Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

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1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Ashley EA, Dhorda M, Fairhurst RM, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 2014;371: DOI: /NEJMoa

2 This supplement contains the following items: 1. Final protocol with changes highlighted from original protocol (page 2), summary of changes (page 45). 2. Original statistical analysis plan (page 46), final statistical analysis plan (page 54-57), summary of changes (page 58) Protocol Approval Process: 1. January 2011: Version 1.1 submitted to the Oxford Tropical Research Ethics Committee (OXTREC). 2. March 2011: Version 1.2 submitted in response to comments from the Committee. This was the protocol version submitted to national ethics committees/irbs and implemented and approved for use at all sites as the original protocol (given OxTREC reference 06-11). 3. May 2011 first patient recruited 4. September 2011: protocol amendment requested. Version 1.3 (see page 2). Used as final protocol for all sites except Kinshasa, DRC 5. September Request to add new site in Kinshasa, DRC and substitute one artesunate treatment arm by artemether-lumefantrine. This was granted as a Chairman s action without the need to submit a revised protocol. The full protocol was revised with this change and approved by Republique Democratique du Congo, Ministere de l Enseignement Superieur, Universitaire et Recherche Scientifique, Universite de Kinshasa, Ecole de Sante Publique. Comite d Ethique. 1

3 Protocol version Sep 2011 shown below with all the changes from the original protocol highlighted in yellow/strikethrough as applicable. A multicentre, randomised trial to detect in vivo resistance of Plasmodium falciparum to artesunate in patients with uncomplicated malaria. A study by the Tracking Resistance to Artemisinin Collaboration (TRAC) Short title: artesunate resistance study Principal Investigator: Sponsor Professor Nicholas J White University of Oxford Funder UK Department for International Development (DFID) Protocol development team: A Dondorp, R Fairhurst, EA Ashley, W Taylor, P Cheah, N Lindegårdh, J Tarning, S Borrmann, K Chotivanich, M Imwong, K Stepniewska, D Bethell, N Day, NJ White on behalf of TRAC. Confidentiality Statement This document contains confidential information that must not be disclosed to anyone other than the Sponsor, the Investigator Team, host institution, relevant ethics committee and regulatory authorities. 2

4 TABLE OF CONTENTS 1. LIST OF ABBREVIATIONS SYNOPSIS BACKGROUND AND RATIONALE Background Study Rationale Proposed activities OBJECTIVES Primary Objective Secondary Objectives TRIAL DESIGN Study sites Summary of Trial Design Study duration Primary and Secondary Endpoints Primary Endpoint Secondary Endpoints Trial Participants Overall Description of Trial Participants Inclusion Criteria Exclusion Criteria PROCEDURES Informed Consent Screening, Eligibility and Baseline Assessments Demographics and Medical History Physical Examination and Vital Signs Drug history Screening blood & urine tests Randomisation and blinding Blood sampling On admission During hospitalisation Pharmacokinetic Study Sampling Blood volumes Urine sample Study drug regimens Artesunate Artemether-lumefantrine Dihydroartemisinin-piperaquine

5 6.6.4 Artesunate-mefloquine Artesunate-amodiaquine Study drug administration Follow up procedures Time windows Additional visits Patients with recurrent parasitaemia Epidemiological follow-up Rescue treatment Analysis Drug assays Analysis of PK data Ex vivo drug sensitivity assay Molecular studies PCR for quantitative parasitaemia Discontinuation/ Withdrawal of Participants from the Study Source Data STUDY DRUGS Storage of Study Drugs Compliance with Study Drugs Accountability of the Study Treatment Concomitant Medication SAFETY REPORTING Definitions Adverse Event (AE) Serious Adverse Event Reporting Procedures for Serious Adverse Events STATISTICAL CONSIDERATIONS Sample size justification Statistical Analyses Proportional data Continuous data Pharmacokinetic data DIRECT ACCESS TO SOURCE DATA/DOCUMENTS QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES Monitoring DSMB ETHICS Declaration of Helsinki

6 12.2 ICH Guidelines for Good Clinical Practice Approvals Risks Risks of Artesunate Risks of dihydroartemisinin+piperaquine Risks of AS-mefloquine Risks of artemether/lumefantrine Risks of AS-amodiaquine Risk of phlebotomy & finger stick Benefits Benefits of Treatment Alternatives to Study Participation Incentives & Compensation Confidentiality SAMPLE SHARING AND STORAGE DATA HANDLING AND RECORD KEEPING SPONSORSHIP AND INSURANCE PUBLICATION POLICY REFERENCES APPENDIX 1. STUDY SCHEDULES D14 & D42 FOLLOW UP APPENDIX 2. DOSING TABLES Artesunate Dihydroartemisinin piperaquine Artemether lumefantrine Artesunate mefloquine loose regimen Artesunate-mefloquine fixed dose Artesunate-amodiaquine- fixed dose APPENDIX 3. LIST OF STUDY SITES & PRINCIPAL INVESTIGATORS APPENDIX 4: WWARN TERMS OF SUBMISSION

7 1. LIST OF ABBREVIATIONS ACT Artemisinin-based combination therapy AE Adverse event A/L Artemether-lumefantrine AS Artesunate AS-AQ Artesunate-amodiaquine AS-MQ Artesunate-mefloquine AUC Area under the (plasma concentration-time) curve CRF Case record form CTSG Clinical Trials Support Group (MORU) DHA Dihydroartemisinin DHA-PPQ Dihydroartemisinin-piperaquine DSMB Data and Safety Monitoring Board EDTA Ethylenediaminetetraacetic acid G6PD Glucose-6-phosphate dehydrogenase GCP Good Clinical Practice GLURP Glutamate-rich protein Hct Haematocrit IC Inhibitory concentration ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use IM Intramuscular IV Intravenous KEMRI Kenya Medical Research Institute MORU Mahidol-Oxford Research Unit MSP Merozoite surface protein OUCRU-VN Oxford University Clinical Research Unit, Viet Nam NIH National Institutes of Health NMCP National Malaria Control Programme PCR Polymerase Chain Reaction PCT Parasite Clearance Time PD Pharmacodynamic PI Principal Investigator PK Pharmacokinetic QA Quality Assurance QC Quality Control SAE Serious Adverse Event SEA South-East Asia SMRU Shoklo Malaria Research Unit SNP Single-nucleotide polymorphism SOP Standard Operating Procedure TRAC Tracking Resistance to Artemisinin Collaboration WHO World Health Organisation WWARN Worldwide Antimalarial Resistance Network 6

8 2. SYNOPSIS Study Title Short Title and ACRONYM Trial Design Trial Participants A multicentre, randomised trial to detect in vivo resistance of Plasmodium falciparum to artesunate in patients with uncomplicated malaria. Tracking Resistance to Artemisinin TRAC Multi-centre, open-label randomised trial to assess the clearance rates of peripheral blood P. falciparum parasitaemias in patients with acute uncomplicated falciparum malaria treated with two different doses of artesunate Patients with acute uncomplicated P. falciparum malaria Sample size 60 patients per drug arm. Estimated total sample size Inclusion Criteria Male or female, aged from 6 months to 65 years old, inclusive Acute uncomplicated P. falciparum malaria, confirmed by positive blood smear with asexual forms of P. falciparum (or mixed with nonfalciparum species) Asexual P. falciparum parasitaemia: 10,000 to 200,000/uL, determined on a thin or thick blood film Fever defined as > 37.5 C tympanic temperature or a history of fever within the last 24 hours Written informed consent (by legally acceptable representative in case of children) Willingness and ability of the patients/guardians to comply with the study protocol for the duration of the study Exclusion Criteria Signs of severe/complicated malaria (WHO, 2000) Haematocrit < 25% or Hb < 8 g/dl at enrollment Acute illness other than malaria requiring treatment For females: pregnancy, breast feeding Patients who have received artemisinin or a derivative or an artemisinin-containing combination therapy (ACT) within the previous 7 days History of allergy or known contraindication to artemisinins, or to the ACT to be used at the site Previous splenectomy Planned Trial Period 18 to 24 months (May 2011 April 2013) Primary Objective To compare the P. falciparum parasite clearance rate as defined by the slope of the linear portion of the natural logarithm parasite clearance curve, at multiple sites, to a reference parasite clearance rate obtained from historical data in artemisinin-sensitive falciparum malaria. Secondary Objectives To measure the incidence and duration of gametocyte carriage in 7

9 patients with uncomplicated malaria after treatment with artesunate To assess PK and PD relationships for artesunate and DHA To compare ex vivo susceptibility profiles of P. falciparum isolates across geographic regions To identify parasite genetic determinants of the slow P. falciparum clearance phenotype (currently called artemisinin resistance) To identify host factors influencing the clearance of P. falciparum, e.g. haemoglobinopathies and glucose-6-phosphate dehydrogenase (G6PD) deficiency To create a repository of P. falciparum isolates with a range of artemisinin susceptibilities to identify a molecular marker of, and develop improved in-vitro tests for artemisinin resistance To develop improved in-vivo tests for artemisinin resistance To assess risk factors for malaria e.g. environmental, behavioural Primary endpoint Parasite clearance rate as defined by the slope of the linear portion of the natural logarithm parasite clearance curve Secondary endpoints Parasite clearance time assessed by microscopy Parasite reduction rates and ratios at 24 and 48 hours assessed by microscopy and quantitative PCR Time for parasite count to fall to 50%, 90% and 99% of initial parasite density Fever clearance time (i.e. the time taken for the tympanic temperature to fall below 37 C and remain there for at least 24 hours) Proportion of patients with gametocytemia before, during and after treatment with artesunate, assessed at admission, on days 3, 7 and 14, stratified by presence of gametocytes at enrolment Gametocyte carriage rates assessed in person weeks up to 14 days IC50, IC90, IC99 of P. falciparum responses to artemisinins ex vivo (at selected sites) Pharmacokinetic profiles of artesunate and DHA (half-life, Cmax, AUC, Tmax) at selected sites Parasite molecular markers of drug resistance Identification of host factors that correlate with slow parasite clearance Efficacy at D42 at selected sites Drugs Arm 1: AS2 - Artesunate 2 mg/kg/day for 3 days followed by a full course of either artemether-lumefantrine or DHA-piperaquine or artesunate-mefloquine or artesunate-amodiaquine Arm 2: AS4 - Artesunate 4 mg/kg/day for 3 days followed by a full course of 8

10 either artemether-lumefantrine or DHA-piperaquine or artesunate-mefloquine or artesunate-amodiaquine Note: In western Cambodia only Arm 2, i.e.4 mg/kg will be used. 9

11 3. BACKGROUND AND RATIONALE The spread of highly artemisinin resistant falciparum malaria would have devastating consequences for malaria control and elimination. It must be stopped. The response to artemisinin resistance in P. falciparum depends critically upon answering one pivotal question: how far has it spread? This research proposal focuses on filling critical gaps in knowledge that are essential to planning an effective response. 3.1 Background Over the last five decades, Southeast Asia (SEA) has been the epicentre for the evolution and spread of resistance to all important classes of antimalarials. Chloroquine and sulfadoxine-pyrimethamine resistance in P. falciparum emerged in the late 1950s and 1960s on the Thai-Cambodian border and spread across Asia and then Africa, contributing to millions of deaths from malaria. Artemisinins are the cornerstone of current antimalarial treatment. Evidence of reduced susceptibility to artemisinins in Western Cambodia was first presented in January 2007 and confirmed in a subsequent detailed pharmacokineticpharmacodynamic study conducted by our group [1]. Artemisinin resistance was manifest by a marked slowing of parasite clearance. In Pailin, in Cambodia, the median parasite clearance time was 84 hours compared to 48 hours on the Thai side of the international border with Myanmar following either 2 mg/kg of artesunate (AS) alone for 7 days or 4 mg/kg AS for 3 days plus 25 mg/kg of mefloquine at both locations [2]. Conventional in vitro drug sensitivity testing was uninformative in identifying the in vivo resistant phenotype [2].There were no molecular markers found in genes associated with resistance against other antimalarials or putatively associated with artemisinin resistance (PfMDR1, PfCRT, and PfSERCA) [1, 2]. Detailed in vivo assessment is therefore the only way to detect the presence of artemisinin resistance. A reduction of the in vivo responsiveness in the presence of adequate concentrations of artesunate and dihydroartemisinin (the active metabolite of artesunate) meets the WHO case definition of antimalarial drug resistance. In addition, a positive parasite count on Day 3, exceeding 3% of a study population of an adequate sample size, is currently being proposed as a marker of artemisinin resistance [3]. A slowing of parasite clearance could result from parasite or host factors, but laboratory research indicates that artemisinin resistance is a stable heritable trait in Plasmodium spp [4]. Artemisinin resistance has probably arisen as a result of unregulated use of artemisinins as monotherapies in the region since the mid-1970s. Further selection may therefore lead to higher levels of resistance and increasing failure rates. Spread across Asia to Africa would be a disaster as the artemisinins are the most potent antimalarial drugs available [5]. The clinical studies proposed here have the primary objective of defining the extent of the spread, so that containment efforts can be planned sensibly. They have the secondary objective of providing a large number of well characterised parasites to support the identification of molecular markers of artemisinin resistance to facilitate global monitoring efforts. 3.2 Study Rationale The in vivo assessment of drug efficacy combined with pharmacokinetic evidence of adequate drug absorption is the optimal way to define drug resistance in patients. Several WHO in vivo monitoring classifications have evolved over time but none are sufficiently sensitive to detect early resistance to the artemisinins. 10

12 Because the artemisinins are the most potent antimalarial drugs, the reduction in parasite numbers is rapid [5]. Therefore, early measures of reducing parasite counts are needed. This study will look at conventional markers of parasite reduction e.g. parasite clearance time, parasite reduction ratio, and the time to achieve a fall of 50%, 90% and 99% of the pre-treatment parasitaemia. Defining artemisinin resistance in vivo requires the use of artesunate alone because it is now appreciated that the partner drug in a combination treatment has a significant impact on the rate of parasite clearance [3]. This study will dose patients for 3 days with artesunate alone and measure the parasite count frequently in order to be able to define an accurate regression line of a graph of the natural logarithm of the parasite count (Y axis) versus time (X axis). Artesunate therapy will be followed by a full course of an ACT. Two different dose regimens of artesunate will be compared at most sites since unpublished observations from the Thai-Myanmar border suggest that the standard lower daily dose of 2mg/kg may enable the earlier detection of low level resistance than a 4mg/kg daily dose. In western Cambodia, only the 4mg/kg dose will be used due to the high failure rates of a 2mg/kg dose observed in a previous study [2]. 3.3 Proposed activities All the organisations in this collaboration will work closely with local counterparts including the National Malaria Control Programmes (NMCPs), non-governmental and other relevant organisations. Training is an integral part of this collaborative working relationship, and the building of local research capacity is an essential component of all research plans. All research-related activities, from study design, planning, implementation through to analysis and writing of reports will be performed jointly with local counterparts. Both on-the-job training and formal training will be provided when needed, in particular for Good Clinical Practice (GCP) laboratory skills. The close interaction between WHO and its regional offices will ensure that new knowledge is disseminated efficiently and effectively throughout the region. The Worldwide Antimalarial Resistance Network website ( will be also used as a medium to disseminate information. The main activity proposed is a large series of detailed in vivo clinical, parasitological and pharmacokinetic assessments in South Asia (N=2), Southeast Asia (N=11), and Africa (N=2), where increases in parasite clearance times were recently reported from one site (Borrmann et al., unpublished data). The proposed sites have been chosen based on current information, presence of established clinical research programmes (Mahidol Oxford Research Unit, United States National Institutes of Health (USNIH), Indian Council of Medical Research, Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Lao PDR, Oxford University Clinical Research Unit, Viet Nam, Department of Medical Research, Myanmar, Medical Action Myanmar, the Kenya Medical Research Institute (KEMRI)-Wellcome Trust Research Programme, Chittagong Medical College, Bangladesh, University of Ilorin, Nigeria) and feasibility to provide a map of likely resistance spread. The trial will be overseen by a Data and Safety Monitoring Board (DSMB) and will be monitored regularly. 11

13 4. OBJECTIVES 4.1 Primary Objective To compare the P. falciparum parasite clearance rate as defined by the slope of the linear portion of the natural logarithm parasite clearance curve, at multiple sites, to a reference parasite clearance rate obtained from historical data in artemisinin-sensitive falciparum malaria. 4.2 Secondary Objectives To measure the incidence and duration of gametocyte carriage in patients with uncomplicated falciparum malaria after treatment with artesunate To assess PK and PD relationships for artesunate and DHA To compare ex vivo susceptibility profiles of P. falciparum isolates across geographic regions To identify parasite genetic determinants of the slow P. falciparum clearance phenotype (currently called artemisinin resistance) To identify host factors influencing the clearance of P. falciparum, e.g. haemoglobinopathies and G6PD deficiency To create a repository of P. falciparum isolates with a range of artemisinin susceptibilities to identify a molecular marker of, and develop improved invitro tests for artemisinin resistance To develop improved in-vivo tests for artemisinin resistance To assess risk factors for malaria e.g. environmental, behavioural 12

14 5. TRIAL DESIGN 5.1 Study sites The study will take place at 17 sites in Asia, SE Asia and Africa (Appendix 3). The following countries may be involved in this study: SE Asia: Thailand (4 sites), Laos, Cambodia (4 sites), Myanmar (2 sites), Viet Nam & China South Asia: India (2 sites), Bangladesh Africa: Kenya, Nigeria. Additional sites may be added from other countries or from other regions within the countries listed above. If so, details will be forwarded to the ethics committees. 5.2 Summary of Trial Design This will be a randomised, open label study of two dose schedules of artesunate monotherapy to determine and compare the parasite clearance rates of P. falciparum parasites in peripheral blood. The parasite clearance rate is defined by the regression line of a plot of the natural log of the parasite count over time and is measured in number of parasites/unit time. 5.3 Study duration The recruitment phase of the study is expected to last 12 months. The first sites intend to start recruiting patients in April Training will precede study execution by up to 1 month. Data management and analysis, sample analysis (PK, in vitro, molecular markers) and report writing are expected to take about 12 months per site. Therefore, the total time to complete the study will be about 2 years. 5.4 Primary and Secondary Endpoints Primary Endpoint Parasite clearance rate as defined by the slope of the linear portion of the natural logarithm parasite clearance curve Secondary Endpoints Parasite clearance time assessed by microscopy Parasite reduction rates and ratios at 24 and 48 hours assessed by microscopy and quantitative PCR Time for parasite count to fall to 50%, 90% and 99% of initial parasite density Fever clearance time (i.e. the time taken for tympanic temperature to fall below 37 C and remain there for at least 24 hours) Proportion of patients with gametocytemia before, during and after treatment with artesunate, assessed at admission, on days 3, 7 and 14, stratified by presence of gametocytes at enrolment Gametocyte carriage rates assessed in person weeks up to 14 days IC50, IC90, IC99 of P. falciparum responses to artemisinins ex vivo (at selected sites) Pharmacokinetic profiles of artesunate and DHA (half-life, Cmax, AUC, Tmax) at selected sites 13

15 Parasite molecular markers of drug resistance Identification of host factors that correlate with slow parasite clearance Efficacy at D42 at selected sites 5.5 Trial Participants Overall Description of Trial Participants Male and non-pregnant female patients aged between 6 months and 65 years with acute uncomplicated falciparum malaria are the target study population. All study patients must meet the applicable inclusion and exclusion criteria Inclusion Criteria Male or female, aged from 6 months to 65 years old, inclusive Acute uncomplicated P. falciparum malaria, confirmed by positive blood smear with asexual forms of P. falciparum (or mixed with non-falciparum species) Asexual P. falciparum parasitaemia: 10,000 to 200,000/uL, determined on a thin or thick blood film Fever defined as > 37.5 C tympanic temperature or a history of fever within the last 24 hours Written informed consent (by legally acceptable representative in case of children) Willingness and ability of the patients/guardians to comply with the study protocol for the duration of the study Note on parasitaemia calculation: Parasitaemia = N parasites per 1000 red blood cells on the thin smear x Hct x Or Parasitaemia- N parasites per x White blood cells on the thick smear by WBC count (OR 8000 if unavailable) / x Exclusion Criteria Signs of severe/complicated malaria (WHO, 2000) Haematocrit < 25% or haemoglobin (Hb) < 8 g/dl at enrollment Acute illness other than malaria requiring treatment For females: pregnancy, breast feeding Patients who have received artemisinin or a derivative or an artemisinincontaining combination therapy (ACT) within the previous 7 days History of allergy or known contraindication to artemisinins, or to the ACT to be used at the site Previous splenectomy 14

16 6. PROCEDURES Study procedures will be performed according to the schedule of assessments (Appendix 1). 6.1 Informed Consent The subject (or witness if illiterate) or the parent or guardian of a minor must personally sign and date the latest approved version of the informed consent form before any study specific procedures are performed. Written and verbal versions of the participant information and informed consent in the local language will be presented to the participants detailing no less than: the exact nature of the study; the implications and constraints of the protocol; the known side effects and any risks involved in taking part. It will be clearly stated that participation is voluntary and that the participant or guardian is free to withdraw from the study at any time for any reason without prejudice to future care, and with no obligation to give the reason for withdrawal. The subject or guardian will be allowed up to two hours to consider the information and the opportunity to question the Investigator, or other independent parties to decide whether they will (or allow his/her charge to) participate in the study. Written informed consent will then be obtained by means of participant or guardian dated signature or thumb print (if unable to write) and dated signature of the person who presented and obtained the informed consent. Children capable of understanding the study (approximately 7 years of age or above) will be asked to sign an assent form. A copy of the signed informed consent/assent document(s) will be given to the participants. 6.2 Screening, Eligibility and Baseline Assessments Patients who present at the participating sites will be screened to assess eligibility. Full consent/assent will be obtained before any enrolment procedures are conducted. It will be made clear from the outset that refusal to participate will not jeopardize subsequent antimalarial treatment. A screening log will be kept Demographics and Medical History Basic demographic and epidemiological data (e.g. sex, age, address, bed net use, malaria risk factors, tobacco smoking, prior treatment and a full medical history will be recorded by the study staff Physical Examination and Vital Signs Physical examination will be conducted by a qualified investigator. Weight, height, pulse, blood pressure, respiratory rate, temperature, spleen and liver size will be recorded Drug history All prescribed or over-the-counter and traditional medications used within the last 7 days will be recorded. Any drug allergies will be recorded Screening blood & urine tests These will be: A parasite count from Giemsa or Field stained thick and thin blood films Full blood count or haematocrit or Hb (depending on local resources) Urine pregnancy test for females of child bearing potential 15

17 6.3 Randomisation and blinding Subjects who fulfil all the inclusion criteria and have none of the exclusion criteria will be randomised 1:1 to one of the two treatment arms, AS2 or AS4 according to a randomisation schedule (except in Western Cambodia where only the AS4 regimen will be used). Randomisation will be in blocks of 20. Allocation will be done by drawing the next sequential numbered opaque envelope, which contains the study number of the patient referring to a study box containing the treatment allocation and necessities for blood sampling. The subjects will be assigned a study arm through a computer-generated randomisation schedule. Individual, sealed and sequentially numbered envelopes will be provided for each trial site with one envelope per subject, indicating the treatment allocation. This is an open-label study so the blinding of investigators and patients is not applicable. However, the randomisation procedure allows for adequate drug allocation concealment before envelopes are opened. All laboratory investigations will be performed without knowledge of the treatment allocation. 6.4 Blood sampling On admission Patients will have an intravenous catheter inserted for the first 24 hours. An SOP will be provided instructing how this is to be kept patent and how to take blood for protocol tests. On study admission, immediately before drug administration, blood will be collected for the following: repeat parasite count (thick and thin films). If it is found subsequently that this parasitaemia no longer meets the inclusion criterion for parasitaemia the patient will be kept in the study. filter paper blood blots (six dots in 3MM filter paper) for: o o o quantitative PCR of parasites PCR parasite genotyping (MSP1, MSP2, GLURP in case of recurrence during follow-up) and host genetics (G6PD deficiency, haemoglobin electrophoresis) full blood count (1 or 3 ml) or haematocrit or haemoglobin 1 ml in an EDTA tube (G6PD, haemoglobinopathies and other human polymorphisms relevant to drug resistant malaria) drug concentrations (1 ml whole blood) special tube ex vivo parasite cultivation (3 ml adult, 2ml child) parasite cryopreservation (3 ml adult, 2 ml child) parasite DNA and RNA (10 ml adult, 5 ml child) EDTA-anticoagulated blood filtered through a CF11 column. (N.B. the reduced volume in children will only yield enough DNA for the SNP chip) Metabolomic studies and antibody levels to blood-stage and gametocyte antigens in human plasma During hospitalisation During hospitalisation, patients will have blood taken for malaria films at 4h, 6h, 8h and 12h and thereafter every 6 hours until parasite clearance (when two consecutive 16

18 malaria slides are negative). After parasite clearance, patients will be discharged. Filter paper blots and haematocrits or Hbs will be done whenever a malaria blood film is made. Three doses of artesunate will be given (Days 0, 1, 2), followed on study Day 3 by the first dose of an ACT. ACT treatment will be given supervised whether the patients are still in hospital or if they have been discharged as outpatients. The ACT given will vary by study site and will follow national policy. Options in Asia include artemether-lumefantrine, dihydroartemisinin-piperaquine and artesunate (AS) combined with mefloquine. In Africa, options include artemether-lumefantrine and artesunate combined with amodiaquine. Patients being treated with twice daily artemether-lumefantrine will be given their evening dose to take unsupervised at home Pharmacokinetic Study Sampling PK sampling using 1 ml of whole blood will be done to evaluate the drug exposure of AS and DHA using a conventional non-compartment analysis. A SOP will be written to detail the technique for specimen collection, handling and storage. Samples will be centrifuged immediately and the plasma stored at minus 80 C in properly labelled cryotubes. There will be a total of 7 samples taken from both adults and children giving a total blood volume of 7 ml. The PK schedule is presented below: Baseline sample at 0 hours Additional 6 samples from patients at 0.5 hr, 1 hr, 1.5 hr, 2 hr, 4 hr and 6 hr after the first dose Blood volumes The blood volumes for the protocol mandated tests are detailed in the study schedule (Appendix 1) and will vary slightly between sites depending on whether sites can perform all of the tests of this protocol e.g. not all sites will be able to do the full blood count, store the plasma PK samples or do the ex vivo parasite culture. Also, it is likely that only a minority of patients will develop a recurrent parasitaemia and be requested to give further blood samples. Maximum blood volumes are presented below for adults and children for 14 and 42 days of follow up. The maximum blood volume means the patients remained in hospital for 6 days, had all blood samples taken and had one recurrent parasitaemia during follow up. For adults, the maximum blood volumes will be 60 ml (14 days of follow up) and 62 ml (42 days of follow up). For children, the corresponding volumes are 46 and 48 ml. Allowing for the possibility that we may need to repeat blood tests, we will add 2 ml to these estimated maximum blood volumes. These volumes become less if: a full blood count is not done (minus 3 ml adults, minus 1 ml children) PK sampling is not done (minus 14 ml for adults and children) there is no recurrent parasitaemia (minus 16 ml adults, minus 10 ml children) 17

19 6.5 Urine sample A 2 ml urine sample will be taken from enrolled patients before treatment. It will be stored at minus 20 0 C for possible later testing of antimalarial drugs e.g. mefloquine and quinine (dipstick), and for chloroquine and sulphadoxine, using a method based on the Saker-Solomon s test [7]. 6.6 Study drug regimens Patients will receive either: OR AS2: Artesunate 2 mg/kg/day for 3 days followed by a full course of either artemether-lumefantrine or DHA-piperaquine or artesunate-mefloquine or artesunate-amodiaquine AS4: Artesunate 4 mg/kg/day for 3 days followed by a full course of either artemether-lumefantrine or DHA-piperaquine or artesunate-mefloquine or artesunate-amodiaquine Each dose administration of artesunate will be observed and recorded. A single recent batch of quality-tested artesunate will be used. In Western Cambodia, only the AS4 regimen will be used due to known high failure rates at the lower dose. After their AS, patients will receive a standard ACT. The first dose will be given in hospital. Patients will be asked to return for supervised ACT administration to complete their course of treatment. The choice of the ACT will be decided by the individual sites in accordance with local guidelines. Only the morning daily dose of artemether-lumefantrine will be supervised Artesunate Artesunate (Guilin Pharmaceutical Company, China). One tablet of artesunate contains 50mg artesunate. The dose will be administered to the nearest quarter tablet (Appendix 2) Artemether-lumefantrine A/L (Coartem, Novartis, Basel, Switzerland). One tablet contains 20mg artemether and 120mg lumefantrine. The regimen is twice daily for 3 days with a delay of at least 8 hours between the first and second dose. It is dosed by weight categories (Appendix 2). The drug will be given with some milk or another suitable source of fat in accordance with the package insert Dihydroartemisinin-piperaquine DHA-PPQ (Duo-Cotecxin Beijing Holley-Cotec Pharmaceuticals Co., Ltd, China) or Arterakine (The 1st National Joint Stock Pharmaceutical company Pharbaco, Vietnam One tablet contains 40mg of dihydroartemisinin and 320 mg piperaquine. A weightbased regimen containing a total dose of approximately 7 mg/kg DHA and 55 mg/kg piperaquine phosphate (see dosing tables in Appendix 2) Artesunate-mefloquine This will be given using either the loose formulation or the fixed dose coformulation, according to the locally recommended dosing schedule (Appendix 2). 18

20 6.6.5 Artesunate-amodiaquine This will be given using either the loose formulation or the fixed dose coformulation, according to the locally recommended dosing schedule (Appendix 2). The dose of loose AQ will follow National recommendations Study drug administration The artesunate will be administered by study medical or nursing staff while the patients are hospitalised. If the patient vomits within half an hour after intake of the antimalarial drugs, the dose will be repeated. If vomiting occurs between half and one hour, half of the dose will be repeated. Repeat doses will be recorded on the CRF. 6.7 Follow up procedures After patients are discharged, they will be followed up daily until Day 7 for: Supervised ACT treatment (for those who did not complete their treatment as inpatients) Malaria blood slide Haematocrit/Hb Filter paper blot After day 7, they will be next seen on Day 14 for: Malaria blood slide Haematocrit/Hb Filter paper blot Selected sites may choose to extend follow to 42 days (e.g. Viet Nam) when patients will be seen once weekly when blood will be taken for Malaria blood slide Haematocrit/Hb Filter paper blot Time windows The time-window for the visit on Day 7 is + 3 days and for the visits on Days is 1 to +3 days. If a patient does not attend, a home-visitor from the study team will try to locate the patient and bring them to the clinic Additional visits Patients presenting to the clinic with a fever or other symptoms on unscheduled days will be assessed by the study physician. Their temperature will be recorded and blood smear will be made for any patient with a documented fever (tympanic temperature 37.5 C) or a history of fever. Patients will be treated as clinically indicated Patients with recurrent parasitaemia Patients with a recurrent falciparum parasitaemia (including mixed with another malaria species) during follow up will have blood taken for the following: ex vivo parasite cultivation parasite cryopreservation parasite DNA and RNA 19

21 PCR to distinguish between a new or a recrudescent infection using three polymorphic genes: MSP1, MSP2 and GLURP. Patients with any recurrent parasitaemia will be given rescue treatment and withdrawn from the study. 6.8 Epidemiological follow-up In order to have a greater understanding of the local transmission and environmental risk factors for malaria, and to relate genetic diversity to geographical location, patients or their guardians will be asked if a field worker can conduct a home visit at the time of hospital discharge. The field worker will carry out a basic situational analysis (e.g. description of local land use, forest cover, access to health care) and will map the home location using the global positioning system (GPS). This will be optional and will be signed for separately in the consent form. 6.9 Rescue treatment The indications for rescues treatment are: the development of any danger signs or signs of severe malaria at any point. the parasitaemia rises after 12 hours i.e. the parasite count at 18h > vs. 12h count parasite count at 24h has not fallen by 75% compared to the baseline value. Rescue treatment will consist of parenteral artesunate, 2.4 mg/kg IV/IM STAT, followed by 2.4 mg/kg IV/IM at 12 hours and 24 hours and then daily until able to take oral medication [6]. In Pailin and Pursat only (where artemisinin resistance is known to have emerged) parenteral quinine will be added given in standard doses: 20 mg/kg (in 5% dextrose) IV over 4 hours, followed by 10 mg/kg every 8 hours until the patient is able to take oral medication. At the Kenyan site the national treatment protocol for severe malaria is still parenteral quinine. Patients with persistent asexual parasitaemia on day 7 or who develop a recurrent parasitaemia on D14 or beyond (sites with extended follow up) with no signs of severity will be treated with either another ACT or a different combination e.g. quinine and doxycycline or clindamycin, in accordance with local recommendations. Patients who develop a non-falciparum parasitaemia during follow up will be treated according to local guidelines Analysis Drug assays Artesunate and dihydroartemisinin concentrations will be measured in plasma samples. These assays will be performed in the MORU laboratories, Bangkok, Thailand. Material transfer agreements will be in place to allow for the transfer of the PK samples Analysis of PK data Pharmacokinetic analyses will be performed by the MORU Department of Pharmacology. 20

22 Ex vivo drug sensitivity assay An ex vivo assay will be performed to measure parasite responses to artesunate and DHA. Parasites will be also be cryopreserved for future studies Molecular studies Parasite DNA will be used for genomic studies including but not limited to microsatellite typing to identify parasite clones and single nucleotide polymorphisms (SNP) typing/whole genome sequencing to generate data for genome-wide association studies of in vivo, ex vivo, and in vitro responses of parasites to artemisinin and controls. Parasite RNA will be used for transcriptome analyses PCR for quantitative parasitaemia One of the objectives of this study is to measure the parasitaemia by PCR and compare clearance rates estimated using this method to microscopy. A filter paper sample will be taken with every blood film while the patient is in hospital. Thereafter, a filter paper sample and a malaria blood film will be taken when the patients return for their supervised ACT treatment Discontinuation/ Withdrawal of Participants from the Study Each participant has the right to withdraw from the study at any time. In addition, the investigator may withdraw a participant if he or she considers it necessary for any reason including: Significant non-compliance with treatment regimen or study requirements An adverse event which requires discontinuation of the study medication or results in inability to continue to comply with study procedures Disease progression which requires discontinuation of the study medication or results in inability to continue to comply with study procedures Blood transfusion while malaria blood smear is still positive Development of severe malaria Recurrent parasitaemia Loss to follow up If an individual is prematurely discontinued from study treatment for any reason, the investigator must make every effort to perform the following evaluations: physical examination and vital signs assessment, haematocrit level, parasite count and AE assessment. The reason for withdrawal will be recorded in the CRF. If the subject is withdrawn due to an adverse event, the investigator will arrange for follow-up visits until the adverse event has resolved or stabilised. Any pregnancy must be reported to the Principal Investigator within one working day of awareness. The PI must take all reasonable efforts to discover the outcome of the pregnancy and fill out the pregnancy form. If there is a congenital abnormality or a still born baby, this needs to be reported as a serious adverse event Source Data Source documents are original documents, data, and records from which participants CRF data are obtained. These include, but are not limited to, hospital records (from which medical history and previous and concurrent medication may be summarised into the CRF), clinical and office charts, laboratory and pharmacy records, diaries, microfiches, radiographs, and CRFs. 21

23 CRF entries will be considered source data if the CRF is the site of the original recording (e.g., there is no other written or electronic record of data). In this study the CRF will be used as the source document for most of the data points. All documents will be stored safely in confidential conditions. On all study-specific documents, other than the signed consent form, the participant will be referred to by the subject number and initials, not by name. 22

24 7. STUDY DRUGS 7.1 Storage of Study Drugs All efforts will be made to store the study drugs in accordance with the manufacturers' recommendations in a secure area. This may be difficult at some sites where air conditioned storage rooms are not available. The artesunate and ACTs should be stored between 15 C to 30 C (59 F to 86 F). Where this is not possible and monitored storage conditions do not meet the recommendations the artesunate content of batches of AS will be retested at the end of the study. 7.2 Compliance with Study Drugs Artesunate will be administered as Directly-Observed-Therapy. If the patient vomits, and is re-dosed; this will be recorded in the CRF. Once the course of AS is completed, patients will be discharged. They will receive their first dose of ACT while in hospital and will be asked to return to receive the other doses. All ACT doses will be recorded in the CRF. 7.3 Accountability of the Study Treatment All movements of study medication will be recorded. Both individual subject and overall drug accountability records will be kept up to date by the study staff. 7.4 Concomitant Medication Throughout the study, investigators may prescribe any concomitant medications or treatments deemed necessary (e.g. antipyretics or anti-emetics) to provide adequate supportive care except for antibiotics with antimalarial activity unless unavoidable (e.g. doxycycline, azithromycin). If these are required the patients will be kept in the study and this will be noted as a protocol deviation. Antimalarials for recurrent infections (see Rescue treatment) and non-falciparum malaria (if applicable) will be prescribed as described above. Haematinics and antihelmintics may be prescribed after day 7 if indicated. Any medication, other than the study medication taken during the study will be recorded in the CRF. 23

25 8. SAFETY REPORTING This trial will be using drugs that have either been registered or evaluated extensively. Therefore, the safety aspects of this trial will be limited to close observation while patients are in hospital, treating any intercurrent illnesses or drug-related side effects and recording and reporting only serious adverse events. 8.1 Definitions Adverse Event (AE) In this study, an AE is either a drug-related side effect or a new or exacerbation of a pre-existing symptom, sign or illness (other than malaria) Serious Adverse Event A serious adverse event is an AE that: results in death is life-threatening i.e. the patient was at risk of death at the time of the AE requires inpatient hospitalisation or prolongation of existing hospitalisation results in persistent or significant disability/incapacity or is a congenital anomaly/birth defect requires acute medical or surgical care to prevent one of the outcomes listed above 8.2 Reporting Procedures for Serious Adverse Events All SAEs must be reported by the site investigator to the Medical Monitor, Dr. Arjen Dondorp, or his designee, within one day of his or her awareness of the SAE. The Medical Monitor will then report the SAE to the DSMB and the TRAC project Executive Committee within one day of his awareness. The site investigator must also report the SAEs to the local ethics committee in accordance with local requirements. 24

26 9. STATISTICAL CONSIDERATIONS 9.1 Sample size justification The sample size was calculated based on an assumption that clearance rates are lognormally distributed. This is based upon the assessment of distributions from 1952 patients [2, 8, Nosten, unpublished observations]. Means will be compared using the t- test. Analysis of frequent sampling parasite counts from these studies shows that the geometric means of clearance rate range between per hour with standard deviations on the log-scale between ; CV% on the original scale 43-49%. A sample size of 50 gives 12, 14, 15 and 17% precision on each side of the 95% CI for the SD of the log-transformed slope values of 0.4, 0.45, 0.5, 0.55, respectively (PASS, 2005) If there are no differences between treatments groups and we combine them the precision would be: 8, 9, 10, 12% depending on the SD. Assuming that the geometric mean of the clearance rates in a "sensitive" parasite population is 0.2 [2, 8, Nosten], with 50 (or 100) patients we will be able to show that the clearance rate is significantly reduced if the true value of the clearance rate was 0.157, 0.164, (0.169, 0.174, 0.179) for SDs equal to 0.55, 0.5, 0.4 of the logtransformed values, respectively (PASS 2005). Finally, with 50 patients in each treatment group, we will have 80% power to detect a 20% (log-transformed SD=0.4), 25% (0.5), 27 %( 0.55) decrease in clearance rate for clearance rates between (PASS, 2005). To allow for 10% loss to follow up or incomplete data, a total of 60 patients per arm will be recruited. 9.2 Statistical Analyses Analysis of other endpoints will be described in a Statistical Analysis Plan. A brief overview is given below Proportional data These will be compared using chi squared or Fisher s exact test, as appropriate. Crude proportions will be calculated with the exact 95% confidence intervals (CI), where relevant Continuous data These will be summarised by medians (IQR, ranges) and means (standard deviations, 95% CIs), as appropriate, and will include the parasite counts and laboratory parameters. Comparisons of continuous data will be assessed using the paired/unpaired t tests or the sign rank/mann Whitney U tests, as appropriate. Analyses of the parasite clearance data will be conducted to look for geographical differences e.g. Asia vs. Africa, Cambodia vs. other Asian countries Pharmacokinetic data The pharmacokinetic parameters of AS and DHA will be estimated with a noncompartment analysis and will include standard PK parameters e.g. Cmax, Tmax, and AUC. 25

27 10. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS Direct access will be granted to authorised representatives from the sponsor and host institution and the regulatory authorities, if applicable, to permit trial-related monitoring and inspections. 26

28 11. QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES The study will be conducted in accordance with the current approved protocol, ICH GCP, any national regulations that may apply to this study and standard operating procedures. The WWARN will be engaged in assuring QA/QC of study execution in collaboration with the MORU Clinical Trials Support Group (CTSG). Their role will include but not be limited to monitoring adherence to SOPs for collection of clinical data and laboratory specimens and quality checks (curation) of clinical and laboratory data according to standard methodologies. Malaria slide QC will be performed by MORU Monitoring Study sites may have in place a system for internal monitoring. In addition, regular external monitoring of all sites will be performed by the MORU CTSG according to ICH GCP and a Monitoring Plan. Data will be evaluated for compliance with the protocol and accuracy in relation to source documents. The monitors will check whether the clinical trial is conducted and data are generated, documented and reported in compliance with the protocol, GCP and the applicable regulatory requirements. Evaluation of on-site monitoring schemes, such as a reciprocal monitoring scheme, may be undertaken at selected sites by CTSG and WWARN. The Clinical Trial Facility at the KEMRI-Wellcome Trust Research Programme in Kilifi conducts routine internal monitoring of all clinical trials in the Programme DSMB An independent Drug Safety and Monitoring Board (DSMB) will be set up consisting of qualified volunteers with the necessary knowledge of clinical trials. The DSMB will review and approve a monitoring plan before the study commences. This will include how often the DSMB should receive summary reports. All data reviewed by the DSMB will be in the strictest confidence. A DSMB charter will outline its responsibilities and how it will operate. The DSMB will be notified within 1 day of the Principal Investigator being aware of the occurrence of a Serious Adverse Event. 27