant during pregnancy where microscopy can be unreliable (Martin et al.
2009).
1.16 ASSESSMENT OF ANTIMALARIAL DRUG
COMBINATIONS
Combination antimalarial therapies play a pivotal role in delaying the onset of
resistance to new agents and in reducing the effects of resistance to current agents
(White 1998). Effective combination drug regimens often achieve a therapeutic efficacy
greater than that achieved with monotherapy (Fivelman et al. 2004). In vitro drug
interaction studies provide essential information for the selection of optimal drug
combinations for further clinical trials. However, in vitro combination efficacy does not
necessarily translate to efficacy in vivo, as therapeutic efficacy is dependent on
pharmacokinetic characteristics of both drugs within the host (Fivelman et al. 2004).
The biological responses of two agents in combination can be assessed in vitro by the
construction of an isobologram, which graphically displays the effects of each agent
alone and in combination (Figure 1.22) (Berenbaum 1978; Czarniecki et al. 1984;
Chawira et al. 1987; Davis et al. 2006). The outcomes of drug interactions are either
synergistic, indifferent (no interaction) or antagonistic. The concentrations of agents,
Chapter 1 General Introduction
53
either in combination or alone, required to achieve 50% inhibition of parasite growth
are calculated and normalised to fractional inhibitory concentrations (FICs)
(Berenbaum 1978). The sum (Σ) of FICs can be calculated by the addition of FICs of
agents A and B, i.e. (IC50 of A in a mixture resulting in 50% inhibition/IC50 of A alone)
+ (IC50 of B in a mixture resulting in a 50% inhibition/IC50 of B alone) (Berenbaum
1978). When the ΣFIC equals 1.0, the combination is additive, or has no interaction. In
this case, the plotted points should fall close to the straight line drawn between the FICs
of 1.0 on the abscissa and ordinate (Figure 1.22). A ΣFIC of <1 indicates synergistic
interaction, the data points from which would form a concave isobole beneath the line
of additivity, and a ΣFIC of >1 is indicative of an antagonistic interaction represented
by a convex isobole (Berenbaum 1978; Chawira et al. 1987; Fivelman et al. 2004;
Davis et al. 2006). A more conservative interpretation of isobolographic results have
been recommended, where synergy is defined as ΣFIC values ≤ 0.5, antagonism as
ΣFIC values ≥ 4.0, and no interaction when ΣFIC >0.5 – 4.0 (Odds 2003).
Figure 1.22 Representation of isoboles.
The original checkerboard method by Berenbaum (1978) has been widely used to
evaluate antimalarial interactions (Hassan Alin et al. 1999; Skinner-Adams et al. 1999;
Gupta et al. 2002). An alternative fixed-ratio isobologram method formerly developed
for studies in bacteria has been applied for P. falciparum (Hall et al. 1983; Fivelman et
al. 2004; Wong et al. 2009). This newer approach demonstrated comparable findings
with the checkerboard method and is less labour intensive with fewer calculation steps
Chapter 1 General Introduction
54
(Fivelman et al. 2004). Both checkerboard and fixed-ratio methods require
predetermination of IC50 values of each drug alone. From these data, a starting
concentration for each agent is selected and drug mixtures are prepared in various ratios
of the initial concentrations and subjected to serial dilution. In the checkerboard
method, the concentration of agent A is fixed whilst that of agent B is varied and vice
versa. The fixed-ratio method on the other hand, uses serial dilutions of fixed ratios of
both agents, so that drug concentrations are varied at the same time over predetermined
sets of concentrations (Fivelman et al. 2004). The fixed-ratio method is more resilient
in terms of inter-day variations in IC50s where inaccurate initial IC50s may cause poor fit
of the sigmoidal growth response curve, resulting in clustering of data points at the
extremities of the isobole axes. In addition, dose-response curves from the fixed-ratio
approach are based on drug concentration ratios, each of which is constructed to range
from 100 to 0% parasite inhibition, thus enabling a more accurate regression curve fit
and IC50 determination (Fivelman et al. 2004).
1.17 IN VIVO DETECTION OF DRUG RESISTANCE IN P.
FALCIPARUM
The level of P. falciparum resistance to antimalarial drugs is often assessed by
therapeutic response (Wongsrichanalai et al. 2002). In vivo response involves the
assessment of clinical and parasitological response over a period of time post-treatment
(WHO 2006). Parasitological responses are classified by the clearance of parasitaemia
and are graded as sensitive (S) and three levels of resistance (RI, RII, and RIII) (Table
1.3). This classification system, though remaining valid in areas with low transmission,
may be difficult to apply in areas with intensive transmission, where new infections and
recrudescences cannot be differentiated on the basis of microscopy and complicates the
outcome (Wongsrichanalai et al. 2002). More recently, the WHO introduced a modified
system based on clinical symptoms and the level of parasitaemia (Table 1.3) (Bloland
2001). The previously established follow-up period of 14 days is considered insufficient
as a significant proportion of recrudescence often appeared after this period and shorter
observation periods have led to the overestimation of treatment efficacy (WHO 2006).
The current recommended duration of follow-up is a minimum of 28 days for most
antimalarial drugs in areas of intense as well as low to moderate transmission. Extended
Chapter 1 General Introduction
55
follow-up periods of 42 days and 63 days are recommended for slowly eliminated drugs
(i.e. lumefantrine and MQ, respectively) to effectively capture recrudescences (WHO
2006). Clinical studies however are costly and often confounded by poor compliance,
difficulty with recruitment and patient follow-up particularly in remote villages (Han et
al. 1976; Karunajeewa et al. 2008b).
Table 1.3 Classifications of in vivo antimalarial susceptibility outcomes. (Talisuna et
Clearance of parasites after treatment without subsequent recrudescence within a defined period
Adequate clinical response (ACR)
Absence of parasitaemia on day 14, irrespective of fever status, without previously meeting any of the criteria for ETF or LTF
Absence of fever irrespective of the parasitaemia status without previously meeting any of the criteria for ETF or LTF
RI parasitological failure Initial clearance followed by recrudescence after day 7
Early treatment failure (ETF) Danger signs or severe malaria on day 1, 2, or 3 in the presence of parasitaemia Fever (axillary temperature, ≥37.5°C) persists on day 2 and the parasite density is greater than that on day 0. Fever and parasitaemia on day 3 Parasite density on day 3 is ≥25% of the day 0 parasite density
RII parasitological failure
Reduction of parasitaemia on day 2 to less than 25% of day 0 parasitaemia, but no complete clearance
RIII parasitological failure
On day 2, either no reduction of parasitaemia or reduction to a level equal to or greater than 25% of the day 0 parasitaemia
Late treatment failure (LTF) Danger signs or severe malaria develop in the presence of parasitaemia on any day from day 4 to day 14 Fever and parasitaemia on any day from day 4 to day 14, and yet the patient could not be classified as ETF
Chapter 1 General Introduction
56
1.18 MOLECULAR MARKERS OF DRUG RESISTANCE
Recent advances have provided powerful molecular tools to detect drug resistance
(Ekland et al. 2007). Molecular methods are renowned for their rapid quantitative
assessment for genetic markers of drug resistance, providing an attractive and relatively
inexpensive alternative to clinical approaches for monitoring the prevalence and spread
of resistant parasites (Ekland et al. 2008). The sequencing and annotation of the P.
falciparum genome in 2002 has greatly enhanced the identification of gene candidates
as genetic markers of drug resistance (Gardner et al. 2002). Conserved polymorphisms
within the genome including microsatellites (repeats of short nucleotide sequence),
single nucleotide polymorphisms (SNPs), and small insertions or deletions, act as
surrogate markers for drug resistance determinants (Ekland et al. 2007). The P.
falciparum genome contains an estimate of 25,000 to 50,000 SNPs that tend to cluster
in blocks known as haplotypes surrounded by recombination hotspots (Mu et al. 2005).
Since the number of polymorphisms is different within different genes, and by tracking
a signature set of SNP tags, various haplotypes associated with drug resistance can be
identified (Mehlotra et al. 2001; Carnevale et al. 2007; Ekland et al. 2007).
The identification of pfcrt as the primary determinant in CQ resistance has enabled the
development of a simple PCR assay that identifies the presence of resistant strains
(Fidock et al. 2000). Mutations in the pfcrt gene, particularly the substitution of lysine
(K) to threonine (T) at residue 76 (K76T), is central to CQ resistance. The K76T
polymorphism is consistently found in CQ-resistant strains regardless of geographic
origin. It can occur within different amino acid haplotypes between residues 72 to 76:
CVIET, CVMNT, CVMET, and SVMNT, all of which are associated with CQ
resistance (Fidock et al. 2000). Higher levels of CQ resistance can be attributed to
changes in the pfmdr1 gene in point mutation 86Y (Foote et al. 1990; Reed et al. 2000;
Babiker et al. 2001; Djimde et al. 2001; Pickard et al. 2003). Clinical resistance to AQ
has been associated with SNPs in pfcrt K76T, pfmdr1 N86Y, F184Y, D1246Y, in
particular the triple tyrosine pfmdr1 haplotype YYY at codons 86, 184 and 1246 has
been selected post AQ monotherapy (Humphreys et al. 2007; Tinto et al. 2008). The
predictive value of pfcrt and pfmdr1 polymorphisms for both CQ and AQ underscores
the similarity in their mode of action, and the predisposition of high level of CQ
Chapter 1 General Introduction
57
resistance in endemic regions may compromise the future use of AQ-artesunate as an
alternative ACT (Thwing et al. 2009). Allelic substitution experiments have also shown
both pfcrt and pfmdr1 polymorphisms contribute to quinine resistance (Reed et al.
2000; Sidhu et al. 2005). Another molecular determinant, the pfnhe1 gene located on
chromosome 13 which encodes a putative sodium/hydrogen exchanger was associated
with reduced quinine sensitivity in culture-adapted P. falciparum (Bennett et al. 2007).
Mutations in the pfmdr1 gene also confer resistance to multiple other antimalarials
including MQ, lumefantrine, halofantrine and quinine (Cowman et al. 1994; Peel et al.
1994; Reed et al. 2000). In a pfmdr1 gene allelic replacement study, Reed et al (2000)
demonstrated pfmdr1 mutations altered artemisinin susceptibility in parasite lines from
PNG (D10) and South America (7G8). This finding has serious implications for future
artemisinin effectiveness in endemic areas such as PNG. Polymorphic changes in the
pfdhfr and pfdhps genes encoding dihydrofolate reductase and dihydropteroate synthase
respectively are good indicators for pyrimethamine and sulfadoxine resistance. The
S108N mutation in pfdhfr has been implicated in pyrimethamine resistance with
additional mutations at positions 16, 50, 51, 59, 140 and 164 contributing to elevated
levels of resistance (Cowman et al. 1988; Peterson et al. 1988). Similarly, the A437G
mutation in pfdhps confers initial resistance to sulfadoxine. Other mutations including
K540E, A581G, S613A, S436A/F lead to higher resistance (Triglia and Cowman 1994;
Triglia and Cowman 1999). Table 1.4 provides a summary of molecular markers
associated with drug resistance.
Chapter 1 General Introduction
58
Table 1.4 Molecular markers for antimalarial drug resistance. (Triglia et al. 1994;
P. falciparum multiplication correlates with a rise of pLDH activity thus this can be
used to assess parasite growth in response to drug treatment (Roth 1988; Makler et al.
1993a). A modification of the colourimetric method was used (Makler et al. 1993b;
Wong et al. 2010). PLDH is produced as a terminal glycolytic enzyme that converts
lactate to pyruvate in the presence of NAD+ (Figure 2.5). Activities of Plasmodium and
human isoforms can be distinguished by using 3-acetyl pyridine adeninedinucleotide
(APAD), an analogue of NAD+ that is specifically utilised by parasite LDH. Colour
intensity was then measured by absorbance. Inclusion of diaphorase in the solution
amplifies colour production (Figure 2.5).
Figure 2.5 Colourimetric detection of pLDH activity. As the reaction proceeds, the
APADH generated reduces nitro blue tetrazolium (NBT), a yellow compound, to nitro
blue formazan (NBF), a purple compound.
2.2.4.2 Assay Set Up
After incubation, the drug susceptibility plates were subjected to three cycles of freeze-
thawing to facilitate the release of pLDH. The haemolysate was homogenised by
pipetting up and down with a multichannel pipette. A 10 μL sample from each well was
added to 200 µL of Malstat solution (Appendix B), 10 µL of NBT solution (Appendix
B) and 10 µL of diaphorase solution (Appendix B). The pLDH reaction was allowed to
proceed at RT for 45 to 90 min to allow development of colour within the wells.
Chapter 2 Methods and Materials
71
Interference by air bubbles was circumvented by directing a blow-dryer over the plate.
Colour intensity was measured by absorbance at 650 nm (FLUOstar OPTIMA, BMG
biotechnologies) and the data were analysed by non-linear regression (Graphpad Prism
4.0) to construct dose-response curves for determination of drug-specific IC50.
Figure 2.6 pLDH reaction in field isolates of P. falciparum.
2.2.5 3H-Hypoxanthine Incorporation Assay
Hypoxanthine is required for DNA synthesis during parasite replication. The radio-
activity of the incorporated tritium (3H) reflects parasite sensitivity to antimalarial
compounds (Desjardin 1979). Briefly, drug susceptibility assays were set up at a final
mixture of 0.5% parasitaemia at 1.5% hct as previously described (Section 2.2). Each
well consisted of 90 µL of RBC suspension, 100 µL of drug diluted in media and 10 µL
of a 3H-hypoxanthine working solution (5 mg/mL) (Appendix B), resulting in a final
concentration of 0.5 µCi per well. After incubation, the plates were subjected to three
cycles of freeze-thawing and harvested onto a 96-well glass-fibre filtermat (Perkin
Elmer) using a Harvester 96 (Tomtec Incorporated, USA) (Figure 2.7). The filtermat
Chapter 2 Methods and Materials
72
was air-dried and sealed in a plastic envelope with 4 mL beta scintillant (Perkin Elmer)
and counted on a Wallac microbeta liquid scintillation counter (1450 Microbeta Plus).
The data generated were analysed by non-linear regression analysis (Graphpad Prism
4.0).
Figure 2.7 Tomtec Harvester 96 system.
2.3 MOLECULAR TECHNIQUES
The multiplex PCR ligase techniques for the detection of Plasmodium species and
genetic markers for drug resistance were recently developed by collaborators at Case
Western Reserve University (McNamara et al. 2004; Carnevale et al. 2007) (Figure
2.8). This platform enables high-throughput, sensitive and simultaneous diagnosis of
infection by different Plasmodium species. In a similar assay, this approach can
simultaneously detect a large number of SNPs from the pfcrt, pfdhfr, pfdhps, and
pfmdr1 genes that are associated with malarial drug resistance.
Chapter 2 Methods and Materials
73
2.3.1 DNA Extraction
P. falciparum DNA was isolated from whole blood from study participants or parasite
cultures using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s
protocol. The resulting DNA extracts (~200 µL) were stored at -20°C.
Figure 2.8 Wolstein Research Building, CWRU, Cleveland, Ohio, USA.
2.3.2 Polymerase Chain Reaction (PCR)
2.3.2.1 PCR for Plasmodium Species
A small-subunit ribosomal RNA gene fragment (491 to 500 bp) was amplified for
Plasmodium species diagnosis using oligoprimers and conditions previously described
(Mehlotra et al. 2000; McNamara et al. 2004). Briefly, PCR plates (ThermoGrid™ C-
18096, Denville Scientific Inc, USA) were irradiated with ultraviolet light in a
Stratalinker 2400 UV Crosslinker (Stratagene, CA, USA) before use to destroy any
Chapter 2 Methods and Materials
74
residual nucleic acid. Each well contained 25 µL of PCR master mix solution
(Appendix B) containing 67 mM Tris-HCl (pH 8.8), 6.7 mM MgSO4, 16.6 mM
(NH4)2SO4, 10 mM 2-mercapto-ethanol, 100 µM of dNTPs (Appendix B), 2.5 units of
thermo-stable DNA polymerase and 3 µL of genomic DNA sample. Sequences for
Plasmodium genus specific upstream and downstream primers were 5’-TTC AGA TGT
CAG AGG TGA AAT TCT-3’ and 3’-AAT TAG CAG GTT AAG ATC TCG TTC-3’
respectively (Integrated DNA Technologies, Iowa). The plates were sealed using
Microseal® ‘A’ film (Bio-Rad, USA) and mixed by centrifugation (3000 rpm for 30
sec) and amplification reactions were performed in a PTC-225 Peltier Thermal Cycler
(MJ Research, Iowa). The specific thermocycling conditions used were 92°C for 2 min
(1 cycle), 92°C for 30 sec and 63°C for 2 min (35 cycles), 63°C for 5 min (1 cycle)
(McNamara et al. 2004). PCR amplicons were stored at -20°C until assayed.
2.3.2.2 PCR for pfcrt, pfdhfr and pfdhps genes
The amplification of target sequences for P. falciparum pfcrt, pfdhfr, and pfdhps were
achieved using oligoprimers as described by Carnevale et al (2007). Primers and
thermocycling conditions for the amplification of pfcrt and pfdhfr were optimised to
eliminate the necessity of performing nested reactions (Table 2.2). The upstream and
downstream primers listed in (Table 2.2) were used to prepare master mixtures
(Appendix B) for each drug resistance gene.
2.3.2.3 Controls
Laboratory-adapted P. falciparum strains were obtained from the Malaria Research and
Reference Reagent Resource (MR4; ATCC, VA) and the haemolysate of various strains
(HB3, Dd2, 3D7, K1, 7G8, VS/1 and FCB) were kindly provided by Dr. Peter
Zimmerman (CWRU, Cleveland, Ohio, USA). DNA extracts from seven strains of P.
falciparum were included in each PCR run as batch controls in the Plasmodium species
and drug resistant SNPs assays. Distilled water used in the PCR reaction served as a
negative control for each amplification assay.
Chapter 2 Methods and Materials
75
Table 2.2 PCR primer sequences and thermocycling conditions for pfcrt, pfdhps
and pfdhfr target sequences. Conditions for pfdhfr and pfcrt (Carnevale et al. 2007)
were optimised to eliminate the necessity for performing nested reactions. apfcrt for
SNPs at codons 72 to 76. bdhfr fragment for SNPs at codons 51, 59, 108 and 164. cpfdhps gene fragment for SNPs at codons 540, 581, 613. dPCR programs were
preceded by an initial denaturation step at 95°C for 2 min.
Gene PCR Primer Sequence Thermocycling Condition
d
pfcrt a 5’ -TAATACGACTCACTATAGGGCCGTTA-3’ 5’-ATTAACCCTCACTAAAGGGACGGATG-3’
35 cycles of 95°C for 30 sec, 56°C for 30 sec, 60°C for 1 min
pfdhfr b 5’-TAACTACACATTTAGAGGTCTA-3’
5’-GTTGTATTGTTACTAGTATATAC-3’
35 cycles of 95°C for 30 sec, 56°C for 30 sec, 72°C for 1 min
pfdhps c 5’-AATGATAAATGAAGGTGCTAGT-3’
5’-ATGTAATTTTTGTTGTGTATTTA-3’
35 cycles of 95°C for 30 sec, 56°C for 30 sec, 60°C for 1 min
2.3.3 Detection of Amplified Products
To evaluate amplification efficiency, 5 µL of PCR product were premixed with 3 µL of
loading dye buffer and loaded (5 µL) on 2% agarose I gels (Appendix B). For a 96-well
gel, electrophoresis was performed at 290V for 30 min. The gel was subsequently
stained in SYBR Gold (Molecular Probes, Eugene, Oreg.) diluted 1:10,000 in 1 X TBE
buffer (Appendix B) on a rocking platform for 20 min and DNA products were
visualised on a Storm 860 PhosphorImager coupled with Image-Quant version 5.2
software (Molecular Dynamics, CA) (Figure 2.9).
Chapter 2 Methods and Materials
76
Figure 2.9 Electrophoresis and Image processing for DNA visualisation for
v:v in TMAC (20 ng/µL) as it binds to the 3’biotin on the conserved sequence primers
at 37°C for 35 - 45 min in 96-well V-bottom plates (Costar 6511 M polycarbonate,
Corning Inc., Corning NY).
Chapter 2 Methods and Materials
79
Table 2.4 LDR primer sequences for drug resistance markers pfcrt, pfdhfr and
pfdhps. aLowercased nucleotides (24 bases) represent tag sequences added to the 5’
ends of each allele-specific LDR primer. bID, identification of Luminex microsphere. cCM, common sequence primer immediately downstream from the allele-specific
Chapter 3 In vitro drug sensitivity of PNG field isolates
100
3.4 DISCUSSION
The high prevalence of in vitro CQ resistance in the present study accords with recent
local molecular and clinical data. A number of genotyping studies have reported near-
fixation of the CQ resistance-associated mutation pfcrt in PNG (Mehlotra et al. 2005;
Carnevale et al. 2007). In addition, the significant rates of in vivo CQ-SP treatment
failure in the Madang area (Marfurt et al. 2007; Karunajeewa et al. 2008b) are also
consistent with the present in vitro findings, although mutations associated with SP
resistance will have contributed (Casey et al. 2004; Mita et al. 2007; Saito-Nakano et
al. 2008).
The IC50s for AQ and its active metabolite dAQ in the present study were much lower
than in previous studies from PNG (Trenholme et al. 1993; Al-Yaman et al. 1996). This
is likely to reflect methodological differences, since microscopic assessment of schizont
maturation produces IC50 values several times higher than those derived from
radioisotope incorporation (Wernsdorfer et al. 1988) and, by implication, from the
pLDH assay. However, consistent with the present IC50 data, a recent study from
neighbouring East Sepik Province found a much lower prevalence of in vitro resistance
to AQ than CQ (Genton et al. 2005).
Clinical studies in PNG have found equivalent high treatment failure rates for AQ-SP
and CQ-SP (Marfurt et al. 2007; Karunajeewa et al. 2008b). However, AQ-SP is used
in younger children (those <19 kg in body weight) than CQ-SP under PNG national
treatment guidelines (PNGDOH 2000). This means that a lack of immunity may offset
relative parasite sensitivity to AQ in this age-group, producing comparable failure rates
to those with CQ-SP in older children. Indeed, AQ is more effective than CQ in African
children of similar age (Brasseur et al. 1999; Oduro et al. 2005; Pradines et al. 2006).
Nevertheless, there may not be a clear relationship between AQ in vitro parasite
sensitivity and clinical outcome (Trenholme et al. 1993; Pradines et al. 2006).
A valid in vitro resistance threshold for PQ remains to be confirmed. An IC50 <100 nM
has been used to identify sensitive strains of P. falciparum by radioisotope uptake
(Deloron et al. 1985; Basco 2003b; Mwai et al. 2009b), while Chinese investigators
Chapter 3 In vitro drug sensitivity of PNG field isolates
101
have reported resistant isolates with IC50 values >300 nM using the schizont maturation
microtechnique (Yang et al. 1999b; Lin et al. 2005). All PQ IC50 values in the present
study were <100 nM. Sixteen of the isolates were from children treated with DHA-PQ
in the recent comparative clinical trial (Karunajeewa et al. 2008b) and one (IC50 19.5
nM) was a late parasitological failure. These various data suggest that further in vivo-in
vitro correlation studies are needed to establish a clinically meaningful resistance
threshold for PQ.
MQ has not been used previously in PNG. All isolates had MQ IC50 values below the
resistance threshold of 108 nM established recently using in vivo responses, 3H-
hypoxanthine uptake and molecular characteristics including the number of copies of
the P. falciparum multidrug resistance 1 (pfmdr1) gene (Price et al. 2004). This
threshold was also employed in a study of field isolates from Laos which were assessed
using the pLDH method (Mayxay et al. 2007). The present in vitro MQ data are
consistent with the results of a recent molecular survey that reported an absence of
multiple copies of pfmdr1 gene in PNG isolates (Hodel et al. 2008).
Data from this current study are the first characterising the in vitro sensitivity of PNG
isolates to LM and NQ, drugs that have both recently become available as part of ACT
in PNG. A previously published resistance threshold for LM of >150 nM was based on 3H-hypoxanthine uptake studies in African isolates without in vivo correlation (Basco et
al. 1998). None of the PNG isolates had an IC50 value above this level. There are no
equivalent published cut-points for NQ but the median IC50 (10.3 nM) was less than
those reported for isolates from Southern China using the micro-test method (mean 88.5
nM for ‘artesunate-sensitive’ and 119.4 nM for ‘artesunate-resistant’ strains) (Yang et
al. 1999a). The IC50 values for DHA were all below the suggested cut-point of 10.5 nM
derived from 3H-hypoxanthine uptake studies in African isolates without in vivo
correlation (Pradines et al. 1998). The AZ IC50s from the present study (mean 13.9 µM)
are also largely below a previously reported range derived from Thai isolates and the
micro-test technique (mean 29.3 µM) (Noedl et al. 2001).
The positive inter-correlations between IC50 values for 4-aminoquinoline and related
Chapter 3 In vitro drug sensitivity of PNG field isolates
102
compounds have been reported in studies from other countries (Fan et al. 1998; Yang et
al. 1999a; Basco et al. 2003a; Pradines et al. 2006). These findings are consistent with
the observation that pfcrt alleles influence parasite susceptibility to drugs other than CQ
such as MQ (Sidhu et al. 2002; Johnson et al. 2004; Sidhu et al. 2005), but other
mechanisms such as common drug-specific effects on parasite haem polymerase may
be involved (Slater et al. 1992; Dorn et al. 1998). It is also possible that the positive
associations in the present study reflect general parasite fitness rather than shared
resistance determinants, but the lack of significant associations involving LM and AZ,
also reported by others (Noedl et al. 2001; Noedl et al. 2007), are against this. The IC50
values for PQ correlates with CQ, AQ and dAQ in the present study. However, this is
not the case in the African data set (Mwai et al. 2009b), raising questions about
mechanisms of action of PQ. In addition, the African study showed consistent inverse
relationship between LM sensitivities and CQ which has also be reported by others
(Pradines et al. 1999b; Price et al. 2006). These differences in the African and PNG
findings may be due to variations in methodologies (i.e. longer assay time of 84 hr,
adaption of field isolates to long-term culture prior to sensitivity testing) and different
histories of parasite drug exposure. As with most antibiotics with antimalarial activity,
the macrolide AZ is relatively weak and slow-acting, and best used as adjunctive
therapy (Anderson et al. 1995; Noedl et al. 2006; Noedl et al. 2007).
Lumefantrine and MQ are aryl-aminoalcohols with related chemical structures and a
similar mode of action (Peel et al. 1994; Basco et al. 1998; Pradines et al. 2006).
However, a significant correlation was found between the LM IC50s and those of MQ
but not with the other long half-life antimalarial drugs. This observation is in accord
with previous reports (Basco et al. 1998; Pradines et al. 2006) and is also consistent
with the significantly better clinical response to artemether-LM than DHA-PQ in a
recent comparative trial (Karunajeewa et al. 2008b). There were generally weak
associations between DHA IC50s and those of other drugs, consistent with the findings
of others (Basco et al. 2003a; Attlmayr et al. 2005; Pradines et al. 2006; Noedl et al.
2007). There is some evidence that pfcrt status influences the antimalarial activity of the
artemisinin derivatives (Sidhu et al. 2002), but the known association between P.
falciparum sensitivity to these drugs and pfmdr1 mutations and copy number (Sidhu et
al. 2005; Sidhu et al. 2006) does not apply in PNG (Hodel et al. 2008).
Chapter 3 In vitro drug sensitivity of PNG field isolates
103
PLDH assessment of P. falciparum drug sensitivity was originally described as a
kinetic assay requiring repeated measurement of absorbance (Makler et al. 1993a). This
method determines the mean Vmax (mOD/min) of pLDH activity reflecting parasite
growth (Makler et al. 1993a; Basco et al. 1995). A recent field study in Malawi
employed a similar single pLDH measurement as used in the present study (Druilhe et
al. 2001; Nkhoma et al. 2007). The IC50s determined by the kinetic approach correlated
positively with those determined by the reference isotopic method (Makler et al. 1993a;
Basco et al. 1995). In the interests of simplicity and efficiency in the field where a
spectrophotometer was not readily accessible, non-kinetic assessment of pLDH activity
was more convenient. The IC50s from this approach correlated well with the isotopic
method although it tended to overestimate the IC50s above 100 nM as shown in the
Bland-Altman analysis.
Unlike culture-adapted strains, P. falciparum isolates from patients varied in their in
vitro growth and consequent pLDH activity. A number of isolates failed to develop over
the 48 hr incubation period, hence little colour contrast was observed between drug-free
controls, antimalarial-dosed and non-parasitised control wells on pLDH assay. Some
isolates developed colour intensities more slowly than others, for which the OD at 180
min into the pLDH reaction was measured. The use of a pre-test was helpful in reducing
reagent wastage and for determining measurement time. This involved first testing the
haemolysate from one drug-free and one non-parasitised well from each sample and
time for colour development prior to running all three 96-well plates.
During validation of the modified pLDH method, efforts were made to ensure
comparability with the reference 3H-hypoxanthine incorporation technique. Factors
underlying between-day variations of in vitro estimates of drug sensitivity include the
time-dependent growth characteristics of the cultures. To minimise such effects on
assay performance, both assays were conducted in parallel and on the same day. In
order to reduce unnecessary transfer and handling of radioactive material, two sets of
drug dilutions were prepared for each method (one lacking hypoxanthine) rather than
taking an aliquot of cell suspension from the isotopic test wells for pLDH assessment,
as has been done previously (Makler et al. 1993a). Two data points showed
Chapter 3 In vitro drug sensitivity of PNG field isolates
104
unexpectedly high IC50s by the isotopic method for MQ (Figure 3.2), the influence of
these outliers may be reduced if more samples were examined for this drug. Agreement
between the two methods may be more substantial if the drug dilutions used or
haemolysate were from the same experiment set. Although it is worth noting that
previous reports have only compared correlations and not the agreement between the
two methods (Basco et al. 1995; Delhaes 1999).
The present study provides baseline data at a time when, as a result of the findings of a
large-scale clinical trial (Karunajeewa et al. 2008b), the treatment of uncomplicated
malaria in PNG will change from AQ-SP or CQ-SP to artemether-LM. Ongoing
assessment of in vitro sensitivity using the same techniques will facilitate assessment of
the adequacy of such treatment. Conventional monitoring involves the WHO micro-test
with labour-intensive visual enumeration of schizonts (Al-Yaman et al. 1996;
Hombhanje 1998b). The colourimetric pLDH assay allows prompt semi-automated
generation of parasite growth data from triplicate experiments involving multiple
antimalarial drugs. The IC50 values generated correlate well with those derived using 3H-hypoxanthine incorporation and there is no issue with disposal of radioisotopes, an
important consideration in countries such as PNG. Assays based on pLDH
quantification have been recently introduced for screening patient isolates against
multiple antimalarial drugs in Africa and Asia (Brockman et al. 2004; Kaddouri et al.
2006; Nkhoma et al. 2007). The assay may serve to monitor the possible reversal of CQ
resistance after its official withdrawal in PNG as evident in Malawi (Laufer et al. 2006).
Rational drug policy in countries such as PNG can only benefit from such convenient,
high-throughput in vitro testing, especially if this is done regularly so that emerging
resistance can be identified with relative confidence at an early stage.
This work for the most part has been published in Tropical Medicine and International
Health, titled “In vitro sensitivity of Plasmodium falciparum to conventional and novel
antimalarial drugs in Papua New Guinea”. Work regarding methodology validation has
been published in Malaria Journal, titled “A comparative study of a flow-cytometry-
based assessment of in vitro Plasmodium falciparum drug sensitivity”. A small portion
of data relating to drug sensitivity has been published in The New England Journal of
Medicine, titled “A trial of combination antimalarial therapies in children from PNG”.
CHAPTER 4
CHARACTERISATION OF DRUG
RESISTANT POLYMORPHISMS OF
P. FALCIPARUM USING A NEW
MOLECULAR ASSAY
Chapter 4 Molecular Characterisation of PNG isolates
106
CHAPTER 4. CHARACTERISATION OF DRUG
RESISTANT POLYMORPHISMS OF P. FALCIPARUM
USING A NEW MOLECULAR ASSAY
4.1 INTRODUCTION
Resistance of Plasmodium species to 4-aminoquinolines emerged in PNG in 1976 and
has since spread across the country (Grimmond et al. 1976; Marfurt et al. 2007). In
addition, mass dosing of pyrimethamine in the 1960’s conveyed continuous drug
pressure on the parasite population that led to the selection of resistant mutations. High-
level resistance has been documented both in vivo (Darlow et al. 1980; Marfurt et al.
2007; Karunajeewa et al. 2008b) and in vitro (Reeder et al. 1996; Mita et al. 2006a).
Chloroquine (CQ) or amodiaquine (AQ) monotherapy was retained as first-line
treatment for uncomplicated malaria until 2000 when sulfadoxine/pyrimethamine (SP)
was added to improve clinical efficacy (Casey et al. 2004). Despite initial success, cure
rates have since declined (Marfurt et al. 2007; Karunajeewa et al. 2008b).
Parasite drug resistance is largely assessed in three ways. The reference assessment is
by clinical efficacy trials where treatment outcome is monitored. However, they are
costly, time consuming, and patient recruitment and follow-up are often difficult (WHO
2005). Parasite drug sensitivity can be tested in vitro, but it can be labour intensive
particularly if multiple drugs are to be screened for each sample. With advancing
technology, molecular surveillance of malaria resistance is increasingly valuable as it
overcomes many challenges associated with clinical and in vitro approaches.
Single nucleotide polymorphisms (SNPs) in parasite genes determining drug effects can
underlie resistance. Mutations in the P. falciparum chloroquine transporter (pfcrt) gene,
in particular K76T, is central to determining the phenotype of CQ resistance and in
predicting treatment failure (Fidock et al. 2000; Basco et al. 2002). The pfcrt K76T
mutation is often associated within different amino acid haplotypes (CVIET, CVMNT,
CVMET, or SVMNT residues 72-76), however the roles of these haplotypes is not well
Chapter 4 Molecular Characterisation of PNG isolates
107
defined except that they are reflective of the parasite’s geographic origin (Fidock et al.
2000).
Higher-levels of CQ resistance result from other SNPs and is inversely associated with
the copy number of the multidrug resistance 1 (pfmdr1) gene (Foote et al. 1990; Reed et
al. 2000; Babiker et al. 2001; Pickard et al. 2003). Pfmdr1 gene polymorphisms also
confer resistance to other antimalarials including quinine, mefloquine, lumefantrine and
halofantrine (Cowman et al. 1994; Peel et al. 1994; Reed et al. 2000). Of particular
concern are the results of a pfmdr1 gene allelic replacement study in which various
polymorphisms reduced artemisinin susceptibility in cloned parasite lines (Reed et al.
2000). The study showed pfmdr1 polymorphisms at codons 86, 1034, 1042 and 1246
altered artemisinin susceptibility in D10 and 7G8, originating from PNG and South
America, respectively. This finding has serious implications for future prospect of
artemisinin effectiveness in endemic areas.
Polymorphic changes in the genes encoding dihydrofolate reductase (dhfr) and
dihydropteroate synthetase (dhps) underlie parasite resistance to pyrimethamine
(Cowman et al. 1988; Peterson et al. 1988) and sulfadoxine (Triglia et al. 1994; Triglia
et al. 1999), respectively. The S108N mutation in dhfr is a primary determinant of
pyrimethamine resistance and additional mutations at codons 16, 50, 51, 59, 140 and
164 cause higher-level resistance (Cowman et al. 1988; Peterson et al. 1988). Similarly,
polymorphisms involving S436A/F, A437G, and K540E in the pfdhps gene confer
initial mutation to sulfadoxine. Other genetic alterations such as A581G and S613A will
lead to higher-level resistance (Triglia et al. 1994; Triglia et al. 1999). Almost all
strains of P. falciparum from patients from Madang Province in PNG who fail CQ-SP
treatment carry pfcrt K76T and pfmdr1 N86Y, while pfdhfr C59R and S108N are also
found at moderate/high levels, reflecting the selective pressure from long periods of CQ
and pyrimethamine usage (Casey et al. 2004; Carnevale et al. 2007).
At present, most molecular techniques for SNP analysis are based on PCR restriction
fragment length polymorphism (RFLP), sequence-specific oligonucleotide probe
hybridisation (SSOPH) and direct sequencing. However, most such methods identify a
Chapter 4 Molecular Characterisation of PNG isolates
108
small number of candidate SNPs regarded as primary predictors of clinical resistance
(Ranford-Cartwright et al. 2002). Mutations that are not directly involved in resistance
but which may have compensatory or modulating effects that contribute to the overall
phenotype are often omitted. An approach based on DNA microarray allows parallel
detection of multiple SNPs (Crameri et al. 2007), but remains relatively expensive. An
alternative technique is a post-PCR ligase detection reaction-fluorescent microsphere
assay (LDR-FMA) that enables cost-effective evaluation of 22 SNPs (Carnevale et al.
2007).
In view of the importance of a low-cost system for large-scale monitoring of drug
resistance in developing countries, the present study further expanded this system to
detect an additional 10 different pfmdr1 allelic variants. In addition to assay
development, this new technique has been applied in a study of key drug resistance
mutations in P. falciparum field isolates from clinical studies conducted in PNG. The
prevalence of different allelic variants of the pfcrt, pfdhfr, pfdhps and pfmdr1 genes are
presented. Associations between these mutations and treatment outcome are also
examined.
4.2 MATERIALS AND METHODS
4.2.1 Field Studies, P. falciparum isolates
The present study utilised a subset of 402 samples for Plasmodium speciation from a
large-scale treatment trial in children aged 6 months to 5 years (mean 36 months) with
uncomplicated malaria (Karunajeewa et al. 2008b) (Australian New Zealand Clinical
Trials Registry ACTRN12605000550606). The study was conducted between 2005 and
2007 in Madang and East Sepik Provinces. Participants were assigned CQ-SP,
artesunate-SP (ART-SP), piperaquine-dihydroartemisinin (PQ-DHA) or artemether-
lumefantrine (AL). Children who had been treated with antimalarial drugs within the
previous 14 days were excluded. The samples used in the present study were those
collected at baseline prior to treatment allocation. Full details of the trial protocol have
been published previously (Karunajeewa et al. 2008b).
Chapter 4 Molecular Characterisation of PNG isolates
109
The number of samples that were assayed for pfcrt, pfdhps and pfdhfr genotypes from
the treatment groups CQ-SP, ART-SP, PQ-DHA and AL were 81, 86, 94 and 90,
respectively (total of 351) and for pfmdr1 were 63, 65, 79 and 72 respectively (total of
279) due to limited sample volume. Efficacy was assessed over 42 days using WHO
definitions (WHO 2003) with correction for re-infections by PCR genotyping
(Karunajeewa et al. 2008b), specifically adequate clinical and parasitological response
(ACPR), early treatment failure (ETF; an inadequate parasitological response and/or
worsening of clinical signs by day 3), late parasitological failures (LPF; emergent
parasitaemia between days 4 and 42), or late clinical failure (LCF; where LPF was
associated with fever). Informed consent was obtained from the parents/guardians
before recruitment. Scientific/ethical approvals for the main study and present sub-
study were obtained from the Medical Research and Advisory Committee of the
Ministry of Health of PNG, the University Hospitals Case Medical Centre and the
University of Western Australia Human Research Ethics Committee.
4.2.2 Genomic DNA
Laboratory-adapted P. falciparum strains including 3D7, Dd2, K1, 7G8 and HB3 were
provided by MR4, American Type Culture Collection. DNA was extracted from 200 µL
whole blood (field samples) or haemolysate (laboratory-adapted strains) using the
QIAmp 96 DNA blood kit or DNeasy Blood and Tissue Kit (Qiagen, CA) under the
manufacturer’s protocol (Section 2.3.1).
4.2.3 Plasmodium Speciation
Detection of Plasmodium species was by amplification of ssu rDNA by a modified
multiplex LDR-FMA (McNamara et al. 2006). Parasite genomic DNA served as
templates for the PCR primers flanking the small-subunit rRNA gene fragment (491-
500 base-pairs). This domain contains sequences conserved within the Plasmodium
genus and those that are species-specific (Section 2.3.2.1). All PCR reactions (25 µL)
were performed using a Peltier Thermal Cycler, PTC-225 (MJ Research, MA)
consisting of 3 µL genomic DNA in a master mix containing 3 pmol of appropriate
Chapter 4 Molecular Characterisation of PNG isolates
110
upstream and downstream primers (Section 2.3.2.1).
To evaluate amplification efficiency, the PCR products were visualised by
electrophoresis on 2% agarose gels stained with SYBR Gold and images were acquired
using a Storm 860 (Section 2.3.3). This was followed by species-specific ligase
detection reaction (LDR) as described previously (Section 2.3.4.1 and Table 2.3). LDR
utilises the ability of DNA ligase to preferentially join adjacent oligonucleotides to the
target PCR amplicon where there is a perfect complementation at the junction during
hybridisation.
The second step involves hybridisation of LDR products with anti-tag oligonucleotides
coupled with Plasmodium species-specific microspheres (Section 2.3.4.3). These
microspheres (Luminex Corporation, TX) are embedded with varying ratios of
red:infra-red fluorochromes and emit unique fluorescent ‘classification’ signatures. The
hybridised mixture is then labelled with a reporter dye (streptavidin-R-phycoerythrin,
Molecular Probes, OR) through binding to the biotin end of the LDR conjugate.
Fluorescent signals are sorted into allele-specific ‘bins’ by the bioplex array reader
(Bio-Rad Laboratories, CA).
4.2.4 Detection of Drug Resistant Polymorphisms
Amplification of target sequences for P. falciparum pfdhps, pfdhfr, pfcrt (Carnevale,
2007) and pfmdr1 were achieved using oligoprimers and conditions described in Table
2.2 and Section 4.3.1.1, respectively. Following PCR, the products were combined in a
multiplexed LDR (Table 2.4 and Section 4.3.1.2). Upstream LDR primers are allele-
specific and contain the complementary base to the SNP of interest at the 3’ end (Figure
4.1). The upstream primers were designed to have unique tag sequences of 24
nucleotides at the 5’ end that enables subsequent identification of specific SNPs.
Downstream LDR primers contain conserved sequence oligonucleotides and were 5’
phosphorylated and 3’ biotinylated.
Chapter 4 Molecular Characterisation of PNG isolates
111
Figure 4.1 Principle of LDR-FMA diagnosis of drug resistant polymorphisms.
Top: Main components in the reaction. Middle: During thermocycling, the LDR primers
hybridise to the PCR products with matching base-pairs. If there is a perfect match
between the junctions of adjacent LDR primers, the gap will be sealed by DNA ligase.
A single base mismatch will not result in ligation. Bottom: LDR products are hybridised
with anti-tag oligonucleotides coupled to microspheres that report signals specific to the
SNPs of interest. This is followed by labelling with streptavidin-R-phycoerythrin
(SAPE) through binding to biotin.
Chapter 4 Molecular Characterisation of PNG isolates
112
During thermocycling, the LDR primers hybridise to PCR products with matching base-
pairs. As a result, the LDR primers specific to the gene and SNP of interest are brought
together in close proximity. If there is a perfect match at the junction of the LDR
upstream and downstream primers, the nick will be sealed by a DNA ligase. A single
base mismatch will not result in ligation. Hence, this step is highly specific (Figure 4.1).
Details of the LDR for pfcrt, pfdhfr, and pfdhps are described in Section 2.3.4.2.
Recipes for PCR and LDR master mix solutions and respective primer sequences are
outlined in Appendix B.
4.2.5 Data Analysis
Statistical analysis was performed using GraphPad PRISM version 4.0 (GraphPad
Software, CA). Fluorescent signals from the field samples were classified positive or
negative for drug susceptibility markers according to thresholds determined by
standardised procedures. Fluorescent signals were first normalised to a mean of 10,000
and SD 1,000 arbitrary units for each codon by subtracting the calculated mean from
every signal within the corresponding codon, then multiplying by 1,000/codon-specific
SD, and finally adding 10,000. The same procedure was applied to fluorescent signals
from culture-adapted strains with known genotypes, thus providing controls within each
SNP assay. Once adjusted, codon-specific cut-points that applied to all control strains
were derived with a value that predicted the highest number of true positives as a
conservative cut-point for distinguishing positive signals from background
fluorescence.
A cut-point of >9600 had 97.5%, 98.8% and 98.6% accuracy for predicting true
positive alleles for codons 540, 581 and 613 in the pfdhps gene in control strains. The
>9600 cut-point also applied to codons 1042 and 1246 in the pfmdr1 gene, while >9800
accurately predicted known alleles at codon 86 in the pfmdr1 gene, codons 51, 59, 108
and 164 in the pfdhfr gene, and in the CVMNK, SVMNT, and CVIET pfcrt haplotypes.
A threshold of >10,000 applied to pfmdr1 codons 184 and 1034. By reversing the
normalisation process, the cut-points were made specific to each drug resistance
marker. A similar approach that uses polar-co-ordinates has also been used to determine
thresholds for the LDR-FMA system (DaRe et al. 2010).
Chapter 4 Molecular Characterisation of PNG isolates
113
Mixed strain infections can be identified when fluorescence signals from both alleles
(i.e. wild type and mutated) from the same codon occur above calculated cut-points.
Previous experiments have shown that strain-specific allele fluorescence signals are in
direct proportion to the ratio of the parasite strain densities within the sample
(Carnevale et al. 2007). Therefore, Day 28 and day 42 post-treatment blood samples
from patients from the clinical trial (Karunajeewa et al. 2008b) that were parasite
negative by both microscopy and PCR were assayed from which very low fluorescence
signals (<200) were found. These observations indicate that multiple P. falciparum
strains and non-falciparum DNA such as that from the human host do not interfere with
SNP detection by LDR-FMA. While haplotypes were assigned based on the dominant
allele signals at each locus, they have masked the presence of a minor clone in the case
of a multiple strain infection. Although multiplicity of infection (MOI) was not
calculated in the analysis of drug resistance haplotypes, efforts were made to exclude
samples showing mixed infection at more than two loci. In addition, previous studies
have shown that multiclonal infections are rare in PNG, with a mean MOI of 1.3 - 1.8
(Felger et al. 1994; Cortes et al. 2004).
Associations between parasite mutations and measures of treatment outcome were
assessed using Fisher’s exact test or ANOVA with Bonferroni post hoc adjustment for
multiple comparisons (SPSS v16.0, Chicago IL).
4.3 RESULTS
4.3.1 Pfmdr1 LDR-FMA Development
4.3.1.1 PCR Optimisation
Pfmdr1 SNPs are clustered in two regions approximately 2000 base-pairs (bp) apart.
Two sets of PCR primers (Integrated DNA Technologies) were designed for the
amplification of pfmdr1 polymorphisms at codons 86 and 184 (a total of 4 alleles)
designated as region 1, and polymorphisms at codons 1034, 1042 and 1246 (a total of 6
alleles) as region 2.
Chapter 4 Molecular Characterisation of PNG isolates
114
Previously designed PCR primers and conditions for pfmdr1 regions 1 and 2
(Carnevale, unpublished) were tested in seven laboratory strains of P. falciparum. This
involved two successive runs of PCRs with the second intended to amplify a target
sequence within the first-run product (i.e. nested PCR). Well defined PCR products of
expected size (~294 bp) were observed for 6 of the 7 control strains from pfmdr1 region
1. The nested 1 amplification of pfmdr1 region 2 was less successful, producing very
light bands. Non-specific amplification including multiple bands and smearing in the
nest 2 reaction was likely due to sub-optimal annealing temperature (TA).
To improve amplification specificity, gradient experiments were employed to select for
the optimal TA. This was tested using genomic DNA from 7G8 (Figure 4.2). PCR
amplification was successful across TA of 40°C to 60°C for region 1. For region 2
however, specificity increased when TA was >48°C. Since 56°C was the optimal TA for
the PCR amplification of other drug resistant genes (pfcrt, pfdhfr and pfdhps), it was
used for pfmdr1 for further validation.
The necessity of a nested PCR was assessed. Firstly, PCR products from the pfmdr1
region 2 nest 1 were used as a template for the nest 2 primers. Secondly, the region 2
nest 2 primers were used directly with genomic DNA. Multiple PCR product bands and
smearing resulted when nest 1 products were used (data not shown). However, nest 2
primers used directly with genomic DNA resulted in well defined bands of ~694bp,
which negated the need for a nested PCR for pfmdr1 region 2.
Despite acceptable PCR amplification, the products (required for subsequent LDR-
FMA) produced high background fluorescent intensities (FI), particularly for alleles
86Y/N and 1034S/C. The HB3 strain carries the pfmdr1 allele 86N (wild type);
however, FI for both 86N and 86Y were high at 18168 and 17758, respectively.
Similarly, in Dd2 which carries the 1034S allele, FI of 25537 and 13628 were obtained
for 1034S and 1034C, respectively. Ideally, the FI of the negative allele should be
<1500, as observed in other LDR-FMA assays (Carnevale et al. 2007). This high
background was suggestive of cross-reactivity or non-specific binding. Closer
examination of the oligonucleotide sequences of respective PCR and LDR primers
Chapter 4 Molecular Characterisation of PNG isolates
115
revealed an overlap of 19 bp. This may have caused partial amplification of the PCR
primers against LDR probes and contributed to the high background signals.
With the aim of enhancing the LDR-FMA FI specificity, new PCR primers were
designed (Table 4.1). Forward and reverse complementary oligonucleotides sequences
were selected from the P. falciparum genome (Genebank accession #X56851).
Amplification of pfmdr1 regions 1 and 2 using the new primers have proven successful
in control strains. Figure 4.3 illustrates well-defined amplicons of expected sizes from
both pfmdr1 regions in all control strains.
Table 4.1 Primer sequences and thermocycling conditions for pfmdr1 assays.
Initial and optimised conditions for the PCR amplification of pfmdr1 regions 1 and 2 (in
parentheses) are shown.
Set
Gene (region)
PCR Primer Sequence
pfmdr1(1) 754 forward 1048 reverse
5’-GTGTTTGGTGTAATATTAAAG-3’
5’-CAAACGTGCATTTTTTATTAATG-3’
pfmdr1(2) Nest 1 3439 forward 4489 reverse
5’-GATCCAAGTTTTTTAATACAGG-3’
5’-TTAGGTTCTCTTAATAATGCAC-3’
Initial
pfmdr1(2) Nest 2 3570 forward 4264 reverse
5’-TATTGTAAATGCAGCTTTATGG-3’
5’-CACTAACTATTGAAAATAAGTTTC-3’
pfmdr1(1) 681 forward 1119 reverse
5’-TGTATGTGCTGTATTATCAG-3’
5’-CTTATTACATATGACACCACA-3’
Optimised
pfmdr1(2) 3499 forward 4311 reverse
5’-TAGAAGATTATTTCTGTAATTTG-3’
5’-CAATGTTGCATCTTCTCTTCCA-3’
Chapter 4 Molecular Characterisation of PNG isolates
116
Figure 4.2 PCR amplification of pfmdr1 regions 1 and 2 in 7G8 over a temperature
gradient. PCR products of expected sizes (294 bp and 694 bp) were observed from
regions 1 and 2, respectively over a range of annealing temperature (TA). Specificity
was enhanced at TA >48°C for the region 2 reaction.
Chapter 4 Molecular Characterisation of PNG isolates
117
Figure 4.3 Gel scan of PCR products generated using new pfmdr1 primers.
PCR products of expected sizes (438 bp and 812 bp) were generated using new pfmdr1
primers. DNA ladder (HyperLadder™ IV, Bioline, London, UK) band size = 100 bp.
Upper: PCR amplicons of pfmdr1 region 1 from laboratory-adapted strains HB3, Dd2,
K1, 3D7, 7G8, empty, PNG 1917, Bk; water blank. Lower: PCR amplicons of pfmdr1
region 2 from control strains HB3, Dd2, K1, 3D7, 7G8, PNG1905, PNG1917 and water
blank.
Chapter 4 Molecular Characterisation of PNG isolates
118
Preliminary LDR-FMA data generated by using new PCR primers showed enhanced
clarity between known positive and negative alleles. A noticeable reduction in
background FI was evident in the HB3 strain, a carrier of the 86N allele. In this
example, signals correspond to 86Y and 86N alleles were 17758 and 18168 using initial
primers, compared with 5457 and 14393 using modified primers, respectively.
The effect of the number of PCR cycles in reducing background FI was also
investigated. DNA from control strains were subjected to PCR using 10, 15, 20, 25, 30,
35 and 40 amplification cycles. As expected, higher number of cycles produced more
PCR products in both regions (Figure 4.4). The optimal number of PCR cycles ranged
from 30 to 40 for pfmdr1 regions 1 and 2 (Figure 4.4).
Optimised pfmdr1 PCR conditions are summarised in Tables 4.2 and 4.3. For pfmdr1
region 1, the reaction begins by preheating at 95°C for 2 min, followed by 32 cycles of
94°C for 30 sec, annealing at 56°C for 30 sec, 72°C for 30 sec, followed by final
extension at 72°C for 4 min. Thermocycling conditions for the amplification of the
pfmdr1 region 2 were similar to that of region 1 except for a longer extension time at
72°C for 1 min and repeated for 40 cycles.
4.3.1.2 LDR Optimisation
Each allele-specific LDR primer was designed with a unique 24-bases tag sequence
added to its 5’ end. These tag sequences are complementary to the anti-tag sequences
that are bound to microspheres and each emits a distinctive classification code (Table
4.4).
Chapter 4 Molecular Characterisation of PNG isolates
119
Figure 4.4 Effect of PCR cycles on pfmdr1 amplification.
PCR amplification of pfmdr1 regions 1 (upper) and region 2 (lower) by different
number of cycles.
Chapter 4 Molecular Characterisation of PNG isolates
120
Table 4.2 Optimised PCR conditions for pfmdr1 region 1. Table 4.3 Optimised PCR conditions for pfmdr1 region 2.
PCR: pfmdr1 region 1
Volume (µL)
Cycling Program
Sterile distilled water 19.6 95°C – 2 min
10 x PCR Buffer 2.5 94°C – 30 sec
2.5mM dNTPs 2.0 56°C – 30 sec
72°C – 30 sec Forward primer (10pmol/µL) pfmdr1
(region 1) 681
0.3
X 32 cycles
72°C – 4 min Reverse primer (10pmol/µL) pfmdr1
(region 1) 1119
0.3
10°C
Mac Taq 0.3
Total volume per well 25.0
PCR: pfmdr1 region 2
Volume (µL)
Cycling Program
Sterile distilled water 19.6 95°C – 2 min
10 x PCR Buffer 2.5 94°C – 30 sec
2.5mM dNTPs 2.0 56°C – 30 sec
72°C – 1 min Forward primer (10pmol/µL) pfmdr1 (region 2) 3499
0.3
X 40 cycles
72°C – 4 min Reverse primer (10pmol/µL) pfmdr1 (region 2) 4311
0.3
10°C
Mac Taq 0.3
Total volume per well 25.0
Chapter 4 Molecular Characterisation of PNG isolates
121
Table 4.4 LDR primers for P. falciparum pfmdr1 molecular markers. aLowercase
nucleotides represent tag sequences added to the 5’ ends of each allele-specific LDR
primer. bID, microsphere fluorescence identification. Luminex microsphere sets are
synthesised to exhibit unique fluorescence. Each microsphere set is coupled to different
anti-tag sequences that are complementary to allele-specific tag sequences. cCom,
common (conserved) sequence primer positioned immediately downstream from the
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APPENDICES
Appendix A: Isolate Information
261
APPENDIX A. ISOLATE INFORMATION
P. falciparum Isolate
Source Drug Sensitivity Comments
3D7 Netherlands CQ-sensitive Derived from NF54 isolated in Amsterdam; presumed of African origin, not confirmed
HB3 Honduras CQ-sensitive Derived from I/CDC (Honduras)
K1 Thailand CQ-resistant Pyrimethamine-resistant
Kanchanaburi
PNG1905 Australia CQ-resistant Origin not confirmed
PNG1917 Australia CQ-resistant Papua New Guinea isolate
W2mef Indochina CQ-resistant Selected from W2 for resistance to mefloquine
Appendix B: Recipes and Solutions
263
APPENDIX B. RECIPES FOR SOLUTIONS
Culture of P. falciparum
5% Albumax II
� Albumax II (11021-045) (Gibco) 5.5 g
� Milli-Q water 110 mL
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at -20°C in 10 mL aliquots.
Complete Maintenance Medium (PNG)
� RPMI Medium 1640 powder (31800-089) (Gibco, Auckland, NZ) 5.735 g
� HEPES (Sigma-Aldrich) 4.47 g
� Hypoxanthine (Sigma) 22.5 mg
� 5% Albumax II (see section below) 50 mL
� 5% NaHCO3 (see section below) 21 mL
� Gentamycin sulfate (10mg/mL) (Calbiochem, Darmstadt, Germany) 5 mL
� Neomycin sulfate (10mg/mL) (Calbiochem, Darmstadt, Germany) 5 mL
� Milli-Q-water 500 mL (final)
Adjust to pH 7.3.
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at 4°C and use within 2 weeks, or supplement with L-glutamine after 2 weeks.
Appendix B: Recipes and Solutions
264
Complete Maintenance Medium
� RPMI Medium 1640 (R5886) (Gibco, liquid) 90 mL
� Human plasma (see section below) 10 mL
� L-glutamine solution (see section below) 1 mL
� Hypoxanthine solution (see section below) 1 mL
� Gentamycin (see section below) 100 µL
Store at 4°C and use within 2 weeks, or supplement with L-glutamine after 2 weeks.
Cryoprotective Solution
� Sorbitol (BDH, England) 37.8 g
� NaCl (BDH, Australia) 8.1 g
� Glycerol (BDH, Australia) 350 mL
� Milli-Q-water 900 mL (final)
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at 4°C.
Gentamycin Stock (50 mg/mL) (PNG)
� Gentamycin sulfate (Calbiochem) 1 g
� Milli-Q-water 20 mL
Protect from light, store at -20°C in 5 mL aliquots
Gentamycin Stock (40 mg/mL)
80 mg/2 mL (Delta West, Pharmacy IV injections).
Store at 4°C.
Appendix B: Recipes and Solutions
265
5% Giemsa Stain
� Giemsa stain (BDH, Australia) 0.5 mL
� PBS (pH 7.2) 9 mL
Stain is prepared fresh before use.
Human Plasma Inactivation
O+, A+, AB+ Plasma units (Fremantle Hospital, Transfusion Medicine) were pooled, defibrined and heat inactivated at 56°C in a waterbath as follows:
1. Plasma was added to sterile 500 mL conical flasks containing 1 - 2 cm of autoclaved (2 mm) glass beads, covered and shaken in a waterbath for 2 - 3 hr at 37°C.
2. Pool plasma and heat-inactivate at 56°C in waterbath for 40 min.
3. Use 10 mL of new plasma to make up test CM (100 mL) and test for support of 3D7 strain of P. falciparum cultures for 2 weeks prior use.
4. Store at -20°C.
Human Tonicity Phosphate Buffered Saline (HTPBS)
� NaCl (BDH, Australia) 7 g
� Na2HPO4 (BDH, Australia) 2.85 g
� NaH2PO4 (BDH, Australia) 0.625 g
� Milli-Q-water 1000 mL
Adjust to pH 7.3.
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at 4°C.
Appendix B: Recipes and Solutions
266
Hypoxanthine Solution (5 mg/mL)
� Hypoxanthine (Sigma, USA) 0.5 g
� Milli-Q-water 100 mL
Heat in microwave until dissolved.
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at 4°C.
L-Glutamine Solution (200mM)
� L-glutamine (Sigma) 2.92 g
� Milli-Q-water 100 mL
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at -20°C in 5 mL aliquots.
5% NaHCO3
� NaHCO3 (Sigma-Aldrich, St Louis, Mo, USA) 2.5 g
� Milli-Q-water 50 mL
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at -20°C in 10 mL aliquots.
PBS (6.7mM) for Giemsa Staining
� K2PO4 (BDH, Australia) 0.41 g
� Na2HPO4 (BDH, Australia) 0.65 g
� Milli-Q-water 1000 mL
Adjust to pH 7.1.
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA) (optional).
Appendix B: Recipes and Solutions
267
12% Sodium Chloride (NaCl) Solution
� NaCl (BDH, Australia) 11.3 g
� HTPBS (see section above) 100 mL
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at 4°C.
1.6% Sodium Chloride (NaCl) Solution
� NaCl (BDH, Australia) 0.9 g
� HTPBS (see section above) 100 mL
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at 4°C.
0.9% Sodium Chloride (NaCl) Solution
� NaCl (BDH, Australia) 0.2 g
� Glucose (Sigma) 0.2 g
� HTPBS (see section above) 100 mL
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at 4°C.
5% Sorbitol for Synchronisation
� Sorbitol (BDH, England) 5 g
� Milli-Q-water 100 mL (final)
Filter sterilise using 0.2 µM membrane (Nalgene Filterware, USA).
Store at 4°C.
Appendix B: Recipes and Solutions
268
LDH Assay
Malstat Solution
� Trizma base (Sigma, Australia) 2.42 g
� Milli-Q-water 180 mL
� Adjust with HCl to pH 9.1
� Triton X-100 (Sigma, Australia) 0.4 mL
� Lithium-L-lactate (Sigma, Australia) 4 g
� APAD (Sigma, Australia) 132 mg
Protect from light and store at 4°C.
Use within one week.
Diaphorase Solution
� Diaphorase (Sigma-Aldrich, USA) 15 mg
� Milli-Q-water 15 mL
Protect from light and store at 4°C.
Nitro Blue Tetrazolium (NBT) Solution
� NBT chloride monohydrate (Sigma-Aldrich, USA) 15 mg
� Milli-Q-water 15 mL
Protect from light and store at 4°C.
Appendix B: Recipes and Solutions
269
Molecular Assays
2% Agarose Gel
� Agarose I ™ (Amresco, USA) 3 g
� 1 X TBE buffer 150 mL
Melt agarose in buffer before pouring into a large 96-well electrophoresis tray and allow 20 min for gel to set.
2.5mM dNTPs Working Solution
� 100 mM dGTP (Denville Scientific Inc, USA) 25 µL
� 100 mM dATP (Denville Scientific Inc, USA) 25 µL
� 100 mM dCTP (Denville Scientific Inc, USA) 25 µL
� 100 mM dTTP (Denville Scientific Inc, USA) 25 µL
� Nuclease free water 900 µL
1.5 X TMAC Hybrisation Buffer
� 5 M Tetramethyl ammonium chloride (TMAC) (Sigma, USA) 30 mL
� 1 M Tris pH 8.0 (Amresco, USA) 2.5 mL
� 0.5 M EDTA (Amresco, USA) 300 µL
� 20% Sodium dodecyl sulfate (Amresco, USA) 250 µL
� Sterile distilled water 16.95 mL
Appendix B: Recipes and Solutions
270
10 x Tris borate (TBE) Buffer
� Tris base 108 g
� Boric acid 55 g
� 0.5M EDTA 40 mL
� Milli-Q-water 1000 mL (final)
10 x PCR Buffer
� 1 M Tris pH 8.8 33.5 mL
� 1 M MgSO4 3.4 mL
� 1 M (NH4)2SO4 8.4 mL
� β-mercaptoethanol (14.3 M) 0.35 mL
� Nuclease-free water 4.4 mL
LDR Master Mix – Plasmodium Species
� Nuclease-free water 11.4 µL
� NEBuffer for Taq DNA (10X) (Biolabs, New England) 1.5 µL
� LDR primers for species assay (x 7) (Table 2.3) 0.15 µL/primer
� Taq DNA ligase (Biolabs, New England) 0.05 µL
� PCR product 1 µL
Appendix B: Recipes and Solutions
271
LDR Master Mix – Drug Resistance SNPs of pfcrt, pfdhfr, pfdhps genes
� Nuclease-free water 7.2 µL
� NEBuffer for Taq DNA (10X) (Biolabs, New England) 1.5 µL
Notes: Compounds have been identified by comparison of mass spectra with the NIST 2005 mass spectral database. Compounds in italic have been
tentatively identified (<90% match) and may indicate the structural class rather than the actual compound. *Siloxanes possibly derived from coating of
the SPME fibre or the GC column stationary phase. **Known contaminants (Middleton 1989).
282
Compounds Common to both P. falciparum Culture and Non-parasitised Control Supernatant Extraction by SPME immersion Supernatant Extraction by Methanol and Sep-Pak