IN VITRO AND GENETIC ASPECTS OF TREATMENTS FOR FALCIPARUM MALARIA: STUDIES OF CONVENTIONAL AND NOVEL DRUGS WITH A PARTICULAR FOCUS ON PAPUA NEW GUINEA

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1 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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3 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

4 DECLARATION FOR THESIS CONTAINING PUBLISHED WORK AND/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) 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) 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) ii

5 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) 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) 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, 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 # AAC ) 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 culturevolatile compounds capture apparatus, performed experiments, GCMS and data analysis, and drafting of manuscript. Overall Contribution: 70%. Candidate Signature: Coordinating Supervisor Signature: iii

6 PUBLICATIONS 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) 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) 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, 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) 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) 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) 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, 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 # AAC ) 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. iv

7 CONFERENCE PRESENTATIONS Rina P. M. Wong & Timothy M. E. Davis. In vitro susceptibility and interrelationships 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) 14 th 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). v

8 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 1 st Annual World Congress of Microbes: 1 st Annual Symposium of Antiparasites, Beijing, China. (Oral, submitted by Invitation). vi

9 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, monodesethyl-amodiaquine (DAQ), piperaquine (PQ), naphthoquine (NQ), mefloquine (MQ), lumefantrine (LM), dihydroartemisinin (DHA) and azithromycin was assessed by colorimetric lactate dehydrogenase growth inhibition assay. Its non-isotopic, semiautomated, high-throughput nature makes it suitable for field use in developing countries. The mean [95% confidence interval] concentration required to inhibit parasite growth by 50% (IC 50 ) was 215 [ ] nm for CQ; 82% of strains were CQresistant. Except for azithromycin, the mean IC 50 s of the other drugs were <27 nm. There were strong associations between the IC 50 s of 4-aminoquinoline (CQ, AQ, DAQ and NQ), bisquinoline (PQ) and aryl-aminoalcohol (MQ) drugs, suggesting crossresistance. The only such correlation for LM was with MQ which, with the low artemisinin IC 50 s, supports artemether-lm as new first-line therapy in PNG. Parasite mutations compromising treatment effectiveness were also evaluated using a modified high-throughput post-pcr multiplexed ligase detection reaction-fluorescent microsphere assay. The assay was used to detect single nucleotide polymorphisms (SNPs) in 402 P. falciparum isolates from PNG children participating in an antimalarial treatment trial. There was fixation of pfcrt K76T, pfdhfr C59R and S108N, and pfmdr1 mutations (92%, 93%, 95% and 91%, respectively). Multiple mutations were frequent, vii

10 88% of isolates possessed a quintuple mutation SVMNT+NRNI+KAA+YYSND in codons for pfcrt, 51, 59, 108, 164 for pfdhfr, 540, 581, 613 for pfdhps, and 86, 184, 1034, 1042, 1246 for pfmdr1, and four carried the K540E pfdhps allele. Pfmdr1 D1246Y was associated with PCR-corrected day 42 treatment failure in children allocated PQ-DHA (P=0.004). Although the pfmdr1 NFSDD haplotype was found in only four isolates, it has been associated with artemether-lm treatment failure in Africa. The assay allowed large-scale assessment of resistance-associated SNPs that reflected previous heavy 4-aminoquinoline/SP use in PNG. Since artemether-lm and PQ-DHA will become first- and second-line treatment, respectively, in PNG, monitoring pfmdr1 SNPs appears a high priority. Desbutyl-lumefantrine (DBL) is a metabolite of LM. Its in vitro activity and interactions were assessed from tritium-labelled hypoxanthine uptake in laboratoryadapted P. falciparum. DBL was more potent than LM. Isobolographic analysis of DBL-LM combinations showed no interaction but mild synergy with DBL-DHA. Mean plasma DBL concentrations in 94 day-7 samples from an antimalarial treatment trial predicted treatment response, suggesting that it could be a useful alternative to LM as part of ACT. Drugs licensed for other indications can sometimes have antimalarial properties, an example being lipid-lowering therapy which is becoming affordable even in malariaendemic developing countries. In vitro drug sensitivity experiments confirmed atorvastatin to have the highest activity of available statins against P. falciparum regardless of strain CQ sensitivity but at an IC 50 well above plasma concentrations after therapeutic doses in vivo. Fibrates have a different mechanism of action to that of statins. Fenofibric acid had a relatively low in vitro IC 50, similar to those of conventional antimalarial drugs. It may act by interfering with parasite P-glycoprotein and ABC-1 mediated transport and/or via a putative peroxisome proliferator-activated receptor-like protein, and could have an adjunctive role in combination antimalarial therapy. The detection of P. falciparum-specific volatile organic compounds (VOCs) in breath/other samples as a way of enhancing diagnosis and therapeutic monitoring was viii

11 explored using culture-capture apparatus. Optimised conditions supported cultures of high parasitaemia (>20%) from which VOCs within the headspace and supernatant were extracted using traditional (solvent) and novel (solid-phase) methods. Gas chromatography-mass spectrometry data revealed the production of a variety of volatile compounds but no unique malarial finger-prints. Future in vivo studies analysing the breath of patients with severe malaria may yet reveal specific clinically-useful volatile biomarkers. ix

12 ACKNOWLEDGEMENTS My PhD journey would not have been successful without the unyielding support of my family, friends and colleagues. To my supervisor Prof. Tim Davis, I have admired your expertise in the field and your driven-nature since day-one. You have taught me to think critically, work independently as well as providing opportunities for me to do research as part of a team abroad. You have amazing writing skills that I can only learn to mimic. Thank you so much for supporting me through the highs and lows. I m grateful for your financial support which helped to sustain my newly established family. It is a privilege to be part of your team and have the chance to travel/work in two very different worlds: Papua New Guinea (PNG) and the United States of America. The experiences gained from these collaborations are extraordinary and most unforgettable, Tenkyu tru! (Pidgin). Special thanks to Drs Wendy Davis, Martin Firth and Shih Ching Fu for your time and invaluable advice on statistical modelling and analyses for the molecular aspect of this project. To Dr Pete Zimmerman, Laurie Gray and many colleagues at Case Western Reserve University, thank you for sharing your cutting edge molecular techniques and being so accommodating when I used your machines up to 12 hours a day! I treasure the time I spent in your lab and thank you for giving me this wonderful opportunity. This project would not have been possible without support from Drs Peter Siba, Pascal Michon, Ivo Mueller, study volunteers and administrative staff from the PNG Institute of Medical Research. Thanks to Livingstone Tavul and Dulcie Lautu for your technical support for the in vitro drug assays. Dr Harin Karunajeewa who led a dedicated team of nurses at the Alexishafen Health Clinic, a big thank you for collecting the field samples and allowing me to take part in this amazing experience. Dr Jane Allan, you have always been there for me (literally) to offer comforting words when I m disheartened and provided tips that often facilitated my trouble shooting. Thank you for looking after the parasites when I was sick and for your continuous x

13 support. I really appreciate your friendly and approachable qualities; you are an awesome lab manager. To my colleagues at the Medical Sciences Lab, Fremantle Hospital Janet, Ross, Debbie, Caryn, Stephan, Eng, Carly, Angela, Yoke Leng, Bruce, Roheeth, Frances, Borut, Kristine, Janina, Anita, Gwen and Chun Wei, thank you for your friendship and amazing support throughout my PhD journey. To our school administrator, Brenda Riley, thank you for your time and care in ironing out the many hiccups and providing a listening ear. Michelle England and the nurses upstairs, thank you for spicing things up and taking my blood every few weeks! I m grateful and indebted to Mr Graham Icke, aka. Pop, my honours supervisor and mentor. Thanks for your encouragement and time in critiquing this thesis. Thanks to Rolf and friends from church, for your encouragements throughout this journey. Shih Ching Fu, my loving husband who is also under the pump with his own thesis, you have been a tremendous support. Thanks for sacrificing countless weekends to accompany me to the lab and giving your red cells for my parasites. Thank you for your patience, encouragement, understanding and lending me your shoulders to cry on. To my Mum (Yolanda) and Dad (Siu Kee), thank you for spurring me on when I m discouraged and pulling me back when I m exhausted. You have supported me so much through words, deeds and prayers. Louis, although you can t talk, I know you are always supporting your sister in your heart. Thank you for being understanding and I will strive to spend more time with you. Most of all I would like to thank God for blessing me with this amazing PhD experience and the love from his son Jesus that has carried me through these years and beyond. The studies herein are supported by grants from the WHO Western Pacific Region, the National Health and Medical Research Council of Australia (grant no , T. Davis as CIA) and the U.S. National Institutes of Health (grants AI52312 and TW ). R. P. M. Wong is supported by the Australian Postgraduate Award, Ad hoc Scholarship (School of Medicine and Pharmacology), the UWA Student Travel Award and the UWA PhD Completion Scholarship. xi

14 PREFACE For the in vitro work presented in Chapter 3 and published in Tropical Medicine and International Health (2010), I went to Papua New Guinea (PNG) for a period of 5 months to collect and test field isolates of P. falciparum. I was based in the Vector Borne Disease Unit in the PNG Institute of Medical Research at Yagaum Hospital, Madang. I took primary responsibility in setting up drug sensitivity assays, data analysis and interpretation of results. A small number of assays were set up by local colleagues Dulcie Lautu and Livingstone Tavul in my absence. Two to three times each week, I helped out at a remote Health Clinic at Alexishafen, where young children presenting with fever were screened for malaria infection and enrolled into a standard treatment trial. The samples included in this thesis were mainly derived from this cohort but a few were from children who presented to Modilon Hospital, Madang Town. I travelled to Case Western Reserve University, Cleveland, Ohio for the molecular analysis of parasite DNA for drug-resistant markers as described in Chapter 4 and published in Antimicrobial Agents and Chemotherapy (2011). I spent three and a half months at the Centre for Global Health and Diseases under the supervision of Dr Peter Zimmerman and learnt a post-pcr technique that has been developed there, namely the Ligase Detection Reaction-Fluorescence Microsphere Assay (LDR-FMA). After familiarisation of the laboratory and equipment, I performed LDR-FMA for Plasmodium species, and single nucleotide polymorphisms (SNPs) detection in the pfcrt, pfdhps, pfdhfr and pfmdr1 genes of blood samples from the treatment trial cohort. Initial PCR primers had been designed for the pfmdr1 LDR-FMA by Eric Carnevale, which required further modifications. I continued to develop and optimise the assay and successfully applied this to the field samples. I took primary responsibility for the generation and analysis of the molecular data. Since there were no standard cutoff points for discriminating between positive and negative SNPs signals, I liaised with Dr Martin Firth from the Mathematics department, UWA. With his advice, I was able to establish a new way of calculating appropriate thresholds. The SNPs data were then used to predict treatment failure rates using the clinical data from the trial. I received xii

15 advice from Dr Wendy Davis regarding statistical methods and software tools, and I took responsibility for performing these analyses. Due to direct and indirect evidence in the published literature that fibrates and statins, as well as being lipid-altering drugs, might also have antimalarial properties, I initiated the investigation into these drug classes with the support of my supervisor W/Prof. Tim Davis. Fibrates showed antimalarial activity in the experiments I conducted. The in vitro studies regarding desbutyl-lumefantrine (DBL) (Chapter 5 and published in Antimicrobial Agents and Chemotherapy (2011), fibrates and statins (Chapter 6, in part published in Antimicrobial Agents and Chemotherapy (2009)) were performed at the Malaria Culture Facilities at the University Department of Medicine, Fremantle Hospital. I took responsibility for parasite culture maintenance, the design of experiments and in establishing a renewed approach to assess drug interactions. Due to the labour intensive nature of this work, I was assisted by a research assistant, Miss Jenny Wong on a casual basis. The pharmacokinetic component of the DBL work (Chapter 5) was performed by my colleague and fellow PhD student Sam Salman. The investigation of volatile organic compounds in P. falciparum was carried out in collaboration with the Chemistry Department, UWA. I was under the supervision of Dr Gavin Flematti, School of Biomedical, Biomolecular and Chemical Sciences. Together we designed two prototypes of culture flasks that enabled the capture and analysis of the head space atmosphere. I was responsible for optimising culture conditions in these and performed various extractions followed by GC-MS analysis. I received technical support with the use of equipments and machinery, and advice regarding the analysis of chromatogram peaks from Dr Gavin Flematti. The work presented in this thesis was performed within the time constraints of my PhD enrolment. xiii

16 ABBREVIATIONS ABC ACPR ACR ACTs AL amu ANOVA APAD ARMD ART-SP AQ AV AZ ATP-binding cassette sub-family A member Adequate clinical and parasitological response Adequate clinical response Artemisinin based combination therapies Artemether-lumefantrine Atomic mass unit Analysis of variance 3-acetylpyridine adenine dinucleotide Accelerated resistance to multidrug Artesunate-sulfadoxine-pyrimethamine Amodiaquine Atorvastatin Azithromycin bp base-pairs CDC cfu CI Centre for Disease Control and Prevention (Atlanta, USA) Colony-forming units Confidence interval xiv

17 CO 2 CPM CSP CQ CYC CQ-SP Carbon dioxide Counts per minute Circumsporozoite protein Chloroquine Cycloguanil Chloroquine-sulfadoxine-pyrimethamine daq DBL DDT DELI Monodesethyl-amodiaquine Desbutyl-lumefantrine Dichloro-diphenyl-trichloroethane Double-site enzyme-linked pldh immunodetection d. H 2 O Distilled water DHA DHFR DHPS DMSO DNA dntps DVB Dihydroartemisinin Dihydrofolate reductase Dihydropteroate synthase Dimethylsulphoxide Deoxyribonucleic acid Deoxynucleotide triphosphate Divinylbenzene xv

18 EDTA ETF Ethylenediaminetetraacetic acid Early treatment failure fmol FI FIC femtomole Fluorescent intensities Fractional inhibitory concentration GC-MS Gas chromatography- mass spectrometry H 2 O HCl hct HDL HEPES HF HMG-CoA HPLC HRP-2 hr HTPBS Water Hydrochloric acid Haematocrit High-density lipoprotein N-2-hydroxyethylipiperazine-N-2ethanesulfonic acid Halofantrine 3-hydroxy-methyl-glutaryl coenzyme A High performance liquid chromatography Histidine-rich protein-2 Hour Human tonicity phosphate buffered saline xvi

19 IC 50 Drug concentration required to inhibit parasite growth by 50% IC 90 Drug concentration required to inhibit parasite growth by 90% IC 99 Drug concentration required to inhibit parasite growth by 99% ICAM-1 IFN-γ IL-10 Intercellular cell adhesion molecule-1 Interferon-gamma Interleukin-10 LCF LC-MS LDH LDL LDR LDR-FMA LM LPF LTF Late clinical failure Liquid chromatography-mass spectrometry Lactate dehydrogenase Low-density lipoprotein Ligase detection reaction Ligase detection reaction-fluorescent microsphere assay Lumefantrine Late parasitological failure Late treatment failure mg min Milligram Minute(s) xvii

20 ml mm MOI MR4 MQ Millilitre Millimolar Multiplicity of infection Malaria research and reference reagent centre Mefloquine ng nm nm NaCl NAD NaOH NBF NBT NQ Nanogram Nanometre Nanomolar Sodium chloride Nicotinamide adenine dinucleotide Sodium hydroxide Nitro blue formazan Nitro blue tetrazolium Naphthoquine O 2 Oxygen OD PBS PCR Optical density Phosphate saline buffer Polymerase chain reaction xviii

21 PDMS PfATP6 Pfcrt Pfdhfr Pfdhps Pfmdr1 Pfserca pirbc pldh pmol PNG PPAR PQ PQ-DHA PV PYR Polydimethylsiloxane (see Pfserca) Plasmodium falciparum chloroquine resistant transporter Plasmodium falciparum dihydrofolate reductase Plasmodium falciparum dihydropteroate synthase Plasmodium falciparum multidrug resistant-1 Plasmodium falciparum sarco-endoplasmic reticulum calcium ATPase6 Packed infected red cells Plasmodium lactate dehydrogenase Picomole Papua New Guinea Peroxisome proliferator-activated receptor Piperaquine Piperaquine-dihydroartemisinin Pravastatin Pyrimethamine RBC rdna Red blood cells Ribosomal deoxyribonucleic acid xix

22 RI RII RIII rpm RPMI RNA rrna RT RV Low grade resistance Moderate resistance High grade resistance Revolutions per minute Roswell Park Memorial Institute ribonucleic acid ribosomal ribonucleic acid Room temperature Rosuvastatin sec SD SDS-PAGE SNPs SP SPME SV Second (s) Standard deviation Sodium dodecyl sulphate polyacrylamide gel electrophoresis Single nucleotide polymorphisms Sulfadoxine-Pyrimethamine (Fansidar) Solid phase micro-extraction Simvastatin T A Annealing temperature TGF-β Transforming growth factor-β xx

23 TNF v/v vs VOCs Tumour necrosis factor Percentage volume per volume Versus Volatile organic compounds WARN WHO w/v World Antimalarial Resistance Network World Health Organisation Percentage weight per volume 3 H Tritium ⁰ ⁰C Degree Celsius % Percentage α β ΣFIC µci Alpha Beta Sum of fractional inhibitory concentrations Microcurie µg Microgram µl Microlitre xxi

24 µm Micron µm Micromolar Nucleotide Adenine Cytosine Guanine Thymine Code A C G T Amino acid Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glycine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Threonine Tyrosine Valine Single letter code A R N D C E G I L K M F S T Y V xxii

25 TABLE OF CONTENTS DECLARATION... I PUBLICATIONS... IV CONFERENCE PRESENTATIONS...V ABSTRACT...VII ACKNOWLEDGEMENTS...X PREFACE...XII ABBREVIATIONS... XIV TABLE OF CONTENTS... XXIII LIST OF TABLES...XXIX LIST OF FIGURES...XXXI CHAPTER 1. GENERAL INTRODUCTION INTRODUCTION DISEASE DISTRIBUTION PLASMODIUM LIFE CYCLE AND BIOLOGY Development in the Human Host Development in the Mosquito PATHOLOGY CLINICAL SIGNS AND SYMPTOMS DIAGNOSIS TRANSMISSION PREVENTION TREATMENT MALARIA IN WESTERN PACIFIC EMERGENCE OF ANTIMALARIAL RESISTANCE IN PAPUA NEW GUINEA ANTIMALARIAL CHEMOTHERAPY Quinoline Related Compounds Quinine Chloroquine Amodiaquine Mefloquine Lumefantrine Naphthoquine xxiii

26 Piperaquine Antifolate Combination Drugs Artemisinin and its Derivatives Antibiotics Summary of Antimalarial Activities LIPID-LOWERING AGENTS AS ANTIMALARIALS Statins Fibrates ANTIMALARIAL DRUG RESISTANCE Definitions Treatment Failure and Drug Resistance Emergence of Resistance to Principal Antimalarials Determinants of Antimalarial Resistance Mechanism of Resistance to 4-Aminoquinolines and Arylaminoalcohols Mechanism of Resistance to Antifolates Mechanism of Resistance to Artemisinin and Derivatives IN VITRO DETECTION OF RESISTANCE IN P. FALCIPARUM Schizont Maturation Macro test Micro test H-Hypoxanthine Incorporation Assay Plasmodium Lactate Dehydrogenase (pldh) Detection Colourimetric pldh microtests Immunocapture of pldh Histidine-Rich Protein II (HRP2) Assay Dual Detection of HRP2 and PLDH ASSESSMENT OF ANTIMALARIAL DRUG COMBINATIONS IN VIVO DETECTION OF DRUG RESISTANCE IN P. FALCIPARUM MOLECULAR MARKERS OF DRUG RESISTANCE BREATH TEST FOR MALARIA SCOPE OF THE STUDIES PRESENTED IN THIS THESIS CHAPTER 2. METHODS AND MATERIALS IN VITRO CULTURE TECHNIQUES Parasites Retrieval from Liquid Nitrogen Maintenance of Cultures Erythrocytes Preparation Determination of Parasitaemia Synchronisation of Parasite Forms xxiv

27 2.1.7 Cryopreservation DRUG SUSCEPTIBILITY ASSAYS Drug/Compound Preparation Preparation of parasitised cells Controls Plasmodium Lactate Dehydrogenase Assay Principle of pldh Assay Assay Set Up H-Hypoxanthine Incorporation Assay MOLECULAR TECHNIQUES DNA Extraction Polymerase Chain Reaction (PCR) PCR for Plasmodium Species PCR for pfcrt, pfdhfr and pfdhps genes Controls Detection of Amplified Products Ligase Detection Reaction Fluorescent Microsphere Assay (LDR-FMA) Ligase Detection Reaction for Plasmodium species Ligase Detection Reaction for pfcrt, pfdhfr, pfdhps SNPs Hybridisation and Reporter Labelling Bio-plex Fluorescent Detection SOLID PHASE MICRO-EXTRACTION (SPME)...80 CHAPTER 3. IN VITRO SENSITIVITY OF P. FALCIPARUM TO NEW AND CONVENTIONAL DRUGS IN PAPUA NEW GUINEA INTRODUCTION MATERIALS AND METHODS Study Site and Sample Collection In vitro Culture of Parasite Isolates Drug Susceptibility Assays Assay Validation Data Analysis RESULTS Comparison of pldh and Isotopic Assays Effect of pldh Reaction Duration on IC 50 Values Field Application of the pldh Assay Antimalarial Susceptibility of PNG P. falciparum Isolates Correlations of in vitro Responses to Nine Antimalarials DISCUSSION CHAPTER 4. CHARACTERISATION OF DRUG RESISTANT POLYMORPHISMS OF P. xxv

28 FALCIPARUM USING A NEW MOLECULAR ASSAY INTRODUCTION MATERIALS AND METHODS Field Studies, P. falciparum isolates Genomic DNA Plasmodium Speciation Detection of Drug Resistant Polymorphisms Data Analysis RESULTS Pfmdr1 LDR-FMA Development PCR Optimisation LDR Optimisation Optimised LDR-FMA for pfmdr1 and Multiplexed Detection of SNPs in pfdhfr, pfdhps and pfcrt genes Assay Validation Comparison between LDR-FMA and RFLP speciation Inter-assay concordance Identification of drug resistance alleles Field Application of the LDR-FMA Speciation and drug resistance genes in PNG field isolates Prevalence of polymorphic alleles in pfcrt, pfmdr1, pfdhfr and pfdhps Parasite drug resistance mutations and treatment outcome DISCUSSION CHAPTER 5. ANTIMALARIAL PROPERTIES OF DESBUTYL-LUMEFANTRINE INTRODUCTION MATERIALS AND METHODS Parasite Cultures Antimalarial Drugs In vitro Drug Susceptibility Drug Interaction Studies Study Site and Sample Collection Liquid Chromatography and Mass Spectrometry Statistical Analysis RESULTS In vitro Antimalarial Potency of DBL DBL Interaction with Conventional Antimalarials DBL Plasma Levels on Day 7 Post-Treatment Influence of DBL Plasma Levels on Clinical Outcome DISCUSSION xxvi

29 CHAPTER 6. STATINS AND FIBRATES: LIPID-MODIFYING DRUGS AS ANTIMALARIALS INTRODUCTION Statins as Lipid-lowering and Antimicrobial Agents Fibrates as Potential Antimalarial Drugs MATERIALS AND METHODS In vitro Parasite Growth Inhibition Drug Interaction Studies Dosed Plasma Bioassay RESULTS In vitro Antimalarial Activities of Statins In vitro Antimalarial Activities of Fibrates Interaction of Atorvastatin with Conventional Antimalarials Interaction of Fibrates with Conventional Antimalarials Bioassay of Atorvastatin Bioassay of Fenofibric Acid BLAST Analysis for PPAR-like Region in Plasmodium BLAST Analysis for ABC-1 transporter in P. falciparum DISCUSSION CHAPTER 7. CHARACTERISATION OF VOLATILE ORGANIC COMPOUNDS OF P. FALCIPARUM IN VITRO INTRODUCTION MATERIALS AND METHODS Parasites Solid Phase Micro-Extraction (SPME) Solvent Extraction Thermal Desorption: Purge and Trap Gas Chromatography and Mass Spectrometry Data Analysis RESULTS Malaria VOCs Assay Development Design of culture-capture apparatus Optimisation of culture conditions Analysis of VOCs DISCUSSION CHAPTER 8. CONCLUDING DISCUSSION OVERVIEW xxvii

30 8.1.1 Major Findings and Contributions THE ROLE OF IN VITRO RESISTANCE AND PARASITE GENETIC MUTATIONS IN TREATMENT OUTCOME Limitations of PNG field studies UNCONVENTIONAL AND NOVEL ANTIMALARIAL AGENTS Desbutyl-lumefantrine and its Potential Implementation Lipid-modifying Agents as Antimalarials A PILOT STUDY OF MALARIA VOCS CONCLUSION AND FUTURE DIRECTIONS Directions for Future Research BIBLIOGRAPHY APPENDICES APPENDIX A. ISOLATE INFORMATION APPENDIX B. RECIPES FOR SOLUTIONS Culture of P. falciparum LDH Assay Molecular Assays APPENDIX C. EFFECTS OF LDR ANNEALING TEMPERATURE AND DILUTION APPENDIX D. ISOBOLOGRAM ANALYSIS APPENDIX E. VOCS ANAYLSIS xxviii

31 LIST OF TABLES TABLE 1.1 ANTIMALARIAL DRUGS AND THEIR SPECIFIC ACTIVITIES TABLE 1.2 DETERMINANTS OF ANTIMALARIAL RESISTANCE...40 TABLE 1.3 CLASSIFICATIONS OF IN VIVO ANTIMALARIAL SUSCEPTIBILITY OUTCOMES TABLE 1.4 MOLECULAR MARKERS FOR ANTIMALARIAL DRUG RESISTANCE...58 TABLE 2.1 SOLVENTS AND OPTIMISED ASSAY CONCENTRATION RANGES FOR DRUG SUSCEPTIBILITY TESTING...69 TABLE 2.2 PCR PRIMER SEQUENCES AND THERMOCYCLING CONDITIONS FOR PFCRT, PFDHPS AND PFDHFR TARGET SEQUENCES TABLE 2.3 LDR PRIMER SEQUENCES FOR PLASMODIUM SPECIES DIAGNOSIS...77 TABLE 2.4 LDR PRIMER SEQUENCES FOR DRUG RESISTANCE MARKERS PFCRT, PFDHFR AND PFDHPS TABLE 3.1 OVERVIEW OF IN VITRO DRUG SENSITIVITY FINDINGS IN PNG...86 TABLE 3.2 IN VITRO SUSCEPTIBILITIES OF P. FALCIPARUM PNG ISOLATES AGAINST 4-AMINOQUINOLINES AND OTHER ANTIMALARIAL DRUGS TABLE 3.3 SPEARMAN CORRELATION CO-EFFICIENTS FOR ASSOCIATIONS BETWEEN IC 50 VALUES...99 TABLE 4.1 PRIMER SEQUENCES AND THERMOCYCLING CONDITIONS FOR PFMDR1 ASSAYS TABLE 4.2 OPTIMISED PCR CONDITIONS FOR PFMDR1 REGION TABLE 4.3 OPTIMISED PCR CONDITIONS FOR PFMDR1 REGION TABLE 4.4 LDR PRIMERS FOR P. FALCIPARUM PFMDR1 MOLECULAR MARKERS TABLE 4.5 OPTIMISED LDR CONDITIONS FOR PFMDR1 REGION TABLE 4.6 OPTIMISED LDR CONDITIONS FOR PFMDR1 REGION TABLE 4.7 CONCORDANCE BETWEEN RFLP AND LDR-FMA DIAGNOSIS OF PLASMODIUM SPECIES IN PNG FIELD SAMPLES TABLE 4.8 LDR-FMA EVALUATION OF PFMDR1 SNPS IN LABORATORY-ADAPTED P. FALCIPARUM STRAINS TABLE 4.9 FLUORESCENCE DETECTION THRESHOLDS AND MAXIMA FOR P. FALCIPARUM PFDHPS, PFDHFR, PFCRT AND PFMDR1 IN PNG FIELD SAMPLES TABLE 4.10 OCCURRENCE OF P. FALCIPARUM ISOLATES CARRYING MULTIPLE MUTATIONS ACROSS 4 GENES ASSOCIATED WITH DRUG RESISTANCE TABLE 5.1 DRUG COMBINATION RATIOS FOR ISOBOLOGRAM ASSAYS TABLE 5.2 IN VITRO SENSITIVITY OF LABORATORY-ADAPTED P. FALCIPARUM TO DESBUTYL-LUMEFANTRINE AND OTHER ANTIMALARIAL DRUGS TABLE 5.3 IN VITRO EFFICACY OF ANTIMALARIAL DRUG COMBINATIONS AGAINST P. FALCIPARUM CLONES 3D7 AND W2MEF AS ASSESSED BY ISOBOLOGRAPHIC ANALYSIS TABLE 6.1 INTERACTION RATIOS OF STATINS, FIBRATES AND CONVENTIONAL ANTIMALARIALS TABLE 6.2 IN VITRO ACTIVITIES OF STATINS AGAINST CQ-SENSITIVE AND CQ-RESISTANT STRAINS OF P. FALCIPARUM xxix

32 TABLE 6.3 IN VITRO ACTIVITIES OF FIBRATES AGAINST CQ-SENSITIVE AND CQ-RESISTANT STRAINS OF P. FALCIPARUM TABLE 6.4 IN VITRO EFFICACY OF FIBRATES AND ANTIMALARIAL DRUG COMBINATIONS TABLE 7.1 VOCS DETECTED IN CULTURE HEADSPACE xxx

33 LIST OF FIGURES FIGURE 1.1 DISTRIBUTION OF MALARIA INCIDENCE BY COUNTRY....4 FIGURE 1.2 LIFE CYCLE OF PLASMODIUM...5 FIGURE 1.3 CHILD WITH MALARIA FIGURE 1.4 ANOPHELES ALBIMANUS TAKING A BLOOD MEAL FIGURE 1.5 MALARIA MORTALITY IN WESTERN PACIFIC...15 FIGURE 1.6 REGIONS AND PROVINCES OF PAPUA NEW GUINEA...16 FIGURE 1.7 ARTEMISIA AND CINCHONA FIGURE 1.8 CHEMICAL STRUCTURE OF QUININE...19 FIGURE 1.9 CHEMICAL STRUCTURE OF CHLOROQUINE...20 FIGURE 1.10 CHEMICAL STRUCTURE OF AMODIAQUINE FIGURE 1.11 CHEMICAL STRUCTURE OF MEFLOQUINE FIGURE 1.12 CHEMICAL STRUCTURE OF LUMEFANTRINE FIGURE 1.13 CHEMICAL STRUCTURE OF NAPHTHOQUINE...25 FIGURE 1.14 CHEMICAL STRUCTURE OF PIPERAQUINE FIGURE 1.15 CHEMICAL STRUCTURES OF SULFADOXINE (LEFT) AND PYRIMETHAMINE (RIGHT)...26 FIGURE 1.16 CHEMICAL STRUCTURE OF ARTEMISININ (LEFT) AND ITS DERIVATIVES (RIGHT) FIGURE 1.17 TARGETS OF ANTI-BACTERIAL DRUGS IN P. FALCIPARUM APICOPLAST...29 FIGURE 1.18 CHEMICAL STRUCTURE OF AZITHROMYCIN FIGURE 1.19 OVERVIEW OF ISOPRENOID BIOSYNTHESIS...34 FIGURE 1.20 RIGHT-WARD SHIFT OF CONCENTRATION-EFFECT RELATIONSHIP DUE TO DRUG RESISTANCE. 37 FIGURE 1.21 EMERGENCE OF RESISTANCE TO PRINCIPAL ANTIMALARIAL DRUGS FIGURE 1.22 REPRESENTATION OF ISOBOLES...53 FIGURE 2.1 NALGENE DESICCATOR USED FOR P. FALCIPARUM CULTURE...63 FIGURE 2.2 GIEMSA-STAINED THIN SMEAR OF SYNCHRONISED P. FALCIPARUM CULTURE FIGURE 2.3 PREPARATION OF A THIN SMEAR...66 FIGURE 2.4 LAYOUT OF A DRUG SUSCEPTIBILITY PANEL...68 FIGURE 2.5 COLOURIMETRIC DETECTION OF PLDH ACTIVITY FIGURE 2.6 PLDH REACTION IN FIELD ISOLATES OF P. FALCIPARUM FIGURE 2.7 TOMTEC HARVESTER 96 SYSTEM FIGURE 2.8 WOLSTEIN RESEARCH BUILDING, CWRU, CLEVELAND, OHIO, USA FIGURE 2.9 ELECTROPHORESIS AND IMAGE PROCESSING FOR DNA VISUALISATION FOR EVALUATING PCR AMPLIFICATION EFFICIENCY FIGURE 2.10 BIO-PLEX ARRAY READER FIGURE 2.11 SOLID PHASE MICRO-EXTRACTION SAMPLER FIGURE 3.1 CANDLE JAR METHOD USED FOR P. FALCIPARUM CULTURE IN PNG...89 FIGURE 3.2 COMPARISON OF PLDH AND 3 H-HYPOXANTHINE INCORPORATION METHODS FOR ANALYSIS OF xxxi

34 ANTIMALARIAL SENSITIVITY IN CULTURE-ADAPTED P. FALCIPARUM FIGURE 3.3 EFFECT OF PLDH REACTION TIME ON IC 50 S IN PNG P. FALCIPARUM FIGURE 3.4 SCATTER PLOT OF IC 50 S DETERMINED FROM THREE PLDH TIME POINTS FIGURE 3.5 DISTRIBUTION OF 50% INHIBITORY CONCENTRATIONS (IC 50 ) OF ANTIMALARIALS AGAINST PNG P. FALCIPARUM ISOLATES FIGURE 4.1 PRINCIPLE OF LDR-FMA DIAGNOSIS OF DRUG RESISTANT POLYMORPHISMS FIGURE 4.2 PCR AMPLIFICATION OF PFMDR1 REGIONS 1 AND 2 IN 7G8 OVER A TEMPERATURE GRADIENT FIGURE 4.3 GEL SCAN OF PCR PRODUCTS GENERATED USING NEW PFMDR1 PRIMERS FIGURE 4.4 EFFECT OF PCR CYCLES ON PFMDR1 AMPLIFICATION FIGURE 4.5 PREVALENCE OF PFCRT, PFMDR1, PFDHFR, PFDHPS ALLELES IN P. FALCIPARUM-INFECTED INDIVIDUALS FROM THE MADANG AND EAST SEPIK PROVINCES, PNG FIGURE 4.6 FREQUENCY DISTRIBUTIONS OF PFCRT, PFDHPS, PFDHFR, PFMDR1 HAPLOTYPES IN P. FALCIPARUM-INFECTED INDIVIDUALS FROM PNG FIELD SITES FIGURE 5.1 IN VITRO SUSCEPTIBILITY OF LABORATORY STRAINS OF P. FALCIPARUM TO CHLOROQUINE, DESBUTYL-LUMEFANTRINE AND LUMEFANTRINE FIGURE 5.2 ISOBOLOGRAMS ILLUSTRATING INTERACTIONS BETWEEN DESBUTYL-LUMEFANTRINE WITH CONVENTIONAL ANTIMALARIALS FIGURE 5.3 BOXPLOTS SUMMARISING DAY 7 PLASMA LEVELS OF LUMEFANTRINE AND DESBUTYL- LUMEFANTRINE FIGURE 6.1 IN VITRO SUSCEPTIBILITY OF LABORATORY STRAINS OF P. FALCIPARUM TO CHOLESTEROL- LOWERING DRUGS AND CHLOROQUINE FIGURE 6.2 INTERACTION BETWEEN ATORVASTATIN AND CONVENTIONAL ANTIMALARIALS FIGURE 6.3 ATORVASTATIN BIOASSAY FIGURE 6.4 FENOFIBRATE BIOASSAY FIGURE 6.5 DISTRIBUTION OF PLASMODIUM BLAST HIT SEQUENCES ON HUMAN PPARΑ FIGURE 6.6 DISTRIBUTION OF P. FALCIPARUM BLAST HIT SEQUENCE ON HUMAN ABC FIGURE 7.1 EXTRACTION OF VOCS FROM CULTURE SUPERNATANT BY AN ORGANIC SOLVENT FIGURE 7.2 PURGE AND TRAP SET-UP FOR THERMAL DESORPTION FIGURE 7.3 PROTOTYPE 1 CULTURE-CAPTURE APPARATUS WITH SPME FIGURE 7.4 DESIGN AND DIMENSIONS OF CULTURE CONTAINER (PROTOTYPE 2) FOR HEADSPACE CAPTURE FIGURE 7.5 CHROMATOGRAMS OF VOCS IN THE HEADSPACE OF CULTURED P. FALCIPARUM xxxii

35 CHAPTER 1 GENERAL INTRODUCTION

36 Chapter 1 General Introduction 1.1 INTRODUCTION CHAPTER 1. GENERAL INTRODUCTION The term malaria was derived from the Italian words Mala aria or bad air, as the sickness was once thought to be associated with inhalation of foul smelling air near swampy areas (Harrison 1978). The disease has been recognised for over 4000 years and has significantly influenced human history (Cox 2002; Rich et al. 2009). Malaria caused more casualties than those due to bullets during the World Wars and other military campaigns, with famous victims including Alexander the Great, Genghis Khan, Ho Chi Minh and George Clooney (MVI 2004; Kakkilaya 2008; News 2011). Malaria inflicts greater detrimental impacts on the human population than other parasitic diseases (CDC 2004). Close to 3 billion people are exposed to malaria world-wide, with the disease accounting for 1-2 million deaths per year, most of which are in pregnant women and children due to their low immunity (Bloland 2001; Snow et al. 2005; WHO 2008). Five Plasmodium species are known to cause human malaria infections: P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. The latter species originated from macaque monkeys in Malaysian Borneo, and has been recently identified in naturally acquired infections with mortality reported in South-East Asia and in European travellers (Singh et al. 2004; Cox-Singh et al. 2008; Luchavez et al. 2008). P. falciparum and P. vivax are the most common but P. falciparum is the most virulent species. It is capable of invading erythrocytes (RBC) of all ages and RBC containing mature forms sequester in the microvasculature of vital organs. P. vivax only infects young RBC and may exhibit relatively weak cytoadherence (Carvalho et al. 2010). P. falciparum has also developed rapid resistance to antimalarial drugs (Al-Yaman et al. 1996; Dondorp et al. 2009; Preechapornkul et al. 2009) and is responsible for approximately 500 million cases annually and the highest morbidity of all infectious diseases (Bloland 2001; Snow et al. 2005; WHO 2009). The impact of malaria varies with local epidemiology. Developed countries such as Australia, European countries and North America do not have local transmission apart 2

37 Chapter 1 General Introduction from occasional autochthonous outbreaks, and virtually all cases are imported. (Gratz 2005; Berry et al. 2008). The most intense transmission occurs in sub-saharan Africa and other areas of the rural tropics such as Papua New Guinea (PNG) where young children with limited immunity are at greatest risk of morbidity and death (Mueller et al. 2003; Ouellette et al. 2003). Although both preventable and curable, malaria remains a substantial social and economic burden in tropical regions (WHO 2009). Mass treatment programs, variable drug compliance and counterfeit antimalarial drugs have all contributed to widespread parasite drug resistance (White 2004; Newton et al. 2008). In particular, P. falciparum has developed resistance to multiple antimalarial agents including, most recently, the potent artemisinin derivatives (Wongsrichanalai et al. 1992a; Bloland 2001; Dondorp et al. 2009). Not only is the arsenal of effective antimalarial drugs diminishing, our current understanding of their mechanisms of action is still limited. The present review outlines the basic epidemiology of falciparum malaria, and the status of parasite drug resistance and its underlying mechanisms. It examines current methods employed for the assessment of drug resistance and pharmacological strategies for limiting its effects and spread, with particular reference to PNG. In addition, several novel compounds with potential antimalarial properties are reviewed. 1.2 DISEASE DISTRIBUTION According to a recent report, malaria is present in 109 countries, with the highest transmission occurring in subtropical and tropical regions in sub-saharan Africa, Central and South America, the Middle East, the Indian subcontinent, South-East Asia and Oceania (Figure 1.1) (WHO 2009). Transmission intensity and risk of infection are dependent on climatic factors such as temperature, humidity, rainfall, altitude, and the presence of the Anopheles mosquito vector. Typically, malaria transmission is rare in the highlands at altitudes above 1500 m and in arid areas with <1,000 mm of rainfall per year (Bloland 2001). However, these tropical areas suffer greater risks of epidemic malaria especially with increased movement of people between these areas and malarious lowlands (John et al. 2005; Mueller et al. 2005). Climatic conditions 3

38 Chapter 1 General Introduction favourable to vector breeding coupled with the lack of, or low, immunity to malaria within a local population have led to devastating epidemics with high mortality rates (Fontaine et al. 1961; Mueller et al. 2005). Malaria intensity is generally higher in regions adjacent to the equator where transmission is year-round. PNG is no exception, with reported cases per year (Figure 1.1). At temperatures below 16 C, P. falciparum cannot complete a normal growth cycle in the mosquito host and hence there is no transmission (Teklehaimanot et al. 2004). Figure 1.1 Distribution of malaria incidence by country. (WHO 2004) 1.3 PLASMODIUM LIFE CYCLE AND BIOLOGY The malaria parasite has developed an intricate relationship with its mammalian and insect hosts. The sexual reproduction of the parasite takes place in the mosquito gut whereas asexual replication occurs within RBC of the human host. Mammalian blood stage of the P. falciparum life cycle starts with rupture and release of merozoites from intra-hepatic schizonts into the blood stream (Figure 1.2). 4

39 Chapter 1 General Introduction Figure 1.2 Life cycle of Plasmodium. (CDC 2006) Development in the Human Host As the female Anopheles mosquito probes for a blood meal, P. falciparum sporozoites are injected with its saliva into the dermis of the host. The sporozoites migrate slowly out of the inoculation site and penetrate dermal capillaries upon contact to enter the circulation (Sidjanski et al. 1997; Matsuoka et al. 2002; Yamauchi et al. 2007). A portion of the sporozoites move through the lymphatics of the host (Amino et al. 2006; Yamauchi et al. 2007). The sporozoites invade the liver (Shin et al. 1982) and pass through the endothelial lining of the sinusoids (Figure 1.2-A). Circumsporozoite protein (CSP) is released to facilitate invasion and exo-erythrocytic development, making it a 5

40 Chapter 1 General Introduction target for vaccine development (Cohen et al. 2009; Kester et al. 2009). The asexual reproduction cycle of P. falciparum occurs at 48 hr intervals. It begins with the release of merozoites from hepatic schizonts into the peripheral circulation (Figure 1.2-B). The merozoite is oval-shaped (1 to 1.5 µm) with a single nucleus and adjacent cytoplasm. It is equipped with erythrocyte binding antigen (EBA 175), merozoite surface protein-1 (MSP-1) and other proteins specific for adherence to the RBC membrane (Camus et al. 1985; Perkins et al. 1988; Sam-Yellowe et al. 1988). During invasion, the merozoite positions its apical end to form a tight junction with the RBC membrane. Invasion is rapid, complete in 30 sec, and is facilitated by anterior organelles such as polar rings, rhoptries and micronemes, and the posterior actinmyosin network that drives the parasite into its host RBC (Kilejian 1976; Aikawa et al. 1978). At entry, the outer structures of the merozoite degrade and the parasite rounds up as a uninuclear trophozoite (Aikawa 1966). The young trophozoite is surrounded by a parasitophorous vacuole membrane originated from the host cell. A vacuole develops, forming the characteristic ring stage of the malaria parasite. The trophozoite continues to develop as it digests host cell cytoplasm and haemoglobin. This process however, produces free haem by-products, the accumulation of which leads to toxicity and parasite death. To circumvent this, the parasite polymerises the free haem into haemazoin crystals, which is easily recognised microscopically as the malaria pigment within the food vacuole in mature parasites (Dorn et al. 1995). The beginning of parasitic nuclear division marks the beginning of the schizont stage. During schizogony, rapid DNA synthesis and nuclear mitosis proceeds as the parasite becomes enlarged and multinucleated. Mitochondrial budding occurs, and merozoite organelles reappear to form 15 to 20 new merozoites within each schizont that are subsequently released for re-invasion (Aikawa 1966). Gametocytogenesis may take place instead of asexual replication where the merozoite develops into gametocytes, sex cells that are subdivided into either microgametocyte (male) or macrogametocyte (female). The events that trigger this process are not well understood. The gametocyte develops through five morphologically distinctive stages to 6

41 Chapter 1 General Introduction reach maturity (Hawking et al. 1971; Carter et al. 1979; Ponnudurai et al. 1982). It features a single unagglomerated nucleus with the male being larger and has a paler cytoplasm compared to the female gametocyte. It develops as a small rounded globule to a triangular body taking up half the RBC to ellipsoidal and finally crescentic forms (Figure 1.2-B7) (Jensen 1979) and is infective to its mosquito host Development in the Mosquito Both sexual and asexual stages of malaria parasites are ingested through the blood meal by a female Anopheles mosquito (Figure l.2-8). The presence of xanthurenic acid and the decrease in temperature within the mosquito triggers gamete formation. The macrogametocyte exits the RBC to form a macrogamete within 10 min, whereas microgametogenesis is less rapid. Mitotic division begins as the nucleus divides into eight portions. Each of these portions enters a projection on the surface of the microgamete and breaks free as an individual microgamete in a process known as exflagellation (Gao 1981; Aikawa et al. 1984). The microgamete promptly fertilises a macrogamete forming a zygote (Figure 1.2-9). Within 24 hr of fertilisation, the zygote develops into a slow moving ookinete (Gao 1981; Aikawa et al. 1984). With structures similar to the merozoite stage (Garnham et al. 1962) the ookinete invades the microvilli of the mosquito gut and secretes digestive enzymes to gain entry into the epithelial cell (Huber et al. 1991). On reaching the cell basal membrane, it develops into a vegetative oocyst (Figure ) (Torii et al. 1992). After 6 days of repeated nuclear division, the oocyst develops into a polypoid measuring 50 µm containing as many as 10,000 sporozoites (Aikawa 1988). The motile sporozoite exits the oocyst and travels to the salivary glands of the mosquito. It contains similar apical organelles as the merozoite stage (Aikawa 1988), equipped with CSP and the thrombospondin-related anonymous protein (TRAP) (Rogers et al. 1992) required for sporozoite formation (Menard et al. 1997) and cell invasion in the mosquito salivary glands and hepatocytes of the human host, respectively (Sultan et al. 1997). The infectivity of the sporozoite to the human host is low then increases with residency in the mosquito salivary glands and gradually 7

42 Chapter 1 General Introduction decreases with age. At the next feeding, sporozoites are injected subcutaneously into the human host. From the site of inoculation, the sporozoites then migrate through the circulation to the liver to begin the asexual cycle. 1.4 PATHOLOGY P. falciparum is responsible for the most severe forms of malaria, causing diverse clinical and pathological conditions in multiple organ systems. Several parasite factors contribute to disease severity and manifestations. The predilection of P. falciparum merozoites for RBC of all ages, augmented by the speed and abundance of schizogony, makes them efficient in re-invasion. Progeny amplification at every 48 hr intervals results in the rapid increase of parasite numbers to as many as per host (Greenwood et al. 2008). Another distinguishing characteristic of P. falciparum is its ability to bind to endothelium during the intra-erythrocytic stage of infection (Aikawa et al. 1980). The sequestration of infected RBC in the microvasculature of organs including the heart, liver and the brain in cerebral malaria (Sachanonta et al. 2008) can alter or occlude blood flow and lead to potentially fatal complications (Luse et al. 1971). A number of studies have shown that P. falciparum infected RBC can bind platelets to form erythrocyte clumps referred to as platelet-mediated clumping, a cytoadherence phenomenon distinct from sequestration and rosette formation of infected RBC, all of which have largely been associated with disease severity (Pain et al. 2001; Miller et al. 2002; Chotivanich et al. 2004). However, a Malian study demonstrated that platelet-mediated clumping of P. falciparum infected RBC is primarily associated with high parasitaemia and not with severe clinical manifestations of malaria (Arman et al. 2007) and the causality of malarial encephalopathy appears multifactorial (Coltel et al. 2004; Mackintosh et al. 2004; Clark et al. 2009; Idro et al. 2010). A systemic host response relating to the release of inflammatory cytokines has been proposed as a primary cause for disease (Clark et al. 2004; Clark et al. 2008). The cyclic destruction of RBC via schizont rupture can result in anaemia and tissue anoxia. The absence of reticulocytes during acute infection suggests defective erythropoiesis which can further aggravate anaemia (Boonpucknavig et al. 1988). 8

43 Chapter 1 General Introduction Recent views regard the primary pathogenesis of malaria as similar to viral and bacterial infectious diseases in which, the host s unbridled response to the invading organism rather than the organism itself is the cause of disease (Clark et al. 2000; Clark et al. 2004). The importance of these cytokine storms in the pathogenesis of cerebral malaria has been consistently championed by Clark since the late 1980s (Clark et al. 1987; Clark et al. 2008). Although this concept was criticised initially, it has gained some support with the recognition that cytokines and immune effector cells may play pivotal roles in severe malaria (Hunt et al. 2003; Kusi et al. 2008). The release of proinflammatory cytokines such as tumour necrosis factor (TNF) by host cells as triggered by malaria antigens has been considered a primary factor in the onset of pathology, especially the pathogenesis of cerebral malaria (Gimenez et al. 2003; Hunt et al. 2003). Clinical studies in African and PNG children have demonstrated a correlation between circulating levels of TNF and disease severity as characterised by fever and parasite density (Butcher et al. 1990; Kwiatkowski 1990a; Al-Yaman et al. 1998; Tchinda et al. 2007). Some clinical observations suggest circulating TNF levels predict death. Serum levels of TNF were twice as high in cerebral malaria survivors, and up to ten-fold higher amongst fatal cases compared to those with uncomplicated malaria (Kwiatkowski et al. 1990b; Al-Yaman et al. 1998). Other evidence in support of the importance of TNF include the in vivo prevention of the onset of neurological syndrome in murine models by neutralisation of cytokines (Grau et al. 1987) and the absence of cerebral malaria in transgenic murine models (Garcia et al. 1995). The current understanding of severe malaria pathogenesis is that there is no longer a clear-cut, single pathogenic correlate (Mackintosh et al. 2004; Haldar et al. 2007). There is likely to be a complex interplay of inflammatory, pro-inflammatory cytokines and cellular responses. TNF increases the expression of intercellular cell adhesion molecule-1 (ICAM-1), particularly on brain endothelial cells and on some sequestered, intravascular leukocytes in human and murine models (Turner et al. 1994). ICAM-1 is a receptor for P. falciparum infected RBC, and both leukocytes and other parasitised RBC have the potential to adhere to capillaries when its expression is up-regulated. Therefore, subsequent blockages may be an outcome secondary to the surge of TNF from host effector cells. Several findings from murine models have also shown the 9

44 Chapter 1 General Introduction activation of platelets in contributing to the formation of neurovascular lesions seen in cerebral cases (Grau et al. 1993a; Grau et al. 1993b). Other cytokines released by natural killer T cells such as interferon-gamma (IFN-γ) and lymphotoxin (LT) have also been implicated in driving the immunopathological progression to cerebral malaria (Amani et al. 2000; Hansen et al. 2003), whilst the anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β) appear to counter this process (Ho et al. 1998). In uncomplicated malaria however, pro-inflammatory cytokines particularly IFN-γ have been associated with a more favourable role, limiting the progression to severe disease (Torre et al. 2002). The literature on IL-10 is generally consistent on its host-protective effect by inhibiting the production of cytokines that are suspected to cause complications of severe and cerebral malaria (Hunt et al. 2003). However, a recent report indicated that IL-10 may indeed down-regulate proinflammatory responses, but at the same time exacerbate the infection by inhibiting anti-parasitic immune function. Hugosson et al found high baseline IL-10 levels were associated with higher parasite densities post treatment after adjusting for initial parasitaemia, age temperature and sex. The authors concluded that the induction of high IL-10 production might be a direct or indirect mechanism whereby the parasite evades host immune response (Hugosson et al. 2004). All in all, severe malaria is not a single and discrete syndrome as coma develops through multiple pathways with many mechanisms of brain injury (Idro et al. 2010). 1.5 CLINICAL SIGNS AND SYMPTOMS The incubation period of malaria varies with the level of host immunity acquired through prior exposure and the use of antimalarial prophylaxis. In non-immune individuals, the time of infection to detectable parasitaemia ranges from 5 to 10 days, and the development of symptoms from 6 to 14 days (Trampuz et al. 2003). Clinical symptoms are primarily due to the destruction of RBC with the release of merozoites. The presentation of malaria may be non-specific, often with symptoms resembling those of a common cold. Infection with P. falciparum can be associated with welldefined febrile paroxysms, consisting of fever, chills and rigors at regular intervals. Other symptoms include headache, diaphoresis, nausea, vomiting, dizziness, malaise, abdominal pain, mild diarrhoea and dry cough. Clinical signs include jaundice, pallor, 10

45 Chapter 1 General Introduction tachycardia, orthostatic hypotension, hepatomegaly, splenomegaly, metabolic acidosis, hypoglycaemia, seizures, coma, renal failure and cerebral oedema (Gilles 1988; Clark et al. 2000; Trampuz et al. 2003; Mackintosh et al. 2004). Figure 1.3 demonstrates a child infected by P. falciparum who was found to have splenomegaly and fever. Figure 1.3 Child with malaria. Father comforting his malaria-infected son whilst waiting outside the Alexishafen Health Care Centre, Madang Province, PNG. 1.6 DIAGNOSIS Light microscopic examination of intracellular parasites on Giemsa stained thick and thin blood smears remain the definitive method of diagnosis of malaria (Bloland 2001). This method enables the quantification and identification of different Plasmodium species, and the presence of mixed-species infection, diagnosis of which will have a bearing on treatment selection. Parasite densities are liable to considerable fluctuation due to the impact of chemo-suppression, development of host immunity, and their cyclic multiplication patterns. In some instances, infection with P. falciparum may produce scanty intra-erythrocytic stages with parasitaemia below detectable threshold, as synchronous mature parasites are sequestered away from the peripheral circulation in the microvasculature. In these circumstances, the identification of schizonts, 11

46 Chapter 1 General Introduction gametocytes and malarial pigments in macrophages are of value (Shute 1988). Before a blood slide is considered negative, at least 200 fields should be examined under 1000 magnification. With positive smears, the level of parasitaemia is reported as a percentage of infected RBC or as the number of parasites per microliter (µl) of blood. Microscopy diagnosis can be time consuming and requires trained personnel to ensure consistent reliability. It has relatively low sensitivity compared to other techniques, especially when parasitaemia is low and in areas of unstable, low transmission (John et al. 2005; Coleman et al. 2006; Menge et al. 2008). Whilst the availability of microscopy has been shown to reduce drug use in trial settings, in practice blood film results are often disregarded by clinicians as presumptive clinical diagnosis is customary and a cost saving approach in malaria endemic areas (Jonkman 1995; Barat et al. 1999; PNGDOH 2000). Alternative diagnostic methods include rapid dipstick immunochromatographic assays that detect species-specific parasite antigens targeting either histidine-rich protein-2 (HRP-2) by, for example, the Parasight F test or Plasmodium lactate dehydrogenase (pldh) by the OptiMAL test (Schiff et al. 1993; Makler et al. 1998). However, persistence of the HRP-2 antigen in the circulation after parasite clearance can give rise to false positives (Makler et al. 1998). The first generation of OptiMAL tests were prone to loss of sensitivity caused by humidity and high temperature. However, this instability has been overcome in the second generation OptiMAL IT tests (Moody et al. 2002). Occasional cases of false-positive results for P. falciparum were observed in samples containing high levels of heterophile antibodies (Moody et al. 2002). One field evaluation of the OptiMAL test showed 96% sensitivity, 100% specificity and with 100% and 97.5% positive and negative predictive values respectively compared to conventional microscopy. However sensitivity decreased when parasitaemia was <300 parasites/µl whole blood (Zerpa et al. 2008). Although dipsticks tests may enhance the speed of diagnosis, microscopy remains the most cost-effective diagnostic method in malaria endemic areas. The use of polymerase chain reaction (PCR) based assays for the detection of speciesspecific Plasmodium genome is highly specific and sensitive, with a detection limit as 12

47 Chapter 1 General Introduction low as 1 parasite/µl (Hanscheid et al. 2002). However, in addition to the cost of reagents, this method requires a stable electricity supply and sophisticated equipment that is often not available in field settings. PCR methods are more useful in epidemiological studies and for the diagnosis of imported malaria in developed countries (Berry et al. 2008; Menge et al. 2008). 1.7 TRANSMISSION Malaria parasites are transmitted through the bite of infected female Anopheles mosquitoes (Figure 1.4). On taking a blood meal as a requirement by the mosquito for ovulation, malarial sporozoites harboured in the salivary glands of the insect are inoculated into the capillary of the human host. At the same time, gametocytes from the human host can be transferred into the mosquito to continue the sexual phase of its life cycle (Section 1.3) (Garnham 1988). In this way, infected populations of both human and Anopheles mosquitoes function as reservoirs for each other. Transmission can also occur congenitally by transplacental transfer of infected RBC into the neonate, and malaria in pregnancy contributes to a significant number of maternal and infant deaths (Malhotra et al. 2006; Brabin 2007). Figure 1.4 Anopheles albimanus taking a blood meal. (Cook et al. 2006) 13

48 Chapter 1 General Introduction 1.8 PREVENTION In malaria endemic areas, the use of insect repellent and clothing that provides covering are important measures to reduce the chance of mosquito biting. Anopheles generally feeds in the evening, hence staying indoors and sleeping within insecticide-treated bed nets are effective night-time preventive measures. The use of chemoprophylaxis is also recommended for travellers to malaria endemic areas. Other community based interventions include indoor residual spraying of insecticides, reducing mosquito breeding sites such as stagnant water and improving drainage. Significant challenges in mosquito control include increasing resistance to key insecticides such as DDT and pyrethroids with the development of new pesticides proving costly and time consuming (WHO 2009). 1.9 TREATMENT The best treatment currently recommended for P. falciparum infection is artemisininbased combination therapy (ACT). Examples of ACTs include artemether-lumefantrine (Coartem ), artesunate-sp, dihydroartemisinin-piperaquine (Duo-Cotecxin ) and artemisinin-naphthoquine (ARCO ). However, the spread of parasite resistance is undermining malaria control efforts and there are no effective alternatives to artemisinins currently on the market or near completion of the drug development process (WHO 2009). Recent clinical and molecular studies from the Cambodia- Thailand border where artesunate-mefloquine is the standard treatment regimen suggest the emergence of ACT-resistant parasite strains (Denis et al. 2006; Vijaykadga et al. 2006; Alker et al. 2007). The reported treatment failures were considered most likely due to the high level of mefloquine resistance rather than to the artemisinin component, as mefloquine was used for monotherapy long before the introduction of ACTs. Molecular findings of increased pfmdr1 copy number in local P. falciparum isolates attested to mefloquine resistant in conjunction with the loss of sensitivity in vivo (Price et al. 2004; Wongsrichanalai and Meshnick 2008; Chaijaroenkul et al. 2010). However, recent studies have also suggested that initial parasite clearance, the hallmark of artemisinin efficacy, is delayed in this area. This suggests that resistance to this valuable group of antimalarial drugs has now started to develop (Dondorp et al. 2010). 14

49 Chapter 1 General Introduction 1.10 MALARIA IN WESTERN PACIFIC Ten of the thirty-seven Western Pacific countries are malaria endemic; China, Philippines, Cambodia, Korea, Malaysia, Laos Peoples Democratic Republic, Viet Nam, PNG, Solomon Islands and Vanuatu, accounting for 340,000 cases per year. Recent statistics showed a substantial reduction in malaria cases and mortality from 760,000 cases in 1990 to half this figure in However, malaria still claims many lives in the Pacific region (Figure 1.5). PNG has the highest mortality within the Pacific region where the up-scaling of vector control has been sluggish (WHO and WPRO 2007). Figure 1.5 Malaria mortality in Western Pacific. Malaria mortality rate (per 100,000 population) in selected countries in the Western Pacific Region, (WHO 2009). 15

50 Chapter 1 General Introduction 1.11 EMERGENCE OF ANTIMALARIAL RESISTANCE IN PAPUA NEW GUINEA Similar to the situation in Africa, young children in PNG carry the major disease burden due to their lack of immunity (Michon et al. 2007). In the 1970s, reports from a missionary station in Milne Bay (Figure 1.6) described cases of P. falciparum infection that apparently did not respond to chloroquine (CQ) treatment. A cascade of in vivo trials soon followed (Saint-Yves 1971; Han et al. 1976). Investigations conducted in Maprik and Popondetta reported on the retained effectiveness of CQ. These studies however, encountered immense difficulties with patient recruitment as only a small number (22 of 1000) of children screened were eligible, all of whom were aged 7 to 12 years old, and thus probably semi-immune. The early Maprik and Popondetta findings may not therefore, have reflected true resistance, especially when younger children are found to harbour resistant parasites (Michon et al. 2007). Figure 1.6 Regions and provinces of Papua New Guinea. ENB = East New Britain, WNB = West New Britain, EH = Eastern Highlands, SH = Southern Highlands, WH = Western Highlands. 16

51 Chapter 1 General Introduction Not long after the initial alert from Milne Bay, two cases of CQ treatment failure in non-immune expatriates were confirmed in Port Moresby, the capital city of PNG. Both patients had travelled to the Kiunga area of the Western Province. Six patients from the same region who used CQ as a prophylaxis also suffered malaria that did not respond to standard treatment (Grimmond et al. 1976). Subsequent studies confirmed the presence of CQ-resistant P. falciparum particularly along the border with Indonesia and nearby provinces in the Northern coast of PNG (Han 1978). The appearance of CQ-resistant P. falciparum coincided with the decreased effectiveness of residual insecticide spraying initiated in the 1950s which further exacerbated the spread of resistant parasites (Colbourne et al. 1970). Three decades on, resistance to other 4-aminoquinolines particularly amodiaquine (AQ) and quinine have been reported under vigilant in vivo and in vitro surveillance (Schuurkamp and Kereu 1989; Trenholme et al. 1993; al- Yaman et al. 1996; Marfurt et al. 2007). The rapid spread of resistant P. falciparum across the provinces of PNG has been documented in clinical (Darlow et al. 1982; Sapak et al. 1991; Genton et al. 2005; Marfurt et al. 2007) and in vitro studies (summarised in Chapter 3). In addition to a long history of monotherapy, mass dosing of pyrimethamine during the era of malaria control/eradication has ensured constant drug pressure on the parasite population thus selecting for resistance. A high level of resistance has been documented in numerous in vivo (Schuurkamp and Kereu 1989; Karunajeewa et al. 2008; Darlow et al. 1980) and in vitro studies (Reeder et al. 1996; Hombhanje 1998). In 2000, PNG health authorities replaced CQ monotherapy with sulfadoxine-pyrimethamine (SP) combination therapy as first-line treatment to improve clinical efficacy (Nsanzabana et al. 2010) and to delay the development of drug resistance (Casey et al. 2004; Genton et al. 2005). The year 2011 marked the beginning of another era in antimalarial drug usage as PNG implemented artemether-lumefantrine as first-line treatment (Mueller, 2010, personal communication). 17

52 Chapter 1 General Introduction 1.12 ANTIMALARIAL CHEMOTHERAPY Quinine and artemisinin derivatives are, respectively, the most employed and most active antimalarial compounds, both of which have a long history as ancient herbal therapies. The famous sweet wormwood (Artemisia Annua) also known as Qinghaosu (Figure 1.7) was recorded as early as 200 BC in a Chinese medical treatise; the Fiftytwo Prescriptions discovered in the Mawangdui Tomb of the Hunan Province. The antimalarial application of Qinghaosu was also documented in "The Handbook of Prescriptions for Emergencies" by Ge Hong in the 4th century. In other parts of the world, Cinchona tree bark had been used by indigenous South American tribes for its antipyretic properties. The use of Cinchona bark (Figure 1.7) for treating fever was subsequently popularised by the 17th century Spanish Jesuit missionaries (Lee 2002). Both natural sources served as prototypes of the potent antimalarials available today. Despite tremendous resources devoted to the search for antimalarial drugs, only a handful of compounds have been discovered. Despite extensive research, our understanding of the molecular basis of antimalarial activity remains incomplete. The following section outlines the history of current antimalarials, their proposed mechanism of action and the development of drug resistance. Figure 1.7 Artemisia and Cinchona. Artemisia annua (left) (NomadRSI 2002). Cinchona bark (right) (KEW 2007). 18

53 Chapter 1 General Introduction Quinoline Related Compounds Quinine Figure 1.8 Chemical structure of quinine. (O'Neill et al. 2006) Quinine (Figure 1.8), derived from the bark of the Cinchona tree, was first employed as an effective malaria treatment in South America in the 17th century. This alkaloid compound was isolated in the early 1800s by Portuguese chemists and its synthesis was described in 1944 (Turner et al. 1953). Under political and military influences, Cinchona plantations outside Peru were established. The demand for quinine intensified with the advent of World Wars I and II, as supplies were inadequate and unstable. The associated neurotoxicity and other side-effects have provided an impetus to research synthetic alternatives. The availability of better tolerated drugs such as CQ has reduced its usage and slowed the development of widespread resistance to this drug (Wongsrichanalai et al. 1992b). Quinine is by far the most commonly used treatment for severe malaria in the world, being the standard treatment for severe disease across Africa and other malaria endemic regions (WHO 2000; Sinclair et al. 2011), however recent findings suggest that this is likely to change in favour of artesunate (Checkley and Whitty, 2007; Dondorp et al. 2005; Dondorp and Day, 2007; Dondorp et al. 2010). Quinine is used with SP as a second-line regimen for uncomplicated malaria and for the treatment of severe malaria in PNG (Genton et al. 2005). Quinine has remained increasingly important for the treatment of CQ-resistant P. falciparum in other parts of the world, particularly for 19

54 Chapter 1 General Introduction treatment during pregnancy (SANDH 2007; Chico et al. 2010) Chloroquine Figure 1.9 Chemical structure of chloroquine. (O'Neill et al. 2006) Originally known as Resochin, CQ (Figure 1.9) was first synthesised in Germany and thought to be too toxic for further development. After World War II, re-evaluation proved its clinical safety, high efficacy and low toxicity (Loeb et al. 1946). CQ has since been used extensively, and has been the drug of choice for uncomplicated malaria and chemoprophylaxis. In 1950s to 1960s, the WHO devised population-based dosing regimens for the Global Eradication Programme in which over 84,000 tons of CQ was supplied to Brazil, parts of Africa and Asia for incorporation in table salt as means of chemoprophylaxis (Greenwood et al. 2008). Resistant P. falciparum emerged rapidly and has since spread to many endemic areas of the world, compromising its usefulness (Peters 1987; Fidock et al. 2004). In spite of this, CQ is still employed as first-line treatment in PNG in combination with SP for the treatment of uncomplicated malaria during pregnancy (Casey et al. 2004; Mueller et al. 2008) despite increasing evidence for the loss of effectiveness (Marfurt et al. 2007; Karunajeewa et al. 2008). Although generally perceived to have timed out, CQ remains effective in some parts of the world. A study has shown that P. falciparum isolates from Malawi have regained their sensitivity to CQ a decade after its withdrawal (Laufer et al. 2006; Nkhoma et al. 2007). A similar trend to reduction in resistance to CQ was also evident in Kilifi, a coastal town in Kenya, following its official withdrawal, although at a much slower rate (Mwai et al. 2009a). Recent microsatellite analyses of resurgent CQ-susceptible parasites revealed they are likely to be a re-expansion of pre-existing CQ-sensitive population, 20

55 Chapter 1 General Introduction rather than a reverse mutation in a previously resistant parasite or a new selective sweep (Laufer et al. 2010). Extensive research has been devoted into elucidating CQ s mechanism of action. Early studies suggested the inhibition of DNA replication and RNA synthesis, albeit at rather high concentrations (Allison et al. 1965; Thelu et al. 1994). Subsequent characterisation of enzymes involved in DNA replication proved that they are unlikely targets of CQ (Chavalitshewinkoon et al. 1993; White et al. 1993). To date, it is widely accepted that CQ acts within the parasite food vacuole and exerts its antiplasmodial effect by interfering with detoxification (Pagola et al. 2000; Ursos et al. 2002; Fidock et al. 2004). In uninfected RBC, the uptake of CQ is minimal (Macomber et al. 1966). In parasitised RBC, its accumulation is several thousand-fold higher, localising within the parasite food vacuole perhaps reflecting ion-trapping due to its dibasic nature (Macomber et al. 1966; Aikawa 1972; Ginsburg et al. 1989; Hawley et al. 1996). As the parasite digests haemoglobin, free haem (ferriprotoporphyrin IX) and reactive oxygen species are produced as toxic by-products. To counter this, the parasite polymerises the toxic haem into non-toxic haemazoin crystals (Figure 2.2) (Blauer et al. 1997). CQ acts by binding to these free haem moieties, thus interfering with detoxification where toxic by-products accumulate, leading to parasite death (Fitch 1998; Foley et al. 1998; Pagola et al. 2000; Fidock et al. 2004) Amodiaquine Figure 1.10 Chemical structure of amodiaquine. (O'Neill et al. 2006) 21

56 Chapter 1 General Introduction During World War II, the US government invested in a massive screening program involving 300,000 compounds, and in 1955, AQ (Figure 1.10) was identified as one of three compounds that were active against P. falciparum (Peters 1987). AQ served as an alternative prophylaxis for falciparum malaria for over 40 years (Foley et al. 1998). However, support for its prophylactic use was withdrawn by the WHO after reports of fatal adverse reactions in the mid 1980s, and associated high incidence of hepatitis and agranulocytosis (Greenwood 1995). However, the rapid spread of CQ resistance has prompted the re-evaluation of AQ for therapeutic use. A subsequent systematic review of AQ treatment in uncomplicated malaria found the rates of adverse events were no higher than that of CQ and SP treatments in controlled trials, without life-threatening events (Olliaro et al. 1996). In vitro studies have shown AQ to be more potent than CQ against less susceptible strains of P. falciparum (Rieckmann 1971; Siddiqui et al. 1972). Despite this, clinical resistance to AQ soon followed and AQ monotherapy is not recommended (Al-Yaman et al. 1996). AQ has found its use as first-line treatment and as seasonal intermittent preventative treatment for malaria as part of ACT regimens (Adjei et al. 2008; Sokhna et al. 2008). AQ (Camoquine) has been used as first-line treatment with SP, but more recently in combination with artesunate for young children in PNG and Africa (PNGDOH 2000; Brasseur 2007; Marfurt et al. 2007). AQ and its active metabolite monodesethyl-aq share structural similarity with CQ and partial cross resistance has been reported (Basco and Ringwald 2003; Wong et al 2010). AQ competitively inhibits CQ accumulation in the parasitic food vacuole (Fitch 1973). As a diprotic weak base with lower pka values compared to CQ, it may accumulate less efficiently in the parasite food vacuole via ion-trapping. However, AQ is found in higher concentrations than CQ in the parasite, indicative of enhanced uptake by an additional pathway (Hawley et al. 1996). Several studies have demonstrated the ability of AQ to bind to haem and inhibit haem polymerisation, analogous to CQ (Chou et al. 1993). 22

57 Chapter 1 General Introduction Mefloquine Figure 1.11 Chemical structure of mefloquine. (O Neill et al. 2006) Mefloquine (MQ) (Figure 1.11) was also selected from a screening program and developed through global collaborative efforts. A distinguishing property of MQ is its long terminal elimination half-life of 2 to 3 weeks, hence it can achieve clinical efficacy with a single dose. It is well tolerated by adults and children. Common side-effects include nausea, dizziness, headache, rash, pruritis, in some instances bradycardia and the well publicised psychiatric disturbances (Palmer et al. 1993). It was first deployed in Thailand in 1984, initially in combination with SP, but resistance developed relatively quickly. It has been more recently incorporated with artesunate as part of ACT. The relative high cost of MQ in addition to concerns regarding toxicity limits its use in many resource-poor malaria endemic countries. 23

58 Chapter 1 General Introduction Lumefantrine Figure 1.12 Chemical structure of lumefantrine. (Basco et al. 1998) Formerly known as benflumetol, lumefantrine is a 2, 3 benzindene that belongs to the amino-alcohol class that also includes quinine, mefloquine and halofantrine (WHO 2006) (Figure 1.12). The compound was first synthesised in China and has high clinical efficacy against multidrug resistant P. falciparum as a co-formulation with artemether (Coartem ) (WHO 2006; Karunajeewa et al. 2008b). The oral formulation is efficacious for the treatment of children with uncomplicated malaria and drug-related adverse events are rare (Adjei et al. 2008; Karunajeewa et al. 2008b). Bioavailability of lumefantrine is variable and its absorption is significantly increased when administrated with fatty foods (Ezzet et al. 1998; White et al. 1999; Borrmann et al. 2010). Lumefantrine-artemether is the current first-line treatment for uncomplicated malaria in Africa (Kabanywanyi et al. 2007; Borrmann et al. 2010) and PNG (WHO 2007; Schoepflin et al. 2010). 24

59 Chapter 1 General Introduction Naphthoquine Figure 1.13 Chemical Structure of Naphthoquine. (Wang et al. 2004) Naphthoquine (NQ) (Figure 1.13) is a relatively new drug first registered in 1993 in China. This drug exhibits potent schizontocidal effects in CQ-resistant parasites. Clinical studies of NQ monotherapy carried out on Hainan Island, China reported 100% and 96.7% cure rate that compared favourably with artemisinin monotherapy which had a cure rate of 73.3% (Pang et al. 1999; Guo et al. 2000). No distinctive adverse reactions have been associated with NQ use, although transient abdominal discomfort has been reported in some patients. Elimination half-life of NQ ranges from 41 hr to 255 hr (Wang et al. 2004; Qu et al. 2010). The long half-life of NQ makes it a suitable partner with artemisinin in the relatively new ARCO TM therapy that is undergoing safety and efficacy trials in PNG (Hombhanje et al. 2009; Davis, unpublished). Parasite susceptibility data outside of Chinese literature are limited, with only one study performed in PNG field isolates (Wong et al. 2010), the findings of which will be discussed in subsequent chapters. 25

60 Chapter 1 General Introduction Piperaquine Figure 1.14 Chemical Structure of Piperaquine. (Davis et al. 2005) Piperaquine (PQ) (Figure 1.14) is a bisquinoline compound first synthesised from screening programs in France and China during the 1960s. It was thought to offer little advantage over CQ and hence development was not pursued except in China. PQ was developed for clinical use and was the drug of choice in China and Indochina for two decades until the emergence of resistance (Chen 1991; Fan et al. 1998). In the 1990s, renewed interest has seen PQ as a long half-life partner in ACTs. Examples include CV8 (PQ-dihydroartemisinin-trimethoprim-primaquine), Artecom (PQ-primaquine) and more recently Artekin or Duo-Cotecxin (PQ-dihydroartemisinin), the latter producing high cure rates and tolerability in areas of CQ-resistant P. falciparum (WHO 2006; Karunajeewa et al. 2008b). A recent in vitro study from Cameroon has also shown PQ to be highly active against both CQ sensitive and resistant strains of P. falciparum isolates (Basco and Ringwald 2003) with a long elimination half-life of 33 days (Tarning et al. 2005) Antifolate Combination Drugs Figure 1.15 Chemical structures of sulfadoxine (left) and pyrimethamine (right). (WHO 2006) 26

61 Chapter 1 General Introduction Antifolate combinations comprise of inhibitors of Plasmodium dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps), both of which are involved in parasite DNA synthesis. Examples of pfdhfr inhibitors include pyrimethamine (Figure 1.15), proguanil, chlorproguanil, and trimethoprim. Sulfa drugs that inhibit pfdhps include sulfadoxine (Figure 1.15), dapsone, sulfalene and sulfamethoxazole. Genetic mutations in parasite pfdhfr and pfdhps genes confer rapid resistance against these drugs, particularly when used alone. When used in combination however, these drugs produce a synergistic effect where they are effective even in the presence of parasite resistance to individual components. Common antifolate combinations include SP, which is useful in pregnant women for placental malaria, sulfamethoxazole-trimethoprim (cotrimoxazole), sulfalene-pyrimethamine (metakelfin) and a more recent combination of dapsone-chlorproguanil (LapDap) that is more affordable and provides higher cure rates (Watkins et al. 1997) but which has now been withdrawn because of adverse effects (haemolytic anaemia) in patients with glucose-6-phosphate dehydrogenase deficiency (Bukirwa et al. 2004; Fanello et al. 2008) Artemisinin and its Derivatives Figure 1.16 Chemical structure of artemisinin (left) and its derivatives (right). Artemether (R = OCH 3 ); Artesunate (R = OCH 2 CH 2 CO 2 Na); Dihydroartemisinin (R = OH). Artemisinin and its derivatives (Figure 1.16) are frontline antimalarial treatments that facilitate rapid parasitaemia clearance and prompt resolution of symptoms. Artemisinin is a sesquiterpine lactone isolated from the plant Artemisia annua or Qinghaosu (Figure 1.7) which has been used for thousands of years by Chinese herbalists for curing febrile 27

62 Chapter 1 General Introduction illnesses. Qinghaosu was re-discovered in 1972 from a mass screening project of traditional medicinal herbs funded by the Chinese military (Tu You-you 1982; Zhang 2007). Artemisinin and its derivatives are the most potent antimalarials to date, characterised by a rapid clearance of parasitaemia (Skinner et al. 1996). These have proven to be efficacious and well tolerated as monotherapy although these are not recommended to be used alone due to their short half-lives with associated high rates of recrudescence related to rapid elimination. Examples of semi-synthetic artemisinin derivatives include artemether and artesunate. Since artemisinin and its derivatives are rapidly eliminated from the body and long courses are required for high long-term cure rates, greater efficacy can be obtained by partnering these potent but short-lived compounds with those with a longer half-life such as in lumefantrine, MQ or AQ as ACTs. These strategies are now recommended in Africa and PNG to delay the development of resistance (WHO 2006; Ekland et al. 2008; O'Neill et al. 2010). These regimens include artemisinin-nq (Wang et al. 2004), artesunate-aq and artemether-lumefantrine (Adjei et al. 2008). Dihydroartemisinin (DHA) is an active metabolite of artemether and artesunate. In comparison to other derivatives, in vitro studies have shown DHA to act on all stages of intra-erythrocytic P. falciparum, and is the most potent in achieving 100% inhibition within 2 to 4 hr of exposure (Skinner et al. 1996) Antibiotics Several antibiotics are currently in use as prophylaxis and in combinations with other antimalarials as treatment for uncomplicated P. falciparum infections (Nakornchai et al. 2006; Ejaz et al. 2007; Noedl et al. 2007). However, the exact mechanism and cellular effects of these parasiticidal anti-bacterial drugs remain largely unknown. As with other apicomplexa parasites such as Babesia spp, Toxoplasma gondii and Cyclospora cayetanensis, Plasmodium species have an apicoplast similar to the plastid structure found in algae and bacteria. This organelle is non-photosynthetic but contains essential house-keeping machineries for DNA replication, transcription, translation and posttranslational modification, and various anabolic pathways (Goodman et al. 2007). 28

63 Chapter 1 General Introduction Recent identification and characterisation of this unique apicomplexan structure has revealed a number of putative targets with the potential to help explain the mysterious activities of antibiotics against malaria (McFadden et al. 1996; Wilson et al. 1996; Goodman et al. 2007b). The absence of a similar structure in mammalian cells renders the Plasmodium apicoplast a valuable drug target. A number of antibiotics have been shown to target the prokaryotic 70S ribosome in Plasmodium apicoplast thus affecting protein synthesis (Figure 1.17). Examples include azithromycin (AZ), clindamycin, tetracycline, doxycycline and thiostrepton. The macrolides (AZ, clindamycin) block the peptide exit tunnel, preventing peptide elongation, lincosamides (clindamycin) and tetracyclines interfere with the translocation and binding of peptidyl trnas to the acceptor site, respectively (Auerbach et al. 2002). Figure 1.17 Targets of anti-bacterial drugs in P. falciparum apicoplast. Antibacterial drugs targeting the house-keeping functions of the P. falciparum apicoplast. (Modified and adapted from Goodman and McFadden, 2007) An interesting phenomenon with antibiotics observed in apicomplexan parasite is the delayed-death effect, where the drug has no apparent effect on the parasite as it continues to grow and divide and its daughter cells re-invade a new host. It is not until the second generation where the progenies fail to develop and eventually die (Fichera et 29

64 Chapter 1 General Introduction al. 1995; Fichera et al. 1997). Various studies have shown the significant reduction of IC 50 values when in vitro antibiotic drug trials against P. falciparum were extended from 48 hr to 96 hr (Yeo et al. 1995; Goodman et al. 2007b). It was hypothesised that drugs targeting prokaryote-like translation, as those mentioned previously, would produce this effect. Antibiotics that target DNA replication, transcription and interfere with fatty acid and isoprenoid pathways such as fosmidomycin and triclosan, cause immediate parasite death within the first intra-erythrocytic cycle (Jomaa et al. 1999; Goodman et al. 2007a; Wiesner et al. 2008) Azithromycin Figure 1.18 Chemical Structure of Azithromycin. Azithromycin (AZ) (Figure 1.18) is a broad spectrum macrolide derived from erythromycin with more favourable pharmacokinetics and longer elimination half-life (Girard et al. 1987). It is used clinically for the treatment of streptococcal, staphylococcal, chlamydial and gonorrhoeal infections. Since the discovery of AZ s activity against CQ-resistant P. falciparum (Gingras et al. 1992; Gingras et al. 1993), this drug has been studied with renewed interest in the campaign against multidrug resistant malaria. Only recently has its mode of action been elucidated. According to this report, AZ binds to the 50S ribosomal subunit in the apicoplast translation machinery, interfering with protein synthesis and subsequently leads to a classic delayed-death (Sidhu et al. 2007). Despite its slow onset and considerably lower potency compared to other antimalarials (Noedl et al. 2001; Noedl et al. 2007; Wong et al. 2010), the safety profile in young 30

65 Chapter 1 General Introduction children and extensive experience with its use during pregnancy (Bace et al. 1999; Arrieta et al. 2003; Sarkar et al. 2006) render it an attractive candidate to partner with fast-acting conventional antimalarials. An earlier study with AZ-artesunate combination in Thai adults with uncomplicated falciparum malaria resulted in an unexpectedly low cure rate (Na-Bangchang et al. 1996). This was likely due to inadequate AZ dosage. With a higher dose, a phase 2 clinical trial demonstrated the safe and efficacious use of AZ-quinine and AZ-artesunate (Noedl et al. 2006) Summary of Antimalarial Activities The following table provides a list of common antimalarial drugs and their specific activities (Table 1.1). 31

66 Chapter 1 General Introduction Table 1.1 Antimalarial drugs and their specific activities. (WHO 2006) Drug Class Compound Specific Activities 4-Aminoquinoline Chloroquine Amodiaquine Naphthoquine Blood schizontocides Interference of haem detoxification in parasite food vacuole 8-Aminoquinoline Primaquine Tafenoquine Intra-hepatic schizontocides Gametocytocides Bis-quinoline Piperaquine Blood schizontocides Arylaminoalcohol Antifolates Artemisinin Antibiotics Quinine/Quinidine Mefloquine Halofantrine Lumefantrine Sulfadoxine Sulfamethoxazole Dapsone Pyrimethamine Proguanil Chlorproguanil Trimethoprim Artemisinin Artesunate Dihydroartemisinin Arteether Artemether Azithromycin Clindamycin Doxycycline Tetracycline Ciprofloxacin Rifampicin Thiostrepton Mature trophozoites Blood schizontocides Early stage gametocytocides Inhibitors of dihydropteroate synthase Inhibitors of dihydrofolate reductase Slow-acting blood schizontocide Sporontocide Possible activity against intra-hepatic forms Fast-acting blood schizontocides Gametocytocides, Inhibitors of calcium adenosine triphosphatase Slow-acting blood schizontocides Inhibitors of aminoacyl-trna binding during protein synthesis Naphthoquinones Atorvaquone Inhibits intra-hepatic forms Inhibits oocyte development in the mosquito Interferes cytochrome electron transport 32

67 Chapter 1 General Introduction 1.13 LIPID-LOWERING AGENTS AS ANTIMALARIALS Recent interest in alternative antimalarial drugs led to the discovery of P-glycoprotein, an efflux transporter encoded by the multidrug resistant gene which actively removes compounds from cells and prevents intracellular drug accumulation. Interestingly, certain statins and fibrates have been shown to interact with P-glycoprotein in mammalian cells expressing multidrug resistance (Wu et al. 2000; Ehrhardt et al. 2004). Although a P-glycoprotein homologue 1 has been identified in P. falciparum, studies investigating the antimalarial activity of these lipid-lowering agents are limited Statins Statins are cholesterol-lowering drugs that are considered first-line therapies for reducing morbidity and mortality associated with atherosclerotic disease (Grundy et al. 2004; Wilt et al. 2004; Thavendiranathan et al. 2006). They are inhibitors of 3-hydroxymethyl-glutaryl coenzyme A (HMG-coA) reductase that decreases cholesterol synthesis and plasma cholesterol and lipoprotein levels (Figure 1.19) (Maron et al. 2000). Examples include atorvastatin (Lipitor ), rosuvastatin (Crestor ), pravastatin (Pravacol ) and simvastatin (Zocor ). Despite a common mechanism of action, statins differ in their chemical structures, pharmacokinetic profiles and lipid-modifying efficacy. Pravastatin and simvastatin are derived from fungal metabolites and have elimination half-lives of 1 to 3 hr. Atorvastatin and rosuvastatin are both synthetic compounds, with elimination half-lives of 14 hr and 19 hr respectively. Rosuvastatin (Crestor ) is a recently developed drug with the most potent LDL-cholesterol-lowering properties, followed by atorvastatin, simvastatin and pravastatin (Soran et al. 2008). In addition to their cholesterol-lowering efficacy and good clinical safety profile (Cilla et al. 1996; Bellosta 2004), statins exhibit a wide range of antimicrobial activities against bacteria, yeasts and protozoan including P. falciparum (Montalvetti 2000; Song et al. 2003; Catron et al. 2004; Pradines et al. 2007; Wong et al. 2009). Atorvastatin and lovastatin reduced the growth of Salmonella enterica in mouse model and in cultured macrophages (Catron et al. 2004). They acted synergistically with fluconazole to inhibit the eukaryotic sterol pathway, as demonstrated by the reduced growth of 33

68 Chapter 1 General Introduction Candida albicans after exposure (Song et al. 2003). In addition, they reduced the growth of Schistosoma mansoni, Trypanosoma cruzi and Leishmania species through inhibition of 3HMG-CoA reductase (Chen et al. 1990; Urbina 1993; Andersson et al. 1996; Montalvetti 2000). Figure 1.19 Overview of isoprenoid biosynthesis. In humans, the biosynthesis of isopenteny-diphosphate and dimethylallyl-diphosphate and ultimately cholesterol proceeds entirely via the mevanlonate pathway. In Plasmodium, these isoprenoid building blocks are supplied by the 1-deoxy-D-xylulose-5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway. Statins inhibit the mevalonate pathway as indicated (Moreno et al. 2008). 34

69 Chapter 1 General Introduction The antimalarial effects of statins have also been described. Lovastatin and simvastatin inhibited intra-erythrocytic development of P. falciparum in vitro (Grellier et al. 1994). In other recent studies, atorvastatin exhibited high in vitro activity against P. falciparum (Pradines et al. 2007; Parquet et al. 2009; Wong et al. 2009). Despite these encouraging findings, neither simvastatin nor atorvastatin given in high doses improved outcome in P. berghei-infected mice (Bienvenu et al. 2008; Kobbe et al. 2008) and there was no effect on parasitaemia (Bienvenu et al. 2008). No study to date has included the most potent statin in clinical use, rosuvastatin (Soran et al. 2008). In addition, despite demonstration of in vitro synergy between mevastatin and the P-glycoprotein inhibitor tunicamycin against P. falciparum (Naik 2001), the interaction between statins and conventional antimalarial drugs has not been assessed Fibrates While statins decrease cholesterol synthesis, fibrates primarily intensify catabolism of triglyceride-rich lipoproteins and increase HDL-cholesterol. Gemfibrozil, fenofibrate, clofibrate are examples of fibrates in clinical use. The fibrates are agonists of peroxisome proliferator-activated receptor alpha (PPARα). Fenofibrate has also been shown to inhibit P-glycoprotein mediated transport with a similar potency to simvastatin (Ehrhardt et al. 2004) and modulate the expression of the lipid-efflux protein, ATP-binding cassette sub-family A member (ABC-1) (Arakawa et al. 2005). If malaria pathology is attributable to systemic inflammation (Clark et al. 2008), the use of fibrates to hinder the release of inflammatory cytokines may constitute a novel treatment approach. A number of studies have investigated the anti-inflammatory effects of fibrates. Gemfibrozil reduced TNF production in monocytes and downregulated the production of superoxide and expression of nuclear factor κb, both of which regulate inflammation (Zhao et al. 2003; Calkin et al. 2006). Furthermore, gemfibrozil and fenofibrate both inhibited TNF, IL-1 beta, IL-6 and nitric oxide production (Xu et al. 2006). In a murine influenza model, where severe systemic disease is thought to arise through overproduction of proinflammatory cytokines, Budd et al. (2007) found gemfibrozil offered some protection against the virus, with an 35

70 Chapter 1 General Introduction increased survival rate from 26% to 52% compared to vehicle-treated mice (Clark et al. 2004; Budd et al. 2007). An early study exploring the effect of plasma free fatty acid concentrations and temperature on parasitaemia in P. berghei infected mice used clofibrate as a lipidlowering agent. Coincidentally, the study reported on a significant retardation in the development of parasitaemia in the clofibrate treatment group, suggesting a direct inhibition on parasite growth (McQuistion 1979). A recent report highlighted the potential use of PPAR agonists as a novel treatment for cerebral malaria (Balachandar et al. 2011). However, no other study to date has investigated the effects of fibrates against malaria. The antimalarial activities of fibrates and statins, and their interactions with conventional antimalarials will be explored in Chapter ANTIMALARIAL DRUG RESISTANCE Definitions The development of drug resistance has been a major obstacle in the battle against malaria. The WHO defines antimalarial drug resistance as the ability of parasite strains to survive and/or multiply despite the administration and absorption of a drug given in doses equal to or higher than those usually recommended but within tolerance of the subject. This was later modified in 1986 to specify that the form of the drug active against the parasite must be able to gain access to the parasite or the infected RBC for the duration of the time necessary for its normal action (Bruce-Chwatt 1986). This refined definition takes into account the variations in drug pharmacodynamics and pharmacokinetics between individuals, such as in the metabolism of sulfonamides and sulfones (Trenholme 1975; WHO 2005). Drug resistance causes a right shift in the dose response curve that can be observed in vitro (Figure 1.20). Confirmation of drug resistance requires evidence of parasite recrudescence in a patient (microscopically or by PCR) after receiving a treatment dose of antimalarial. 36

71 Chapter 1 General Introduction Simultaneous demonstration of effective concentrations of the drug or its metabolite in the blood using established laboratory methods such as HPLC is also required. Results from in vitro drug susceptibility tests and detection of molecular markers associated with drug resistance are additional indicators of resistance. In practice however, these tests are seldom performed alongside in vivo studies. For this reason, inadequate parasitological clearance is conventionally considered to be associated with resistance (WHO 2005). Figure 1.20 Right-ward shift of concentration-effect relationship due to drug resistance. Changes in the concentration-effect relationship may occur as a parallel shift (B) from the normal profile (A) or, the slope changes and/or a reduction in the maximum parasite killing effect (C) (WHO treatment guideline 2006-Annex 6) Treatment Failure and Drug Resistance A distinction must be made between antimalarial drug resistance and treatment failure, the latter of which is a failure to clear parasitaemia and/or resolve clinical symptoms after treatment. While drug resistance may cause treatment failure, other factors may contribute. These include incorrect dosage, patient non-compliance regarding duration of dosing, poor drug quality, drug interactions, poor absorption or rapid elimination (diarrhoea and vomiting), and misdiagnosis (WHO 2005). Many of these factors may also accelerate the development and spread of drug resistance as parasites are exposed 37

72 Chapter 1 General Introduction to suboptimal drug levels Emergence of Resistance to Principal Antimalarials Despite an apparently replete list of antimalarials (Table 1.1), the emergence of P. falciparum exhibiting resistance to multiple antimalarials has resulted in a gradual degradation of the effectiveness of all available drugs. This along with the failure of multifaceted approaches to eradicate and control the spread of malaria has seen resurgence in the incidence of this life threatening infection on a global scale. For comprehensive reviews on the evolution and selection of malarial drug resistance, see (Plowe 2009) and (Mackinnon et al. 2010). Resistance to CQ first observed in South-East Asia and South America about half a century ago, now occurs almost everywhere that P. falciparum is transmitted. It is important to recognise that the acquisition of resistance to quinine and CQ took many years (278 and 12, respectively) compared with the rapid appearance of resistance to proguanil, SP, MQ, and atovaquone, all within 5 years of their introduction (Wongsrichanalai et al. 2002). Figure 1.21 illustrates the emergence of resistance to principal antimalarials. Quinine, CQ and SP have remained effective for a considerable length of time even after the initial reports of resistance. AQ was withdrawn by the WHO in 1990 as denoted by the dotted line (Figure 1.21), but was re-introduced for therapeutic usage after confirmation of its clinical safety and efficacy. Recent reports from the Cambodia-Thailand border has indicated modest increase in P. falciparum resistance to ACTs (artesunate-mq) (Wongsrichanalai et al. 2008; Carrara et al. 2009; Rogers et al. 2009). 38

73 Chapter 1 General Introduction Figure 1.21 Emergence of resistance to principal antimalarial drugs. (Modified and adapted from Ekland and Fidock 2008). Coloured bars represent antimalarial regimen in combination and as monotherapy along with the year since its first introduction and the first report of resistance. Rectangles signify the approximate time when resistance has spread to various geographical regions. Timelines were derived from (Al-Yaman et al. 1996; Wongsrichanalai et al. 2002; Ekland et al. 2008; Wongsrichanalai et al. 2008) and references therein. ACTs = artemisinin-based combination therapies; Atov/Prog = atovaquone/proguanil; AQ = amodiaquine; CQ = chloroquine, Q = quinine; R = resistance; MQ = mefloquine; SP = sulfadoxine/pyrimethamine; PNG = Papua New Guinea; E = East; S = South Determinants of Antimalarial Resistance Numerous factors contribute to the advent, spread and intensification of malaria resistance, although their relative contribution remains unknown. Gene mutations conferring resistance occur naturally in low frequencies as parasite populations show 39

74 Chapter 1 General Introduction heterogeneity. Therefore, selection of the most fit parasites occurs in the face of chemotherapy, where parasites harbouring single or multiple point mutations that provide survival advantage would recover and multiply (Wongsrichanalai et al. 2002; Mackinnon et al. 2010). Other aspects attributing to resistance concern the dynamic interplay of drug characteristics and usage, human behaviour and host factors, the mosquito vector and environmental factors (Table 1.2). Drug characteristics are important determinants of antimalarial resistance. Firstly, drugs with a long elimination half-life, such as MQ and SP have the benefit of simpler, singledose regimen which can greatly improve compliance and dosing. However, the subtherapeutic drug concentrations in plasma during slow elimination exert substantial selection on parasites from new infections acquired after the initial treatment (Wernsdorfer 1994; Bloland 2001). This is particularly common in areas with intense malaria transmission (Wongsrichanalai et al. 2002). Host factors such as immunity increase the efficacy of chemotherapy and may offset the spread of resistance. A semi-immune individual may be cured by a drug despite partial drug resistance of the infecting parasites. The specific immune response elicited by repeated exposure to the pathogen is more effective in parasite clearance compared to the non-specific response generated by those naïve to malaria. Introduction of resistant parasites in migrant and refugee populations may thus increase the opportunity for manifestation and spread of resistance (Wongsrichanalai et al. 2002). Table 1.2 Determinants of antimalarial resistance. (Modified and adapted from Wongsrichanalai et al * ). pfcrt = Plasmodium falciparum chloroquine resistance transporter; pfmdr1 = P. falciparum multidrug resistance 1; pfdhps, P. falciparum dihydropteroate synthase; pfdhfr, P. falciparum dihydrofolate reductase; pfserca, P. falciparum sarco-endoplasmic reticulum calcium ATPase6. Examples were obtained from (Wernsdorfer et al ** ; Jonkman 1995 # ; Molyneux et al ; PNGDOH 2000 ; Winstanley 2001^; Bloland ; 2001 Newton et al. 2008^^). (Wernsdorfer et al. 1991; Jonkman 1995; Molyneux et al. 1999; PNGDOH 2000; Winstanley 2001; Newton et al. 2008). 40

75 Chapter 1 General Introduction Factor and characteristic Example Drug Half-life Resistance to chlorproguanil-dapsone (short halflife) develops more slowly than that to SP (long half-life)^. Dosing Non-target drug pressure Pharmacokinetics 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 **. Presumptive use of antimalarial drugs without laboratory diagnosis or for indications other than malaria #,. 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 Maintenance of resistant parasite reservoir Malnourished and HIV infected individuals have. response significantly poorer parasitological Non-detection of drug failure *. Parasite Genetic mutations Polymorphisms in the genes: pfcrt, pfmdr1, pfdhps, pfdhfr, pfserca *. Vector and environment Transmission level Mosquito affinity of parasites 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. Increased infectivity and productivity of CQresistant parasites in Anopheles dirus and the propagation of CQ resistance in South-East Asia and Western Oceania *. 41

76 Chapter 1 General Introduction 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 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 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 foodvacuole (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- 42

77 Chapter 1 General Introduction 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 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 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 43

78 Chapter 1 General Introduction 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). 44

79 Chapter 1 General Introduction 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% (IC 50 ) (Rieckmann et al. 1978; WHO 2001; Noedl et al. 2007). The IC 50 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 IC 50 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, IC 50 s 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 45

80 Chapter 1 General Introduction 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 Schizont Maturation 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 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). 46

81 Chapter 1 General Introduction 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 47

82 Chapter 1 General Introduction produce IC 50 values that are two to three times higher than by the isotopic method (Wernsdorfer et al. 1988) H-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 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 surveillance of P. falciparum drug resistance Plasmodium Lactate Dehydrogenase (pldh) Detection 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 48

83 Chapter 1 General Introduction 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 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 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 IC 50 s 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 49

84 Chapter 1 General Introduction no advantage over the unmodified version 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 nonspecific 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 IC 50 s 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 worldwide implementation for epidemiological monitoring of drug resistance. 50

85 Chapter 1 General Introduction 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 IC 50 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 3 H-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 51

86 Chapter 1 General Introduction 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 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) 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, 52

87 Chapter 1 General Introduction 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. (IC 50 of A in a mixture resulting in 50% inhibition/ic 50 of A alone) + (IC 50 of B in a mixture resulting in a 50% inhibition/ic 50 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 > (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 53

88 Chapter 1 General Introduction (Fivelman et al. 2004). Both checkerboard and fixed-ratio methods require predetermination of IC 50 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 IC 50 s where inaccurate initial IC 50 s 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 IC 50 determination (Fivelman et al. 2004) 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 54

89 Chapter 1 General Introduction 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 al. 2004) Parasitological classifications Sensitive Clearance of parasites after treatment without subsequent recrudescence within a defined period RI parasitological failure Initial clearance followed by recrudescence after day 7 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 Treatment failure classifications 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 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 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 55

90 Chapter 1 General Introduction 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 56

91 Chapter 1 General Introduction 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. 57

92 Chapter 1 General Introduction Table 1.4 Molecular markers for antimalarial drug resistance. (Triglia et al. 1994; Reed et al. 2000; Chaijaroenkul et al. 2010). Antimalarial Gene mutation Remarks Chloroquine Sulfadoxinepyrimethamine Mefloquine, Quinine pfcrt 76T pfmdr1 86Y, 184F, 1034C, 1042D, 1246Y pfdhfr 108, 51, 59, 164 pfdhps 436, 437, 540, 581, 623 pfmdr1 Strong association Possible association pfdhfr 108 essential for pyrimethamine resistance, degree of resistance increases with additional mutations Leu51 and Arg59 or triple mutation. Absolute resistance conferred by the addition of Leu164, thus quadruple mutation, irrespective of dhps mutations Not clearly understood, pfmdr1 copy number is associated with reduced sensitivity to mefloquine, quinine Artemisinin pfmdr1 pfatpase6 pfmdr1 copy number is associated with reduced sensitivity to artesunate 1.19 BREATH TEST FOR MALARIA Breath tests have been developed to assist in the early diagnosis of infections with Mycobacterium tuberculosis and Helicobacter pylori (Lechner et al. 2005b; Phillips et al. 2007). The procedure involves the trapping of a sample of a patient s breath in a sorbent column followed by extraction and analysis of the retained volatile organic compounds (VOCs). The profile or fingerprint of VOCs that is generated can be used in determining whether a particular disease is present (Lechner et al. 2005a). Recent developments of breath-collection apparatus allow portability of equipment and thus testing may be performed promptly and more cost-effectively wherever the patient presents (Phillips 1997; Lindstrom et al. 2002; Martin et al. 2010). The identification of P. falciparum using conventional microscopy, antigen detection and PCR are inadequate in circumstances where the peripheral parasitaemia is low. This includes cases of severe malaria in which the majority of parasites are sequestered 58

93 Chapter 1 General Introduction within the microvasculature (Sachanonta et al. 2008). In addition, antigen detection methods and PCR in this context may not, if positive, differentiate between viable and non-viable parasites, an important consideration for therapeutic monitoring. In such instances, breath tests may provide a novel, non-invasive approach for the sensitive detection of an active malaria infection SCOPE OF THE STUDIES PRESENTED IN THIS THESIS The preceding literature review has provided an overview of malaria infection in humans, the use of conventional and novel antimalarial agents in malaria management, the role of in vitro and in vivo methods in the detection of parasite drug resistance, and how these factors contribute to the understanding of malaria pathophysiology and treatment outcome. The proximity of PNG to Australia and the political and economic relationships between the two countries mean that malaria control in PNG should be of concern to Australia. A significant proportion of imported malaria in Australia originates in PNG (Charles et al. 2005) while malaria outbreaks in the Torres Strait Islands are regularly reported (Needham 2011). The studies reported in Chapters 3 and 4 of this thesis include a multifaceted investigation into the efficacy of treatment for falciparum malaria, including mechanisms underlying P. falciparum resistance, in PNG children. Although this is an extremely important public health issue in PNG with clear implications for Australians visiting PNG or living close to the southern part of the country, there have been few studies of this type conducted to date. The data from the present PNG paediatric malaria studies and the evolution of resistance of P. falciparum and P. vivax to available antimalarial drugs in other parts of the tropics underscore the need for the identification of novel treatments. This can include compounds within existing or new classes of antimalarial drugs. Studies in Chapters 5 and 6 of this thesis address both these possibilities, providing pre-clinical and some clinical data that are essential to the further development and clinical application of desbutyl-lumefantrine and fenofibrate. Desbutyl-lumefantrine is the active metabolite of lumefantrine which is part of the recommended first-line ACT 59

94 Chapter 1 General Introduction treatment of uncomplicated malaria in PNG. The impetus to examine lipid-modifying drugs was provided by the recognition that non-communicable diseases, especially cardiovascular disease, represent an increasing disease burden in developing countries such as PNG (Ōtsuka et al. 2007). Drugs such as statins and fibrates will be increasingly used as a result and antimalarial effects could provide significant health and economic benefits to the individual and community in PNG and other countries with similar epidemiology. This has been investigated in the case of the blood glucoselowering drug rosiglitazone (Boggild et al. 2009) but lipid-altering therapies have even wider potential therapeutic application. During the course of the foregoing studies, the deficiencies in available techniques for diagnosis and antimalarial therapeutic monitoring provided the impetus to evaluating VOCs as a surrogate sensitive biomarker of viable parasites in the circulation. At the same time as data collection for the studies presented in Chapters 3 and 4 was proceeding, a separate investigation done in Madang, PNG, by another group identified the VOCs methyl nicotinate in the breath of tuberculosis patients (Syhre et al. 2009). This suggested that a similar breath test might be feasible in malaria. A corollary of parasite-specific VOCs production might be that these compounds have biological activity including acting as anaesthetic agents in patients with cerebral sequestration and consequent coma. The experiments in Chapter 7 detail attempts to determine, as the first step in this line of investigation, whether cultured P. falciparum generates specific VOCs as quantified using the most sensitive chemical assay techniques. The main implications of these various studies, their inter-relationships and prospects for future related research are discussed in Chapter 8. 60

95 CHAPTER 2 METHODS AND MATERIALS

96 Chapter 2 Methods and Materials CHAPTER 2. METHODS AND MATERIALS 2.1 IN VITRO CULTURE TECHNIQUES Culture techniques for P. falciparum described in the following section have been adapted from previously published methods (Trager et al. 1976) Parasites PNG field isolates of P. falciparum were collected from children with uncomplicated malaria (Chapter 3). Laboratory-adapted strains 3D7, Dd2, W2mef, K1 and E8B used in other in vitro studies were obtained from The Walter and Eliza Hall Institute in Melbourne and from Dr. Tina Skinner-Adams and colleagues of the Queensland Institute of Medical Research, Australia Retrieval from Liquid Nitrogen Intra-erythrocytic stages of P. falciparum preserved in cryo-suspensions were retrieved from long term liquid nitrogen storage as described previously (Meryman et al. 1972; Skinner-Adams 1999). Briefly, parasite stock vials were thawed at 37 C in a waterbath and transferred into a centrifuge tube. Pre-warmed 12% NaCl solution (Appendix B) was added drop-wise (1 drop/sec) with gentle mixing until a fifth of the original volume was added (i.e. 200 µl to 1 ml cell suspension) and allowed to stand at RT for 3 min. With gentle mixing, pre-warmed 1.6% NaCl solution (Appendix B) was added dropwise to reach 10 times the volume (i.e. 10 ml to 1 ml) then centrifuged (1200 rpm for 5 min). The supernatant was removed and pre-warmed 0.9% NaCl solution (Appendix B) was added drop-wise to reach 10 times pellet volume whilst ensuring the RBC were fully resuspended. After centrifugation, the supernatant was removed and the cells were resuspended in complete media (Appendix B) at 5% haematocrit (hct) using RBC (Section 2.1.4) and cultured in a microaerophilic environment (Section 2.1.3). 62

97 Chapter 2 Methods and Materials Maintenance of Cultures P. falciparum cultures were cultured in sterile lidded-dishes (5 ml, 10 ml or 20 ml) at 5% hct in a Nalgene desiccator (Figure 2.1). This provided a controlled microaerophilic atmosphere for the culture of P. falciparum (Trager et al. 1976; Scheibel et al. 1979). Non-infected RBC (Section 2.1.4) were added to the cultures at 2-3 day intervals to maintain parasitaemia between 0.5 to 5%. To prevent accumulation of metabolic byproducts, high parasitaemia were reduced by expanding the culture to a larger dish or by aspirating out part of the culture and re-adjusting the hct by replenishing with nonparasitised RBC. Figure 2.1 Nalgene desiccator used for P. falciparum culture. Parasite cultures were maintained at 37⁰C in an airtight chamber (modified Nalgene desiccator cabinet, Nalgene, U.S.A) flushed with a gas mixture of 1% (or 5%) O 2 and 5% CO 2 balanced in N 2 (BOC gases, Australia) for 90 sec daily or when opened. The oxygen concentration within the chamber ranged between 3-9% over 24 hr as indicated by an O 2 monitor (ToxiRAE II, San Jose, CA USA) Erythrocytes Preparation 63

98 Chapter 2 Methods and Materials Blood from a healthy volunteer was collected into sodium heparin Vacuette and centrifuged (1300 rpm for 5 min). Buffy coat was removed and plasma was stored at - 20⁰C for later use as media supplement (Appendix B). Packed RBC was transferred into a 50 ml tube topped with 3 times cell volume of media. After centrifugation (1300 rpm for 15 min), the supernatant was discarded and the process repeated twice. After the final wash, the cells were resuspended 1:1 (i.e. 50% RBC) in non-supplemented media. The stock was refrigerated and used within 3 weeks for cultures and assays Determination of Parasitaemia Parasite viability and development can be visually monitored by light microscopy (Figure 2.2). A thin blood smear was prepared by taking 3-10 µl of RBC in an equal volume of media and applied as a drop on a glass slide (Menzel-Glӓser, Lomb Scientific) (Figure 2.3). After air drying, the thin smear was fixed in methanol and stained for 10 min using freshly prepared 5% Giemsa Solution (Appendix B). After rinsing under tap water, it was air-dried and examined under oil or anisole (Fluka) immersion (100 objective). The parasitaemia was determined by counting the number of infected RBC in at least 1000 RBC and expressed as a percentage of total RBC Synchronisation of Parasite Forms One developmental cycle prior to use, parasite cultures were synchronised by sorbitol lysis to achieve 90% ring forms (Lambros 1979). RBC harbouring mature forms are more fragile and lyse under osmotic stress. Briefly, cultures with >40% rings were centrifuged and the pellet was resuspended in 10 ml of pre-warmed 5% sorbitol (Appendix B) and incubated at 37 C for 10 min. This was centrifuged and resuspended in complete media for culture. 64

99 Chapter 2 Methods and Materials Figure 2.2 Giemsa-stained thin smear of synchronised P. falciparum culture. Cultured parasites under anisole immersion ( 1000 magnification). From top to bottom: rings, trophozoites and schizonts. Note the rupture of a schizont with two newly released merozoites (M) about to invade an adjacent erythrocyte. Characteristic malaria brown pigment or haemazoin crystals (H) were also observed within RBC or as free crystals released into the culture space during schizont rupture. 65

100 Chapter 2 Methods and Materials Figure 2.3 Preparation of a thin smear. Thin smear is useful for detailed examination of parasite structures and developmental stages. This was prepared by spreading a spot of blood along the edge of another glass slide at a 45 angle and bringing it forward by a smooth gliding motion Cryopreservation Field isolates or culture-adapted parasite strains were cryopreserved for future use. Cryopreservation was performed on a routine basis to maintain stocks of various parasite strains in liquid nitrogen. Cultures should be of high parasitaemia i.e. (>5%) and mostly in the ring stage, as RBC infected with young parasites are more resilient to lysis during the procedure. Briefly, after centrifugation (1300 rpm for 5 min) and removal of the supernatant, the pellet was resuspended in an equal volume of cryoprotective solution (Appendix B) and transferred into a cryopreservation vial (Nunc, U.S.A.) and snap frozen in liquid nitrogen 66

101 Chapter 2 Methods and Materials 2.2 DRUG SUSCEPTIBILITY ASSAYS Drug/Compound Preparation Drug susceptibility assays were carried out in 96-well plates (Sarstedt) where parasites were treated with low to high doses of drug. Most assays were carried out over 48 hr, with the exception of AZ for 72 hr. A typical lay-out of a drug susceptibility panel is shown in Figure 2.4. The antimalarial drugs used in this study consisted of chloroquine (CQ), amodiaquine (AQ) and its metabolite monodesethyl-amodiaquine (daq), piperaquine (PQ), mefloquine (MQ), naphthoquine (NQ), lumefantrine (LM) and its metabolite desbutyllumefantrine (DBL), dihydroartemisinin (DHA) and azithromycin (AZ). Details for statins and fibrates are described in subsequent chapters. On solubilisation, stock standards of 1 mm concentration were prepared in compatible solvents (Table 2.1) and stored in aliquots protected from light at -20 C. Due to the slow-acting nature of AZ, it was tested on a different panel. On the day of assay, drug stocks were thawed and further diluted in media to prepare 5 µm working standards. These were used for twofold serial dilutions in media at double assay concentrations (Table 2.1) and dispensed in triplicates at 100 µl per well for pldh and 3 H-hypoxanthine incorporation assays Preparation of parasitised cells Prior to assay, parasite cultures were centrifuged with the supernatant removed and packed RBC were used directly. For field isolates, whole blood samples were centrifuged with plasma and buffy coat removed. RBC were washed 3 times in nonsupplemented media before use. Each drug panel comprised of four drugs tested in triplicates hence a minimum of 10 ml suspension of cells standardised to 3% hct at 0.5 to 1% parasitaemia (depending on the method of growth assessment) was required. For pldh assessment, 100 µl per well of the suspension was added giving a final 1% parasitaemia and 1.5% hct in a 200 µl mixture of drug/rbc/media. If growth was assessed by 3 H-hypoxanthine incorporation, 90 µl suspension/ well was used instead. 67

102 Chapter 2 Methods and Materials Figure 2.4 Layout of a drug susceptibility panel. The top row of the panel consisted of drug-free parasitised and non-parasitised controls. Antimalarial drugs were serially diluted from the bottom row (highest concentration) to the second row (lowest concentration) in triplicates Controls For parasitised drug-free controls, 100 µl (or 90 µl for 3 H-hypoxanthine incorporation) of infected RBC suspension were added to 100 µl of assay media per well. These wells should exhibit the highest parasite growth. For non-parasitised control wells, uninfected RBC were used instead. 68

103 Chapter 2 Methods and Materials Table 2.1 Solvents and optimised assay concentration ranges for drug susceptibility testing. Drug (Supplier) Solvent Assay Range Chloroquine diphosphate (Sigma Chemicals, St Louis, USA) Amodiaquine dihydrochloride (Sigma) Monodesethyl-amodiaquine (Sapec Fine Chemicals, Lugano, Switzerland) Naphthoquine phosphate (ZYF Pharm Chemical, Shanghai, China) Piperaquine tetraphosphate (Yick-Vic Chemicals and Pharmaceuticals, Hong Kong) Mefloquine hydrochloride (Sigma) Sterile d.h 2 O Sterile d.h 2 O Sterile d.h 2 O 12.5 nm 1600 nm 5 nm 320 nm 5 nm 320 nm 50% v/v ethanol 3.13 nm 200 nm 0.5% w/v lactic acid 6.25 nm 400 nm 70% v/v ethanol 0.78 nm 200 nm Dihydroartemisinin (Sigma) 70% v/v ethanol 0.78 nm 51.2 nm Lumefantrine (Novartis Pharma, Basel, Switzerland) Desbutyl-lumefantrine (Novartis Pharma, Basel, Switzerland) Azithromycin (Pfizer, NSW, Australia) Pravastatin sodium (Bristol-Myers Squibb, Australia) Simvastatin (Alphapharm, QLD, Australia) Rosuvastatin (AstraZeneca, NSW, Australia) Atorvastatin calcium (Pfizer, NSW, Australia) 1:1:1 v/v/v mixture of linoleic acid, ethanol and Tween 80 1:1:1 v/v/v mixture of linoleic acid, ethanol and Tween 80 Sterile d.h2o 3.12 nm 400 nm 3.12 nm 400 nm 1250 nm nm DMSO 3.12 µm 400 µm DMSO 3.12 µm 400 µm DMSO 3.12 µm 400 µm DMSO 3.12 µm 400 µm 69

104 Chapter 2 Methods and Materials Plasmodium Lactate Dehydrogenase Assay Principle of pldh Assay 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 Assay Set Up After incubation, the drug susceptibility plates were subjected to three cycles of freezethawing 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. 70

105 Chapter 2 Methods and Materials 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 IC 50. Figure 2.6 pldh reaction in field isolates of P. falciparum H-Hypoxanthine Incorporation Assay Hypoxanthine is required for DNA synthesis during parasite replication. The radioactivity of the incorporated tritium ( 3 H) 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 3 H-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 71

106 Chapter 2 Methods and Materials 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. 72

107 Chapter 2 Methods and Materials 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 Polymerase Chain Reaction (PCR) 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 , Denville Scientific Inc, USA) were irradiated with ultraviolet light in a Stratalinker 2400 UV Crosslinker (Stratagene, CA, USA) before use to destroy any 73

108 Chapter 2 Methods and Materials 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) 2 SO 4, 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 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 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. 74

109 Chapter 2 Methods and Materials 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. a pfcrt for SNPs at codons 72 to 76. b dhfr fragment for SNPs at codons 51, 59, 108 and 164. c pfdhps gene fragment for SNPs at codons 540, 581, 613. d PCR programs were preceded by an initial denaturation step at 95 C for 2 min. Gene PCR Primer Sequence Thermocycling Condition d pfcrt a pfdhfr b pfdhps c 5 -TAATACGACTCACTATAGGGCCGTTA-3 5 -ATTAACCCTCACTAAAGGGACGGATG-3 5 -TAACTACACATTTAGAGGTCTA-3 5 -GTTGTATTGTTACTAGTATATAC-3 5 -AATGATAAATGAAGGTGCTAGT-3 5 -ATGTAATTTTTGTTGTGTATTTA-3 35 cycles of 95 C for 30 sec, 56 C for 30 sec, 60 C for 1 min 35 cycles of 95 C for 30 sec, 56 C for 30 sec, 72 C for 1 min 35 cycles of 95 C for 30 sec, 56 C for 30 sec, 60 C for 1 min 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). 75

110 Chapter 2 Methods and Materials Figure 2.9 Electrophoresis and Image processing for DNA visualisation for evaluating PCR amplification efficiency Ligase Detection Reaction Fluorescent Microsphere Assay (LDR-FMA) The multiplex LDR-FMA facilitates both rapid and specific detection of Plasmodium species in a single reaction, avoiding cumbersome, separate procedures (McNamara et al. 2004; McNamara et al. 2006). Similarly, this platform has been adapted to analyse simultaneously an assortment of SNPs associated with CQ and SP resistance (Carnevale et al. 2007). The development of a LDR-FMA for pfmdr1 SNPs is described in Chapter Ligase Detection Reaction for Plasmodium species Briefly, PCR products from the Plasmodium species assay were used directly for LDR in a master mix solution (Appendix B) containing 20 mm Tris-HCl buffer (ph 7.6), 25 mm potassium acetate, 10 mm magnesium acetate, 1 mm NAD +, 10 mm dithiothreitol, 0.1% Triton X-100, 10 nm (200 fmol) of each LDR primer, 1 µl PCR product and 2 Units of Taq DNA ligase (New England Biolabs, MA). Thermocycling conditions involved initial heating at 95 C for 1 min, followed by 32 cycles of denaturation at 95 C for 15 sec and annealing/ligation at 58 C for 2 min. LDR primer sequences for species diagnosis are shown in Table 2.3 (McNamara et al. 2006). 76

111 Chapter 2 Methods and Materials Table 2.3 LDR primer sequences for Plasmodium species diagnosis. a Lowercased nucleotides are nonspecific and were added to the rrna gene LDR primers to reach a desired specific length. Nucleotide W and R correspond to T or A and G or A degeneracy, respectively. Pf1 and Pf2, P. falciparum 1 and 2; Pv, P. vivax; Pm, P. malariae; Po, P. ovale; Common 1 and 2, common sequence. b ID, identification. One hundred unique Luminex microsphere sets are synthesised to exhibit unique fluorescence. Each microsphere set is coupled to different anti-tag sequences. Anti-tag sequences are complementary to species-specific tag sequences (adapted from McNamara et al 2006). Primer Sequence a ID b Pf1 5 -tacactttctttctttctttctttaaa AGT CAT CTT TCG AGG TGA CTT-3 12 Pf2 Pv Pm 5 -ctatctatctaactatctatacatgt AGC ATT TCT TAG GGA ATG TTG ATT TTA TAT-3 5 -cttttcatcttttcatctttcaataaa ATA AGA ATT TTC TCT TCG GAG TTT ATT C-3 5 -ttacctttatacctttctttttacaag AGA CAT TCT TAT ATA TGA GTG TTT CTT- 3 Po 5 -ctactatacatcttactatacttttaa GAA AAT TCC TTT CGG GGA AAT TTC-3 14 Common1 5 -phosphate-tag AAT TGC TTC CTT CAG TAC CTT ATG-biotin-3 2 Common2 5 -phosphate-tta GAT WGC TTC CTT CAG TRC CTT ATG-biotin Ligase Detection Reaction for pfcrt, pfdhfr, pfdhps SNPs Following the PCR amplifications of the pfcrt, pfdhps and pfdhfr genes carrying drug resistance associated SNPs, the products were combined by transferring 5 µl of each gene product into a fresh 96 well plate. Pooled PCR products were mixed by centrifugation and 1 µl was used to prepare the LDR master mixture (Appendix B) and sealed (sealing film B , Denville Scientific Inc) prior to the multiplexed LDR. Primer sequences for each drug resistance gene are detailed in Table 2.4 (Carnevale et al. 2007). The LDR thermocycling conditions used were as for Plasmodium species. 77

112 Chapter 2 Methods and Materials Hybridisation and Reporter Labelling The second stage of the reaction initiates specific classification of the LDR products, where hybridisation occurs between anti-tag oligonucleotides probes that are bound to fluorescent microspheres (Luminex Corporation, TX) specific for the various tag sequences at the 5 end of the LDR products. The hybridisation and reporter labelling protocols are the same for Plasmodium species and drug resistance SNPs diagnosis. This required adding 5 µl of LDR products to 60 µl of pre-warmed hybridisation solution (Appendix B) containing 250 Luminex FlexMAP microspheres from each allelic set (total of 18 alleles). The reaction mixture was heated to 95 C for 90 sec and incubated at 37 C for min. Reporter labelling of the conjugate followed with the addition of 6 µl streptavadin-r-phycoerythrin dye (Molecular Probes, OR) diluted 1:50 v:v in TMAC (20 ng/µl) as it binds to the 3 biotin on the conserved sequence primers at 37 C for min in 96-well V-bottom plates (Costar 6511 M polycarbonate, Corning Inc., Corning NY). 78

113 Chapter 2 Methods and Materials Table 2.4 LDR primer sequences for drug resistance markers pfcrt, pfdhfr and pfdhps. a Lowercased nucleotides (24 bases) represent tag sequences added to the 5 ends of each allele-specific LDR primer. b ID, identification of Luminex microsphere. c CM, common sequence primer immediately downstream from the allele-specific primer. Gene Primer c Sequence a ID b pfcrt CVMNK 5'-aatctacaaatccaataatctcatATTTAAGTGTATGTGTAATGAATAA-3' 60 CVIET 5'-tcataatctcaacaatctttctttAATTAAGTGTATGTGTAATTGAAAC-3' 68 SVMNT 5'-aatcctttctttaatctcaaatcaATTTAAGTGTAAGTGTAATGAATAC-3' CM 5'-phosphate-AATTTTTGCTAAAAGAACTTTAAAC-biotin-3' pfdhfr 51 I 5'-caatttcatcattcattcatttcaGAGTATTACCATGGAAATGTAT-3' N 5'-ctactatacatcttactatactttGAGTATTACCATGGAAATGTAA-3' CM 5'-phosphate-TTCCCTAGATATGAAATATTTT-biotin-3' 59 R 5'-cttttcatcttttcatctttcaatTCACATATGTTGTAACTGCACG-3' C 5'-tacactttctttctttctttctttTCACATATGTTGTAACTGCACA-3' CM 5'-phosphate-AAAATATTTCATATCTAGGGAAWTA-biotin-3' 108 T 5'-ctaactaacaataattaactaacTTGTAGTTATGGGAAGAACAAC-3' S 5'-ctataaacatattacattcacatcTTGTAGTTATGGGAAGAACAAG-3' N 5'-tcatcaatcaatctttttcactttTTGTAGTTATGGGAAGAACAAA-3' CM 5'-phosphate-CTGGGAAAGCATTCCAAAAAAA-biotin-3' 164 I 5'-ctttctatctttctactcaataatGAAATTAAATTACTATAAATGTTTTATTA-3' L 5'-ctatctttaaactacaaatctaacGAAATTAAATTACTATAAATGTTTTATTT-3' CM 5'-phosphate-TAGGAGGTTCCGTTGTTTATC-biotin-3' pfdhps 540 K 5'-ctacaaacaaacaaacattatcaaGGAAATCCACATACAATGGATA-3' E 5'-ctttaatcctttatcactttatcaGAAATCCACATACAATGGATG-3' CM 5'-phosphate-AACTAACAAATTATGATAATCTAG-biotin-3' 581 A 5'-aatctaacaaactcctctaaatacTTGATATTGGATTAGGATTTGC-3' G 5'-ctttcaattacaatactcattacaTTGATATTGGATTAGGATTTGG-3' CM 5'-phosphate-GAAGAAACATGATCAATCTATTA-biotin-3' 613 A 5'-tacactttaaacttactacactaaGATATTCAAGAAAAAGATTTATTG-3' S 5'-aaacaaacttcacatctcaataatGGATATTCAAGAAAAAGATTTATTT-3' CM 5'-phosphate-CCCATTGCATGAATGATCAAA-biotin-3' 79

114 Chapter 2 Methods and Materials Bio-plex Fluorescent Detection The SNP or species-specific LDR products with microsphere-labelled anti-tag probes were detected by dual-fluorescence flow cytometry in the Bio-Plex array reader (Bio- Rad Laboratories, CA). The chamber temperature was set to 37 C as reporter signals were collected into allele-specific or species-specific classification bins via the Bio-Rad software, Bio-Plex Manager 3.0 (Figure 2.10). Figure 2.10 Bio-plex array reader. LDR-FMA Fluorescence signals of the Plasmodium species and drug resistance SNPs are detected and sorted into classification bins by the Bio-plex reader. 2.4 SOLID PHASE MICRO-EXTRACTION (SPME) Solid phase micro-extraction (SPME) is a sensitive and simple method for the extraction of volatile organic compounds (VOCs) from gaseous or liquid samples. This technique was used to detect VOCs in the headspace of P. falciparum cultures. The SPME fibre (SUPELCO, Sigma Aldrich) is bonded to a plunger inside a protective needle (Figure 2.11). The fibre was conditioned initially according to the manufacturer s instructions (PDMS phase 250 C for 0.5 hr and triple fibre phase 270 C 80

115 Chapter 2 Methods and Materials for 1 hr. Before each analysis, the fibre was activated in the injector port of the gas chromatography (GC) at 250 C for 5 min and repeated after each sampling. The SPME fibre was introduced into the headspace of the flask by gently pushing the protective needle through the septum that sealed the sample flask. The plunger was lowered to expose the adsorbent fibre to the gaseous phase for one hour at 35⁰C. During this time, equilibrium between the atmosphere and the fibre was achieved, and the volatile and semi-volatile organic compounds were adsorbed onto the coating of the fibre. After retracting the fibre and withdrawing the needle, the syringe sampler was taken to the gas chromatography-mass spectrometry (GC-MS) for desorption and subsequent analysis of VOCs. Details of VOCs experiments are described in Chapter 7. Figure 2.11 Solid phase micro-extraction sampler. 81

116 Chapter 2 Methods and Materials 82

117 CHAPTER 3 IN VITRO SENSITIVITY OF P. FALCIPARUM TO CONVENTIONAL AND NEW DRUGS IN PAPUA NEW GUINEA

118 Chapter 3 In vitro drug sensitivity of PNG field isolates CHAPTER 3. IN VITRO SENSITIVITY OF P. FALCIPARUM TO NEW AND CONVENTIONAL DRUGS IN PAPUA NEW GUINEA 3.1 INTRODUCTION Resistance of P. falciparum to CQ first emerged in PNG in the 1970 s (Saint-Yves 1971; Grimmond et al. 1976; Yung et al. 1976; Han 1978). As these treatment failures were infrequent and low grade (RI), CQ or amodiaquine (AQ) were initially retained as recommended therapy for uncomplicated malaria. However, because higher grade (RII and RIII) in vivo resistance to CQ and AQ became more widespread (Darlow et al. 1981; Dulay et al. 1987; Schuurkamp et al. 1989; Sapak et al. 1991; Trenholme et al. 1993; Al-Yaman et al. 1996), the PNG Health Department added sulfadoxinepyrimethamine (SP) in 2000 to improve clinical efficacy (Casey et al. 2004). This approach provided relatively brief respite. Further in vivo studies (Marfurt et al. 2007) and a recently-published large-scale comparative efficacy trial (Karunajeewa et al. 2008a) demonstrated that neither CQ-SP nor AQ-SP met WHO criteria for retention as first-line treatment in PNG (WHO 2006). As a result, the ACT artemether-lumefantrine replaced these regimens in An overview of in vitro and treatment outcome studies conducted in PNG is presented (Table 3.1) Because of their relative simplicity and low cost compared with in vivo assessment, in vitro tests of parasite drug sensitivity can serve as an early warning system for the emergence of drug resistance (WHO 2005). This includes artemisinin and the longer half-life partner components of ACT (Price et al. 2004; Ekland et al. 2008; Noedl et al. 2008). The antimalarial activity of the partner drug appears especially important for selection of an appropriate ACT in countries such as PNG (Karunajeewa et al. 2008b), but there is often a lack of in vitro data to facilitate this choice. In PNG, for example, the most recent parasite sensitivity data are from the study period for CQ, AQ and antifolate drugs (Genton et al. 2005). In this study, the in vitro antimalarial activities of a range of conventional and novel antimalarial drugs was assessed in P. falciparum isolates collected from Madang Province where malaria transmission is 84

119 Chapter 3 In vitro drug sensitivity of PNG field isolates hyperendemic (Cattani et al. 1986; Mueller et al. 2003) and where there has been evidence of progression of parasite drug resistance (Darlow et al. 1981; Trenholme et al. 1993; Al-Yaman et al. 1996; Al-Yaman et al. 1997; Casey et al. 2004; Marfurt et al. 2007). 85

120 Chapter 3 In vitro drug sensitivity of PNG field isolates Table 3.1 Overview of in vitro drug sensitivity findings in PNG. IVR = In vitro resistance reported as percentage, where no inhibitory concentrations were available. Reported median or mean IC 50 s of antimalarials; chloroquine (CQ), amodiaquine (AQ), quinine (Q), mefloquine (MQ), halofantrine (HF) and Pyrimethamine (PYR), Cycloguanil (CYC); Schizont Maturation = microtechnique where drug sensitivity was determined by schizont maturation. (Al-Yaman et al. 1996; Reeder et al. 1996; Hombhanje et al. 1998a; Hombhanje 1998b; Genton et al. 2005; Mita et al. 2006b; Wong et al. 2010) 86

121 Chapter 3 In vitro drug sensitivity of PNG field isolates Authors Province Study Period Samples (n) CQ AQ Q MQ HF PYC CYC Method Mita et al 2006 East Sepik IVR 82% Mean 88 to 107nM Schizont Maturation Genton et al 2005 East Sepik IVR 50% IVR 27% IVR 0% IVR 8% IVR 0% IVR 4% IVR 6% Schizont Maturation Hombhanje Central IVR 50% - IVR 10% IVR 0% IVR 0.7% - - Schizont 1998b Median Median Median Median Maturation 1150nM 2760nM 350nM 1.0nM Hombhanje et al 1998a Central Not stated Median 1.5nM - - Schizont Maturation al-yaman et al 1996 Madang IVR 86% IVR 86% IVR 7% Schizont Maturation Reeder et al 1996 East Sepik IVR 38% IVR 34% Schizont Maturation 87

122 Chapter 3 In vitro drug sensitivity of PNG field isolates 3.2 MATERIALS AND METHODS Study Site and Sample Collection The present study was carried out at the PNG Institute of Medical Research, Yagaum Hospital, Madang, PNG. The study utilised blood samples taken in 2006 and 2007 from children aged 6 months to 10 years as part of clinical studies conducted at the Alexishafen Health Centre in Madang Province and at Modilon Hospital in Madang town. In all cases, informed consent was obtained from the parents or legal guardians before recruitment and blood sampling. Scientific and ethical approval for each study was obtained from the Medical Research and Advisory Committee of the Ministry of Health of PNG. Of the 64 samples collected, 45 (70.3%) were from children recruited to a randomised trial of uncomplicated malaria (Karunajeewa et al. 2008b). The remaining 18 samples (29.7%) were from pharmacokinetic studies conducted at Alexishafen or studies of severe malaria in progress at Modilon Hospital In vitro Culture of Parasite Isolates In vitro culture of P. falciparum isolates from paediatric patients followed similar methods to those described in section 2.1.3, with modifications to suit field conditions. After initial diagnosis of P. falciparum infection from a finger-prick blood smear, 4 ml venous blood were collected into a heparin-containing tube and transported to the laboratory within 24 hr. After centrifugation, both plasma and buffy coat were removed. The packed RBC were washed twice in culture medium and blood smears were prepared for confirmation of P. falciparum mono-infection and quantification of parasitaemia by microscopy. Parasites were cultured at 37 C using a modified candle jar method to create a CO 2 -rich microaerophilic atmosphere (Trager et al. 1976) (Figure 3.1). The composition of the culture medium was also different under field conditions (Appendix B). Parasites were maintained in type O human RBC from a non-immune individual at 5% hct in RPMI 1640 media supplemented with neomycin as well as gentamycin. Since non-immune human plasma was not available, Albumax II (Gibco) was used in the complete media (Appendix B). 88

123 Chapter 3 In vitro drug sensitivity of PNG field isolates Figure 3.1 Candle jar method used for P. falciparum culture in PNG. A container into which a lit candle is placed prior sealing the airtight lid. The flame burns until extinguished by O 2 deprivation, creating a CO 2 -rich microaerophilic environment Drug Susceptibility Assays Stock solutions of CQ diphosphate (Sigma Chemicals, St Louis, USA), AQ dihydrochloride (Sigma), monodesethyl-aq (daq) (Sapec Fine Chemicals, Lugano, Switzerland), piperaquine tetraphosphate (PQ) (Yick-Vic Chemicals and Pharmaceuticals, Hong Kong), naphthoquine phosphate (NQ) (ZYF Pharm Chemical, Shanghai, China), mefloquine hydrochloride (MQ) (Sigma), lumefantrine (LM) (Novartis Pharma, Basel, Switzerland), dihydroartemisinin (DHA) (Sigma) and azithromycin (AZ) (Pfizer, NSW, Australia) were prepared as described in Section Drug sensitivity was assessed in triplicate in 96-well plates, with each well containing 100 µl drug-containing media and 100 µl parasitised RBC suspension at % parasitaemia and a final hct of 1.5% (Section 2.2). With the exception of AZ, which 89

124 Chapter 3 In vitro drug sensitivity of PNG field isolates was incubated for 72 hr due to its slower antimalarial activity (Noedl et al. 2006), all other plates were incubated for 48 hr at 37 C. The plates were then subjected to four freeze-thaw cycles to achieve complete haemolysis. The haemolysate were kept frozen until assayed. A modification of a Plasmodium lactate dehydrogenase (pldh) detection method was used to assess parasite growth as detailed in Section (Makler et al. 1993a; Makler et al. 1993b) Assay Validation For validation purposes, the pldh assay was compared to the reference 3 H- hypoxanthine incorporation method (Chulay et al. 1983) using culture-adapted CQsensitive and CQ-resistant strains of P. falciparum 3D7, W2mef and E8B and a panel of antimalarial drugs. Briefly, two sets of CQ, MQ and DHA drug dilutions in either complete media or complete media without hypoxanthine as appropriate for the pldh and 3 H-hypoxanthine assays, respectively. To allow for between-day variability in assay performance the drug sensitivity of each P. falciparum strain was assessed simultaneously using the two methods Data Analysis Statistical analyses were performed using GraphPad PRISM version 4.0 (GraphPad Software, CA) and Microsoft Excel for Windows. The concentration of drug required to inhibit parasite growth by 50% (IC 50 ) and 90% (IC 90 ) for each antimalarial drug as measured by pldh assay were determined by non-linear regression analysis of logarithmically-transformed dose-response curves using HN NonLin v.1.1, a free tool for malaria in vitro drug sensitivity analysis (Noedl 2002). Comparisons between the IC 50 values in laboratory-adapted strains obtained by pldh and 3 H-hypoxanthine incorporation assays were made using regression analysis (Bablok et al. 1988) and the Bland and Altman method (Bland et al. 1986). One-way analysis of variance (ANOVA) was used to compare differences between the IC 50 values obtained from the three different measurement time-points of the pldh reaction. Associations between IC 50 and IC 90 values of drug pairs for evaluation of in vitro cross resistance were assessed using Spearman s rank correlation co-efficient. Because of the number of comparisons, 90

125 Chapter 3 In vitro drug sensitivity of PNG field isolates a significant P-value <0.05 was used throughout. Correlations between the IC 50 s of CQ, PQ and LM in a subset of the present patients have been reported previously (Karunajeewa et al. 2008b). 3.3 RESULTS Comparison of pldh and Isotopic Assays The pldh assay was compared to the reference 3 H-hypoxanthine incorporation method in cultured-adapted P. falciparum (Figure 3.2). There was a significant linear correlation between the data obtained by the two IC 50 assay methods (r 2 =0.97, n=26; P=0.001), with a slope and intercept ([95% confidence intervals]) of 1.13 [1.00 to 1.25] and 7.83 [-3.3 to 18.98] nm, respectively. The Bland-Altman plot showed that the pldh assay sometimes significantly underestimated the IC 50 at high values and provided the least reliable estimations at IC 50 s >200 nm Effect of pldh Reaction Duration on IC 50 Values For simplicity and efficiency, a single endpoint was used to interpret the results of the pldh assay; specifically measurement of the OD when substantial colour contrast had developed during the enzyme reaction, rather than multiple measurements at 30 sec intervals over 30 min as originally described (Makler et al. 1993a). In laboratoryadapted P. falciparum, 45 to 60 min of incubation was adequate for visual evaluation of dye reduction. At this point in the reaction, the colouration in drug-free wells is intense (purple), reflecting heavy parasite growth, whilst non-parasitised control wells show minimal colour change (pink) (see Figure 2.6). In field isolates however, the rate of pldh dye reduction varied between samples, with some requiring up to 2 hr to produce maximal contrast. 91

126 Chapter 3 In vitro drug sensitivity of PNG field isolates Figure 3.2 Comparison of pldh and 3 H-hypoxanthine incorporation methods for analysis of antimalarial sensitivity in culture-adapted P. falciparum. Upper panel: correlation plot of IC 50 s obtained by both methods, red circles for CQ, open circles for MQ and triangles for DHA. Lower panel: Bland-Altman plot of difference between the pldh and 3 H-hypoxanthine incorporation data vs the mean of the two methods. 92

127 Chapter 3 In vitro drug sensitivity of PNG field isolates To investigate whether the IC 50 values calculated for antimalarial drugs were influenced by the duration of pldh incubation, dose-responses curves for six antimalarial agents assessed against four field isolates were determined from OD measurements at 80, 120 and 180 min, and their respective IC 50 s compared (Figure 3.3). The majority of isolates tested produced similar dose-response curves to each respective antimalarial drug over a 3 hr period (data not shown). In some isolates (e.g. 106 and 112 in Figure 3.3), an increase in CQ IC 50 was observed with reaction time. This increase was due to greater colour contrast as the reaction proceeded, causing changes in the slope of the doseresponse curve. Overall, there were no significant differences between IC 50 s calculated from data measured at 80 to 180 min (n=93; P=0.60). When IC 50 s were plotted against pldh reaction time (Figure 3.4), the general distribution of data points was similar between groups, with collective median IC 50 s of 15.7, 15.9 and 16.7 nm at 80, 120 and 180 min, respectively Field Application of the pldh Assay The in vitro activities of nine antimalarial drugs against P. falciparum isolates from paediatric patients with uncomplicated malaria were evaluated. Blood samples with parasitaemia ranged between 0.3 to 14% were included in the drug sensitivity assay. Samples with inadequate RBC were only screened against 4-aminoquinolines drugs. Of 125 field isolates obtained, 64 (51%) were both cultured successfully and provided valid data drug sensitivity data by pldh assay. Loss of isolates reflected mainly logistic issues with transportation, and laboratory-related problems including power supply reliability, reagent availability, clotting of blood samples due to inadequate mixing with anticoagulant and bacterial contamination. Experiments involving LM, DHA and AZ were only undertaken during the latter part of the study period. The distribution of IC 50 s in response to antimalarial drugs is illustrated in Figure

128 Chapter 3 In vitro drug sensitivity of PNG field isolates Figure 3.3 Effect of pldh reaction time on IC 50 s in PNG P. falciparum. Four samples of P. falciparum were tested against six antimalarial drugs; chloroquine (CQ); piperaquine (PQ); desethyl-amodiaquine (daq); amodiaquine (AQ); naphthoquine (NQ) and mefloquine (MQ). Optical measurements were taken at 80, 120 and 180 min into the pldh reaction. IC 50 s were calculated for each time point for comparison. 94

129 Chapter 3 In vitro drug sensitivity of PNG field isolates Figure 3.4 Scatter plot of IC 50 s determined from three pldh time points. IC 50 values for CQ, PQ, AQ, daq and MQ at three different time points were compared in 18 field isolates of P. falciparum. Optical measurements were collected at 80, 120 and 180 min into the pldh reaction from which IC 50 s were determined and plotted. The median for each time point is shown. 95

130 Chapter 3 In vitro drug sensitivity of PNG field isolates Figure 3.5 Distribution of 50% inhibitory concentrations (IC 50 ) of antimalarials against PNG P. falciparum isolates. Piperaquine (PQ), amodiaquine (AQ), desethyl-amodiaquine (daq), chloroquine (CQ), naphthoquine (NQ), mefloquine (MQ), dihydroartemisinin (DHA), lumefantrine (LM) and azithromycin (AZ). 96

131 Chapter 3 In vitro drug sensitivity of PNG field isolates Antimalarial Susceptibility of PNG P. falciparum Isolates The mean IC 50 and IC 90 values for the nine antimalarial drugs are summarised in Table 3.2. The accepted in vitro resistance threshold for CQ ( 100 nm) is based on the in vitro response of African isolates obtained from malaria-infected, non-immune individuals taking CQ prophylaxis and semi-immune patients failing to respond to CQ treatment (Le Bras et al. 1990; Cremer et al. 1995; Ringwald et al. 1996; Basco et al. 2002). This threshold was obtained using the reference 3 H-hypoxanthine incorporation method but has been employed in many subsequent studies using different techniques for in vitro drug susceptibility assessment (Attlmayr et al. 2006; Kaddouri et al. 2006; Mayxay et al. 2007; Nkhoma et al. 2007). Because there was close agreement between the pldh and 3 H-hypoxanthine methods in the present study at this concentration, a 100 nm cut-point was used, with 82% of isolates tested exhibiting an IC 50 above this level. In vitro resistance thresholds for the other antimalarial drugs tested have not been established through valid correlative in vivo studies. 97

132 Chapter 3 In vitro drug sensitivity of PNG field isolates Table 3.2 In vitro susceptibilities of P. falciparum PNG isolates against 4- aminoquinolines and other antimalarial drugs. IC 50 = 50% inhibitory drug concentrations and IC 90 = 90% inhibitory drug concentrations, CI = confidence interval. Isolates tested (n) IC 50 (nm) Mean (95% CI) Correlations of in vitro Responses to Nine Antimalarials IC 90 (nm) Mean (95% CI) Chloroquine ( ) 431 ( ) Amodiaquine ( ) 54.3 ( ) Desethyl-amodiaquine ( ) 48.8 ( ) Piperaquine ( ) 48.8 ( ) Naphthoquine ( ) 31.0 ( ) Mefloquine ( ) 21.2 ( ) Lumefantrine ( ) 12.7 ( ) Dihydroartemisinin ( ) 11.1 ( ) Azithromycin 15 13,895 (6,277 21,513) 39,471 (21,300 57,642) Correlations between IC 50 values for the panel of antimalarial drugs are shown in Table 3.3. Apart from LM, there were strong associations between the IC 50 s of 4- aminoquinoline (CQ, AQ, DAQ and NQ), bisquinoline (PQ) and aryl-aminoalcohol (MQ) compounds. Although the numbers of isolates tested were low, AZ activity did not correlate significantly with that of any other drug. The artemisinin derivative DHA IC 50 values showed positive associations with those of the 4-aminoquinoline and related compounds. 98

133 Chapter 3 In vitro drug sensitivity of PNG field isolates Table 3.3 Spearman correlation co-efficients for associations between IC 50 values. The number of drug pairs analysed are shown in parentheses. *P<0.05, **P<0.01, ***P< Amodiaquine 0.46*** (60) Piperaquine Naphthoquine Mefloquine Lumefantrine Dihydroartemisinin Chloroquine Amodiaquine Desethylamodiaquine Desethylamodiaquine 0.45*** (58) 0.61*** (57) Piperaquine 0.51*** 0.59*** 0.51*** (54) (53) (55) Naphthoquine 0.56*** 0.61*** 0.64*** 0.74*** (37) (36) (37) (36) Mefloquine 0.61*** 0.52*** 0.37** 0.52*** 0.52** (54) (57) (52) (48) (32) Lumefantrine * (22) (24) (19) (17) (7) (23) Dihydroartemisinin 0.45* (27) 0.37* (29) 0.43* (24) 0.22 (20) 0.84** (10) 0.57** (28) 0.31 (23) Azithromycin (12) (13) (9) (9) (3) 99 (12) (12) (12)

134 Chapter 3 In vitro drug sensitivity of PNG field isolates 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 nearfixation 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 IC 50 s 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 IC 50 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 IC 50 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 IC 50 <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 100

135 Chapter 3 In vitro drug sensitivity of PNG field isolates have reported resistant isolates with IC 50 values >300 nm using the schizont maturation microtechnique (Yang et al. 1999b; Lin et al. 2005). All PQ IC 50 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 (IC 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 IC 50 values below the resistance threshold of 108 nm established recently using in vivo responses, 3 H- 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 3 H-hypoxanthine uptake studies in African isolates without in vivo correlation (Basco et al. 1998). None of the PNG isolates had an IC 50 value above this level. There are no equivalent published cut-points for NQ but the median IC 50 (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 nm for artesunate-resistant strains) (Yang et al. 1999a). The IC 50 values for DHA were all below the suggested cut-point of 10.5 nm derived from 3 H-hypoxanthine uptake studies in African isolates without in vivo correlation (Pradines et al. 1998). The AZ IC 50 s 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 IC 50 values for 4-aminoquinoline and related 101

136 Chapter 3 In vitro drug sensitivity of PNG field isolates 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 IC 50 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 IC 50 s 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 IC 50 s 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). 102

137 Chapter 3 In vitro drug sensitivity of PNG field isolates 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 V max (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 IC 50 s 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 IC 50 s from this approach correlated well with the isotopic method although it tended to overestimate the IC 50 s 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 3 H-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 103

138 Chapter 3 In vitro drug sensitivity of PNG field isolates unexpectedly high IC 50 s 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 IC 50 values generated correlate well with those derived using 3 H-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-cytometrybased 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. 104

139 CHAPTER 4 CHARACTERISATION OF DRUG RESISTANT POLYMORPHISMS OF P. FALCIPARUM USING A NEW MOLECULAR ASSAY

140 Chapter 4 Molecular Characterisation of PNG isolates 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. Highlevel 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 106

141 Chapter 4 Molecular Characterisation of PNG isolates 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 107

142 Chapter 4 Molecular Characterisation of PNG isolates 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 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 ACTRN ). 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 artemetherlumefantrine (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). 108

143 Chapter 4 Molecular Characterisation of PNG isolates 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 substudy 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 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) 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 ( base-pairs). This domain contains sequences conserved within the Plasmodium genus and those that are species-specific (Section ). 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 109

144 Chapter 4 Molecular Characterisation of PNG isolates upstream and downstream primers (Section ). 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 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 ). 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) 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 , respectively. Following PCR, the products were combined in a multiplexed LDR (Table 2.4 and Section ). Upstream LDR primers are allelespecific 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. 110

145 Chapter 4 Molecular Characterisation of PNG isolates 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. 111

146 Chapter 4 Molecular Characterisation of PNG isolates During thermocycling, the LDR primers hybridise to PCR products with matching basepairs. 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 Recipes for PCR and LDR master mix solutions and respective primer sequences are outlined in Appendix B 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 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). 112

147 Chapter 4 Molecular Characterisation of PNG isolates 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 (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 Pfmdr1 LDR-FMA Development 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

148 Chapter 4 Molecular Characterisation of PNG isolates 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 (T A ). To improve amplification specificity, gradient experiments were employed to select for the optimal T A. This was tested using genomic DNA from 7G8 (Figure 4.2). PCR amplification was successful across T A of 40 C to 60 C for region 1. For region 2 however, specificity increased when T A was >48 C. Since 56 C was the optimal T A 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 and 17758, respectively. Similarly, in Dd2 which carries the 1034S allele, FI of and 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 114

149 Chapter 4 Molecular Characterisation of PNG isolates 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 Initial Optimised pfmdr1(1) 754 forward 1048 reverse pfmdr1(2) Nest forward 4489 reverse pfmdr1(2) Nest forward 4264 reverse pfmdr1(1) 681 forward 1119 reverse pfmdr1(2) 3499 forward 4311 reverse 5 -GTGTTTGGTGTAATATTAAAG-3 5 -CAAACGTGCATTTTTTATTAATG-3 5 -GATCCAAGTTTTTTAATACAGG-3 5 -TTAGGTTCTCTTAATAATGCAC-3 5 -TATTGTAAATGCAGCTTTATGG-3 5 -CACTAACTATTGAAAATAAGTTTC-3 5 -TGTATGTGCTGTATTATCAG-3 5 -CTTATTACATATGACACCACA-3 5 -TAGAAGATTATTTCTGTAATTTG-3 5 -CAATGTTGCATCTTCTCTTCCA-3 115

150 Chapter 4 Molecular Characterisation of PNG isolates 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 (T A ). Specificity was enhanced at T A >48 C for the region 2 reaction. 116

151 Chapter 4 Molecular Characterisation of PNG isolates 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. 117

152 Chapter 4 Molecular Characterisation of PNG isolates 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 and using initial primers, compared with 5457 and 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 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). 118

153 Chapter 4 Molecular Characterisation of PNG isolates 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. 119

154 Chapter 4 Molecular Characterisation of PNG isolates 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 C 2 min 10 x PCR Buffer C 30 sec 2.5mM dntps C 30 sec Forward primer (10pmol/µL) pfmdr1 (region 1) 681 Reverse primer (10pmol/µL) pfmdr1 (region 1) Mac Taq 0.3 Total volume per well C 30 sec X 32 cycles 72 C 4 min 10 C PCR: pfmdr1 region 2 Volume (µl) Cycling Program Sterile distilled water C 2 min 10 x PCR Buffer C 30 sec 2.5mM dntps C 30 sec Forward primer (10pmol/µL) pfmdr1 (region 2) 3499 Reverse primer (10pmol/µL) pfmdr1 (region 2) Mac Taq 0.3 Total volume per well C 1 min X 40 cycles 72 C 4 min 10 C 120

155 Chapter 4 Molecular Characterisation of PNG isolates Table 4.4 LDR primers for P. falciparum pfmdr1 molecular markers. a Lowercase nucleotides represent tag sequences added to the 5 ends of each allele-specific LDR primer. b ID, 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. c Com, common (conserved) sequence primer positioned immediately downstream from the allele-specific primer. Gene LDR Primer Sequences a ID b pfmdr1 region 1 86N 86Y Com86 184Y 184F Com184 5'tacactttctttctttctttctttTTGGTGTAATATTAAAGAACATGA-3 5'atcatacatacatacaaatctacaTTGGTGTAATATTAAAGAACATGT-3 5 phosphate-atttaggtgatgatattaatccta-biotin-3 5'tcaaaatctcaaatactcaaatcaGCCAGTTCCTTTTTAGGTTTATA-3 5'ctacaaacaaacaaacattatcaaGCCAGTTCCTTTTTAGGTTTATT-3 5 phosphate-tatttggtcattaataaaaaatgca-biotin pfmdr1 region S 1034C Com N 1042D Com D 1246Y Com1246 5'ttacctttatacctttctttttacATGCAGCTTTATGGGGATTCA-3 5'caatttcatcattcattcatttcaATGCAGCTTTATGGGGATTCT-3 5 phosphate-gtcaaagcgctcaattatttatt-biotin-3 5'cttttcatcttttcatctttcaatCCAAACCAATAGGCAAAACTATT-3 5'ctttttcaatcactttcaattcatCAAACCAATAGGCAAAACTATC-3 5 phosphate-aataaataattgagcgctttgac-biotin-3 5'tcatcaatcaatctttttcactttAATATATGTGATTATAACTTAAGAG-3 5'tcataatctcaacaatctttctttTAATATATGTGATTATAACTTAAGAT-3 5 phosphate-atcttagaaacttattttcaatag-biotin A series of comprehensive experiments were set up to optimise pfmdr1 LDR conditions. To test whether annealing temperature would improve signal clarity, parallel LDR with T A at 58 C, 60 C and 62 C were performed. FI ratios between positive and negative alleles were compared in control strains for different annealing temperatures (Appendix C). For pfmdr1 region 1, 60 C produced the best clarity as higher temperature dampened the FI for positive alleles. The opposite was observed in the LDR for pfmdr1 region 2 in which higher T A enhanced signal contrast. Further 121

156 Chapter 4 Molecular Characterisation of PNG isolates investigation for region 2 using annealing conditions at 62 C, 63 C, 64 C and 65 C, indicated 65 C was optimal. Secondly, the effect of PCR amplicon concentration on FI was investigated in P. falciparum strains HB3, K1 and 7G8. Briefly, PCR products from pfmdr1 region 1, region 2 and regions 1 plus 2 together, were subjected to LDR undiluted, diluted 1:2, 1:5, 1:10 and 1:100 in sterile distilled water. Although signal clarity was improved at some dilutions, no consistent trend was found. In addition, DNA yield from field samples would likely be lower than those derived from cultured parasites, hence standardised dilutions may not be applicable. Optimised LDR for pfmdr1 involves two separate reactions for each region. For pfmdr1 region 1, the reaction mixture was initially heated at 95 C for 1 min, followed by 32 cycles of denaturation at 95 C for 15 sec and annealing/ligation at 60 C for 2 min (Table 4.5). Similar thermocycling conditions were used for LDR of pfmdr1 region 2, with the exception of the final step performed at 65 C for 2 min (Table 4.6) Optimised LDR-FMA for pfmdr1 and Multiplexed Detection of SNPs in pfdhfr, pfdhps and pfcrt genes The first step of the optimised LDR-FMA involves PCR amplification of the pfmdr1 gene from genomic DNA extracts. Briefly, sample DNA was centrifuged (3000 rpm for 30 sec) and 3 µl was combined with PCR master mix (25 µl/well) for pfmdr1 regions 1 and 2. The plate was sealed using Microseal A film (Bio-Rad, USA) and centrifuged prior to PCR. Following PCR, the products were subjected to two separate LDR for both pfmdr1 regions due to different T A requirements. LDR master mixes (14 µl/well) were prepared (Appendix B) and combined with 1 µl of PCR product from corresponding regions. The plates were firmly sealed (sealing film B , Denville Scientific Inc), centrifuged and subjected to thermocycling (Table 4.5 and 4.6) in a PTC-225 Peltier Thermal Cycler (MJ Research, Iowa). On completion, 2.5 µl of LDR products from each pfmdr1 regions 1 and 2 were combined into 60 µl of pre-warmed hybridisation solution (Appendix B) containing microspheres from each 122

157 Chapter 4 Molecular Characterisation of PNG isolates Table 4.5 Optimised LDR conditions for pfmdr1 region 1. Table 4.6 Optimised LDR conditions for pfmdr1 region 2. LDR: pfmdr1 region 1 Volume (µl) Cycling Program Sterile distilled water C 1 min Taq Ligase Buffer C 15 sec Com C 2 min Com x 32 cycles of steps 2 and 3 only 86Ytag Ntag Ytag Ftag Taq DNA Ligase 0.1 PCR product (region 1) 1 Total volume per well 15.0 LDR: pfmdr1 region 2 Volume (µl) Cycling Program Sterile distilled water C 1 min Taq Ligase Buffer C 15 sec Com C 2 min Com x 32 cycles of steps 2 and 3 only Com Stag Ctag Dtag Ntag Dtag Ytag Taq DNA Ligase 0.1 PCR product (region 2) 1 Total volume per well

158 Chapter 4 Molecular Characterisation of PNG isolates allelic set (total of 10 alleles). From this point on, the LDR products (5 µl) from pfcrt, pfdhfr, and pfdhps genes can be multiplexed into a single well with the hybridisation solution provided that it contains microspheres from their respective allelic set (total of 18 alleles). Hybridisation, reporter dye labelling and fluorescent signal measurement were performed as described previously (Sections 2.3.4). Further validation and application of the new LDR-FMA for pfmdr1 SNP screening of culture-adapted and patient isolates of P. falciparum are described in the subsequent section. Positive thresholds for each allele were established as detailed in Section Assay Validation Comparison between LDR-FMA and RFLP speciation Plasmodium speciation using LDR-FMA and the reference RFLP method were compared in 374 samples. The sensitivity and specificity of LDR-FMA compared to the reference RFLP method for P. falciparum speciation were 96.6% and 100%, respectively. Concordances between the two methods are presented in Table 4.7. There were high concordances for the diagnosis of P. falciparum (97%), P. vivax (90%) and P. malariae (98%). 11 of 12 cases that were P. falciparum negative by LDR-FMA, exhibited strong P. vivax fluorescent signals (>20,000), yet they have been classified as P. falciparum positive by the RFLP approach. The remaining case proved to be a nonfalciparum mixed infection by the LDR-FMA characterised by strong positive signals (23,145) for P. malariae and weak positive (667) for P. vivax. However, it was classified as a P. falciparum mono-infection by the RFLP method. The LDR-FMA method identified a total of 10 cases of P. malariae infections compared with 3 cases by RFLP Inter-assay concordance If a sample is positive for P. falciparum by LDR-FMA then the pfcrt, pfdhps, pfdhfr and pfmdr1 genes should also be detectable. There were 304 samples in which speciation and all four specific gene assays were performed using LDR-FMA. In 241 (79%), each of the pfcrt, pfdhps, pfdhfr and pfmdr1 genes were identified. For discordant samples, 124

159 Chapter 4 Molecular Characterisation of PNG isolates the pfdhps and pfdhfr assays were more often PCR negative in the amplification of Plasmodium genome. Table 4.7 Concordance between RFLP and LDR-FMA diagnosis of Plasmodium species in PNG field samples. P. falciparum LDR (+) LDR (-) Total RFLP (+) RFLP (-) RFLP & LDR-FMA Concordance Total % P. vivax RFLP (+) RFLP (-) Total % P. malariae RFLP (+) RFLP (-) Total % Identification of drug resistance alleles The specificities of the pfcrt, pfdhfr and pfdhps assays have been reported previously with 100% concordance with haplotypes from a variety of laboratory-adapted strains (Carnevale et al. 2007). The specificities of the pfmdr1 LDR-FMA probes are shown in Table 4.8. The results for the pfmdr1-specific LDR-FMAs show that allele-specific background median fluorescence intensity (MFI) signals ranged from 168 to 6,464 and positive allele-specific signals ranged from 1421 to 22,008. Strain-specific LDR-FMA results were 100% concordant with published haplotypes (Mehlotra et al. 2008). 125

160 Chapter 4 Molecular Characterisation of PNG isolates Field Application of the LDR-FMA Speciation and drug resistance genes in PNG field isolates In 402 samples with microscopy-confirmed P. falciparum, the Plasmodium species LDR-FMA identified 28 patients with P. vivax co-infections and 13 with P. malariae co-infections. No P. ovale co-infections were found. The maximum median FI obtained for field isolates and thresholds used to determine the presence of an allele are shown in Table 4.9. Table 4.8 LDR-FMA evaluation of pfmdr1 SNPs in laboratory-adapted P. falciparum strains. The fluorescence signal is expressed as median fluorescence intensity (MFI) units. Boldfacing indicates positive allele-specific signals. a pfmdr1 haplotypes for P. falciparum strains are for 3D7, NYSND; for 7G8, NFCDY; for Dd2, YYSND; for HB3, NFSDD; K1, YYSND. Fluorescence signal for the Pfmdr1 a gene, codon and allele: Y N Y F S C D N D Y 3D G Dd HB K

161 Chapter 4 Molecular Characterisation of PNG isolates Prevalence of polymorphic alleles in pfcrt, pfmdr1, pfdhfr and pfdhps Overall, 9 mutant alleles were detected. These were at pfmdr1 codons N86Y (91%), Y184F (2%), N1042D (2%), D1246Y (4%), pfcrt codons C72S and K76T (both 92%), pfdhfr codons C59R (93%), S108N (95%) and pfdhps codon K540E (1.5%) (Figure 4.5). The pfcrt haplotype SVMNT was found to be at fixation in the sample (92%), with only 7% of P. falciparum strains retaining the CQ-sensitive haplotype CVMNK. Two children were infected with mixed strains carrying CVIET and SVMNT mutations. The majority of the P. falciparum isolates carried the CQ resistance-associated YYSND (93%) haplotype of the pfmdr1 gene (Figure 4.6). One patient was infected with an isolate carrying a single pfdhfr S108N mutation. However, most children (97%) had parasites with double mutations in the NRNI haplotype corresponding to amino acids at codons 51, 59, 108 and 164. A few isolates (3%) retained the wild haplotype NCSI. No isolates carried the pfdhfr I164L allele. The pfdhps haplotype KAA which harbours a single mutation at codon 613 was predominant (98%). Four isolates carried the pfdhps K540E mutation. When the isolates with completely defined haplotypes were assessed for multiple-gene mutations, it was found that 88% (100/113) of children were infected with P. falciparum carrying quintuple mutations across the pfcrt, pfdhfr, pfmdr1 and pfdhps genes characterised by the respective haplotypes SVMNT+NRNI+YYSND+KAA. Four patients (one per treatment group) were infected with parasites carrying the six-fold mutated haplotype SVMNT+NRNI+YYSND+EAA. One of these patients was treated with PQ-DHA and had a LPF, whilst the other three had P. falciparum ACPR. Table 4.10 details the number of P. falciparum isolates with multiple combinations of mutations across drug resistance genes. 127

162 Chapter 4 Molecular Characterisation of PNG isolates Table 4.9 Fluorescence detection thresholds and maxima for P. falciparum pfdhps, pfdhfr, pfcrt and pfmdr1 in PNG field samples. *Alleles not detected in the field isolates. Gene and allele Fluorescence intensity signal dhps Threshold Maximum 540K , E 9, S * - 581A , S * - 613A ,403 dhfr 51I * N 23,429 59R ,368 59C 23, T * S 16, N 22, L * - 164I ,695 pfcrt CVIET * CVMNK 20,657 SVMNT pfmdr1 22,523 86Y ,994 86N 18, Y , F 20, C * S , D , N 23, D , Y 3,

163 Chapter 4 Molecular Characterisation of PNG isolates Figure 4.5 Prevalence of pfcrt, pfmdr1, pfdhfr, pfdhps alleles in P. falciparuminfected individuals from the Madang and East Sepik Provinces, PNG. Figure 4.6 Frequency distributions of pfcrt, pfdhps, pfdhfr, pfmdr1 haplotypes in P. falciparum-infected individuals from PNG field sites. The number of samples demonstrating complete haplotype for each gene were 376, 254, 273 and 195, respectively. 129

164 Chapter 4 Molecular Characterisation of PNG isolates Table 4.10 Occurrence of P. falciparum isolates carrying multiple mutations across 4 genes associated with drug resistance. Wild-type alleles of pfcrt, pfdhfr, and pfmdr1 are indicated by boldfacing. Allele mutations are underlined. Pfcrt haplotypes CVMNK, CVIET and SVMNT correspond to codons 72 to 76. Pfdhfr haplotypes NCSI, NRNI and NCNI correspond to codons 51_59_108_164. Pfmdr1 haplotypes NYSND, YYSND and NFSDD correspond to codons 86_184_1034_1042_1246. Double gene mutation Triple gene mutation (+pfmdr1) pfcrt pfdhfr (pfcrt+pfdhfr only)* NYSND YYSND NFSDD NCSI CVMNK with NRNI NCNI* NCSI CVIET with NRNI 1^ 0 1^ 0 NCNI* NCSI SVMNT with NRNI ^^ 3 NCNI *Only assayed for pfcrt and pfdhfr due to inadequate sample volume. ^Sample with P. falciparum carrying both CVIET and SVMNT mutations. ^^Four of these isolates were also carrying the 540E mutation, whilst all other P. falciparum sampled carried 540K allele of the pfdhps gene Parasite drug resistance mutations and treatment outcome In evaluating the association of parasite genes with treatment outcome, the mean number of parasite mutations in ACPR, ETF, LPF and LCF groups in 351 P. falciparum cases was compared. Overall, there were 308 cases of ACPR, 3 cases of ETF (one from CQ-SP; two from ART-SP), 3 cases of LCF (one case each from CQ- SP, PQ-DHA and AL) and 37 cases of LPF (eleven from CQ-SP; twelve from ART-SP; twelve from PQ-DHA and two from AL). There were 4 mutations in 41.6%, with 0.6%, 6.8%, 16%, 27.1%, 7.1%, 0.9% carrying no mutation and single, double, triple, 130

165 Chapter 4 Molecular Characterisation of PNG isolates quintuple and sextuple mutations, respectively. There was no significant difference between the mean number of mutations within the ACPR group (mean 3.3 [95% CI ]) and those in the ETF, LCF, LPF groups (means 2.3 [0-6.1], 3.0 [ ] and 3.4 [ ], respectively, P>0.05 in each case). For the purposes of subsequent analyses, ETF, LCF and LPF were grouped as treatment failure. The polymorphisms pfdhfr N51I, C59R, S108N and pfdhps K540E did not predict treatment failure in 81 children allocated CQ-SP and in 86 allocated ART-SP (P>0.05). In analyses of pfmdr1 N86Y, Y184F, N1042D and pfcrt K76T, pfmdr1 D1246Y was a significant predictor of treatment failure in 79 children treated with PQ-DHA therapy; 40% (4 of 10) of such children carried D1246Y strains while only 3 of 69 (4%) who responded to treatment harboured parasites with this mutation (P=0.004). In none of the other three groups was there a significant association between the presence of D1246Y and treatment failure (P 0.10). Similar analyses did not reveal any associations between haplotypes of pfcrt, pfdhfr and pfdhps and treatment failure. 4.4 DISCUSSION The consequences of past antimalarial treatment policies in PNG were evident in these data. Only 7% of the parasites carried the wild-type pfcrt CVMNK (codons 72 to 76) associated with a CQ-sensitive phenotype. The high prevalence of the resistant K76T polymorphism concurred with studies conducted between 2000 and 2005 in Madang and East Sepik Provinces (Casey et al. 2004; Mehlotra et al. 2005; Schoepflin et al. 2008). The SVMNT haplotype (codons 72 to 76) is at fixation in the PNG parasite population with an increase in prevalence from 83% from the early 1990s to 92.3% in (Mehlotra et al. 2001; Marfurt et al. 2008; Schoepflin et al. 2008). As well as being associated with CQ resistance, a transfection study has shown that parasites carrying the SVMNT haplotype has reduced sensitivity to AQ and its metabolite (Sidhu et al. 2002). Since AQ is recommended in place of CQ for treatment of malaria in PNG children <19 kg (PNGDOH 2000), this may have implications for the most vulnerable patients. Another CQ-resistant haplotype, CVIET (codons 72 to 76) found commonly in Africa 131

166 Chapter 4 Molecular Characterisation of PNG isolates and South-East Asia (Lim et al. 2003; Keen et al. 2007; Nsobya et al. 2007; Yang et al. 2007) was detected in two isolates as a mixed infection with the SVMNT strain, confirming its recent emergence in PNG (DaRe et al. 2007). Differences between the pfcrt intronic microsatellite diversity haplotypes of the SVMNT and CVIET parasites from the province adjacent to Madang suggest that they have been imported (DaRe et al. 2007), presumably by economic migrants from near-by Asian countries (Mehlotra et al. 2001; Mehlotra et al. 2005; Mehlotra et al. 2008). Although assessed in limited numbers of isolates (n=195) due to inadequate sample volume, the CQ-sensitive pfmdr1 NYSND (codons 86_184_1034_1042_1246) was present in 5.1%. Most others carried the N86Y polymorphism with the YYSND haplotype (codons 86_184_1034_1042_1246) associated with CQ resistance. These results reflect an increase of this haplotype since the mid 1990s (Mehlotra et al. 2005; Mehlotra et al. 2008). It was also observed that four isolates (2%) carried the double point mutations Y184F and N1042D, which confirmed the recent emergence of these pfmdr1 polymorphisms in PNG (Marfurt et al. 2008). Although none of the four patients with parasites carrying the pfmdr1 NFSDD haplotype (codons 86_184_1034_1042_1246) were assigned to the AL treatment group in this study. In a Nigerian paediatric study (Happi et al. 2008), the presence of these SNPs with the wildtype 86N allele was significantly associated with AL treatment failure. Since introduction of this treatment in PNG is imminent, monitoring changes in the NFSDD pfmdr1 haplotype should be a high priority. It was found that signal intensities for the pfmdr alleles were relatively weak even in the controls. This was unlikely to reflect poor PCR amplification, as signal intensities for codons 1034 and 1042 derived from the same region were unaffected. Poor hybridisation with the LDR probe is possible. The D1246Y allele, present in 12% of isolates in association with the wild-type allele 1246D, has not been previously detected in PNG (Marfurt et al. 2008; Mehlotra et al. 2008). A recent African study observed an increased prevalence of pfmdr1 N86Y and D1246Y alleles post AQ exposure (Nawaz et al. 2009), but sporadic mutations at the 1246 codon have also been documented in Thailand (Rungsihirunrat et al. 2009) and it may also have been imported into PNG (Mehlotra et al. 2005). Both D1246Y and N86Y have been 132

167 Chapter 4 Molecular Characterisation of PNG isolates associated with diminished in vitro sensitivity to CQ and AQ (Reed et al. 2000; Pickard et al. 2003). The concomitant occurrence of pfmdr1 N86Y and pfcrt K76T mutations was observed in many isolates, with increased frequency compared to a decade earlier (Mehlotra et al. 2008). Concurrent mutations may reflect the involvement of CQ pressure in selecting for the predominant pfmdr1 YYSND haplotype. This hypothesis was supported by the selection of pfmdr1 N86Y allele after CQ and AQ therapy (Duraisingh et al. 1997; Djimde et al. 2001). Concurrent mutations in pfcrt K76T, and pfdhfr C59R and S108N were observed in 92.7% of the PNG isolates. Of those with pfmdr1 genotyping, 88.5% also harboured N86Y mutations. Parasites with multiple mutations were more common than found in another study conducted several years earlier in the same region (Schoepflin et al. 2008). The pfmdr1 haplotypes NFCDD, NFSND, and YFSND haplotypes (codons 86_184_1034_1042_1246) were not found in PNG isolates as found in those from Thailand (Pickard et al. 2003), or the NFCDY and NFSDY haplotypes found in Brazil and Colombia (Mehlotra et al. 2008). In vitro studies by Sá et al. showed 7G8 and hybrid crosses with its pfmdr1 haplotype (NFCDY) resulted in higher 50% growth inhibitory concentrations (IC 50 s) for AQ and its metabolite (Sá et al. 2009). However, the CDY (i.e. S1034C, N1042D, D1246Y) mutation of the pfmdr1 gene has not been detected in PNG. Collective polymorphisms in the 3 coding region (N86Y and Y184F) can confer resistance to quinine, mefloquine and halofantrine and modulate parasite sensitivity to artemisinin drugs (Reed et al. 2000), and are found in South America, Africa and Asia. Their geographical distribution suggests the requirement of drug pressure for their maintenance and spread (Foote et al. 1990), as may occur with the future use of ACTs in PNG. However, the frequency of the wild-type 86N may increase with adoption of ACT as has been seen in African studies (Dokomajilar et al. 2006; Humphreys et al. 2007). This study demonstrated that 97% of the present isolates carried both the C59R and S108N SNPs which constitute the major pfdhfr resistance alleles. Since the prevalence 133

168 Chapter 4 Molecular Characterisation of PNG isolates of this double mutant in East Sepik Province was 72% - 91% in (Mita et al. 2006b; Carnevale et al. 2007), this suggests that there has been continuing consistent selection pressure for alleles conferring pyrimethamine resistance in northern PNG. Carnevale et al. noted that a number of isolates from this area carried a single mutation at codon 108 with the NCNI (n=13) and NCTI (n=1) haplotypes (Carnevale et al. 2007). In the present study, only one isolate had a single pfdhfr mutation with the NCNI haplotype (codons 51_59_108_164) and, consistent with Marfurt et al. (Marfurt et al. 2008), the S108T mutant that was reported to be present in PNG in 1996 (Reeder et al. 1996) was not detected in the current study. In the evaluation of pfdhps polymorphisms at codons 540, 581 and 613, only wild-type alleles at loci 581 and 613 were detected. Four isolates were found to carry the K540E variant first detected in studies in East Sepik and Simbu Provinces in (Mita et al. 2006b; Marfurt et al. 2008). Most of the present isolates (98.4%) carried the KAA haplotype and 1.6% carried the EAA haplotype. SNPs at codons 436 and 437 were not evaluated, as the respective LDR probes were unavailable at the time of study. The lack of association between well known mutations (pfcrt K76T, pfdhfr S108N, pfdhfr C59R, pfmdr1 N86Y) and treatment failure is largely due to fixation levels of these mutations in Madang Province. Although carriers of multiple mutations were more often observed in treatment failure cases, this did not reach statistical significance. In the present study there was limited statistical power because of the relatively small number of treatment failures (43 of 351), but the high baseline number of mutations per isolate and the emergence of immunity in this age group in a high transmission setting may also have attenuated the relationship. Although pfdhfr K540E has been shown to predict SP treatment failure (Talisuna et al. 2004; Bacon et al. 2009), there was a low prevalence of this allele in the isolates and it was unrelated to outcome. These data are consistent with those of Marfurt et al. who showed that pfcrt K76T, and pfdhfr C59R and S108N, did not predict treatment failure after CQ/SP treatment in a region northwest of Madang (Marfurt et al. 2008), reflecting fixation of these mutations in PNG. Interestingly, the association between pfmdr1 D1246Y allele with PQ-DHA and overall treatment failure highlights the increasingly recognised role of this transporter gene in 134

169 Chapter 4 Molecular Characterisation of PNG isolates modulating resistance to antimalarial drugs from different classes (Cowman et al. 1994; Peel et al. 1994; Reed et al. 2000). A novel approach to determine presence or absence of mutations using the LDR-FMA system was adopted. Determination of signal positivity proved challenging. Initially, allele-specific thresholds that have been established previously were applied for the pfcrt, pfdhfr and pfdhps alleles (Carnevale et al. 2007). However, a number of samples were plainly mis-classified when compared by visual discrimination via 2-dimensional and 3-dimensional modelling. Various statistical models were tested but, in the absence of valid malaria-negative samples, it was found that bimodal and gamma distributions were inappropriate. Subsequent use of allelic signal ratios within each codon appeared robust when applied to controls with known haplotypes, especially in cases where strong positive signals were accompanied by high background. However, this approach was based on the assumption of single strain infections, which can only be circumvented by establishing a specific threshold. It was found that, through normalisation of the data, application of gene-specific cut-points from the control strains, and reversing the process to obtain codon-specific cut-offs, prediction of positivity was relatively accurate. Unless better resolution of positive from negative signal is possible, this method or related approaches (DaRe et al. 2010) can be adopted for future LDR-FMA studies. The multiplex LDR-FMA technique proved a cost-effective tool for epidemiologic studies. Taking into consideration the reagents required for each step (DNA extraction, plasticware, PCR and LDR primers, and Fleximap microspheres), the total cost per sample for the analysis of 28 SNPs was AUD $4.14 ($0.15 per SNP), with over 60% of the cost due to DNA extraction. This amounts to less than half the expense of the recently-described microarray SNP detection approach (Crameri et al. 2007). However, as with other expensive equipments in the PNG settings, maintenance of the bioplex array reader and shipment of reagents maybe costly. Compared to PCR-based approaches that have been used to evaluate polymorphisms in the pfdhfr, pfdhps, pfcrt and pfmdr1 genes, most of which involve DNA probe hybridisation and post-pcr RFLP methods (Duraisingh et al. 1998; Casey et al. 2004; Alifrangis et al. 2005; Farcas 135

170 Chapter 4 Molecular Characterisation of PNG isolates et al. 2006; Veiga et al. 2006), the LDR-FMA system offers greater efficiency and objectivity when analysing multiple mutations. Given these considerations and the increase in key parasite mutations in resource-poor malaria-endemic countries such as PNG, the LDR-FMA SNP assay represents an excellent tool for molecular surveillance. The present LDR-FMA platform enables assessment of 18 SNPs in the pfcrt, pfdhps and pfdhfr genes in a single-tube multiplex assay, a task that is beyond the capabilities of existing real-time PCR and RFLP methodology. The inclusion of 10 additional SNPs in the pfmdr1 gene can be easily accommodated through modified microsphere set selection with all 28 SNPs having their own unique classification codes. Monitoring the spread of resistance to CQ, sulfadoxine and pyrimethamine using this or equivalent methodology is a high priority in countries such as PNG where these drugs are still used. In addition, there will be an increasing need for monitoring pfmdr1 SNPs since polymorphisms encompass diverse effects on parasite sensitivity to a range of antimalarial drugs including those used in artemisinin-based combination therapy (Reed et al. 2000). This work has been published in Antimicrobial Agents and Chemotherapy, titled Molecular Assessment of Plasmodium falciparum Resistance to Antimalarial Drugs in Papua New Guinea Using an Extended Ligase Detection Reaction Fluorescent Microsphere Assay. 136

171 CHAPTER 5 ANTIMALARIAL PROPERTIES OF DESBUTYL-LUMEFANTRINE

172 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine CHAPTER 5. ANTIMALARIAL PROPERTIES OF 5.1 INTRODUCTION DESBUTYL-LUMEFANTRINE Desbutyl-lumfantrine (DBL), formerly known as desbutyl-benflumetol is a 2,3- benzindene compound with antimalarial activity. Although previously considered only a putative metabolite of lumefantrine because of a lack of supportive pharmacokinetic data (Samal et al. 2005; Starzengruber et al. 2007), recent analytical developments have enabled the reliable detection of relatively low concentrations of DBL in samples of plasma from small numbers of patients treated with conventional doses of artemetherlumefantrine combination therapy (McGready et al. 2006; Hatz et al. 2008; Ntale et al. 2008). The ratio of maximum plasma concentration (C max ) of the parent compound to that of the metabolite in this situation has varied substantially, from 6 (Ntale et al. 2008) to >270 (Hatz et al. 2008). DBL is more potent in vitro against chloroquine (CQ)-resistant P. falciparum and P. vivax field isolates than lumefantrine (Noedl et al. 2001; Kyavar et al. 2006; Starzengruber et al. 2008). There is evidence of in vitro synergy between lumefantrine and DBL against P. falciparum, but at ratios (999:1 and 995:5) that were presumably selected at a time when plasma concentrations of DBL were assumed to be very much lower than those of the parent compound (Starzengruber et al. 2007; Starzengruber et al. 2008). Interactions between DBL and artemisinin were assessed in a study of schizont maturation in Thai P. vivax field isolates in which antagonism was found at low concentrations and apparent synergy at much higher concentrations (Kyavar et al. 2006). A subsequent similar field study confirmed concentration-dependent synergy in strains of P. falciparum (Muller et al. 2008). Because of its relative in vitro potency and evidence of low cardiac toxicity (Traebert et al. 2004), DBL has been suggested as an antimalarial drug in its own right (Starzengruber et al. 2007). There is, however, a need for further assessment of its interactions with other antimalarial drugs, especially lumefantrine and the artemisinin derivatives, in strains of P. falciparum of differing drug sensitivities. In addition, and 138

173 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine given that artemether-lumefantrine is recommended as first-line treatment for uncomplicated malaria (WHO 2009), there is also a need to confirm the relative plasma concentrations of lumefantrine and DBL together with their therapeutic implications. The present chapter reports on in vitro antimalarial activity of DBL in comparison to lumefantrine and CQ in CQ-resistant and CQ-sensitive laboratory-adapted strains of P. falciparum. It also provides a comprehensive evaluation of DBL interaction with DHA and lumefantrine across 17 combination-ratios. Plasma levels of DBL and lumefantrine in 127 clinical samples and their relationship with artemether-lumefantrine treatment outcome are also presented. 5.2 MATERIALS AND METHODS Parasite Cultures The laboratory-adapted P. falciparum strains 3D7 (CQ-sensitive) and W2mef (CQresistant) were cultured as described previously (Section 2.1.3) (Trager et al. 1976; Scheibel et al. 1979) Antimalarial Drugs Stock solutions of CQ diphosphate, piperaquine tetraphosphate (PQ), mefloquine hydrochloride (MQ), lumefantrine, DBL and dihydroartemisinin (DHA) were prepared as described in Section Stocks and working standards of lumefantrine and DBL were sonicated for 90 sec in an ultrasonic waterbath in pre-warmed media to facilitate dissolution. On the day of assay, aliquots were thawed and further diluted in RPMI to a working standard, and further two-fold serial dilutions in complete RPMI (without hypoxanthine) at double assay concentrations were prepared for CQ ( nm), PQ ( nm), MQ ( nm), DHA ( nm), and lumefantrine and DBL ( nm). 139

174 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine In vitro Drug Susceptibility Drug susceptibility was tested in triplicate as described in Section 2.2. To each well of a 96-well plate was added 100 µl of dosed or drug-free media without hypoxanthine, 90 µl of RBC suspension (final 0.5% parasitaemia, 1.5% hct) and 10 µl of 5 mg/ml 3 H- labelled hypoxanthine. After 48 hr incubation, the plates were harvested and processed as detailed in Section Drug Interaction Studies The traditional checkerboard method (Berenbaum 1978; Canfield et al. 1995; Hassan Alin et al. 1999) and a recently described fixed-ratio approach (Fivelman et al. 2004) were used to develop the following assay. Solutions (5 µm) of each drug were used to prepare 17 fixed molar combination ratios in 12-well plates. The molar ratios tested for lumefantrine:dbl and DHA:DBL are detailed in Table 5.1. Two hundred microlitres of each drug combination were dispensed into row H of a 96-well plate. Rows A to G were filled with 100 µl of media without hypoxanthine. Preparations of one drug alone were assayed in triplicate, and other combination ratios in duplicates. Two-fold serial dilutions were performed using an electronic multichannel pipette (Labnet excel, Fisher Biotec, Australia) from rows H to B. Drug-free control wells were included (row A). All wells contained a final volume of 200 µl including 3 H-labelled hypoxanthine, were standardised to 1.5% parasitaemia at 1.5% hct and were incubated for 48 hr and processed as described previously (Section 2.2.5). Table 5.1 Drug combination ratios for isobologram assays. Molar ratios and drug concentrations of desbutyl-lumefantrine (DBL) paired with either lumefantrine (LM) or dihydroartemisinin (DHA) are shown. Within-well concentration shown in the table is that of the prepared drug combination. Further two-fold serial dilutions of each combination were performed to give six additional tests at lower doses. For simplicity, drug concentrations are displayed only for the first assay well (i.e. the one containing the highest concentration of drugs) for a given combination. 140

175 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine Drug Combination Molar Ratio Within-well assay concentration (nm) DBL LM DHA :0 0:1 1:1 1:3 1:10 1:30 1:100 1:300 1:1000 1: :1 1000:1 300:1 100:1 30:1 10:1 3: Study Site and Sample Collection Plasma concentrations of DBL and lumefantrine were measured in samples taken from children participating in an intervention trial carried out in the Madang Province of Papua New Guinea (PNG) (Karunajeewa et al. 2008b). Children aged 0.5 to 5 years and with uncomplicated falciparum or vivax malaria were randomised to one of four treatments, one of which was artemether-lumefantrine (Coartem, Novartis Pharma, Basel, Switzerland) at a dose of 10 mg/kg lumefantrine twice daily for three days (total dose of 60 mg/kg). Scientific and ethical approval was obtained from the Medical Research and Advisory Committee of the Ministry of Health of PNG and informed consent was obtained from the parents or legal guardians before recruitment and blood sampling. A venous blood sample was taken at 7 day post-treatment for drug assay since this is regarded as a useful surrogate marker of the area under the plasma concentration-time curve and the time above the minimal inhibitory concentration for long-acting antimalarial drugs (Price et al. 2007). Whole blood was centrifuged on site and separated plasma was stored at -80 C until analysis. For the purposes of the present 141

176 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine study, therapeutic response was considered to be either an adequate parasitologic and clinical response (ACPR) or treatment failure (early treatment failure, late parasitologic failure or late clinical failure) during a follow-up of 42 days without PCR correction for reinfection (WHO 2003; Karunajeewa et al. 2008b) Liquid Chromatography and Mass Spectrometry Lumefantrine was analysed using a validated high performance liquid chromatography method (Mansor et al. 1996). Plasma samples of 1.0 ml were spiked with atovaquone as an internal standard and mixed with 7 ml of hexane:diethylether (70:30). After centrifugation, the organic layer was separated, evaporated and reconstituted with 200 µl methanol:acetic acid (98:2). Aliquots of 15 µl were injected onto a Phenomenex C6-phenyl 4.6 x 150 mm column (Phenomenex, CA, USA). A mobile phase of acetonitrile:0.05 M phosphate buffer at ph=2.0 (62:38) with 0.03 M sodium perchlorate was pumped at 1 ml/min. Lumefantrine and atovaquone were measured at 335 nm and quantified using Chemstation Software (Version 9, Agilent Technology, Waldbronn, Germany). The linear range for lumefantrine was 20-20,000 ng/ml. Inter-day variability was 4.94%, 4.93%, 7.16% and 11.23% and intraday variability 2.83%, 4.41%, 4.11%, 9.55% for 20,000, 2000, 200 and 20 ng/ml respectively. DBL was analysed using a validated ultra high-performance liquid chromatographytandem mass spectrometry (UPLC-LCMS-MS) method using a hexyl-analogue as an internal standard (IS). To facilitate protein precipitation, 40 µl of a 0.1 M ZnSO 4 was added to 20 µl sample and briefly vortexed. A 200 µl aliquot of acetonitrile containing the internal standard was added before the sample was centrifuged and 10 µl of the supernatant was injected onto a 2795/Quattro Premier XE UPLC-MS/MSWaters Corp, MA) using a Waters XTerra MS C18, 2.5 x 50 mm 3.5 µm column. Gradient elution was performed using aqueous 2 mm ammonium acetate/0.1% formic acid and methanolic 2 mm ammonium acetate/0.1% formic acid as mobile phases at 0.4 ml/min. Transitions were monitored using positive electrospray ionisation with multiple-reaction monitoring for DBL and the IS which were m/z 472.1/346.0 and 500.1/346.0, respectively. The linear range for DBL was ng/ml with the lower end taken as the limit of quantitation. Inter-day variability was 3.36%, 3.47%, 142

177 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine 9.98% and 6.74% and intraday variability 2.47%, 3.46%, 8.16% and 3.48% for 50, 10, 1 and 0.5 ng/ml, respectively. When matrix effects were assessed, between-subject variability was 3.37%, 4.47% and 9.43% at 50, 10 and 1 ng/ml, respectively Statistical Analysis Drug susceptibility and interactions were analysed by non-linear regression of logarithmically-transformed concentrations. The concentration that inhibited 50% parasite growth (IC 50 ) was determined for each drug, as was the concentration that inhibited 99% of growth (IC 99 ). The fractional inhibitory concentrations (FICs) representing the concentration of each drug alone or in combination resulting in 50% inhibition were used to construct isobolograms (Berenbaum 1978). Two analytical approaches were employed (Davis et al. 2006). First, the sum of fractional inhibitory concentrations (ΣFICs) was calculated using the formula (IC 50 of A in a mixture resulting in a 50% inhibition/ic 50 of A alone) + (IC 50 of B in a mixture resulting in a 50% inhibition/ic 50 of B alone) (Berenbaum 1978). The combination has an indifferent interaction when the ΣFIC is close to 1.0. A ΣFIC of <1 indicates synergy with data points forming a concave isobole beneath the line of additivity (Figure 1.22). A ΣFIC of >1 indicates antagonism as represented by a convex isobole (Berenbaum 1978; Chawira et al. 1987; Fivelman et al. 2004; Davis et al. 2006). Second, the function (y = 1 - x/(x + (1-x)*exp(-I))) (Brueckner et al. 1991; Canfield et al. 1995) was fitted to the data where y is the IC 50 of drug A combined with drug B; x is the IC 50 of drug B when combined with drug A, and I is the interaction value. Positive I values indicate synergy, negative values antagonism and values close to zero an indifferent interaction. Values of I and FIC were considered significantly different from no interaction if both 95% confidence intervals (CIs) of the estimates did not span zero and unity, respectively (Davis et al. 2006). 143

178 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine 5.3 RESULTS In vitro Antimalarial Potency of DBL The geometric mean IC 50 and 95% CIs for DBL, lumefantrine and other drugs are shown in Table 5.2. W2mef had, as expected, a higher CQ IC 50 than 3D7 but there were no other strain-specific differences. Consistent with the IC 50 data, the geometric mean IC 99 values for DBL against 3D7 and W2mef were 78 and 56 nm, respectively, and, for lumefantrine, 239 and 226 nm. Based on both the IC 50 and IC 99 values, the in vitro activity of DBL was at least 3 times that of lumefantrine regardless of CQ sensitivity (Figure 5.1). Table 5.2 In vitro sensitivity of laboratory-adapted P. falciparum to desbutyllumefantrine and other antimalarial drugs. IC 50 values are geometric means. Data represent at least six experiments performed in triplicate. 3D7 IC 50 (nm) 95% CI W2mef IC 50 (nm) 95% CI Chloroquine Lumefantrine Desbutyllumefantrine Piperaquine Dihydroartemisinin

179 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine Figure 5.1 In vitro susceptibility of laboratory strains of P. falciparum to chloroquine, desbutyl-lumefantrine and lumefantrine. 145

180 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine DBL Interaction with Conventional Antimalarials Isobolographic analysis of DBL and lumefantrine combinations showed no interaction in both laboratory-adapted strains (see Table 5.3 and Figure 5.2). Drug interactions between DBL and dihydroartemisinin were mildly synergistic as assessed from both I and ΣFIC. Table 5.3 In vitro efficacy of antimalarial drug combinations against P. falciparum clones 3D7 and W2mef as assessed by isobolographic analysis. Data are the interaction factor (I) and the summed fractional inhibitory concentration (ΣFIC) with 95% confidence intervals. The assessment of interaction is based on both I and ΣFIC data (see text). I (95% CI) ΣFIC (95% CI) Interaction 3D7 DBL-lumefantrine 0.41 (-0.24 to 1.05) 0.99 (0.93 to 1.05) Indifferent DBL-dihydroartemisinin 0.99 (0.71 to 1.28) 0.92 (0.87 to 0.98) Mildly synergistic W2mef DBL-lumefantrine 0.79 (0.02 to 1.56) 1.06 (0.97 to 1.14) Indifferent DBL-dihydroartemisinin 0.92 (0.73 to 1.10) 0.94 (0.90 to 0.99) Mildly synergistic 146

181 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine Figure 5.2 Isobolograms illustrating interactions between desbutyl-lumefantrine with conventional antimalarials. Isobolograms showing effect of desbutyllumefantrine in combination with lumefantrine against P. falciparum 3D7 (top left), W2mef (top right). Effect of desbutyl-lumefantrine in combination with dihydroartemisinin against P. falciparum 3D7 (bottom left) and W2mef (bottom right). The isoboles are representative of three or four experiments in which each of the 17 drug ratios was tested in duplicate. Degree of interaction (I) with 95% CI are represented by red and dotted lines, respectively. 147

182 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine DBL Plasma Levels on Day 7 Post-Treatment DBL and lumefantrine were quantified in 94 available day 7 plasma samples from the 127 children with falciparum malaria recruited to the artemether-lumefantrine arm of the intervention trial (Karunajeewa et al. 2008b). The mean (range) DBL concentrations were 15.5 ( ) ng/ml or 31.9 ( ) nm all children had a plasma concentration above the 0.5 ng/ml lower limit of quantitation. For lumefantrine, the mean (range) plasma concentration was 370 (26-1,720) ng/ml or 699 (49-3,251) nm. The lumefantrine:dbl ratio ranged from 7.0 and with a mean of Influence of DBL Plasma Levels on Clinical Outcome The relationship between plasma lumefantrine and DBL concentrations, and treatment outcome at 42 days is shown in Figure 5.3. The mean plasma concentrations of lumefantrine in those children who failed treatment were lower than in those with an ACPR, but this difference was not significant (P=0.22). In the case of DBL, there was a similar result but at borderline significance (P=0.053). There was no difference in the case of the lumefantrine:dbl ratio (P=0.97). 148

183 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine Figure 5.3 Boxplots summarising day 7 plasma levels of lumefantrine and desbutyl-lumefantrine. Plasma lumefantrine (left panel) and desbutyl-lumefantrine (right panel) concentrations in children who had an adequate clinical and parasitological response (ACPR) or who failed treatment with artemether-lumefantrine 149

184 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine 5.4 DISCUSSION The present observations confirm and extend previous published data relating to in vitro and in vivo aspects of the antimalarial activity of DBL. The two laboratory-adapted strains of P. falciparum were more sensitive in vitro to DBL than lumefantrine, consistent with previous studies using field isolates (Noedl et al. 2001; Starzengruber et al. 2007; Starzengruber et al. 2008). This effect was independent of CQ sensitivity. The lack of in vitro interaction between DBL and lumefantrine appears to contradict previous reports of synergy in field isolates of P. falciparum using limited numbers of drug combinations (Starzengruber et al. 2007; Starzengruber et al. 2008). However, the synergy between DBL and DHA observed using a comprehensive method of isobolographic assessment parallels data from studies of field isolates of P. vivax (Kyavar et al. 2006) and P. falciparum (Muller et al. 2008). Both lumefantrine and DBL were detected at mean concentrations that were well above the IC 99 for both laboratory-adapted strains of P. falciparum in day 7 plasma of children treated with artemether-lumefantrine for uncomplicated malaria. The day 7 plasma DBL was a stronger predictor of subsequent therapeutic outcome than plasma lumefantrine or the plasma lumefantrine:dbl ratio. These various observations have potential clinical implications. When artemether-lumefantrine is administered to patients with malaria, plasma lumefantrine concentrations rise after each of the six doses given over 3 days and then decline with an elimination half-life of around 4 days (Ezzet et al. 1998). There are, as yet, no equivalent pharmacokinetic data for DBL. The day 7 plasma concentrations from this study show that both plasma lumefantrine and DBL concentrations remain above the IC 99 in most patients for at least three parasite life cycles. Even if there were synergy between lumefantrine and DBL at these concentrations as found by others (Starzengruber et al. 2007; Starzengruber et al. 2008), its relevance at this stage in treatment is unclear as initial parasite clearance had occurred in all patients allocated this therapy in the trial (Karunajeewa et al. 2008b) and the presence of one or other compound at a concentration >IC 99 should inhibit low-level (sub-microscopic) parasite replication. In addition, the variable day 7 lumefantrine:dbl ratios in PNG children, which are consistent with the marked apparent between-dose variability in lumefantrine 150

185 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine bioavailability (Ezzet et al. 1998; van Agtmael et al. 1998), would lead to inconsistent interactions. Indeed, the data relating day 7 lumefantrine and DBL concentrations to therapeutic outcome suggest that DBL has a stronger role than the parent compound in suppressing recrudescence and/or preventing reinfection. This may reflect the fact that, even at relatively low plasma concentrations, DBL has more potent antimalarial activity than lumefantrine as exemplified by the present in vitro sensitivity data. The fact that the lumefantrine:dbl ratio had no relationship with outcome is unlikely to translate into a clinically important synergistic interaction. Initial parasite clearance is considered due primarily to the artemisinin component of ACT and, conversely, its prolongation is taken as evidence of artemisinin resistance (Dondorp et al. 2010). It is, however, possible that the longer half-life partner and interactions between the component drugs enhance the relatively rapid parasiticidal effects of artemisinin derivatives. Synergy between artemether and lumefantrine has been reported (Hassan Alin et al. 1999) and the present data and those of others (Kyavar et al. 2006; Muller et al. 2008) suggest a similar interaction for DBL/DHA. However, the clinical importance of such effects is unclear. The synergy observed in the present study was mild. The relatively short half-lives of artemether and its active metabolite DHA (1-2 hr) (Ezzet et al. 1998; Ezzet et al. 2000) limit the time-window for such effects. Interactions can be antagonistic, such as that between artemisinin and DBL at some concentrations (Kyavar et al. 2006) and between DHA and 4- aminoquininolines and related drugs (Davis et al. 2006). The fact that a range of different ACTs show equal efficacy (Sinclair et al. 2009) suggests that acute effects on the parasite other than those from the artemisinin drug are minor. According to the manufacturer, DBL is a degradation product of lumefantrine, and Coartem tablets contain <0.1% of DBL both at the time of manufacture, and at the end of the expiry date (H. Gruninger, personal communication, Novartis Pharma). The occurrence of such low relative concentrations of DBL in pure lumefantrine powder was confirmed by UPLC-LCMS/MS (unpublished data), and excluded DBL:lumefantrine at 1:10000 from the drug interaction analysis as a result. Although a small amount of DBL is present at the time of Coartem administration, it is 151

186 Chapter 5 Antimalarial Properties of Desbutyl-Lumefantrine quantitatively far below the level detected in plasma. Thus, this exogenous contribution is insufficient to influence the plasma DBL concentrations in the present study, with the corollary that DBL is a true metabolite. The present study confirms that DBL has potential as an antimalarial drug in its own right. Its in vitro potency relative to the parent compound, its synergy with DHA, and the positive relationship between day 7 plasma concentrations and ACPR suggest that it could be a useful alternative to lumefantrine as part of ACT. Although the presence of DBL at parasiticidal concentrations during conventional artemether-lumefantrine therapy suggest that it is as safe as lumefantrine, further pharmacokinetic and safety assessment after DBL administration would be required to facilitate the development of optimal dose regimens. In contrast to N-desbutyl-halofantrine, the active metabolite of halofantrine (Traebert et al. 2005), preliminary in vitro cardiotoxicity studies have not raised any significant concerns (Traebert et al. 2004). This work has been published in full in Antimicrobial Agents and Chemotherapy, titled Desbutyl-lumefantrine is a metabolite of lumefantrine with potent in vitro antimalarial activity that may influence artemether-lumefantrine treatment outcome. 152

187 CHAPTER 6 STATINS AND FIBRATES: LIPID-MODIFYING DRUGS AS ANTIMALARIALS

188 Chapter 6 Statins and Fibrates as Antimalarials CHAPTER 6. STATINS AND FIBRATES: LIPID- MODIFYING DRUGS AS ANTIMALARIALS 6.1 INTRODUCTION The increase in non-communicable diseases of affluence in developing countries such as PNG may see a significant shift from heavy use of anti-infective agents to therapies for cardiovascular risk factor reduction (Martin et al. 1981; Iser et al. 1993; Hodge et al. 1996; Lindeberg et al. 1997; Kende 2001; Lesley et al. 2001; Yamauchi et al. 2001; Yamauchi et al. 2005). Drugs used for one indication can sometimes have wider application and there is evidence that some cardiovascular therapies such as the antidiabetic drug rosiglitazone (Boggild et al. 2009) could have antimalarial properties. This is of particular interest given the recent emergence of P. falciparum resistant to artemisinin (Wongsrichanalai et al. 2008; Carrara et al. 2009; Dondorp et al. 2010). Statins and fibrates are distinct classes of lipid-modifying drugs that reduce morbidity and mortality associated with cardiovascular disease. This chapter investigates the potential of these two groups of agents as novel antimalarials Statins as Lipid-lowering and Antimicrobial Agents Statins are well tolerated and widely used for reducing cardiovascular morbidity and mortality (Wilt et al. 2004; Thavendiranathan et al. 2006). They inhibit 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase (Figure 1.19), a key enzyme in cholesterol biosynthesis, which consequently lower serum concentrations of lowdensity lipoprotein cholesterol. There has been interest in alternative applications of statins, with studies demonstrating their abilities to inhibit the growth of bacteria (Catron et al. 2004), yeasts (Song et al. 2003), and protozoa (Chen et al. 1990; Urbina 1993; Andersson et al. 1996; Montalvetti 2000). The first study of their antimalarial effects found that lovastatin and simvastatin inhibited in vitro intra-erythrocytic development of P. falciparum (Grellier et al. 1994). More recently, the in vitro susceptibilities of P. falciparum to six statins revealed atorvastatin had the greatest activity (Pradines et al. 2007). Despite these encouraging findings, neither simvastatin nor atorvastatin in high doses improved the outcome in Plasmodium berghei-infected 154

189 Chapter 6 Statins and Fibrates as Antimalarials mice (Bienvenu et al. 2008; Kobbe et al. 2008) and there was no effect on parasitaemia (Bienvenu et al. 2008). However, no study to date has included the most-potent statin in clinical use, rosuvastatin (Soran et al. 2008). In addition, despite the demonstration of in vitro synergy between mevastatin and the glycoprotein inhibitor tunicamycin against P. falciparum (Naik 2001), the interaction between statins and conventional antimalarial drugs has not been evaluated. Therefore, the relative in vitro antimalarial activity of rosuvastatin and atorvastain, against P. falciparum and the in vitro interactions between statins and both chloroquine (CQ) and dihydroartemisinin (DHA) are presented in this chapter Fibrates as Potential Antimalarial Drugs Fibrates such as gemfibrozil and fenofibrate are agonists of peroxisome proliferatoractivated receptor alpha (PPARα). Although active via a different pathway to that of the statins, fibrates exhibit potent lipid-modifying properties (Derosa et al. 2009). They have well-characterised pharmacokinetic and pharmacodynamic profiles. An early study on the effect of plasma free fatty acid concentrations and temperature on parasitaemia in P. berghei-infected mice employed clofibrate as a lipid-lowering agent (McQuistion 1979). Although not a primary interest of the study, clofibrate directly inhibited the development of parasitaemia. However, the authors did not extend these observations and no other subsequent study has examined the role of fibrates in the treatment of malaria. It is possible that Plasmodium species have PPARα-like motifs which could mediate such fibrate antimalarial effects. There is further indirect evidence of a link between fibrates and malaria. In a biochemical assay, Ehrhardt et al. assessed the in vitro influence of fibrates on P- glycoprotein activity. Of gemfibrozil, fenofibric acid, clofibrate and fenofibrate, the latter drug was the only member of the class to inhibit P-glycoprotein mediated transport (Ehrhardt et al. 2004). A P-glycoprotein homologue 1 in P. falciparum has been implicated in CQ and mefloquine resistance (Reed et al. 2000) and it is possible that fenofibrate may decrease drug efflux mediated by this protein and thus reverse resistance. Fibrates exhibit pleotropic effects that could also modify the effects of 155

190 Chapter 6 Statins and Fibrates as Antimalarials malaria infection. An example of this is the attenuation of brain tissue injury associated with inflammation (Bordet et al. 2006) and vascular occlusion (Guo et al. 2009). In a murine influenza study, where severe systemic disease is thought to arise through overproduction of proinflammatory cytokines, treatment with gemfibrozil doubled survival compared to vehicle-treated mice (Budd et al. 2007). Similar host inflammatory responses have been proposed for malaria pathology, particularly in coma and other clinical manifestations associated with cerebral malaria (Clark et al. 2005; Clark et al. 2008; Clark et al. 2009). In the present study, the direct antimalarial activity of gemfibrozil, clofibrate, fenofibrate, and its metabolite fenofibric acid against P. falciparum are investigated. In vitro interactions between these fibrates and both CQ and DHA are also examined. 6.2 MATERIALS AND METHODS In vitro Parasite Growth Inhibition The laboratory-adapted P. falciparum strains 3D7 (Africa; CQ-sensitive), E8B (Brazil; CQ-resistant), K1 (Thailand; CQ-resistant), W2mef and Dd2 (Indochina; CQ-resistant) were maintained as previously described (Section 2.1). Stock solutions of CQ diphosphate (Sigma Chemicals, St Louis, USA), atorvastatin (Waterstonetech, Carmel, IN), rosuvastatin (Waterstonetech, Carmel, IN), fenofibrate (Sigma), clofibrate (Sigma), gemfibrozil (Sigma-Aldrich) and fenofibric acid (Tyger Scientific Inc, NJ, USA) were prepared in distilled water (CQ) and DMSO (statins and fibrates). Serial dilutions of each drug were prepared in RPMI and added in triplicate to 96-well plates with final concentrations of 12.5 to 1600 nm (CQ), 0.3 to 200 µm (atorvastatin) and 0.6 to 400 µm (rosuvastatin, pravastatin and simvastatin), 6.2 to 800 µm (gemfibrozil, clofibrate, fenofibrate) and 39 to 5000 nm (fenofibric acid). Synchronous parasite suspensions ( 90% rings) were adjusted to 1.0% for non-isotopic and 0.5% parasitaemia for isotopic assays and a final hct of 1.5% in the drug-parasite mixture. Following 48 hr incubation, parasite growth was measured initially by a modification of the pldh assay as described previously (Section 2.2.4) (Makler et al. 1993a) and 156

191 Chapter 6 Statins and Fibrates as Antimalarials subsequently by the 3 H-hypoxanthine incorporation assay (Section 2.2.5) (Desjardin 1979). IC 50 s and IC 90 s were determined by non-linear regression analysis (Graphpad Prism 4.0) Drug Interaction Studies A modified fixed-ratio isobologram method was used to assess drug interactions (Fivelman et al. 2004). Inhibition assays using 3 H-hypoxanthine incorporation (Section 2.) were first carried out to determine individual IC 50 s for statins, fibrates, CQ and DHA (Desjardin 1979). These were used to establish test concentration ranges in the combination assays. A total of six and eleven solutions containing fixed-ratios of conventional antimalarials with statins and fibrates, respectively, were prepared as shown in Table 6.1. The FICs of each drug in each combination determined from doseresponse curves were used to construct isobolograms from which the sum of each FIC was calculated (Berenbaum 1978). 157

192 Chapter 6 Statins and Fibrates as Antimalarials Table 6.1 Interaction ratios of statins, fibrates and conventional antimalarials. Molar ratios and drug concentrations of atorvastatin or fibrates (gemfibrozil, fenofibrate or fenofibric acid) paired with either chloroquine (CQ) or dihydroartemisinin (DHA) are shown. Each within-well concentration shown is that of the prepared drug combination. Further two-fold serial dilutions of each combination were performed to give six additional tests at lower doses. For simplicity, drug concentrations are displayed only for the first assay well (i.e. contains the highest concentration of drugs) for a given combination. *Fenofibric acid concentrations are in nmol. Drug pair Ratio Top concentrations within a combination Statin/Fibrate (µm) CQ (nm) DHA (nm) Atorvastatinantimalarials 5:0 0:5 4:1 3:2 2:3 1: Fibrates-antimalarials Gemfibrozil Fenofibrate Fenofibric acid* 1:0 0:1 1:1 1:3 1:30 1:300 1:3000 3:1 30:1 300:1 3000: * Dosed Plasma Bioassay Due to the possibility that active metabolites and other in vivo factors might contribute to enhanced antimalarial activity of fenofibrate and atorvastatin, such as those observed for atorvaquone (Butcher et al. 2003; Edstein et al. 2005), bioassays were performed. For atorvastatin bioassay, a pre-treatment venous blood sample was taken from a 158

193 Chapter 6 Statins and Fibrates as Antimalarials healthy volunteer who was then given atorvastatin 80 mg (Lipitor, Pfizer, NY) once daily for 4 consecutive days. A second venous blood sample drawn 3 hr after the last dose at the time of the predicted maximal plasma concentration (Cilla et al. 1996). The samples were centrifuged promptly and aliquots of separated plasma stored at -20 C. Aliquots of pre- and post-treatment plasma were used for atorvastatin assay using HPLC (Nirogi et al. 2006). For fenofibrate bioassay, a pre-treatment blood sample was taken from a healthy volunteer who was then given fenofibrate at 145 mg (Lipidil, Solvay Pharmaceuticals) once daily for 6 consecutive days. A second blood sample was drawn on day 6 at the time of the predicted maximal plasma concentration of fenofibric acid at steady state (i.e. 4 hr after the final dose) (Keating et al. 2002). The heparinised blood samples were centrifuged promptly and aliquots of separated plasma were stored at - 20 C. Plasma fenofibric acid levels from pre- and post-treatment samples were measured, after extraction in hexane/chloroform/isopropanol, (18:80:2, v/v/v), by validated high performance liquid chromatography assay with tandem mass spectrometric detection and 2-(2,4,5-trichlorophenoxy)-propionic acid as internal standard (Laboratoires Fournier S.A., Daix, France) (Zhu et al. 2010). Approval for these procedures was obtained from the South Metropolitan Area Health Service Human Research Ethics Committee. A modified microdilution isotopic technique (Desjardin 1979; Kotecka et al. 2003) was used to determine the antimalarial activities of pre- and post-treatment plasma. For atorvastatin bioassay, pre- and post-treatment plasma were spiked with CQ (concentration range, 3.9 to 250 nm), and DHA (0.9 to 60 nm), or an equivalent volume of drug-free RPMI. For fenofibrate bioassay, the drug levels in the posttreatment plasma and inhibitory properties from preliminary in vitro data indicated that these were adequate for a direct assessment using two-fold serial dilutions in drug-free RPMI without the need to spike with an active antimalarial drug. In triplicate experiments, aliquots of 100 µl of plasma were added to 90 µl of parasite suspension (1% parasitaemia, 1.5% hct) and 10 µl of 3 H-hypoxantine (0.5 µci) in 96-well plates, 159

194 Chapter 6 Statins and Fibrates as Antimalarials and the mixture was incubated, harvested and counted (Karl et al. 2009). 6.3 RESULTS In vitro Antimalarial Activities of Statins The in vitro inhibitory effects of atorvastatin, rosuvastatin, simvastatin, pravastatin and CQ are summarised in Table 6.2. All statins showed antimalarial activity, but atorvastatin was more potent than rosuvastatin. The IC 50 and IC 90 values for each statin did not differ between CQ-sensitive and CQ-resistant strains and were well above those for CQ, even against CQ-resistant strains. Simvastatin and pravastatin were weak against P. falciparum (IC 50 >200 µm) In vitro Antimalarial Activities of Fibrates The in vitro inhibitory effects of gemfibrozil, clofibrate, fenofibrate, fenofibric acid and CQ are summarised in Table 6.3. All fibrates showed antimalarial activity but fenofibric acid was most potent. With the exception of gemfibrozil, the IC 50 values of other fibrates did not differ between CQ-sensitive and CQ-resistant strains and were well above those for CQ. 160

195 Chapter 6 Statins and Fibrates as Antimalarials Table 6.2 In vitro activities of statins against CQ-sensitive and CQ-resistant strains of P. falciparum. The means (and 95% CIs) shown are from at least four independent triplicate experiments. *Inhibitory concentrations for chloroquine are in nanomoles. Data from CQ-resistant strains Dd2 and E8B were similar and have been pooled Strain Atorvastatin Rosuvastatin Pravastatin Simvastatin Chloroquine IC 50 (µm) IC 90 (µm) IC 50 (µm) IC 90 (µm) IC 50 (µm) IC 50 (µm) IC 50 (nm) 3D7 25 (16-39) 68 (38-121) 80 (47-137) 205 ( ) >200 > (23-29) Dd2/E8B 17 (8-37) 39 (24-62) 80 (37-174) 232 ( ) >200 > ( ) 161

196 Chapter 6 Statins and Fibrates as Antimalarials Table 6.3 In vitro activities of fibrates against CQ-sensitive and CQ-resistant strains of P. falciparum. Data represent at least six experiments performed in triplicate. *Inhibitory concentrations for chloroquine and fenofibric acid are in nanomoles. 3D7 IC 50 (µm) IC 90 (µm) W2mef IC 50 (µm) IC 90 (µm) Clofibrate 184 (98-345) 441 ( ) 256 ( ) 708 ( ) Gemfibrozil 311 ( ) 541 ( ) 245 ( ) 511 ( ) Fenofibrate 69 (54-88) 228 ( ) 72 (65-80) 178 ( ) Fenofibric acid 152 ( )* 250 ( )* 1120 ( )* 2436 ( )* Chloroquine 23 (15-34)* 48 (23-99)* 230 ( )* 495 ( )* 162

197 Chapter 6 Statins and Fibrates as Antimalarials Figure 6.1 In vitro susceptibility of laboratory strains of P. falciparum to cholesterol-lowering drugs and chloroquine. 163

198 Chapter 6 Statins and Fibrates as Antimalarials Interaction of Atorvastatin with Conventional Antimalarials The potential use of atorvastatin as a partner drug with conventional antimalarials was investigated by means of isobolograms. In vitro interactions between atorvastatin with CQ and DHA are shown in Figure 6.2. The interaction was indifferent in each case with a mean ΣFIC [95% confidence interval] of 1.05 [0.97 to 1.14] for atorvastatin-cq and 1.08 [0.99 to 1.17] for atorvastatin-dha Interaction of Fibrates with Conventional Antimalarials There were no synergistic combinations identified from the drug interaction studies involving fibrates, CQ, DHA and atorvastatin. For fenofibric acid-cq there was no interaction present for 3D7 but antagonism for W2mef. In the case of gemfibrozil-cq against 3D7 there was antagonism and indifferent interaction in W2mef. The remaining combinations including that for fenofibric acid-atorvastatin showed indifferent interactions. 164

199 Chapter 6 Statins and Fibrates as Antimalarials Figure 6.2 Interaction between atorvastatin and conventional antimalarials. Isoboles showing the FICs of atorvastatin plotted against those for CQ (left-hand panel) and DHA (right-hand panel). The isoboles are representative of three independent experiments in which each of the 6 drug ratios was tested in duplicate. The degree of interaction (I) with 95% CI is represented by grey and dotted lines, respectively. 165

200 Chapter 6 Statins and Fibrates as Antimalarials Table 6.4 In vitro efficacy of fibrates and antimalarial drug combinations. Fenofibrate, fenofibric acid and gemfibrozil in combination with CQ, DHA and atorvastatin were assessed against P. falciparum clones 3D7, W2mef and Dd2 using isobolographic analysis. Data are the interaction factor (I) and the summed fractional inhibitory concentration (ΣFIC) with 95% CIs. The assessment of interaction is based on both I and ΣFIC data (see text). Interaction between fenofibrate-dihydroartemisinin was not determined. I (95% CI) ΣFIC (95% CI) Interaction 3D7 Fenofibric acid-chloroquine 0.23 (-0.06 to 0.51) 0.99 (0.95 to 1.02) Indifferent Fenofibric acid-dihydroartemisinin (1.02 to 0.50) 1.11 (0.99 to 1.21) Indifferent Fenofibric acid-atorvastatin (-1.92 to 1.26) 1.19 (1.02 to 1.35) Indifferent Fenofibrate-chloroquine (-1.40 to 0.05) 1.08 (0.98 to (1.12) Indifferent Gemfibrozil-chloroquine (-2.44 to -0.62) 1.12 (1.01 to 1.23) Antagonistic Gemfibrozil-dihydroartemisinin (-3.34 to 0.66) 1.20 (0.95 to 1.45) Indifferent W2mef/Dd2 Fenofibric acid-chloroquine (-2.08 to -0.07) 1.19 (1.02 to 1.36) Antagonistic Fenofibric acid-dihydroartemisinin (-1.31 to 0.36) 1.11 (1.02 to 1.21) Indifferent Fenofibric acid-atorvastatin (-1.52 to -0.09) 1.08 (0.96 to 1.19) Indifferent Fenofibrate-chloroquine (-1.01 to 0.19) 1.01 (0.93 to 1.10) Indifferent Gemfibrozil-chloroquine (-1.80 to 0.73) 1.19 (0.99 to 1.40) Indifferent Gemfibrozil-dihydroartemisinin (-2.10 to 1.61) 1.15 (0.95 to 1.40) Indifferent 166

201 Chapter 6 Statins and Fibrates as Antimalarials Bioassay of Atorvastatin The ability of atorvastatin-containing plasma to inhibit P. falciparum in vitro was assessed by bioassay. Plasma atorvastatin concentrations in the healthy volunteer were undetectable at pre-treatment and were 118 µg/l (0.1 µm) post-treatment. Neither prenor post-treatment plasma inhibited growth of 3D7 (Figure 6.3). The 3D7 IC 50 s for CQ alone, CQ plus pre-treatment plasma, and CQ plus post-treatment plasma were similar (20.5 nm, 20.5 nm, and 20.4 nm, respectively), as were those for DHA alone, DHA plus pre-treatment plasma, and DHA plus post-treatment plasma (12.7 nm, 20.1 nm, and 19.2 nm, respectively) Bioassay of Fenofibric Acid The antimalarial activity of fenofibric acid generated in vivo as a result of fenofibrate dosing was assessed by bioassay (Figure 6.4). Plasma fenofibric acid concentrations in the healthy volunteer were undetectable at pre-treatment and were 13.0 mg/l posttreatment (i.e. plasma fenofibric acid concentration 40,816 nm). Post-treatment plasma inhibited growth of 3D7 at dilutions between 8 to 1024-fold. 167

202 Chapter 6 Statins and Fibrates as Antimalarials Figure 6.3 Atorvastatin bioassay. Dose response of laboratory-adapted P. falciparum subjected to conventional antimalarials with pre- and post- Lipitor (atorvastatin) treatment plasma. No significant differences were indicated in response to pre- and post-treatment plasma added to chloroquine (CQ) (top) and to dihydroartemisinin (DHA) (bottom). 168

203 Chapter 6 Statins and Fibrates as Antimalarials Figure 6.4 Fenofibrate bioassay. Growth response of laboratory-adapted 3D7 to twofold dilutions of plasma pre- and post-treatment of Lipidil (fenofibrate). Data points were obtained from three independent bioassays performed in triplicate. 169

204 Chapter 6 Statins and Fibrates as Antimalarials BLAST Analysis for PPAR-like Region in Plasmodium To elucidate possible mechanisms underlying the activity of fenofibric acid against malaria parasites, similarity searches were conducted between mrna and protein sequences of human PPARα and P. falciparum using basic local alignment search tool (BLAST, NCBI). Nucleic acid alignments between human and Plasmodium mrna produced 102 blast hits of short sequences (<40 bp). One hit had non-random matches (expected value (E) <0.02) identified as a region encoding for a P. falciparum 3D7 conserved Plasmodium protein, however its function is unknown. Comparisons between the human PPARα protein (NP_ ) and reference proteins for P. falciparum taxid (5833) resulted in three blast hits (Figure 6.5). Although the alignment scores were <40 for all three sequences, two were located at the PPARα DNA binding domain and one at the ligand binding domain. The latter, identified as a conserved Plasmodium protein (XP_ ) is of particular interest since it aligns with both the ligand binding site and the heterodimer interface, with 17% coverage of the human PPARα protein sequence. In addition, the same Plasmodium protein was one of five hits with other apicomplexans in comparison with the human PPARα protein. There were two hits from Babesia bovis showing 22 and 27% coverage of the human protein sequence (E = 1.4 and 1.9), and two from Toxoplasma gondii with 11 and 17% sequence coverage. The region ( ) of the Plasmodium protein identified as Plasmodium 1 in Figure 6.5, showed similarity with hypothetical proteins in other Plasmodium species including P. knowlesi, P. vivax, P. yoelii, P. chabaudi and P. berghei. It also weakly aligned with the PPARα protein sequence from Rhesus Macaque, Common Chimpanzee, Guinea pig and human BLAST Analysis for ABC-1 transporter in P. falciparum Proteins within the ATP-binding cassette sub-family A member (ABC-1) mediate cholesterol and lipid transport, are within the same superfamily of ATP-binding cassette 170

205 Chapter 6 Statins and Fibrates as Antimalarials (ABC) transporters as the multidrug resistant P-glycoprotein. Nucleic acid alignments between human and Plasmodium mrna produced 103 blast hits of short sequences (up to 50 bp). Similarity searches between reference protein sequences of human ABC-1 and P. falciparum revealed the presence of a parasite homolog of this lipid efflux pump. There were 49 protein alignment hits, 13 of the P. falciparum nucleotide sequences spanned across two regions (900 to 1116 and 1927 to 2110) with a coverage up to 19% (E <0.006) of the human ABC-1 query sequence (Figure 6.6). Two significant alignments were found in the P. falciparum ABC transporter protein (XP_ ) and the P. falciparum multi-drug resistance protein 2 (XP_ ) with E values of 8-16 and 1-12, respectively. 171

206 Chapter 6 Statins and Fibrates as Antimalarials Figure 6.5 Distribution of Plasmodium BLAST hit sequences on human PPARα. Protein-protein alignments between human PPARα (468bp) and P. falciparum was analysed by BLAST. Three P. falciparum protein sequences (green) have 10-17% coverage of the human PPARα sequence (grey). These specific hits span over the PPARα DNA binding (blue) and ligand binding (red) domains. Plasmodium 1: conserved Plasmodium protein (E = 0.18) (XP_ ), Plasmodium 2: erythrocyte membrane protein, PfEMP1 (E = 1.6) (XP_ ), Plasmodium 3: conserved Plasmodium protein (E = 8.4) (XP_ ). 172

207 Chapter 6 Statins and Fibrates as Antimalarials Figure 6.6 Distribution of P. falciparum BLAST hit sequence on human ABC-1. Protein-protein alignments between human ABC-1 (2261 bp) and P. falciparum was analysed by BLAST. Thirteen of forty-nine P. falciparum sequences from the reference nucleotide database aligned at the same two regions (green) with an overall of 10-19% coverage of the human ABC-1 protein sequence (grey). These specific hits spanned over the ABC-1 conserved domains (CD, in red). Two significant alignments were the P. falciparum ABC transporter protein (E = 8-16 ) (XP_ ), P. falciparum multi-drug resistance protein 2 (E = 1-12 ) (XP_ ), both have alignment scores of 50 to

208 Chapter 6 Statins and Fibrates as Antimalarials 6.4 DISCUSSION The present data confirm that atorvastatin inhibits the growth of P. falciparum in vitro (Grellier et al. 1994; Pradines et al. 2007). Consistent with previous findings (Pradines et al. 2007), pravastatin and simvastatin had minimal activity against P. falciparum. The antimalarial activity of atorvastatin is greater than that of rosuvastatin, but the atorvastatin IC 50 (17 to 25µM; Table 6.2) is approximately 100 times above that achievable in plasma with repeated maximal (80 mg) doses (0.1 to 0.3 µm) (Borek- Dohalsky et al. 2006). As might have been predicted from this observation, and consistent with the hypothesis that the metabolism of atorvastatin does not generate compounds with antimalarial activity, the bioassay showed that therapeutic plasma concentrations had no inhibitory effect against cultured P. falciparum. In addition, there was no synergy with conventional antimalarial drugs. The ability of the malaria parasite to synthesise cholesterol de novo (i.e. via the mevalonate pathway) appears limited (Vial et al. 1984; Wunderlich et al. 1991), and the presence of an HMG-CoA homolog was not revealed by BLASTX analysis of the P. falciparum sequence with other protozoan HMG-CoA protein sequences (Pradines et al. 2007). These observations and the greater antimalarial potency of atorvastatin versus rosuvastatin (the reverse of their ability to inhibit cholesterol synthesis in humans (Jones et al. 2003)) suggest an alternative, albeit low-potency, mechanism of antimalarial action to inhibition of HMG-CoA reductase. The present study demonstrates that gemfibrozil, fenofibrate and clofibrate have weak activity relative to conventional antimalarial drugs in vitro. Nonetheless, their IC 50 s are similar to antibiotics that are used for malaria prophylaxis and adjunctive therapy (Nakornchai et al. 2006). This activity may have accounted for the lower infection rate in the clofibrate-treated group of animals observed previously (McQuistion 1979), although the inflammation modulating effects of clofibrate may have also contributed. Fenofibric acid, the major metabolite of fenofibrate in vivo (Kirchgassler et al. 1998), has greatest activity at nm media concentrations similar to those of conventional agents. As might have been predicted from this observation, and consistent with the hypothesis that the metabolism of fenofibrate would generate sufficient fenofibric acid with 174

209 Chapter 6 Statins and Fibrates as Antimalarials antimalarial activity, the bioassay showed that therapeutic plasma concentrations (13 mg/l or 40,816 nm) had inhibitory effect against cultured P. falciparum with IC 90 s of CQ-sensitive and resistant strains at 250 nm and 2436 nm, respectively. An inhibitory effect was also observed in pre-treatment plasma against P. falciparum. Plasma from healthy individuals can interfere with parasite survival via complementmediated cell lysis. Non-heat treated sera was shown to reduce parasite growth as much as 25% compared to heat-treated control (Teja-Isavadharm et al. 2004). Therefore, heat inactivation of pooled human plasma is required to reduce this growth inhibitory activity prior supplementation in culture medium (Appendix B). Plasma fenofibric acid is stable for up to 8 hr at room temperature (Dubey et al. 2010); however, its stability in plasma at 50 C is unknown. Therefore, to minimise the risk of drug degradation, plasma samples in the bioassays were not heat-treated. This may explain the apparent dose-response of pre-treatment plasma against the parasite. Treatment of bioassay plasma with non-heat approaches such as the use of Affingel Protein-A may reduce this inhibition by removal of human immunoglobulin G (Teja-Isavadharm et al. 2004). For a drug to be clinically useful, the plasma concentration achievable in vivo should be a number of magnitudes higher than the in vitro inhibitory concentration. The steadystate maximum plasma concentration for fenofibric acid is approximately 25.5 mg/l (i.e. 80 µm) (Miller et al. 1998), whilst the maximum plasma concentration of fenofibric acid is in the range 3 15 mg/l ( µm) after an oral dose of fenofibrate of 100 or 200 mg (Bhavesh et al. 2009) with levels >0.4 mg/l (>1.2 µm) sustained over 2 days. The plasma fenofibric acid level available with repeated therapeutic doses is approximately 100 times more than that required to inhibit 50% of cultured P. falciparum. A newer formulation of micronised fenofibrate may further enhance bioavailability (Keating et al. 2002). Although its mode of action remains to be elucidated, fenofibric acid may act by interfering with P-glycoprotein (Ehrhardt et al. 2004) and ABC-1 mediated transport, and/or via a putative PPARα-like protein. BLAST analysis revealed partial similarities between a conserved Plasmodium protein sequence and that of the PPARα ligand 175

210 Chapter 6 Statins and Fibrates as Antimalarials binding domain in humans. Despite the low alignments score, there may be sufficient similarities at key amino acid positions for the tertiary protein conformation to interact with fenofibric acid, resulting in subsequent metabolic interference. In human and murine cells, fenofibric acid interferes with the expression of ABC-1 (Jaye et al. 2003; Arakawa et al. 2005), thus altering lipid accumulation. Fenofibric acid effects on the Plasmodium ABC-1 homolog may disturb the development of P. falciparum by similar mechanisms, depriving the growing parasite of lipid components of membranes and other cellular structures. In conclusion, this study confirmed statins to have antimalarial properties but at a concentration higher than that achieved by therapeutic doses (Borek-Dohalsky et al. 2006). In addition, even at supra-therapeutic doses, no synergy with CQ or DHA was observed. Although atorvastatin has been shown to prevent cytoadherence of P. falciparum to endothelial cells in co-culture models (Taoufiq et al. 2011), outcome data from animal models of severe malaria showed no protection (Bienvenu et al. 2008; Kobbe et al. 2008). These in vivo studies together with the present in vitro findings and do not support calls for clinical trials of statins as adjuvant antimalarial therapy (Bienvenu et al. 2008). The present experiments also revealed fenofibric acid to be the most active lipidmodifying agent against P. falciparum in vitro. The favourable pharmacokinetics of fenofibrate (Miller et al. 1998; Bhavesh et al. 2009) along with the high antimalarial in vitro activity of its major metabolite fenofibric acid warrants confirmatory in vivo investigation of this promising therapeutic application. The statin component of this work has been published in Antimicrobial Agents and Chemotherapy, titled Statins as potential antimalarial drugs: Low relative potency and lack of synergy with conventional antimalarial drugs. The fenofibrate component is undergoing review for a patent. 176

211 CHAPTER 7 DETECTION OF VOLATILE ORGANIC COMPOUNDS OF PLASMODIUM FALCIPARUM IN VITRO

212 Chapter 7 Volatile Organic Compounds CHAPTER 7. CHARACTERISATION OF VOLATILE ORGANIC COMPOUNDS OF P. FALCIPARUM IN VITRO 7.1 INTRODUCTION The detection of viable parasite forms is an essential requirement for malaria diagnosis and subsequent monitoring of the response to antimalarial therapy. For diagnosis, microscopic examination of a peripheral blood smear remains the investigation of choice in a wide variety of clinical situations. However, the sensitivity of microscopy is limited even when expert microscopists view high quality slides. In addition, the diagnosis may be missed in cases of severe falciparum malaria in which the majority of parasites are sequestered within the microvasculature of major organs (Coltel et al. 2004; Safeukui et al. 2008) or in the placenta in infected expectant mothers (Duffy 2007; Goyal et al. 2009). Antigen detection kits can be used where reliable microscopy is unavailable but they have even lower sensitivity and specificity (Section 1.6). PCR increases diagnostic sensitivity but its timely availability is limited largely to specialised laboratories in developed countries. In addition, the sensitivity of PCR (down to 1 parasite/µl) means that even a child weighing only 15 kg and with a circulating blood volume of approximately 1 litre who is PCR-negative may still harbour up to a million malaria parasites. The monitoring of the response to antimalarial therapy in individual patients depends on the availability of serial blood smears complemented by PCR where available. Antigen detection methods cannot be used because of the persistence of antigen after parasite clearance, while PCR does not differentiate between DNA from viable and non-viable parasites. There is a need for the development of alternative diagnostic tests that detect viable parasites before and after treatment with greater specificity and sensitivity than currently available methods. The human breath contains a large number of volatile organic compounds (VOCs) derived from the blood by passive diffusion in the lungs (Phillips et al. 1992c). VOCs in the breath are directly related to concentrations in blood and other tissues as they flow from compartments with higher vapour pressure to those with lower pressure (Phillips 1992b). Breath tests have been used to assist in the early diagnosis of conditions such as heart disease, rheumatoid arthritis and lung cancer 178

213 Chapter 7 Volatile Organic Compounds (Gordon et al. 1985; Humad et al. 1988; Weitz et al. 1991) as they detect increased VOCs released as a result of disease-specific cellular injury. More recently, exogenous VOCs produced by microorganisms such as Mycobacterium tuberculosis have been found in the breath of infected patients (Phillips et al. 2007). Plasmodium species may, in the same way, produce a characteristic VOCs fingerprint that can facilitate diagnosis and therapeutic monitoring. In the case of P. falciparum and perhaps P. vivax (Anstey et al. 2007), the cytoadherence of mature parasite forms in the pulmonary microvasculature may facilitate detection of Plasmodium-specific VOCs in breath samples. The cause of altered consciousness in severe malaria remains unknown. VOCs are used as general anaesthetics in clinical practice (Soukup et al. 2009) and it is possible that coma complicating malaria may result from elaboration of VOCs by malaria parasites in the cerebral microcirculation that have anaesthetic properties. In any case, malaria may, through indirect pathogenic tissue effects such as oxidative stress, alter the VOCs content of human breath in ways that are characteristic of the infection. A number of extraction techniques are used for the capture and analysis of VOCs from human breath and the microbial culture atmosphere (Phillips 1997; Lechner et al. 2005a; Syhre et al. 2008; Martin et al. 2010; Risticevic et al. 2010). Sampling and sample preparation involve pre-concentrating the analytes of interest by purge and trap, headspace, liquid-liquid or solid phase extractions. These conventional techniques consist of multiple labour-intensive procedures and/or require organic solvents. Solid phase micro-extraction (SPME) is an adsorption/desorption technique that circumvents most of the drawbacks to sample preparation (Risticevic et al. 2010). SPME can be used to concentrate volatile and non-volatile compounds in liquid samples or headspace without the use of solvents with sensitivities down to parts per trillion (Risticevic et al. 2010), where the target compounds are subsequently separated and quantified by gas chromatography-mass spectrometry (GC-MS). This chapter outlines the design and optimisation of a P. falciparum culture-sampling system suitable for VOCs headspace capture and analysis. Mass spectra of VOCs emitted by P. falciparum in vitro using GC-MS are also reported. Identification and 179

214 Chapter 7 Volatile Organic Compounds evaluation of these chemical finger-prints may have useful indications for diagnosis and may yield insights into the metabolism and pathogenicity of P. falciparum. 7.2 MATERIALS AND METHODS Parasites The laboratory-adapted P. falciparum strains 3D7 (chloroquine-sensitive) and W2mef (chloroquine-resistant) were maintained in RPMI 1640 HEPES as previously described (Section 2.1). Once the parasitaemia was >5%, synchronous cultures at the trophozoite stage were transferred into custom-designed containers (Section 7.3.1) at 1% hct and purged with a mixture of 1% O 2 and 5% CO 2 in nitrogen at 5 psi for 4 sec and 30 sec for prototypes 1 and 2, respectively. Subsequent optimisation used 5% O 2 and 5% CO 2 in nitrogen at 15 psi for 40 sec in the prototype 2 culture-sampling apparatus. The volume of media required to sustain high parasitaemia was calculated using the formula: volume of media (ml)/24hr = x (µl RBC pellet) x (% parasitaemia) (Radfar et al. 2009). This equation takes into account the nutrient requirements for nonparasitised as well as parasitised RBC. A control was set up with non-infected RBC using similar conditions and incubated for 24 hr at 37 C Solid Phase Micro-Extraction (SPME) After incubation, samples were double-contained and transported to the School of Biomedical, Biomolecular and Chemical Sciences (UWA) for extraction and analysis. Volatile and semi-volatile compounds within the headspace of non-parasitised control and malaria cultures were pre-concentrated onto a polydimethylsiloxane (PDMS, 100 µm) coated SPME fibre (SUPELCO, Bellefonte, PA, USA, #57300-U or portable field sampler #504823) for 1 hr in a heated waterbath (Section 2.4). In subsequent experiments, a triple fibre, 50/30 µm Divinylbenzene/Carboxen/PDMS StableFlex fibre (SUPELCO, Bellefonte, PA, USA, #57328-U) was also used. After sampling was completed, the fibre was retracted and the SPME holder was manually loaded onto the GC injector port where VOCs were desorbed for 5 min in splitless mode for 2 min. 180

215 Chapter 7 Volatile Organic Compounds Solvent Extraction Supernatants from 3D7 and W2mef cultures at high parasitaemia (5% to 13.2%) were pooled together (100 ml and 150 ml, respectively). Cell pellets of each strain were lysed by sonication (Microson ultrasonic cell disruptor, Misonix Inc, NY) for 30 sec and diluted with distilled water prior to extraction. The aqueous supernatant was transferred into a separation funnel and partitioned 3 times with ⅓ volume of an organic solvent (hexane, dichloromethane or ethyl acetate). The procedure involved gentle swirl-mixing with occasional depressurisation via the outlet valve of the funnel. After repositioning the funnel to the retort stand, the aqueous and organic phases gradually separated (Figure 7.1) Thermal Desorption: Purge and Trap For purge and trap extraction, non-parasitised control and P. falciparum infected RBC were cultured in prototype 2 containers (Figure 7.2). Air was drawn through the side inlets containing a loosely fitted cap and through the flask over the surface of the samples and the VOCs were trapped using a Tenax trap (200 mg, SUPELCO, Bellefonte, PA, USA). The headspace was collected for 1 hr at an airflow rate of 1.5 L/min using a portable air sampling pump (224-PCXR8, SKC Inc.) The trap was inserted into a short path thermal desorption injector (TD-2, Scientific Instrument Services, Inc.) and desorbed for 5 min at 200 C using a flow of helium (2 ml/min) into the GC-MS injection port that was also set at 200 C. The desorbed VOCs were collected on the column during the desorption process by cooling a small section of the capillary column with an ethanol/dry ice bath (-20 C to -40 C). The column was then equilibrated to 35 C and the temperature program on the GC-MS was started. The compounds were separated using a 30 m 0.25 mm i.d., 0.25 µm BPX-5 column (SGE), which was set at 35 C for 2 min and increased at 7 C/min until 250 C, and held for 10 min. The mass spectrometer was set to record between 45 and 400 amu. 181

216 Chapter 7 Volatile Organic Compounds Figure 7.1 Extraction of VOCs from culture supernatant by an organic solvent. The aqueous phase was set aside whilst the organic phase was collected into a clean, acetone-rinsed flask. The aqueous phase was subjected to repeated extraction into an organic solvent (a). The organic phase was then extracted against distilled water and collected into a conical flask followed by drying over anhydrous magnesium sulphate. After filtration (Whatman filter papers) (b), the extract was transferred into a round bottom flask where the solvent was evaporated under reduced pressure by means of a rotary evaporator (Rotavapor-R, Buchi Labortechnik, Flawil, Switzerland) (c). For analysis by GC-MS, extracts were evaporated to dryness under nitrogen (d) and resuspended in 100 µl of the extraction solvent. 182

217 Chapter 7 Volatile Organic Compounds Figure 7.2 Purge and trap set-up for thermal desorption. The prototype 2 sampling apparatus was kept warm (37 C) using a waterbath heated with a hotplate/magnetic stirrer. A stirrer bar was used to equilibrate the temperature in the waterbath which was measured with a thermometer. Air was drawn at a steady rate by vacuum through the flask for 1 hr through a loose inlet cap (C). The headspace VOCs were passed through an adsorptive trap (Tenax ) (B) fitted between Teflon tubing (A) where the VOCs were retained Gas Chromatography and Mass Spectrometry SPME and solvent extracted samples were subjected to GC-MS (Shimadzu GCMS- QP2010, Kyoto, Japan). The separation of emitted components was achieved using a 30 m 0.25 mm i.d., 0.1 µm Rt-Stabilwax (Restek, Bellefonte, PA) column in splitless injection mode with ultra-high purity helium as the carrier gas at a constant flow rate of 1 ml/min. The initial oven temperature was set to 35 C and held for 5 min then ramped at 7 C/min to 250 C at which compounds were desorbed. The desorption time for SPME was 5 min, while for solvent injections 1 µl was injected. The ion source was set at 200 C, and the spectrometer was set to record between 45 and 400 amu (Flematti 183

218 Chapter 7 Volatile Organic Compounds et al. 2009) Data Analysis Data collection and mass spectra generation were performed using the GC-MS Real Time Analysis software and Postrun application (GCMSsolution, version 2.40 Shimadzu Corporation). For compound identification, mass fragments of the target molecule were screened interactively against commercial Mass Spectral Libraries (NIST05, NIST05s, Gaithersburg, MD). 7.3 RESULTS Malaria VOCs Assay Development Design of culture-capture apparatus Preliminary studies employed T25 flasks coupled with a rubber stopper with two inlets for the culture and capture of headspace atmosphere (Skinner-Adams T, data unpublished). However, the use of plastic containers and rubber introduces organic contaminants which may interfere with the assay. Therefore, custom-designed glass flasks were created for the in vitro capture of headspace VOCs experiments. The prototype 1 sampling unit for VOCs analysis consisted of a 250 ml conical flask modified with a B-40 joint (Figure 7.3). The flask was fitted with an custom built aluminium stopper coupled with a delrin-polymer holder that served as the SPME holder. A small machined section at the base of the delrin holder allowed the placement of a polytetrafluoroethylene (PTFE) septum above a small opening in the metal stopper, which was pierced during SPME sampling. The prototype 1 container allowed the culture of 18 ml of parasite-cell-medium suspension. The culture flask and metal stopper were autoclave-sterilised prior to use. To maximise parasite mass and VOCs yield, a second design was proposed featuring a shallow container with a large base-area (Figure 7.2 and Figure 7.4). The prototype 2 184

219 Chapter 7 Volatile Organic Compounds sampling units were custom-made and equipped with two Duran screw thread tube connections (GL14, Vel, Leuven, Belgium) useful for purge and trap, and SPME sampling. A B-24 joint with a fitted glass stopper improved access to the parasite cultures and media changing. This was particularly important during the optimisation phase of the study in which parasite growth was monitored daily by microscopic examination of blood smears. The new design allowed a larger volume set-up (50 ml) of parasite-cell-media suspension. Cultures of high parasitaemia (>20%) were achieved under the conditions specified in Section 7.2.1, as facilitated by the maximised air/medium contact surface available for gas-exchange in the new design. Figure 7.3 Prototype 1 culture-capture apparatus with SPME. The prototype 1 design consisted of a conical flask modified with a B-40 joint that served as a receptacle for an aluminium stopper. A delrin polymer SPME holder was screwed into the aluminium stopper and between the mating surfaces, a syringe piercable PTFE septum was placed to seal the sample before analysis. 185

220 Chapter 7 Volatile Organic Compounds Figure 7.4 Design and dimensions of culture container (prototype 2) for headspace capture. The wide-base (154 cm 2 ) and shallow design maximises culture efficiency and total parasite yield. The B-24 joint allows for easy access during RPMI addition and parasite sampling. Two side inlets facilitate the ease of low-oxygen gasreplacement and act as an access point for purge and trap procedures and SPME of the headspace atmosphere Optimisation of culture conditions Headspace capture requires an enclosed culture system, where the atmosphere within the culture container must be optimised for parasite growth. The gas mixture of 1% O 2 and 5% CO 2 in nitrogen used for routine cultures (i.e. in a 28.4 L Nalgene dessicator) was suboptimal to sustain P. falciparum growth in the custom-designed containers. On macroscopic examination, RBC became much darker post incubation, likely attributable to inadequate oxygenation. A series of down-sized, volume vs duration of gas injection experiments were conducted with an oxygen monitor to determine optimal gas balance (data not shown). Although conditions for normal parasite replication were achieved, optimal growth was subsequently attained with a new gas mixture of 5% O 2, and 5% CO 2 in nitrogen (Mlambo et al. 2007). This gas composition allowed complete replacement of the air within the vessel with an atmosphere that is more suited for P. falciparum culture. This adaptation negated the need to find the intricate balance of gaspurging duration to lower the O 2 content in the air as previously performed. 186

221 Chapter 7 Volatile Organic Compounds Routine in vitro culture of P. falciparum usually maintains a maximum of 5% parasitaemia at 5% hct. Prototype 2 container was more suitable for malaria culture than its predecessor. The initial period for VOCs capture was over 48 hr (i.e. one parasite life cycle), however, the system must remain enclosed during this time without the required media changes. This approach produced stressed parasites as indicated by the presence of gametocytes by microscopy. Therefore, subsequent capture experiments were set-up over the later 24 hr of the developmental cycle where there is maximal parasite metabolic activity. Synchronised P. falciparum were cultured at high parasitaemia and pooled into prototype 2 containers at the trophozoite stage (>20% parasitaemia at 1% hct). To promote liberation of VOCs from the parasite-media matrix to the headspace, sample containers were incubated in a shaking incubator at slow rotation (40 rpm) for 24 hr Analysis of VOCs This part of the present study employed a number of extraction approaches to capture and analyse VOCs from the culture samples. Headspace VOCs were extracted by SPME (PDMS and combined Carboxen/DVB/PDMS phases) and purge and trap coupled with thermal desorption. Direct immersion of the SPME fibre, and traditional extractions with organic solvents with various polarities were used to extract VOCs trapped in the culture supernatant and cell lysate matrix. Overall, mass spectra of over 100 different compounds were detected in the headspace, supernatant and cell lysates of both non-parasitised control and P. falciparum cultures (Appendix E). Most masses represent hydrocarbons such as alkanes and alkenes, alcohols, benzene-derivatives and organic acids. Table 7.1 presents a selection of commonly observed compounds in the samples. Although minor differences in compound quantities were detected on occasions, no unique biomarker for P. falciparum was identified. Similarities between VOCs liberated from non-parasitised control and infected samples are demonstrated in the chromatograms from various extraction methods (Figure 7.5). 187

222 Chapter 7 Volatile Organic Compounds Table 7.1 VOCs detected in culture headspace. Compound Intensity Relative abundance (%) 1-Hexanol, 2-ethyl-* 23,423, Heptane, 2,2,4,6,6-pentamethyl- 2,665, Benzene, 1,3-bis(1,1-dimethylethyl)- 2,532, Decane 1,794, Dodecanol 3,7,11-trimethyl- 1,005, Cyclohexanone 893, Octen-3-ol 649, Octane, 3,3-dimethyl- 634, Decane, 3,7-dimethyl- 334, Decane, 5,6-dipropyl- 298, ,4-Dimethyl-1-heptene 290, ,2,4,4-Tetramethyloctane 273, Decane, 3,7-dimethyl- 271, Undecane 218, Nonane, 4-methyl- 213, *Suspected organic contaminant. 188

223 Chapter 7 Volatile Organic Compounds Figure 7.5 Chromatograms of VOCs in the headspace of cultured P. falciparum. GC-MS total ion chromatograms; black line: VOCs detected in non-parasitised control; pink line: VOCs detected in culture-adapted P. falciparum. Data collected from (a) solid phase micro-extraction (SPME); (b) SPME-triple fibre; (c) purge and trap thermal desorption; (d) solvent (dichloromethane) extraction of culture supernatant. 189

224 Chapter 7 Volatile Organic Compounds 7.4 DISCUSSION Despite substantial attempts following a step-wise approach, the present in vitro study revealed no specific patterns of VOCs released by P. falciparum cultures. Various forms of solvent extractions (hexane, dichloromethane, ethylacetate) of supernatants and cell lysates showed minimal differences compared to control non-parasitised cultures. When used with a pre-concentration device such as SPME, GC-MS has sufficient sensitivity in the low ppt range (Chambers et al. 2009; Chambers et al. 2010). Despite this sensitivity, data from SPME using conventional (PDMS) and triple fibre (PDMS+divinylbenzene+carbowax) revealed production of a variety of VOCs in cultured P. falciparum but no unique compounds were identified. Thermal desorption of purge and trap samples also showed no significant differences with a similar VOCs profile to that observed with SPME, suggesting that both techniques were detecting the majority of released VOCs. Previous studies of VOCs generated by other infectious agents have shown positive associations between VOCs liberated in vitro and those detected in the breath from patients. This in vitro vs in vivo relationship is exemplified by studies of respiratory infections with Aspergillus fumigatus (Syhre et al. 2008; Preti et al. 2009; Chambers et al. 2010) and pulmonary tuberculosis (Phillips et al. 2007). There were, however, a number of important differences between these studies and the present series of experiments. Firstly, bacterial and fungal colonies are cultured on solid medium. Any VOCs produced by bacteria and fungi are released directly into the culture headspace. By contrast, P. falciparum are enveloped by two extra layers, being within RBC that settle at the bottom of liquid medium in the culture plate. The possibility of loss of VOCs of malarial origin in cell membranes and the culture medium prompted repeated extractions of supernatant and cell lysate using various organic solvents. No obvious differences between malaria and control cultures were found in these experiments. Secondly, the biomass of bacteria and fungi in vitro assessed from colony-forming units (cfu), viable bacterial or fungal cells per visible colony is much greater than that of P. falciparum in a typical culture. The number cfu per visible colony is substantially higher than the number of malaria parasites in a comparable volume of RBC. For 190

225 Chapter 7 Volatile Organic Compounds Mycobacterium tuberculosis culture, an inoculum of 0.5 ml of a 1.0 McFarland standard contains 1.5 x 10 8 cfu) (Phillips et al. 2007) compared to a 50 ml P. falciparum culture of 20% parasitaemia at 1% hct which contains approximately 1.1 x 10 7 parasitised cells. In addition, the greater biomass for bacteria housed within a smaller culture vessel (1 ml headspace) considerably increases the concentration and thus likelihood of VOCs detection compared with the larger headspace (500 ml) of an intra-erythrocytic parasite culture. The analysis of VOCs released from in vitro malaria cultures presented a substantial challenge because of the fastidious nature of P. falciparum in culture including microaerophilic requirements, daily medium changes and the need for a closed-system for headspace capture. Although the use of glass culture-capture apparatus minimised the presence of contaminants related to the use of plastics, a number of external VOCs were present. Compounds such as diethy-phthalate and ethylhexanol (Appendix E) may have been derived from bis-2-ethyl-hexyl-phthalate, a common additive to plastics which renders the plastic more flexible. Siloxane derivatives were also prevalent such as decamethyl-cyclopentasiloxane and dimethyl-silanediol, and were most likely derived from the PDMS coating of the SPME fibre or the GC column stationary phase. A number of different gas mixtures have been used to support malaria culture. Atmospheres with combinations of 0.5 to 21% O 2 mixed with 1 to 7% CO 2 diluted in nitrogen and microbial gas sachets have been employed (Trager et al. 1976; Mirovsky 1989; Taylor-Robinson 1998; Onda et al. 1999; Radfar et al. 2009). High oxygen concentrations are considered to cause deleterious effects on parasites and reduce yields (Scheibel et al. 1979), however this has been debated (Radfar et al. 2009). A mixture of 5% O 2 with 5% CO 2 in nitrogen supports malaria growth better than 5% CO 2 with 95% air (Cohen et al. 1969), although both mixtures have been successful (Miyagami et al. 1985; Mirovsky 1989; Flores et al. 1997; Onda et al. 1999; Radfar et al. 2009). Therefore, although gas composition is an important consideration, it should not be singled out as the determining factor for successful cultures, particularly for high parasitaemias. In addition to the use of premixed gas, the transition from 5% hct for routine P. falciparum culture to a lower hct (1%) proved an important modification to 191

226 Chapter 7 Volatile Organic Compounds support high parasitaemia for the VOCs experiments. An appropriate ratio of medium to cell pellet volume prevents parasite toxicity and helps maintain viability (Radfar et al. 2009). As SPME is based on attaining equilibrium in the sample or headspace, the GC-MS chromatographs may not directly correspond to the actual composition of the detected compounds. Quantitative information would require inclusion of a calibration curve for each component (Gorecki et al. 1997). The aim of the study was, however, to investigate the qualitative capacity of SPME to extract and simultaneously concentrate VOCs released at both low and high quantities thus enabling comparisons of GC-MS profiles of different samples under similar experimental conditions. There were slight inter-assay differences in relative abundance and retention time of VOCs between controls and P. falciparum samples. The difference in relative abundance probably reflects differences in metabolic requirements of batches of RBC and intra-erythrocytic parasites. The minor differences in retention time were probably due to use of the instrument for unrelated interspersed experiments with resulting shifts in calibration. However, these differences were minor, inconsistent and did not underlie unique signals or chemical patterns. Although VOCs may be more readily detected in vivo in respiratory diseases (Gordon et al. 1985; Chambers et al. 2009; Preti et al. 2009; Chapman et al. 2010), VOCs generated as part of host response to a systemic disease may also serve as biomarkers. Examples include increased production of pentane and carbon disulfide in the breath of patients with schizophrenia (Phillips et al. 1993). Nevertheless, their specificity remains questionable, as breath carbon disulfide has been detected in both smokers and nonsmokers (Phillips 1992a) and has been linked with myocardial infarction (Weitz et al. 1991). An assessment of a characteristic VOCs fingerprint in the context of malaria (including severe and non-severe cases) was beyond the scope of the present in vitro experiments. The present study used optimised experimental conditions to enable the capture, extraction and analysis of VOCs liberated from P. falciparum cultures. Even at high 192

227 Chapter 7 Volatile Organic Compounds parasitaemia, VOCs unique to P. falciparum cultures were not detected using solvent extraction, purge and trap-thermal desorption, or by SPME. GC-MS data revealed a variety of VOCs but no unique malarial finger-prints. Future in vivo studies analysing the breath of patients with severe malaria may yet reveal specific clinically-useful volatile biomarkers. The in vivo parasite biomass is substantially greater than achievable in vitro and there may be VOCs generated as a specific in vivo response to the disease. 193

228 Chapter 7 Volatile Organic Compounds 194

229 CHAPTER 8 CONCLUDING DISCUSSION

230 Chapter 8 Concluding Discussion CHAPTER 8. CONCLUDING DISCUSSION 8.1 OVERVIEW With new national treatment regimens being introduced in PNG, this thesis presents a timely assessment of the in vitro and genetic aspects underlying treatment efficacy in PNG children with falciparum malaria. Through the implementation of two highthroughput resistance surveillance techniques (pldh and LDR-FMA), valuable baseline data were obtained prior to the adoption of artemether-lumefantrine (AL) as first-line treatment in PNG In addition, studies in this thesis explore novel antimalarial agents that could become useful partners with artemisinin derivatives for the treatment of uncomplicated falciparum malaria. The detection of P. falciparumspecific volatile organic compounds (VOCs) in breath/other samples as a way of enhancing diagnosis and therapeutic monitoring was also explored but this technique does not appear to be a viable alternative to conventional methodologies for parasite detection and enumeration Major Findings and Contributions i. Most of the PNG isolates of P. falciparum tested (n=64) were resistant to chloroquine (CQ) but not to other ACT partner drugs (lumefantrine (LM), piperaquine (PQ), mefloquine (MQ) and naphthoquine (NQ)). ii. In PNG P. falciparum isolates, strong associations were observed between their in vitro responses to 4-aminoquinolines (CQ, amodiaquine (AQ) and NQ), bisquinoline (PQ) and aryl-aminoalcohol (MQ) compounds suggesting crossresistance, but LM IC 50 s only correlated with that of MQ. iii. There was fixation of pfcrt K76T, pfdhfr C59R and S108N, and pfmdr1 mutations in PNG isolates of P. falciparum (n=402). Multiple mutations were frequent with 88% of isolates possessing quintuple mutations. 196

231 Chapter 8 Concluding Discussion iv. The pfmdr1 D1246Y mutation was associated with PCR-corrected day 42 in vivo treatment failure in children allocated dihydroartemisinin (DHA) plus PQ (P=0.004). v. A non-isotopic semi-automated, high-throughput drug assay (using pldh) was implemented for the first time in PNG. The assay can be adapted to a basic laboratory setting and facilitates serial assessment of local parasite sensitivity so that emerging resistance can be identified. vi. A novel extension of a multiplex, high-throughput ligase detection reactionfluorescence microsphere assay (LDR-FMA) was developed and implemented for the screening of pfmdr1 mutations in PNG P. falciparum isolates. vii. Desbutyl-lumefantrine (DBL) was found to be several folds more potent than its parent compound against reference strains of P. falciparum and was mildly synergistic with DHA. viii. Mean plasma DBL concentrations were lower in children who failed AL treatment than in those with an adequate clinical and parasitological response (ACPR; P=0.053 vs P>0.22 for plasma LM and plasma LM:DBL ratio). ix. Atorvastatin was more active than rosuvastatin, pravastatin and simvastatin against culture-adapted P. falciparum, although it had weak activity (mean IC µm) and an indifferent interaction with CQ and DHA. x. Fenofibric acid was the most potent lipid-modifying drug against CQ-sensitive and resistant P. falciparum (mean IC 50 s 152 and 1120 nm, respectively). As confirmed by the bioassay, the plasma fenofibric acid level available with therapeutic dosing was 100 times that required to inhibit growth of P. falciparum by 50%. xi. A culture-capture apparatus for P. falciparum headspace VOCs was developed. 197

232 Chapter 8 Concluding Discussion Even at relatively high parasitaemia (>20%), in vitro parasite VOCs production was undetectable, possibly due to loss of VOCs in the components of the experimental system including erythrocytic forms and/or insufficient parasite biomass. The following sections highlight the major findings of the studies in this thesis and discuss their relevance to the understanding of parasite resistance and their potential applications. The first section examines the in vitro and genetic aspects of resistance in PNG isolates of P. falciparum and considers their contribution to the prediction of treatment efficacy. Limitations of these studies are also discussed. The second section examines three different classes of agents as novel antimalarial treatments. The screening of headspace biomarkers of P. falciparum cultures and insights into possible underlying mechanisms of cerebral malaria and potential drug targets are also discussed. Lastly, this chapter outlines directions for future research. 8.2 THE ROLE OF IN VITRO RESISTANCE AND PARASITE GENETIC MUTATIONS IN TREATMENT OUTCOME The results presented in this thesis provided timely baseline data on in vitro (Chapter 3) and genetic (Chapter 4) aspects on drug resistance of P. falciparum isolated from Madang children, prior to the change of national treatment policy from CQ-sulfadoxine pyrimethamine (SP) to AL in In vitro drug sensitivity profiles of 64 isolates to nine conventional (CQ, AQ, daq, DHA) and relatively novel (NQ, PQ, MQ, LM, AZ) antimalarials were obtained. As expected from a history of heavy 4-aminoquinoline usage, the majority (82%) of isolates were resistant to CQ in concordance with previous PNG in vitro studies (Hombhanje 1998b; Mita et al. 2006a). The high level of in vitro resistance was also consistent with near-fixation of the CQ resistance-associated pfcrt allele as revealed by subsequent genotyping of parasite DNA (Chapter 4). Strong associations were noted between the IC 50 s of 4-aminoquinoline (CQ, AQ, DAQ and NQ), bisquinoline (PQ), and aryl aminoalcohol (MQ) compounds suggesting crossresistance, as observed in other countries (Fan et al. 1998; Basco et al. 2003a; Pradines et al. 2006). These findings are consistent with the observation that pfcrt and pfmdr1 198

233 Chapter 8 Concluding Discussion alleles influence parasite susceptibility to drugs other than CQ such as MQ (Sidhu et al. 2002; Johnson et al. 2004; Mita et al. 2006a). LM and NQ have recently become available in PNG as part of ACTs. In vitro sensitivities of PNG field isolates to both of these drugs are described for the first time and could be used as a baseline for future monitoring of resistance to these agents. Since microscopic assessment of parasite response to drug exposure is time-consuming and laborious, a validated high-throughput enzyme method (pldh) was modified and implemented in the field laboratory setting (Chapter 3). This approach allowed for testing of multiple antimalarial drugs in triplicate with relative ease. The non-isotopic semi-automated nature of the pldh assay facilitated convenient serial assessment of local parasite sensitivity so that emerging resistance could be identified with relative confidence at an early stage. Although the pldh assay was readily adaptable in this setting, its relative high cost (AUD $30/plate, assaying four drugs in triplicate) compared to microscopy limits its use for routine screening. Alternative non-isotopic growth detection methods such as Sybr Green staining for DNA, although non-specific to Plasmodium, are much more affordable and would be more likely to be employed for future routine sensitivity testing in PNG (Karl et al. 2009). Surveillance for parasite drug resistance mutations is becoming an established tool in predicting treatment effectiveness, especially as it overcomes the challenges and costs associated with in vivo testing. To investigate underlying molecular mechanisms, a post-pcr, multiplexed ligase detection reaction-fluorescent microsphere assay (LDR- FMA) was used to detect relevant single nucleotide polymorphisms (SNPs) in P. falciparum drug resistance genes (Chapter 4). This technique enables simultaneous identification of 18 SNPs in pfcrt, pfdhfr and pfdhps (Carnevale et al. 2007), and was extended for the purposes of the present studies to screen for 10 allelic variants in pfmdr1. The method is cost-effective (the analysis of 28 SNPs per sample cost AUD $4.14), has a high output format suitable for large-scale epidemiological studies and extends current PCR-based methods. Molecular findings from the studies in this thesis are consistent with previous heavy use 199

234 Chapter 8 Concluding Discussion of 4-aminoquinolines and SP in PNG (Chapter 4). Field isolates collected from children with falciparum malaria showed a fixation of pfcrt 76T, pfdhfr 59R and 108N and pfmdr1 mutations (>90%), consistent with previous PNG studies (Carnevale et al. 2007; Mita et al. 2006a; Mita et al. 2006b; Schoepflin et al. 2008). A high prevalence of multiple mutations across these genes was noted, with majority of the isolates (88%) harbouring a quintuple mutation SVMNT+NRNI+KAA+YYSND in codons for pfcrt, 51, 59, 108, 164 for pfdhfr, 540, 581, 613 for pfdhps, and 86, 184, 1034, 1042, 1246 for pfmdr1. In determining underlying molecular factors that may influence in vivo outcomes, the presence of the pfmdr1 1246Y mutation was associated with treatment failure in all treatment groups combined (i.e. CQ-SP, artesunate-sp, PQ-DHA and AL) and in the PQ-DHA group in particular. However, the association between pfmdr1 1246Y and treatment failure is likely to be location specific. Parasite mutations are largely selected by drug pressure exerted over a period of time with genetic haplotypes reflecting parasite lineage and type of treatment employed in the region (Wongsrichanalai et al. 2002; Mita et al. 2007; Plowe 2009). Although the present findings relating to pfmdr1 1246Y may not be generalisable to other countries even within Oceania, they may inform future surveillance strategies in epidemiologically similar areas. This situation also pertains to PNG. A Peruvian study has shown that the P. falciparum SNPs pfdhfr 164L and pfdhps 540E predict SP treatment failure (Bacon et al. 2009). Despite the previous widespread use of SP, these two mutations are rare in PNG in the present and other studies (Carnevale et al. 2007; Mita et al. 2007) but the Peruvian experience would support active surveillance for these parasite mutations if SP remains part of nationally recommended treatment regimens. Although the pfmdr1 haplotype NFSDD was found in only four isolates, it has been associated with AL treatment failure in Africa (Happi et al. 2008). Therefore, monitoring changes in the pfmdr1 gene should be of high priority due to the recent introduction of this treatment in PNG Limitations of PNG field studies The assessment of the relationship between parasite in vitro drug sensitivity and clinical outcome was limited by a restricted number of isolates available for testing. Overall, 200

235 Chapter 8 Concluding Discussion there were 40 isolates with in vitro drug sensitivity profiles from children allocated randomly to one of the four treatment arms in the large comparative trial (Karunajeewa et al., 2008). Sixteen were from children treated with DHA-PQ and eight were from subjects who received each of CQ-SP, artesunate-sp and AL. Unfortunately, the in vitro component of the trial started when most children had been recruited while a number of isolates that were successfully tested in vitro were from children who were excluded post-hoc from the trial because of protocol violations. There were only three cases of treatment failure in this in vitro-in vivo dataset. Of these, two were late parasitological failures (PCR-confirmed recrudescences on day 28 and or day 42) in the CQ-SP group, and one in the DHA-PQ group. The CQ IC 50 s of isolates from the CQ-SP treatment failures were 197 and 126 nm, within the range obtained for successfully-treated children in the same group and for all isolates tested (Table 3.2). Therefore, in vitro CQ sensitivity alone is unlikely to be a useful predictor of treatment outcome in the PNG setting, especially when in vitro CQ resistance is at fixation. For the single DHA-PQ treatment failure, the PQ IC 50 was 19.5 nm. Subsequent molecular analysis revealed that all three parasite isolates possessed the pfcrt K76T allele and the pfdhfr N51 R59 N108 I164 double mutation. Albeit in small numbers in this subset, the presence of pfcrt and pfdhfr (markers for 4-aminoquinoline and pyrimethamine resistance) in association with treatment failure is consistent with previous findings (Cowman et al. 1988; Peterson et al. 1991; Casey et al. 2004). Although the measurement of IC 50 s provides an acceptable indicator of drug sensitivity trends in parasite populations, an isolate showing in vitro resistance may not equate to treatment failure and vice versa (Wongsrichanalai et al. 2002; Ekland et al. 2008). This is mainly attributed to host immunity and other in vivo factors. Pharmacokinetic profiles are required to confirm that an adequate drug concentration has been achieved to substantiate true resistance and treatment failure. Although these data are available through the main clinical trial, the very low number of children with in vitro drug sensitivity data that failed treatment limited their application. Despite these limitations, in vitro drug susceptibility testing remains an important part 201

236 Chapter 8 Concluding Discussion of the monitoring of resistance in the ACT era. The emergence of parasite resistance to individual drugs employed in ACTs may not be clinically apparent due to the effectiveness of the partner drug (Laufer et al. 2007). Although candidate molecular markers for artemisinin resistance have been proposed (Price et al. 1999; Jambou et al. 2005; Uhlemann 2005), these cannot be validated unless true clinical resistance to artemisinin drugs occurs. Therefore, in vitro drug assays are at the front-line of surveillance for resistance to artemisinin derivatives and ACTs. 8.3 UNCONVENTIONAL AND NOVEL ANTIMALARIAL AGENTS There is a pressing need for new drugs and drug combinations that facilitate prompt resolution of the symptoms of malarial infection, improve treatment success rates, and limit the development of parasite resistance. To address this need, the studies in this thesis have been directed towards identifying a number of novel antimalarial drugs. These include DBL, a metabolite of an ACT partner-drug, and two classes of lipidmodifying drugs, statins (i.e. atorvastatin, rosuvastatin, pravstatin, simvastatin) and fibrates (i.e. fenofibrate, fenofibric acid, gemfibrozil and clofibrate). Their antimalarial activities and possible therapeutic implementation are discussed in the following section Desbutyl-lumefantrine and its Potential Implementation With the recent change to AL as first-line treatment for uncomplicated malaria in PNG, it seems relevant to investigate the antimalarial activity of related drugs (Chapter 5). Despite speculation that DBL is not a metabolite of LM (Starzengruber et al. 2007; Starzengruber et al. 2008) and consistent with other reports (Ntale et al. 2008; Hodel et al. 2009), data from this study confirmed that DBL is indeed a metabolite, being present in significant concentrations in the plasma of children after AL treatment. The superior antimalarial activity of DBL (mean IC 50 9 nm) to LM (mean IC nm) reported in this thesis (Table 5.2) is consistent with previous reports using field isolates (Noedl et al. 2001; Starzengruber et al. 2008). In addition to its potent activity against P. falciparum, DBL also effectively inhibits P. vivax (Pirker-Krassnig et al. 2004). This 202

237 Chapter 8 Concluding Discussion broad-spectrum activity makes DBL a useful candidate for implementation in the field, as co-endemicity of these two major Plasmodium species is common in Asia, the Middle East and Oceania (Mehlotra et al. 2000; Dhangadamajhi et al.; Douglas et al. 2011; Wong et al. 2011). The difference in antimalarial activity between the parent and metabolite is also consistent with the relationship between plasma LM, plasma DBL and treatment outcome (Figure 5.3). AL treatment failure cases had significantly lower plasma DBL concentrations than ACPR cases. Although plasma LM concentrations were also lower in treatment failure, this did not reach statistical significance. This suggests that DBL has a stronger role than the parent compound in suppressing recrudescence/and or reinfection. The in vitro synergy between DBL and DHA (arguably the most potent antimalarial drug discovered to date) highlights DBL-DHA as a promising novel ACT. With antimalarial activities greater than their respective parent compounds and pending detailed pharmacokinetic characterisation, the DBL-DHA formulation may be able to be given in a simpler dosing regimen than the recommended 6-doses of AL. African and PNG studies show that the greatest cure rates after AL are when dosing is directly supervised with fat supplementation given to improve drug absorption (Mutabingwa et al. 2005; Piola et al. 2005; Karunajeewa et al. 2008b; Schoepflin et al. 2010). Poor compliance with the number of doses and coadministered fat may lead to sub-curative drug concentrations and treatment failure, with the likely development of parasite drug resistance (Wongsrichanalai et al. 2002; White et al. 2009). The lipophilicity of DBL means that, as with LM (Ezzet et al. 1998), its bioavailability is likely to be enhanced by concomitant food intake, particularly fat. The fat content of PNG meals tends to be relatively low, as local diets are typically based on a carbohydrate staple and vegetables (Iser et al. 1993). Important sources of dietary fat include oil crops and cooking oil (e.g. peanuts, mature coconuts, palm oil). Given the abundancy and availability of fresh tuna in coastal PNG where malaria is holoendemic, consumption of this oily fish should be encouraged as a dietary supplement with AL dosing to maximise absorption and decrease the chance of recrudescence. Breast- 203

238 Chapter 8 Concluding Discussion feeding mothers can use breast milk as a fat source if their young children are being treated with AL. The education of patients and healthcare providers as to the importance of taking therapy according to the prescribed regimen should increase understanding and compliance. These considerations would extend to potential ACTs involving DBL Lipid-modifying Agents as Antimalarials Drugs licensed for other indications can sometimes have antimalarial properties, an example being lipid-modifying therapy which is becoming affordable even in malariaendemic developing countries. Modernisation and lifestyle changes have seen an increasing need for these agents to reduce cardiovascular diseases, (McMurry et al. 1991; Hodge et al. 1996; Gill 2001). Two classes of commonly used lipid-modifying drugs, namely fibrates and statins, were tested against P. falciparum (Chapter 6). The present in vitro drug sensitivity data have confirmed atorvastatin to have the highest activity of available statins against P. falciparum regardless of strain CQ sensitivity (Pradines et al. 2007), but at an IC 50 well above plasma concentrations after therapeutic doses in vivo. Although its mechanism of action against P. falciparum remains unclear, atorvastatin may act via P-glycoprotein (Holtzman et al. 2006), an efflux protein implicated in 4-aminoquinoline resistance. Fibrates have different lipid-modifying and possibly antimalarial mechanisms of action to those of statins. Gemfibrozil and clofibrate proved to have weak antimalarial activity, whilst fenofibrate in the form of fenofibric acid had a relatively low in vitro IC 50, similar to those of conventional antimalarial drugs (Table 6.3). As evident in the bioassay (Figure 6.4), plasma fenofibric acid levels generated in vivo after therapeutic doses of Lipidil inhibited cultured P. falciparum (Figure 6.4). Although therapeutic plasma levels of fenofibric acid after both single and repeated doses are well above the concentrations required to produce 90% growth inhibition in CQ-resistant P. falciparum (10-fold and 3-fold, respectively), it should not be used as antimalarial monotherapy but in partnership with a more rapidly-acting antimalarial agent. The elimination half-life of fenofibric acid is 20 hr (Bhavesh et al. 2009) which means it is cleared more promptly 204

239 Chapter 8 Concluding Discussion than most 4-aminoquinolines (Krishna et al. 1996; Tarning et al. 2005; Qu et al. 2010). Depending on the completeness of the initial parasiticidal effect, this may be advantageous as an ACT partner drug with a shorter elimination half-life reduces the time of parasite exposure to subcurative levels thus limiting subsequent selection of resistant strains (Wongsrichanalai et al. 2002). Fenofibric acid has indifferent in vitro interactions with DHA (Table 6.4) when assessed using two mathematical analyses (Berenbaum 1978; Brueckner et al. 1991). Visual assessment of isobolographic plots suggests, however, that the equations utilised by these methods may not accurately characterise drug interactions with the result that clinically important interactions may be missed. This visual-mathematical discordance is further discussed in Appendix D. Although its mode of action remains to be elucidated, fenofibric acid may act by interfering with P-glycoprotein (Ehrhardt et al. 2004) and ABC-1 mediated transport, and/or via a putative PPARα-like protein. P-glycoprotein prevents intracellular drug accumulation which results in a multidrug resistance phenotype in both Plasmodium and mammalian cells. Evidence from in vitro drug-uptake assays and confocal laser scanning microscopy have confirmed fenofibrate as an inhibitor of P-glycoprotein activity with a similar potency to verapamil at 7.1 and 4.7 µm, respectively (Ehrhardt et al. 2004). Therefore, fenofibrate may reverse CQ resistance and produce synergistic effects when combined with CQ. However, the present drug-interactions studies revealed indifferent interactions in both CQ-sensitive and resistant P. falciparum. Despite the superior antimalarial activity exhibited by fenofibric acid, biochemical data show that it is neither a substrate nor an inhibitor of human P-glycoprotein (Ehrhardt et al. 2004). The lack of synergistic interaction or potentiation of CQ by fenofibric acid in this study supports previous observations (Ehrhardt et al. 2004). Fenofibric acid affects the expression of ABC-1 lipid transport protein in mammalian cells (Jaye et al. 2003; Arakawa et al. 2005) and may similarly affect the Plasmodium ABC-1 homolog. This could inhibit the development of P. falciparum by depriving the growing parasite of lipid components of membranes and other cellular structures. The relative importance of the effects of fenofibric acid on the Plasmodium homolog of P-glycoprotein, ABC-1 205

240 Chapter 8 Concluding Discussion and a putative PPARα-like protein, together with possible interactions between these effects, cannot be ascertained from the present data. 8.4 A PILOT STUDY OF MALARIA VOCS The use of a non-invasive breath test for the detection of falciparum malaria would provide a unique tool for diagnosis and therapeutic monitoring that could be conveniently carried out at the bed-side. This thesis describes, for the first time, a study of the potential of P. falciparum cultures to release signature VOCs. In addition to possibly permitting detection of viable parasites, these in vitro data could provide insights into the metabolism and pathogenicity of the organism. VOCs are used as general anaesthetics in clinical practice (Soukup et al. 2009) and it is possible that coma complicating malaria may result from elaboration of VOCs by malaria parasites in the cerebral microcirculation that have anaesthetic properties. Challenges encountered in this proof of concept study began with the determination of a microenvironment that optimally supports a high parasite yield within a system that also permits the capture, extraction and analysis of headspace VOCs (Chapter 7). Some important considerations for the design of the culture-capture apparatus included i) the width and height of the culture flask, ii) the provision of sealable-access openings and fittings adapted for VOCs extraction, iii) the parasite stage used and duration of incubation, iv) the culture gas mixture and purging time, and v) initial parasitaemia and haematocrit. Once optimised, VOCs were collected and concentrated by SPME, solvent extraction and thermal desorption. Various steps were also taken to enhance the liberation of VOCs from the liquid matrix and to attain gas-fibre equilibrium. Despite substantial efforts to define the optimal conditions for the in vitro capture of P. falciparum VOCs, GC-MS data revealed the production of a variety of volatile compounds but no unique malarial finger-prints (Chapter 7). A major limitation is the P. falciparum biomass achievable within the in vitro system. Unlike bacterial or fungal colonies that are directly grown on solid medium with direct exposure to the culture headspace, malaria parasites have little access to the headspace, being enveloped within phosphobilayered-host cells within a liquid medium. The use of 206

241 Chapter 8 Concluding Discussion a shaking incubator and the assessment of supernatant and cell lysate for VOCs revealed minimal difference compared to control. Even with the attainment of a 20% parasitaemia, the difference in VOCs is dependent on a minority of infected cells versus the non-infected control cells. A recent publication described means to obtain a synchronous culture of P. falciparum at 60% parasitaemia (Radfar et al. 2009). However, the methodological requirements for achieving such high parasite yields are not compatible with VOCs capture. This includes the need for medium changes at 12 hr intervals, which would result in the escape of any VOCs produced. The use of largebase culture containers and a low haematocrit are common key considerations noted in this and other studies (Radfar et al. 2009). Progress with culture techniques of other protozoans may yet promote novel approaches to P. falciparum cultivation (Hijjawi et al. 2004; Hijjawi et al. 2010). Recent studies have reported on the successful propagation of Cryptosporidium hominis, an intracellular Apicomplexan that shares similar life-cycle stages with P. falciparum, in host cell-free culture (Hijjawi et al. 2010). Such advancements, if possible for Plasmodium, would bring a new dimension to malaria research. 8.5 CONCLUSION AND FUTURE DIRECTIONS Despite past efforts to control malaria, resistance of P. falciparum to antimalarial drugs continues to be a threat to the global community, especially in tropical developing countries such as PNG. The response has been to change from monotherapy to combination therapy and, more recently, to the wide deployment of ACTs to ensure efficacy and retard the spread of parasite resistance (PNGDOH 2000; WHO 2007). However, P. falciparum drug resistance reflects a complex interplay of parasite and host factors (Wongsrichanalai et al. 2002; Mackinnon et al. 2010). The development of novel antimalarials has been slow, despite a surge in research funding in recent years (McCoy et al. 2009; Kazatchkine 2010; Muller et al. 2010). There remains an urgent need for novel, effective and affordable antimalarial agents that can be used with artemisinin derivatives to counter the rapid development of parasite resistance to conventional drugs. 207

242 Chapter 8 Concluding Discussion This thesis has contributed to the understanding of the in vitro and genetic aspects underlying treatment outcome in PNG children. This was achieved via the culture and testing of parasite sensitivity to antimalarial agents, molecular detection of parasite mutations and their associations with treatment outcomes. The antimalarial efficacy findings presented here may become useful for inclusion in the World Antimalarial Resistance Network (WARN), a global database showcasing collaborative efforts in the malaria research community to monitor and counter the development of resistance (Plowe et al. 2007; Sibley et al. 2008; Sibley et al. 2010). Both molecular and in vitro drug susceptibility profile from this study provided useful baseline data for monitoring and evaluation of treatment regimens in coastal PNG and supports the need for adoption of AL as first-line treatment for uncomplicated falciparum malaria Directions for Future Research i. The present studies provide a platform for the continuation of parasite resistance surveillance through the routine assessment of in vitro drug sensitivity and molecular markers. The pldh and LDR-FMA methods established from this work enable the high-throughput monitoring of changes in parasite drug sensitivity (particularly in pfmdr1 polymorphisms) under a new wave of ACT selection. Ongoing assessment using the same techniques will facilitate assessment of the adequacy of such treatment. ii. The high in vitro antimalarial activity of fenofibric acid at media concentrations which can be achieved in vivo may have therapeutic application. Confirmatory preclinical studies in an animal model (perhaps the well recognised Plasmodium berghei murine model) would be useful as the next step in assessing the in vivo efficacy of fenofibric acid. iii. The present studies have highlighted a number of technical challenges with in vitro identification of VOCs from the headspace of P. falciparum cultures. Future in vivo studies analysing the breath of patients with severe malaria may yet reveal specific, clinically useful volatile biomarkers. 208

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293 APPENDICES

294

295 Appendix A: Isolate Information 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 7G8 Brazil CQ-resistant Pyrimethamine-resistant Dd2 Indochina CQ-resistant Pyrimethamine-resistant MQ-resistant E8B Australia CQ-resistant MQ-sensitive Derived from IMTM22 (Brazil) Derived from W2mef Derived from ItG2F6 (Brazil) 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 261

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297 Appendix B: Recipes and Solutions APPENDIX B. RECIPES FOR SOLUTIONS Culture of P. falciparum 5% Albumax II Albumax II ( ) (Gibco) Milli-Q water 5.5 g 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 ( ) (Gibco, Auckland, NZ) HEPES (Sigma-Aldrich) Hypoxanthine (Sigma) 5% Albumax II (see section below) 5% NaHCO 3 (see section below) Gentamycin sulfate (10mg/mL) (Calbiochem, Darmstadt, Germany) Neomycin sulfate (10mg/mL) (Calbiochem, Darmstadt, Germany) Milli-Q-water g 4.47 g 22.5 mg 50 ml 21 ml 5 ml 5 ml 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. 263

298 Appendix B: Recipes and Solutions Complete Maintenance Medium RPMI Medium 1640 (R5886) (Gibco, liquid) Human plasma (see section below) L-glutamine solution (see section below) Hypoxanthine solution (see section below) 90 ml 10 ml 1 ml 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) NaCl (BDH, Australia) Glycerol (BDH, Australia) Milli-Q-water 37.8 g 8.1 g 350 ml 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) Milli-Q-water 1 g 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. 264

299 Appendix B: Recipes and Solutions 5% Giemsa Stain Giemsa stain (BDH, Australia) PBS (ph 7.2) 0.5 ml 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) Na 2 HPO 4 (BDH, Australia) NaH 2 PO 4 (BDH, Australia) Milli-Q-water 7 g 2.85 g g 1000 ml Adjust to ph 7.3. Filter sterilise using 0.2 µm membrane (Nalgene Filterware, USA). Store at 4 C. 265

300 Appendix B: Recipes and Solutions Hypoxanthine Solution (5 mg/ml) Hypoxanthine (Sigma, USA) Milli-Q-water 0.5 g 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) Milli-Q-water 2.92 g 100 ml Filter sterilise using 0.2 µm membrane (Nalgene Filterware, USA). Store at -20 C in 5 ml aliquots. 5% NaHCO 3 NaHCO 3 (Sigma-Aldrich, St Louis, Mo, USA) Milli-Q-water 2.5 g 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 K 2 PO 4 (BDH, Australia) Na 2 HPO 4 (BDH, Australia) Milli-Q-water 0.41 g 0.65 g 1000 ml Adjust to ph 7.1. Filter sterilise using 0.2 µm membrane (Nalgene Filterware, USA) (optional). 266

301 Appendix B: Recipes and Solutions 12% Sodium Chloride (NaCl) Solution NaCl (BDH, Australia) HTPBS (see section above) 11.3 g 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) HTPBS (see section above) 0.9 g 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) Glucose (Sigma) HTPBS (see section above) 0.2 g 0.2 g 100 ml Filter sterilise using 0.2 µm membrane (Nalgene Filterware, USA). Store at 4 C. 5% Sorbitol for Synchronisation Sorbitol (BDH, England) Milli-Q-water 5 g 100 ml (final) Filter sterilise using 0.2 µm membrane (Nalgene Filterware, USA). Store at 4 C. 267

302 Appendix B: Recipes and Solutions LDH Assay Malstat Solution Trizma base (Sigma, Australia) Milli-Q-water 2.42 g 180 ml Adjust with HCl to ph 9.1 Triton X-100 (Sigma, Australia) Lithium-L-lactate (Sigma, Australia) APAD (Sigma, Australia) 0.4 ml 4 g 132 mg Protect from light and store at 4 C. Use within one week. Diaphorase Solution Diaphorase (Sigma-Aldrich, USA) Milli-Q-water 15 mg 15 ml Protect from light and store at 4 C. Nitro Blue Tetrazolium (NBT) Solution NBT chloride monohydrate (Sigma-Aldrich, USA) Milli-Q-water 15 mg 15 ml Protect from light and store at 4 C. 268

303 Appendix B: Recipes and Solutions Molecular Assays 2% Agarose Gel Agarose I (Amresco, USA) 1 X TBE buffer 3 g 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) 1 M Tris ph 8.0 (Amresco, USA) 30 ml 2.5 ml 0.5 M EDTA (Amresco, USA) 300 µl 20% Sodium dodecyl sulfate (Amresco, USA) 250 µl Sterile distilled water ml 269

304 Appendix B: Recipes and Solutions 10 x Tris borate (TBE) Buffer Tris base Boric acid 0.5M EDTA Milli-Q-water 108 g 55 g 40 ml 1000 ml (final) 10 x PCR Buffer 1 M Tris ph M MgSO4 1 M (NH4) 2 SO4 β-mercaptoethanol (14.3 M) Nuclease-free water 33.5 ml 3.4 ml 8.4 ml 0.35 ml 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 270

305 Appendix B: Recipes and Solutions 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 LDR primers for pfcrt, pfdhfr, pfdhps (x 18) (Table 2.4) Taq DNA ligase (Biolabs,New England) 0.20 µl/primer 0.10µL PCR product 1 µl PCR Master Mix Plasmodium Species, pfcrt, pfdhfr, pfdhps Nuclease-free water 19.6 µl 10 x PCR buffer 2.5 µl 2.5 mm dntps 2.0 µl Primers upstream (Section 2.3.2) Primers downstream (Section 2.3.2) 0.3 µl/primer 0.3 µl/primer MacTaq (DNA Taq polymerase, M&C Gene Technology, Beijing) 0.3 µl Keep MacTaq on ice at all times. SYBR Gold Cybergold (Molecular Probes, Eugene, OR) 15 µl 1 X TBE Buffer (see section above) 150 ml 271

306 Appendix C: LDR Optimisation APPENDIX C. EFFECTS OF LDR ANNEALING TEMPERATURE AND DILUTION 272

307 273 Appendix C: LDR Optimisation

308 274 Appendix C: LDR Optimisation

309 275 Appendix C: LDR Optimisation