Conditions: Malaria, Falciparum
Keywords: Malaria, Falciparum Malaria Protozoan Infections Lumefantrine Artemether Amodiaquine Piperaquine Artemether-lumefantrine combination Artemisinins Dihydroartemisinin Mefloquine Artemisinin Antimalarials Antiparasitic Agents Anti-Infective Agents ACT TACT Triple ACT(s) Resistance Antimalarial resistance Cardiotoxicity Safety Tolerability Efficacy
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 310
Healthy volunteers: no

Brief summary:
This study is a multi-centre, open-label randomised trial to assess the efficacy, safety and tolerability of the Triple ACT artemether-lumefantrine+amodiaquine (AL+AQ) compared to the ACT artemether-lumefantrine (AL) in uncomplicated falciparum malaria in Cambodia and Vietnam. The estimated total sample size is 600 patients from 2 sites in Cambodia and 2 sites in Vietnam. There are 2 treatment arms Arm 1: Artemether-lumefantrine for 3 days Arm 2: Artemether-lumefantrine for 3 days plus Amodiaquine for 3 days. According to the World Health Organization guideline, all patients except children under 10 kilograms will also be treated with a single dose of primaquine as a gametocytocidal treatment.

Funder :Bill \& Melinda Gates Foundation (BMGF) Grant reference number: OPP1132628

Detailed description:
"The study of artemether-lumefantrine or artemether-lumefantrine combined with amodiaquine will be a two-arm randomized open label comparative study.

The main activity proposed is a series of detailed in vivo clinical, parasitological and pharmacological assessments in 600 subjects across 2 sites in Cambodian (400 subjects) and 2 sites in Vietnam (200 subjects). The subjects will be randomized between the ACT artemether-lumefantrine and the TACT artemether-lumefantrine+amodiaquine.

Parasite clearance rates will be assessed by repeated assessments of the parasite counts after the start of the antimalarial treatments. Efficacy, safety and tolerability of ACTs and TACTs will be assessed through weekly follow up visits where vital signs, symptom questionnaires, physical examinations, blood smears, biochemistry assays and full blood counts will be performed.

Ex vivo assessments of parasite susceptibility to artemisinins and partner drugs will be measured and compared to historical data, clinical phenotype and other sites in an effort to identify artemisinin and partner drug resistance.

This study will obtain data on the effect of antimalarials on the corrected QT intervals. In addition, the effects of antimalarials on factors such as post-treatment haematocrit and haemoglobin levels will be assessed. Extensive pharmacokinetic analysis will allow for an assessment of drug-drug interactions.

Plasma histidine-rich protein 2 (HRP2) levels (a marker of parasite biomass) that could potentially serve for the estimation of parasitaemia dynamics before and after treatment will be measured and subsequently modelled."

Arms:
arm 1: Artemether-lumefantrine for 3 days plus primaquine at hour 24 arm 2: Artemether-lumefantrine for 3 days plus Amodiaquine for 3 days plus primaquine at hour 24

Interventions: intervention 1: ACT intervention 2: TACT
Intervention descriptions: intervention 1: Artemether-lumefantrine (20/120 mg) as a fixed dose combination twice daily for 3 days according to weight plus low dose primaquine at hour 24 intervention 2: Artemether-lumefantrine (20/120 mg) as a fixed dose combination twice daily for 3 days according to weight plus Amodiaquine (150mg) twice daily for 3 days according to weight plus low dose primaquine at hour 24

Protocol document:
Prot_000.pdf: 
 
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 A multi-centre, open-label randomised trial to assess the efficacy, safety and 
 tolerability of the Triple ACT artemether-lumefantrine+amodiaquine (AL+AQ) 
 compared to the ACT artemether-lumefantrine (AL) in uncomplicated  
falciparum malaria in Cambodia and Vietnam 
 
A study by the Triple Artemisinin Combination Treatments (TACTs) Collaboration 
 
Short title: Study to compare the Triple ACT AL+AQ with the ACT AL in Cambodia and Vietnam 
ACRONYM: TACT-Cambodia-Vietnam 
Protocol no.: MAL 17008 
OxTREC ref: 32-17 
NCT Number: NCT03355664 
 
Principal Investigator:  
Prof. A.M.  Dondorp 
Project coordinator 
Dr. R.W. van der Pluijm (Vietnam) 
Dr. R. Tripura (Cambodia) 
 
 
Sponsor 
 
University of Oxford 
 
 
Funder 
 
 
Bill & Melinda Gates Foundation (BMGF) 
 
 
 
 
 
 
 
Protocol development team:  
AM Dondorp, TT Hien, NJ White, RW van der Pluijm, R Tripura, TJ Peto, JJ Callery, NPJ Day, CJ 
Woodrow, J Tarning, M Imwong, MJ Dhorda, Z Bozdech, K Chotivanich and O Miotto 
 
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 authori-
ties. 
 


 
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Investigator Agreement and Conflict of Interest 
 
“I have read this protocol and: 
- 
agree to abide by all provisions set forth therein. 
- 
agree to comply with the principles of the International Conference on Harmonisation Tripar-
tite Guideline on Good Clinical Practice. 
- 
and declare no conflict of interest, according to the current version of the Declaration of 
Helsinki”  
 
 
 
 
 
 
Prof. Arjen M.  Dondorp                                      
Principal Investigator 
 
 
Principal Investigator’s Signature 
 
 
 
Date :  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
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TABLE OF CONTENTS 
1. 
LIST OF ABBREVIATIONS  
 
 
 
 
 
 
6 
2. 
SYNOPSIS  
 
 
 
 
 
 
 
 
7 
3. 
BACKGROUND AND RATIONALE  
 
 
 
 
 
11 
3.1 
 
Background 
 
 
 
 
 
 
 
 
11 
3.2 
 
Study Rationale  
 
 
 
 
 
 
 
12 
3.3 
 
Proposed activities 
 
 
 
 
 
 
 
12 
4. 
OBJECTIVES 
 
 
 
 
 
 
 
 
14 
4.1.  
 
Primary Objective 
 
 
 
 
 
 
 
14 
4.2.  
 
Secondary Objectives 
 
 
 
 
 
 
 
14 
5. 
TRIAL DESIGN 
 
 
 
 
 
 
 
 
15 
5.1 
 
Study sites 
 
 
 
 
 
 
 
 
15 
5.2 
 
Summary of trial design  
 
 
 
 
 
 
15 
5.3 
 
Study duration 
 
 
 
 
 
 
 
 
15 
5.4 
 
Primary and secondary endpoints 
 
 
 
 
 
15 
5.4.1. 
  
Primary Endpoint 
 
 
 
 
 
 
 
15 
5.4.2. 
  
Secondary Endpoints  
 
 
 
 
 
 
15 
5.5 
 
Trial Participants  
 
 
 
 
 
 
 
16 
5.5.1 
  
Overall Description of Trial Participants 
 
 
 
 
16 
5.5.2 
  
Inclusion criteria 
 
 
 
 
 
 
 
16 
5.5.3 
  
Exclusion criteria 
 
 
 
 
 
 
 
17 
6. 
PROCEDURES 
 
 
 
 
 
 
 
 
18 
6.1. 
 
Informed Consent 
 
 
 
 
 
 
 
18 
6.2. 
 
Screening, Eligibility and Baseline Assessments 
 
 
 
18 
6.2.1. 
  
Demographics and Medical History  
 
 
 
 
18 
6.2.2. 
  
Physical Examination and Vital Signs 
 
 
 
 
18 
6.2.3. 
  
Drug history 
 
 
 
 
 
 
 
 
18 
6.2.4. 
  
Screening tests 
 
 
 
 
 
 
 
18 
6.3. 
 
Randomisation and blinding 
 
 
 
 
 
 
19 
6.4. 
 
Blood sampling  
 
 
 
 
 
 
 
19 
6.4.1 
  
On admission  
 
 
 
 
 
 
 
19 
6.4.2. 
  
During hospitalisation  
 
 
 
 
 
 
19 
6.4.3. 
  
Pharmacokinetic Study Sampling 
 
 
 
 
 
20 
6.4.4. 
  
Plasma HRP2 measurements 
 
 
 
 
 
20 
6.4.5. 
  
Host genotyping 
 
 
 
 
 
 
 
21 
6.4.6. 
  
Blood volumes 
 
 
 
 
 
 
 
21 
6.5 
 
Study drug regimens 
 
 
 
 
 
 
 
22 
6.5.1 
  
Artemether-lumefantrine 
 
 
 
 
 
 
22 


 
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6.5.2. 
  
Amodiaquine  
 
 
 
 
 
 
 
22 
6.5.3. 
  
Primaquine 
 
 
 
 
 
 
 
 
22 
6.6. 
 
Procedures during hospitalisation 
 
 
 
 
 
22 
6.7. 
 
Follow up procedures 
 
 
 
 
 
 
 
22 
6.7.1 
  
Time windows  
 
 
 
 
 
 
 
23 
6.7.2 
  
Additional visits 
 
 
 
 
 
 
 
23 
6.7.3 
  
Patients with recurrent parasitaemia  
 
 
 
 
23 
6.8. 
 
Epidemiological data on place of residence, work, travel history  
 
 
and mobile phone use 
 
 
 
 
 
 
 
24 
6.9. 
 
Rescue treatment 
 
 
 
 
 
 
 
24 
6.10. 
 
Analysis  
 
 
 
 
 
 
 
 
25 
6.10.1   
Drug assays  
 
 
 
 
 
 
 
25 
6.10.2   
Analysis of PK data 
 
 
 
 
 
 
 
25 
6.10.3   
Ex vivo drug sensitivity assay 
 
 
 
 
 
25 
6.10.4   
Molecular studies 
 
 
 
 
 
 
 
25 
6.10.5   
PCR for quantitative parasitaemia 
 
 
 
 
 
25 
6.11. 
 
Discontinuation/ Withdrawal of Participants from the Study 
 
 
25 
6.12. 
 
Source Data 
 
 
 
 
 
 
 
 
26 
7. 
STUDY DRUGS 
 
 
 
 
 
 
 
 
26 
7.1 
 
Storage of Study Drugs  
 
 
 
 
 
 
26 
7.2 
 
Compliance with Study Drugs 
 
 
 
 
 
 
26 
7.3 
 
Accountability of the Study Treatment  
 
 
 
 
26 
7.4 
 
Concomitant Medication  
 
 
 
 
 
 
26 
8.  
SAFETY REPORTING 
 
 
 
 
 
 
 
28 
8.1 
 
Definitions 
 
 
 
 
 
 
 
 
28 
8.1.1. 
  
Adverse Event (AE)  
 
 
 
 
 
 
28 
8.1.2 
  
Serious Adverse Event 
 
 
 
 
 
 
28 
8.1.3 
  
QTc Interval 
 
 
 
 
 
 
 
 
29 
8.1.4 
  
Biochemical assessments 
 
 
 
 
 
 
29 
8.2 
 
Reporting Procedures for Serious Adverse Events 
 
 
 
30 
8.3 
 
Evaluating Adverse Events and Serious Adverse Events 
 
 
30 
8.3.1  
 
 Assessment of Intensity 
 
 
 
 
 
 
30 
8.3.2  
 
 Clarification of the difference in meaning between ‘severe’ and ‘serious’ 
30 
8.3.3 
 
 Assessment of relatedness  
 
 
 
 
 
30 
8.3.4 
  
Outcome 
 
 
 
 
 
 
 
 
30 
9. 
STATISTICAL CONSIDERATIONS 
 
 
 
 
 
31 
9.1 
 
Sample size justification  
 
 
 
 
 
 
31 
9.2 
 
Statistical Analyses 
 
 
 
 
 
 
 
31 
9.2.1 
  
Proportional data 
 
 
 
 
 
 
 
31 
9.2.2 
  
Continuous data 
 
 
 
 
 
 
 
31 


 
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9.2.3 
  
Pharmacokinetic data  
 
 
 
 
 
 
31 
9.2.4 
  
Safety analysis 
 
 
 
 
 
 
 
31 
9.2.5 
  
Adverse events 
 
 
 
 
 
 
 
32 
9.2.6 
  
Assessment of relationship between PK parameters and ECG finding 
32 
9.2.7 
  
Interim analyses 
 
 
 
 
 
 
 
32 
10. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
 
 
 
33 
11. 
QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES 
 
34 
11.1 
 
Monitoring 
 
 
 
 
 
 
 
 
34 
11.2 
 
DSMB 
 
 
 
 
 
 
 
 
 
34 
12. 
ETHICS 
 
 
 
 
 
 
 
 
 
35 
12.1 
 
Declaration of Helsinki 
 
 
 
 
 
 
 
35 
12.2 
 
ICH Guidelines for Good Clinical Practice 
 
 
 
 
35 
12.3 
 
Approvals  
 
 
 
 
 
 
 
 
35 
12.4 
 
Risks 
 
 
 
 
 
 
 
 
 
35 
12.4.1   
Risks of artemether-lumefantrine 
 
 
 
 
 
35 
12.4.2   
Risks of amodiaquine  
 
 
 
 
 
 
35 
12.4.3   
Risks of new partner drug combinations 
 
 
 
 
35 
12.4.4   
Risk of phlebotomy & finger stick 
 
 
 
 
 
35 
12.5 
 
Benefits  
 
 
 
 
 
 
 
 
35 
12.6 
 
Alternatives to Study Participation 
 
 
 
 
 
35 
12.7 
 
Incentives & Compensation 
 
 
 
 
 
 
35 
12.8 
 
Confidentiality 
 
 
 
 
 
 
 
 
36 
13. 
SAMPLE SHARING AND STORAGE 
 
 
 
 
 
37 
14. 
DATA HANDLING AND RECORD KEEPING 
 
 
 
 
38 
15. 
SPONSORSHIP AND INSURANCE 
 
 
 
 
 
39 
16. 
PUBLICATION POLICY 
 
 
 
 
 
 
 
40 
17.  
REFERENCES 
 
 
 
 
 
 
 
 
41 
18. 
APPENDIX 1. STUDY SCHEDULES D42 FOLLOW UP  
 
 
42 
19. 
APPENDIX 2. DOSING SCHEDULES 
 
 
 
 
 
45 
19.1 
 
Artemether-lumefantrine  
 
 
 
 
 
 
45 
19.2 
 
Amodiaquine 
 
 
 
 
 
 
 
 
45 
19.3 
 
Primaquine 
 
 
 
 
 
 
 
 
45 
20. 
APPENDIX 3.  LIST OF STUDY SITES & PRINCIPAL INVESTIGATORS 
46 
21. 
APPENDIX 4: WWARN TERMS OF SUBMISSION 
 
 
 
47 
 


 
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1. 
LIST OF ABBREVIATIONS 
ACPR  
Adequate clinical and parasitological response 
ACT 
 
Artemisinin-based combination therapy 
AE 
 
Adverse event 
A/L 
 
Artemether-lumefantrine 
AQ 
 
Amodiaquine 
AUC 
 
Area under the (plasma concentration-time) curve 
CRF 
 
Case record form 
CTSG  
Clinical Trials Support Group (MORU) 
CYP3A4 
Cytochrome P450 3A4  
DHA 
 
Dihydroartemisinin 
DNA                Deoxyribonucleic Acid 
DSMB  
Data and Safety Monitoring Board 
EDC                Electronic data capture 
EDTA  
Ethylene-diamine-tetra-acetic acid  
G6PD  
Glucose-6-phosphate dehydrogenase 
GCP 
 
Good Clinical Practice 
Hb                   Haemoglobin 
Hct 
 
Haematocrit 
IM 
 
Intramuscular 
IV 
 
Intravenous 
MDR1  
Multi-Drug Resistance Gene 1 
MQ 
 
Mefloquine 
MORU  
Mahidol-Oxford Research Unit 
NMCP  
National Malaria Control Programme 
PCR 
 
Polymerase Chain Reaction 
PCT 
 
Parasite Clearance Time 
PD 
 
Pharmacodynamic 
PK 
 
Pharmacokinetic 
QA 
 
Quality Assurance 
QC 
 
Quality Control 
RNA 
 
Ribonucleic acid 
SAE 
 
Serious Adverse Event 
SNP 
 
Single-nucleotide polymorphism 
SOP 
 
Standard Operating Procedure 
TACT  
Triple Artemisinin-based Combination Therapy 
TRAC  
Tracking Resistance to Artemisinin Collaboration 
WHO  
World Health Organisation 
WWARN 
Worldwide Antimalarial Resistance Network 
 
 
 
 


 
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2. 
SYNOPSIS 
Study Title 
A multi-centre, open-label randomised trial to assess the efficacy, safety and 
tolerability of the Triple ACT artemether-lumefantrine+amodiaquine (AL+AQ) 
compared to the ACT artemether-lumefantrine (AL) in uncomplicated falciparum 
malaria in Cambodia and Vietnam 
ACRONYM 
TACT-Cambodia-Vietnam 
Trial Design 
An open-label randomised trial comparing the Triple ACT (TACT) artemether-lu-
mefantrine+amodiaquine with artemether-lumefantrine ACT), evaluating efficacy, 
safety, tolerability and artemisinin and partner drug resistance in four sites.  
Trial          
Participants 
Patients with acute uncomplicated P. falciparum malaria  
Sample size 
2 sites in Cambodia each recruiting 200 subjects 
2 sites in Vietnam that will recruit a cumulative 200 subjects through competitive-en-
rollment  
Estimated total sample size 600 patients. 
Inclusion    
Criteria 
• Male or female, aged from 2 years to 65 years old  
• 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: 16 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 parent/guardian in case of children) 
• Willingness and ability of the patients or parents/guardians to comply with the study 
protocol for the duration of the study 
Exclusion  
Criteria 
• Signs of severe/complicated malaria (see chapter 5) 
• Haematocrit < 25% or Hb < 8 g/dL at screening  
• 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, lumefantrine or amodia-
quine 
• Previous splenectomy 
• QTc-interval > 450 milliseconds at moment of presentation 
• Documented or claimed history of cardiac conduction problems 
• Previous participation in the current study or another study in the previous 3 months  


 
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Planned Trial 
Period 
30 months (October 2017 – March 2020) 
Primary      
Objective 
To compare the efficacy of the TACT artemether-lumefantrine+amodiaquine versus the 
ACT artemether-lumefantrine as defined by the 42-day PCR corrected adequate clini-
cal and parasitological response (ACPR). 
Secondary 
Objectives 
• To compare the efficacy of the TACT artemether-lumefantrine+amodiaquine versus 
ACT artemether-lumefantrine as defined by the 42-day PCR ACPR according to 
site/geographical region.  
• To assess and compare P. falciparum parasite clearance rates of the standard ACT 
and study TACT 
• To assess and compare fever clearance rates of the standard ACT and study TACT  
• To compare the safety and tolerability of TACT versus standard ACT 
• To compare changes in the electrocardiogram (such as prolongation of the QTc-
interval) in patients treated with TACT versus ACT 
• To assess the spread of genetic markers of artemisinin (such as Kelch13 mutations) 
and partner drug resistance and identify additional genetic determinants of drug re-
sistance. 
• To assess and increase the representability/accuracy of parasite genome sequenc-
ing from dry blood spots for the genome sequencing results from leukocyte depleted 
blood samples 
• To identify differences at the transcriptome level in artemisinin and partner drug sen-
sitive and resistant P. falciparum in order to increase the understanding of mecha-
nisms of resistance 
• To compare clearance dynamics estimated with quantitative PCR measurements of 
parasite loads versus microscopy 
• To measure and compare the incidence and duration of gametocyte carriage in pa-
tients with antimalarial sensitive and resistant malaria before and after treatment with 
TACT and ACT 
• To develop DNA and RNA measurement methods for quantification of male and fe-
male gametocytes 
• To compare ex vivo susceptibility profiles of P. falciparum isolates across geographic 
regions 
• To assess pharmacokinetic and pharmacodynamic interactions between AL and AQ  
• To obtain additional safety data (in particular incidence and rate of haemolysis) on 
the deployment of single low dose primaquine, stratified according to G6PD status 
• To obtain additional data on the effect of the host genotype on the pharmacokinetics 
and pharmacodynamics of antimalarials. 
• To compare clearance dynamics estimated with plasma HRP2 levels of parasite 
loads versus microscopy 


 
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• To obtain data on the place of residence, work, recent travel history and mobile 
phone usage in order to improve the understanding of locations of malaria transmis-
sion and possible routes spread of malaria and artemisinin resistance. 
• To obtain data and GPS mapping on a select group of participants and their peers 
relating to their understanding of the behaviours and risk factors associated with ma-
laria infection in order to improve understanding of local malaria transmission 
Primary   
endpoint 
42-day PCR corrected efficacy defined as adequate clinical and parasitological re-
sponse (ACPR). WHO definition: absence of parasitaemia at day 42 irrespective of 
axillary temperature and without previously meeting any of the WHO criteria for early 
or late treatment failure, or late parasitological failure. 
 
Secondary 
endpoints 
• 42-day PCR corrected efficacy defined as adequate clinical and parasitological re-
sponse (ACPR) according to site/geographic region. 
• Parasite clearance half-life assessed by microscopy as primary parameter to deter-
mine parasite clearance 
• Additional parameters of parasite clearance dynamics 
• Fever clearance time (i.e. the time taken for the tympanic temperature to fall below 
37.5˚C and remain there for at least 24 hours) 
• Incidence of adverse events and serious adverse events by study arms within the 
first 42 days. 
• Incidence of adverse events concerning markers of hepatic or renal toxicity such as 
bilirubin, ALT, AST, Alkaline Phosphatase and creatinine 
• Proportion of patients that reports completing a full course of observed TACT or ACT 
without withdrawal of consent or exclusion from study because of drug related seri-
ous adverse event 
• Incidence of prolongation of the QTc-interval above 500 ms or > 60 ms above base-
line values. 
• Prolongation of QTc-interval compared to baseline at timepoint H4, H24, H28, H48, 
H52, H60 and H64 and between these timepoints 
• Change in haematocrit on day 1 to 7, 14, 21, 28, 35 and 42 according to geographical 
location and study arm, stratified for G6PD status 
• Correlation between the host genotype and the pharmacokinetics and pharmacody-
namics of antimalarials 
• Prevalence of Kelch13 mutations of known functional significance 
• Prevalence/incidence of other genetic markers of antimalarial drug resistance such 
as MDR1 copy number and MDR1 mutations 
• Genome wide association with in vivo/in vitro sensitivity parasite phenotype 
• Correlation between SNPs measured in dry blood spots and whole genome se-
quencing in leukocyte depleted blood samples 
• Transcriptomic patterns at t=0 and t=6h after start of treatment comparing sensitive 
and resistant parasites 


 
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• Correlation between qPCR based versus microscopy based assessments of para-
site clearance dynamics up to day 14 
• Proportion of patients with gametocytemia before, during and after treatment with 
TACT or ACT, assessed at admission, up to day 14, stratified by presence of ga-
metocytes at enrolment 
• Levels of RNA transcription coding for male or female specific gametocytes at ad-
mission up to day 14, stratified by the presence of gametocytes at enrolment 
• In vitro sensitivity of P. falciparum to artemisinins and partner drugs according to 
study sites and genotype 
• Pharmacokinetic profiles and interactions (Cmax and AUC) of artemisinin-deriva-
tives and partner drugs in 20 ACT treated and 20 TACT treated patients of both 
study arms in Vietnam 
• Day 7 drug levels of partner drugs in association with treatment efficacy and treat-
ment arm 
• Correlation between HRP2 based versus microscopy based assessments of para-
site clearance dynamics 
• Data on the place of residence, work, recent travel history and mobile phone use. 
• Data and GPS mapping for a select group of participants and their peers in relation 
to behaviours and risk factors associated with malaria infection.  
Drugs 
 
See  
Appendix 2 
for dosing 
regimens 
  
 Artemether-lumefantrine for 3 days. 
VERSUS 
 Artemether-lumefantrine for 3 days. 
plus: 
Amodiaquine for 3 days. 
 
Primaquine 
According to the WHO guideline, all patients except children under 10 kilograms will 
also be treated with a single dose of primaquine according to the local requirement 
and WHO treatments schedule as a gametocytocidal treatment. 
 


 
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3. 
BACKGROUND AND RATIONALE 
Artemisinin combination therapies (ACTs) have been a major driving force behind substantial re-
ductions in global malaria morbidity and mortality over recent years. However, further gains are 
threatened by the recent emergence of artemisinin resistance in Southeast Asia, a region which has 
been the epicentre for the evolution and spread of resistance to every important class of antimalar-
ials.  
3.1 
Background  
The Tracking Resistance to Artemisinin Collaboration (TRAC) study was focused on mapping the 
spread of artemisinin resistance by conducting a multi-centre clinical trial, coordinated by the Mahi-
dol Oxford Research Unit (MORU) at sites in Asia (N=13) and Africa (N=3). This study measured 
individual parasite clearance rates, identifying locations where artemisinin resistance was present, 
as evidenced by slow parasite clearance (clearance half-life >5 hours) in a high proportion of 
cases[1]. This reduction in artemisinin sensitivity has left partner drugs within ACTs exposed to a 
much larger biomass of parasites and unsurprisingly partner drug resistance has followed [2]. 
Consequently, treatment failures after artemisinin combination therapies are becoming more wide-
spread in Southeast Asia. Failures rates of up to >60% for dihydroartemisinin-piperaquine have 
been documented in Cambodia, Thailand and Vietnam[3-5](TRACII unpublished data).  
Failure of first line ACTs will damage current control and elimination efforts and accelerate the emer-
gence and spread of resistance. A major concern is that artemisinin and partner drug resistance 
may spread across a wider geographic area, as chloroquine resistance did in the 1960s and 1970s, 
moving from Southeast Asia to the Indian subcontinent and Africa. Furthermore, the resistance itself 
could ‘deepen’ by extending beyond the ring stage of the asexual cycle (to which the resistance 
phenotype is currently limited). 
There is an urgent need to evaluate alternative treatments where standard courses of ACTs are 
failing, and to develop combinations of existing drugs which will not fall rapidly to resistance and can 
be deployed immediately. New drugs are at least five years away. The TRACII study has examined 
the safety, tolerability and efficacy of different TACT combinations such as DHA-piperaquine+mef-
loquine and artemether-lumefantrine+amodiaquine. The preliminary results of this study show full 
efficacy of the TACTs DHA-piperaquine+mefloquine in Cambodia, Thailand and Vietnam, even 
though the efficacy of the ACT DHA-piperaquine is poor. In other sites, the TACT artemether-lu-
mefantrine+amodiaquine is as effective as the standard ACT artemether-lumefantrine. Furthermore, 
the preliminary results indicate a good safety and tolerability profile of both the TACTs. 
Artemisinin and piperaquine resistant parasites are now well established in Cambodia, Vietnam and 
Thailand and most likely all originate from the same region on the Thai-Cambodian border [6]. The 
first line treatment for the uncomplicated P. falciparum malaria has recently been changed to ar-
tesunate-mefloquine. Hopefully this change of partner drug will contribute to a diminishing of the 
resistance to piperaquine. However, it is possible that mefloquine resistance will develop on top of 
the existing artemisinin and piperaquine resistance, which would lead to the development and most 
likely spread of truly multi-resistant P. falciparum in Southeast Asia. 
Therefore, although the efficacy of the TACT DHA-piperaquine+mefloquine in Vietnam and Cambo-
dia so far has been excellent, there is the potential of the occurrence of parasites that are resistant 
to artemisinin, mefloquine and piperaquine, which could lead to treatment failures after treatment 
with the TACT DHA-piperaquine+mefloquine. 
We propose to trial the efficacy of artemether-lumefantrine, an ACT that has not been used in Vi-
etnam and Cambodia yet. Furthermore, we propose to assess the efficacy, safety and tolerability of 
the TACT artemether-lumefantrine+amodiaquine. Both this ACT and the TACT could potentially play 
a role in the treatment of multidrug resistant malaria in this region if found to be effective, safe and 
tolerable. 
 


 
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3.2 
Study Rationale 
The principle that multiple drugs with independent mechanisms of action prevent the emergence of 
drug resistance is proven in a range of human diseases. In HIV and tuberculosis for example, the 
occurrence and spread of drug resistance can be prevented by use of a combination of three or 
more antiretroviral or antimycobacterial therapies respectively, but until now this was not thought 
necessary in malaria. In malaria, there is a fortuitous inverse correlation between susceptibility to 
amodiaquine and lumefantrine which will be exploited in the TACTs. 
The global spread of chloroquine (CQ) resistance was also associated with cross-resistance to 
amodiaquine. However, amodiaquine continued to be used in some areas of Southeast Asia, such 
as Myanmar, owing to its low cost. Relatively few trials of the ACT artesunate-amodiaquine have 
been conducted. The largest showed borderline efficacy in Myanmar with recrudescence rates of 
about 10%. Studies in Vietnam, as well as India and Africa, have reported cure rates above 95%[7]. 
 
Transfection data provide the most reliable means of understanding the molecular basis of amodia-
quine resistance. Fidock and colleagues introduced two major patterns of mutations (Dd2 and 7G8) 
into wild-type Pfcrt-encoding parasites and were able to confirm that these mutations were causative 
factors in CQ resistance.[8]. Notably there was a clear inverse correlation between the IC50 values 
for CQ and mefloquine (MQ) as well as for amodiaquine and MQ. In other words, resistance to 
chloroquine/amodiaquine caused by specific Pfcrt-mutations was associated with mefloquine hyper-
sensitivity. 
A second locus, Pfmdr1, also appears to be involved in amodiaquine resistance, with the N86Y 
mutation associated with resistance in vitro and increased clinical failures with amodiaquine mono-
therapy[9]. A paper by Venkatesan et al. suggests that recurrent infection after artemether-lumefan-
trine and artesunate-amodiaquine is associated with specific mutations in the Pfmdr1 gene, but that 
the two partner drugs select alternative and drug resistance opposing alleles[10].  
In theory, the concurrent use of lumefantrine combined with amodiaquine exploits these opposing 
selection pressure effects. Potentially, this will prevent or delay emergence of high-level resistance 
to both drugs at the same time. This would be expected to prolong or maintain the overall efficacy 
of both partner drug components [11]. 
  
3.3 
Proposed activities  
Artemether-lumefantrine+amodiaquine TACT study  
The study of artemether-lumefantrine or artemether-lumefantrine combined with amodiaquine will 
be a two-arm randomised open label comparative study. Based on the relatively safe and limited 
side effect profiles of both drugs, no life-threatening interactions between lumefantrine and amodia-
quine are expected. In fact, the preliminary data of the TRACII trial indicates that the TACT arteme-
ther-lumefantrine+amodiaquine has a comparable safety profile to the use of artemether-lumefan-
trine alone. Currently, recrudescence rates after treatment with artemether-lumefantrine are low in 
Laos, India, Myanmar and Bangladesh.  
If it transpires that the efficacy of artemether-lumefantrine in Cambodia and Vietnam is high, it may 
be too early to see improvement in efficacy after the addition of amodiaquine. However, it is im-
portant to rule out a negative effect of amodiaquine on the overall efficacy, as well as obtaining 
further knowledge of the safety and tolerability of the artemether-lumefantrine+amodiaquine. 
Once safety and tolerability are verified, studies can be conducted to assess the long-term efficacy 
of this novel TACT. 
 
Activities/outcomes 
The main activity proposed is a series of detailed in vivo clinical, parasitological and pharmacological 
assessments in 600 subjects across 2 sites in Cambodian and 2 sites in Vietnam. In Cambodia 400 
subjects will be recruited and in Vietnam, the 2 sites will recruit a cumulative 200 subjects (the 
sample size for this country) through competitive-recruitment due to the expected low recruitment 


 
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rates at each site. The subjects will be randomized between the ACT artemether-lumefantrine and 
the TACT artemether-lumefantrine+amodiaquine. 
 
The sites have been chosen based on current information on resistance patterns and incidence of 
malaria, presence of established clinical research programme and feasibility to perform the pro-
posed research activities. 
Parasite clearance rates will be assessed by repeated assessments of the parasite counts after the 
start of the antimalarial treatments. Efficacy, safety and tolerability of ACTs and TACTs will be as-
sessed through weekly follow up visits where vital signs, symptom questionnaires, physical exami-
nations, blood smears, biochemistry assays and full blood counts will be performed. 
Ex vivo assessments of parasite susceptibility to artemisinins and partner drugs will be measured 
and compared to historical data, clinical phenotype and other sites in an effort to identify artemisinin 
and partner drug resistance. 
The in vivo and ex vivo data on artemisinin and partner drug sensitivity will be used to identify new 
genetic or transcriptomic markers/patterns of artemisinin or partner drug resistance. Also efforts will 
be made to identify a ‘genetic backbone’, which is thought to be a specific characteristic of South 
East Asian malaria parasites leading to an increased tendency to develop artemisinin or partner 
drug resistance[12]. 
This study will obtain data on the effect of antimalarials on the QTc-intervals. In addition, we will 
assess the effects of antimalarials on factors such as post-treatment haematocrit and haemoglobin 
levels. Extensive pharmacokinetic analysis will allow for an assessment of drug-drug interactions. 
 
The pharmacogenetics of antimalarial agents are poorly known despite the fact that application of 
pharmacogenetics might be critical in optimizing treatment of malaria in individuals but also popula-
tions at large. Blood samples (dried blood blots) for human genotyping will be obtained and stored 
from all subjects recruited with subject’s consent. Data that could be indicative of (abnormal) phar-
macokinetics (i.e. low/high drug blood levels) resulting in altered pharmacodynamics such as hae-
molysis and/or prolongation of the QTc-interval will be obtained. For instance, abnormal absorption, 
distribution and/or clearance of the study drug (hence altered pharmacokinetics) might lead to in-
creased drug blood levels and a measured prolongation of the QTc-interval. Genotyping will be 
performed on the samples of subjects with suspected abnormal pharmacokinetics or pharmacody-
namics (for instance in case of unexpected adverse events such as substantial prolongation of the 
QTc-interval). Targeted genotyping could provide insight in the genetic origins of abnormal pharma-
cokinetics and/or pharmacodynamics in patients participating in the study. In addition, samples from 
all individuals will be used for whole genome association studies of the clinical phenotype and the 
subjects’ genotype. For example, changes in the QTc-interval of the individual subjects could be 
correlated with the genotype of all subjects in the form of a whole genome association study in order 
to identify genes that are potentially involved in an abnormal prolongation of the QTc-interval after 
administration of the antimalarials. Alternative examples of whole genome association studies are 
the correlation of blood drug levels with the host genotype. 
 
Plasma HRP2 levels (a marker of parasite biomass) could potentially serve for the estimation of 
parasitaemia dynamics before and after treatment. Plasma that is left over after antimalarial drug 
level measurements will be used for HRP2 measurements and subsequent modelling of parasite 
dynamics. 
 
All the organisations in this collaboration will work closely with local counterparts including the Na-
tional 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 re-
search 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 
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The close interaction between WHO and its regional offices will ensure that new knowledge is dis-
seminated efficiently and effectively throughout the region. The Worldwide Antimalarial Resistance 
Network website (www.wwarn.org) will be also used as a medium to disseminate information. 
The trial will be overseen by a Data and Safety Monitoring Board (DSMB) and will be monitored 
regularly. (See section 11.2) 
 
4. 
OBJECTIVES 
4.1.  
Primary Objective 
To compare the efficacy of the TACT artemether-lumefantrine+amodiaquine versus the ACT arte-
mether-lumefantrine as defined by the 42-day PCR corrected adequate clinical and parasitological 
response (ACPR). 
4.2.  
Secondary Objectives 
• To compare the efficacy of the TACT artemether-lumefantrine+amodiaquine versus ACT 
artemether-lumefantrine as defined by the 42-day PCR ACPR according to site/geo-
graphical region.  
• To assess and compare P. falciparum parasite clearance rates of the standard ACT and 
study TACT 
• To assess and compare fever clearance rates of the standard ACT and study TACT  
• To compare the safety and tolerability of TACT versus standard ACT 
• To compare changes in the electrocardiogram (such as prolongation of the QTc-interval) 
in patients treated with TACT versus ACT 
• To assess the spread of genetic markers of artemisinin (such as Kelch13 mutations) and 
partner drug resistance and identify additional genetic determinants of drug resistance. 
• To assess and increase the representability/accuracy of parasite genome sequencing 
from dry blood spots for the genome sequencing results from leukocyte depleted blood 
samples 
• To identify differences at the transcriptome level in artemisinin and partner drug sensitive 
and resistant P. falciparum in order to increase the understanding of mechanisms of re-
sistance 
• To compare clearance dynamics estimated with quantitative PCR measurements of par-
asite loads versus microscopy 
• To measure and compare the incidence and duration of gametocyte carriage in patients 
with antimalarial sensitive and resistant malaria before and after treatment with TACT 
and ACT 
• To develop DNA and RNA measurement methods for quantification of male and female 
gametocytes 
• 
To compare ex vivo susceptibility profiles of P. falciparum isolates across geographic 
regions 
• 
To assess pharmacokinetic and pharmacodynamic interactions between AL and AQ  
• 
To obtain additional safety data (in particular incidence and rate of haemolysis) on the 
deployment of single low dose primaquine, stratified according to G6PD status 
• 
To obtain additional data on the effect of the host genotype on the pharmacokinetics and 
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• To compare clearance dynamics estimated with plasma HRP2 levels of parasite loads 
versus microscopy 
• To obtain data on the place of residence, work, recent travel history and mobile phone 
usage in order to improve the understanding of locations of malaria transmission and 
possible routes spread of malaria and artemisinin resistance. 
• To obtain data and GPS mapping on a select group of participants and their peers relat-
ing to their understanding of the behaviours and risk factors associated with malaria in-
fection in order to improve understanding of local malaria transmission.   
 
5. 
TRIAL DESIGN 
5.1 
Study sites 
The study will take place at 2 sites in Cambodian and 2 sites in Vietnam (Appendix 3). 
5.2 
Summary of trial design 
An open-label randomised trial comparing the Triple ACT (TACT) artemether-lumefantrine+amodi-
aquine with artemether-lumefantrine ACT treatment, evaluating efficacy, safety, tolerability and ar-
temisinin and partner drug resistance in four sites. 
5.3 
Study duration 
The recruitment phase of the study is expected to last 24 months once a site starts to recruit. The 
first sites intend to start recruiting patients in October 2017. 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 6 months per site. Therefore, the total time to complete 
the study will be about 30 months. 
5.4 
Primary and secondary endpoints 
5.4.1. 
Primary Endpoint 
42-day PCR corrected efficacy defined as adequate clinical and parasitological response (ACPR). 
WHO definition: absence of parasitaemia at day 42 irrespective of axillary temperature and without 
previously meeting any of the WHO criteria for early or late treatment failure, or late parasitological 
failure. 
5.4.2. 
Secondary Endpoints 
• 
42-day PCR corrected efficacy defined as adequate clinical and parasitological re-
sponse (ACPR) according to site/geographic region. 
• 
Parasite clearance half-life assessed by microscopy as primary parameter to determine 
parasite clearance 
• Additional parameters of parasite clearance dynamics 
• 
Fever clearance time (i.e. the time taken for the tympanic temperature to fall below 
37.5˚C and remain there for at least 24 hours) 
• Incidence of adverse events and serious adverse events by study arms within the first 
42 days. 
• Incidence of adverse events concerning markers of hepatic or renal toxicity such as bili-
rubin, ALT, AST, Alkaline Phosphatase and creatinine 
• Proportion of patients that reports completing a full course of observed TACT or ACT 
without withdrawal of consent or exclusion from study because of drug related serious 
adverse event 


 
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• Incidence of prolongation of the QTc-interval above 500 ms or > 60 ms above baseline 
values. 
• Prolongation of QTc-interval compared to baseline at timepoint H4, H24, H28, H48, H52, 
H60 and H64 and between these timepoints 
• Change in haematocrit on day 1 to 7, 14, 21, 28, 35 and 42 according to geographical 
location and study arm, stratified for G6PD status 
• 
Correlation between the host genotype and the pharmacokinetics and pharmacodynam-
ics of antimalarials 
• 
Prevalence of Kelch13 mutations of known functional significance 
• 
Prevalence/incidence of other genetic markers of antimalarial drug resistance such as 
MDR1 copy number and MDR1 mutations 
• 
Genome wide association with in vivo/in vitro sensitivity parasite phenotype 
• 
Correlation between SNPs measured in dry blood spots and whole genome sequencing 
in leukocyte depleted blood samples 
• 
Transcriptomic patterns at t=0 and t=6h after start of treatment comparing sensitive and 
resistant parasites 
• 
Correlation between qPCR based versus microscopy based assessments of parasite 
clearance dynamics up to day 14 
• 
Proportion of patients with gametocytemia before, during and after treatment with TACT 
or ACT, assessed at admission, up to day 14, stratified by presence of gametocytes at 
enrolment 
• 
Levels of RNA transcription coding for male or female specific gametocytes at admission 
up to day 14, stratified by the presence of gametocytes at enrolment 
• 
In vitro sensitivity of P. falciparum to artemisinins and partner drugs according to study 
sites and genotype 
• 
Pharmacokinetic profiles and interactions (Cmax and AUC) of artemisinin-derivatives 
and partner drugs in 20 ACT treated and 20 TACT treated patients of both study arms in 
Vietnam 
• 
Day 7 drug levels of partner drugs in association with treatment efficacy and treatment 
arm 
• 
Correlation between HRP2 based versus microscopy based assessments of parasite 
clearance dynamics 
• 
Data on the place of residence, work, recent travel history and mobile phone use. 
• 
Data and GPS mapping for a select group of participants and their peers in relation to 
behaviours and risk factors associated with malaria infection.  
5.5 
Trial Participants 
5.5.1 
Overall Description of Trial Participants 
Male and non-pregnant female patients aged between 2 years and 65 years with acute uncompli-
cated falciparum malaria are the target study population. All study patients must meet the applicable 
inclusion and exclusion criteria. 
5.5.2 
Inclusion criteria 
• 
Male or female, aged from 2 years to 65 years old. 


 
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• 
Acute uncomplicated P. falciparum malaria, confirmed by positive blood smear with asex-
ual forms of P. falciparum (or mixed with non-falciparum species) 
• 
Asexual P. falciparum parasitaemia: 16 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 parents/guardian in case of children) 
• 
Willingness and ability of the patients or parents/guardians to comply with the study pro-
tocol for the duration of the study 
Note on parasitaemia calculation: 
Parasitaemia = N parasites per 2000 red blood cells on the thin smear x Hct x 62.8 
Or 
Parasitaemia = N parasites per 500 White blood cells on the thick smear by WBC count x16. 
5.5.3 
Exclusion criteria 
• 
Signs of severe/complicated malaria 
• 
Haematocrit < 25% or Hb < 8 g/dL at screening  
• 
Acute illness other than malaria requiring treatment 
• 
For females: pregnancy, breast feeding  
• 
Patients who have received artemisinin or a derivative or an artemisinin-containing com-
bination therapy (ACT) within the previous 7 days 
• 
History of allergy or known contraindication to artemisinins, or to the ACT or TACT to be 
used at the site  
• 
Previous splenectomy 
• 
QTc-interval > 450 milliseconds at moment of presentation 
• 
Documented or claimed history of cardiac conduction problems 
• 
Previous participation in the current study or another study in the previous 3 months 
 
Criteria for severe malaria (Adjusted from criteria used in SEAQUAMAT trial) 
• 
Glasgow coma scale <11/15 in adults, or Blantyre coma scale ≤3/5 in children. 
• 
Severe prostration 
• 
Shock, assessed by admitting physician (low blood pressure and cool peripheries) 
• 
Lactate >4.0 mmol/L (if measured through point of care test) 
• 
Blood bicarbonate <15 mmol/L (if measured) 
• 
Haematocrit <20% and P.  falciparum parasitaemia >100000/μL 
• 
Visible jaundice and P. falciparum parasitaemia >100000/μL 
• 
Blood urea nitrogen >17 mmol/L (if measured) 
• 
Asexual P.  falciparum parasitaemia >10% 
• 
Plasma glucose <2.2 mmol/L (if measured) 
• 
Respiratory distress (>32 breaths per min) 


 
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6. 
PROCEDURES 
Study procedures will be performed according to the schedule of assessments (Appendix 1). This 
will require that participants are admitted to the hospital for at least 72 hours and continue to be 
followed up on a weekly basis for 6 weeks. This differs from what would be standard management 
of uncomplicated malaria in Cambodia and Vietnam.   
6.1. 
Informed Consent 
The patient (or witness if illiterate) or the parent/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 guard-
ian 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 patient or parent/guardian will be allowed as much time as possible to consider the information 
and take 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. However, no more than two hours 
should elapse between presentation and treatment either on or off the study. Written informed con-
sent 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 con-
sent.  
If required by local ethics committees, children will be asked to sign an assent form. A copy of the 
signed informed consent/assent document(s) will be given to the patients/parents.    
6.2. 
Screening, Eligibility and Baseline Assessments 
Patients who present at the participating sites will be screened to assess eligibility.   Full consent 
(and assent if required) will be obtained before any enrolment procedures are conducted. It will be 
made clear from the outset that refusal to participate will not jeopardise subsequent antimalarial 
treatment. A screening log will be kept.   
6.2.1. 
Demographics and Medical History 
Basic demographic and epidemiological data (e.g. sex, age, address, bed net use, malaria risk fac-
tors, prior treatment and previous participation in this or previous studies), and a full medical history 
will be recorded by the study staff.  
6.2.2. 
Physical Examination and Vital Signs  
Physical examination will be conducted by a qualified study team member. Weight, height, pulse, 
blood pressure, respiratory rate, temperature, spleen and liver size will be recorded if palpable. 
6.2.3. 
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.  
6.2.4. 
Screening tests 
These will be EDTA-anticoagulated blood for: 
• 
A parasite count from Giemsa or Field stained thick and thin blood films 
• 
Haematocrit 
Urine pregnancy test for females of child bearing potential 
Electrocardiograph to assess the QTc-interval 


 
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6.3. 
Randomisation and blinding 
Patients 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 according to a randomisation schedule. Randomisation will be 
in blocks of 8-12. Allocation will be done by drawing the next sequential numbered opaque envelope, 
which contains the study number and treatment allocation.  
The patients 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 patient, 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 alloca-
tion.   
6.4. 
Blood sampling 
6.4.1 
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. 
• 
Dry blood blots (400 microlitres, 4 spots collected on filter papers for:  
o Parasite DNA genotyping for development of genetic surveillance. 
o PCR parasite barcoding (using 24-200 SNPs) to compare genotypes in case of re-
currence of infection. 
o host genetics (G6PD genotyping) and haemoglobin electrophoresis 
o host genetics (genotyping of host factors affecting pharmacokinetics and pharmaco-
dynamics of antimalarial drugs) 
 
Full blood count or WBC differential and/ Hct and Hb if possible. (EDTA-anticoagulated) 
• 
Parasite DNA and RNA measurements (Up to 10 ml for adult, up to 5 ml for child) EDTA-
anticoagulated blood.  
• 
Validation of qPCR measurements by microscopy results. (2 ml of EDTA-anti-coagulated 
blood for adult and child). 
• 
DNA/RNA for gametocyte quantification and characterisation (3 ml of heparinised blood for 
adult, 2.0 ml for child, some sites). 
• 
Ex vivo parasite cultivation for drug sensitivity and transcriptomic assays (3 ml adult, 2 ml 
child, heparinised blood, some sites). 
• 
Parasite cryopreservation (3 ml adult, 2 ml child, heparinised blood, all sites). 
• 
Baseline biochemistry assessments including bilirubin, ALT, AST, Alkaline Phosphatase and 
creatinine levels (heparinised blood)  
6.4.2. 
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 malaria slides are negative). 
In every case, malaria films will be performed at H4, H6, H8, H12, H24, H48 and H72 (even if two 
consecutive negative blood smears have been seen before these timepoints) Patients may be dis-
charged if the final ECG has been performed and parasite clearance has been achieved, but not 
earlier than HR72. Haematocrits will be done whenever a malaria blood film is made.  
During hospitalisation, blood will also be taken for: 


 
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• 
Day 0 H12, day 1 H24 and day 2 H48: Dry blood blots (100 microlitres, 1 spots collected on 
filter papers)  
o Parasite DNA genotyping for development of genetic surveillance. 
• 
D0 H6: Parasite cryopreservation for transcriptomic analysis: EDTA-anticoagulated blood, 
(1 ml for adult, 1 ml for child, all sites). 
• 
Day 1 H24 and Day 2 H48: Parasite DNA/RNA measurements: EDTA-anticoagulated blood 
(1 ml for adult, 1 ml for child). 
• 
Day 3 H72: Validate qPCR measurements versus microscopy results: EDTA-anticoagulated 
blood (2 ml for adult and child). 
• 
Day 3 H72: DNA/RNA for gametocyte quantification and characterization: heparinized blood 
(3 ml of for adult, 2.0 ml for child). 
• 
Day 3 H72: biochemistry assessments including bilirubin, ALT, AST, Alkaline Phosphatase 
and creatinine levels: heparinised blood. 
 
Day 3 H72: Full blood count or WBC differential and Hct and Hb if possible. (EDTA-antico-
agulated) 
6.4.3. 
Pharmacokinetic Study Sampling 
PK sampling using EDTA-anticoagulated blood (1 ml per sampling occasion). Venous plasma will 
be used to evaluate the drug exposure to artemether, lumefantrine, and amodiaquine (and metab-
olites) using a conventional non-compartment analysis (first dose pharmacokinetics). All collected 
samples will also be analysed using nonlinear mixed-effects modelling to assess the possible drug-
drug interactions and the relationship between drug concentrations and treatment outcome. Drug 
levels collected at 4 hours after the first and last dose will also be used to evaluate a direct relation-
ship between possible QTc-prolongation and drug exposures. 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. 
Pharmacology analysis can be divided into two different approaches: 
1. 
Pharmacokinetic profiles and interactions of artemisinin-derivatives and partner drugs in 20 
ACT treated and 20 TACT treated patients of both study arms in Vietnam 
Up to 15 samples taken from both adults and children giving a total blood volume of up to 15 ml. 
The PK schedule is presented below:  
• 
Dense samples will be collected through an indwelling venous cannula during the first 24 
hours: at 0 hr (pre-dose), 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr, 24 hr. Additional samples will be 
collected by venipuncture at 4 hours after last dose (52 hours) and at day 4, 7, 14, and 28.  
 
2.  
A venous day 0 (pre-dose) and day 7 sample will be collected from all patients. EDTA blood 
(1 ml in adults and children).  
Additional PK blood samples (1 ml EDTA) will be taken in the following situations:  
 
In case of an abnormal QTc-interval. In that case a blood sample will be obtained at the 
moment of the first identification of the abnormal QTc-interval and 6 and 12 hours after the 
first abnormal QTc-interval in order to assess the correlation between the prolonged QTc-
interval and the drug levels that could be leading to the prolonged QTc-interval. (Maximum 
3 ml EDTA blood).  
 
In case of a recurrent infection at the day of recurrence. 
6.4.4. 
Plasma HRP2 measurements 
Plasma HRP2-levels will be measured on enrollment and subsequent timepoints and used for mod-
elling of parasite dynamics on the residues of samples obtained for PK analysis (some sites). There-
fore, no additional blood sampling will be needed for these measurements and analysis.  


 
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6.4.5. 
Host genotyping 
Blood samples (dried blood blots) for human genotyping will be obtained and stored from all subjects 
recruited with subject’s consent. Genotyping will be performed on the samples of subjects with sus-
pected abnormal pharmacokinetics or pharmacodynamics (for instance in case of unexpected ad-
verse events such as severe prolongation of the QTc-interval). In addition, all samples will be used 
for whole genome association studies of the clinical phenotype (QTc-interval prolongation, drug lev-
els, haemolysis (among others) and the subjects’ genotype.  
Potential targets for targeted genotyping are polymorphisms of Cytochrome 450 (e.g. CYP2D6 and 
CYP3A4) and other enzymes related to drug metabolism and genes and mutations predisposing to 
long QT-syndrome such as but not limited to the genes KCNQ1 (LQT1), KCNH2 (LQT2), and 
SCN5A (LQT3).   
Genetic samples (in the form of dried blood blots or extracted DNA) will be stored (for a maximum 
of 10 years) and genotyped at the Molecular Tropical Medicine Laboratory, Bangkok, Thailand. In 
case a particular genetic test cannot be performed at the Molecular Tropical Medicine Laboratory, 
Bangkok, Thailand, the genetic samples may be transferred to another institute (which might be a 
foreign country) for genotyping. The subject will be asked for consent for this transfer during the 
initial informed consent process. A material transfer agreement will be in place if required before 
any samples are shipped. 
The results of the genotyping will not be reported back to the subjects unless the abnormality is 
found through a method with a clear diagnostic certificate and the finding is judged to be of clear 
clinical importance to the subject. 
6.4.6. 
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. 
Concerning blood volumes, an age of above 12 years will be referred to as ‘adult’, an age of 12 
years and younger will be referred to as ‘child’.  
Maximum blood volumes are presented below for adults and children for 42 days of follow up. The 
maximum blood volume is the total amount taken if the patients remained in hospital for 3 days, 
had all blood samples taken and where part of the pharmacokinetic as well as other subsets (like 
qPCR validation and sampling for gametocyte genetics) and had one recurrent parasitaemia dur-
ing follow up. The maximum blood volume will be approximately 97.9 ml for adults and 75.9 ml 
for children with a weight above 20 kg (less than 10% of total blood volume taken over 8 weeks 
as recommended by WHO- Bulletin of the World Health Organization 2011:89:46-53). 
 
We will exclude children below 20 kilograms from dense PK, DNA and RNA measurements on day 
3, day 5, day 7, day 14, day 21, day 28, day 35 and day 42 thereby decreasing their maximum drawn 
blood volume approximately to 33.9 ml over 42 days. 
The maximum blood volume that will be drawn in children below the age of 12 (above a weight of 
20 kilograms) is 25.3 ml in the first 24 hours of the study. This is below the 3 ml/kg in the first 24 
hours as recommended by the WHO Bulletin 2011. 
 
Allowing for the possibility that we may need to repeat blood tests, we may add 2 mL to these 
estimated maximum blood volumes.  
These volumes become less if:  
• 
There is no recurrent parasitaemia (minus 20 mL adults, minus 12 mL children) 
 


 
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6.5 
Study drug regimens 
 
 
 
 
 
 
 
 
Patients will be treated with weight-based doses according to the schedule in appendix 2. 
The study drugs will be administered by study medical or nursing staff while the patients are hospi-
talised. The study drug will be taken with at least 1.2 grams of fat to optimize absorption (comparable 
to 80-100 ml of a MILO milk carton or an equivalent fat containing beverage).   
If the patient vomits within half an hour after intake of the antimalarial drugs, the dose will be re-
peated. If vomiting occurs between half and one hour, half of the dose will be repeated. If vomiting 
occurs more than one hour after drug administration, no repeat dosing will be done. Repeat doses 
will be recorded on the CRF. If vomiting within 1 hour occurs more than one time, no repeat dosing 
is allowed. The patient will be treated at the discretion of the investigator. 
6.5.1 Artemether-lumefantrine  
Currently available as dispersible or standard tablets containing 20 mg of artemether and 120 mg 
of lumefantrine, in a fixed-dose combination formulation. The flavoured dispersible tablet paediatric 
formulation facilitates use in young children.  
 
Target dose/range: 
The dose of artemether-lumefantrine is administered according to the treatment schedule in appen-
dix 2, thereby approaching the WHO-recommended target ranges of artemether 5-24 mg/kg and 
lumefantrine 29-144 mg/kg over 3 days. 
6.5.2. Amodiaquine 
Amodiaquine is available in tablets of 150, 300 and 600 mg. The weight-based treatment schedule 
in appendix 2 aims for a dosage of approximately 10mg (4.5-15mg)/kg/day amodiaquine for three 
days, thereby approaching the WHO target dose and range of 10 mg (7.5-15mg)/kg/day.  
6.5.3. Primaquine 
Primaquine is available in tablets of 7.5 and 15 base mg. The weight-based treatment schedule in 
appendix 2 aims for a dosage of approximately 0.15-0.375 mg/kg on day one thereby approaching 
the 0.25 mg base/kg single dose recommended by the WHO. 
6.6. 
Procedures during hospitalisation 
A physical examination, measurement of vital signs will be performed daily and every six hours on 
indication. A symptom questionnaire will be taken daily to help identify adverse events. Follow up 
procedures  
After patients are discharged, they will be followed up at Day 5 and 7 for:  
 
Day 5 and 7: Malaria blood slide  
 
Day 5 and 7: Haematocrit/Hb (EDTA-anticoagulated blood). 
 
Day 5 and 7: Validation of qPCR measurements versus microscopy results: EDTA-antico-
agulated (2 ml for adults, 2 ml for child). 
Overview TRACII drug regimens 
TACT-arm 
ACT-arm 
Artemether-lumefantrine x 3 days 
PLUS 
Amodiaquine x 3 days 
Artemether-lumefantrine x 3 days 
 
Patients will receive Primaquine on day 1 according to the WHO guidelines and local 
requirement 


 
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 
Day 5 and 7: DNA/RNA for gametocyte quantification and characterization: Heparinised 
blood (3 ml for adults, 2 ml for child). 
 
Day 7: Basic biochemistry assessments including bilirubin, ALT, AST, Alkaline Phosphatase 
and creatinine levels: heparinised blood  
 
Day 7: Full blood count or WBC differential and Hct and Hb if possible (EDTA-anticoagulated) 
 
Day 7: ECG 
 
Physical examination and recording of symptoms and adverse events.  
 
After day 7, they will be next seen weekly until Day 42 for: 
 
Malaria blood slide  
 
Haematocrit/Hb (EDTA-anticoagulated blood).  
 
Day 14, 28 and 42: Validation of qPCR measurements versus microscopy results: EDTA-
anticoagulated (2 ml for adults, 2 ml for child). 
 
Day 14, 21, 28, 35 and 42: DNA/RNA for gametocyte quantification and characterization (3 
ml for adults, 2 ml for child, heparinised blood). 
 
Day 28: Basic biochemistry assessments including bilirubin, ALT, AST, Alkaline Phospha-
tase and creatinine levels. (Heparinised blood) 
 
Day 28: Full blood count or WBC differential and Hct and Hb if possible (EDTA-anticoagu-
lated) 
 
Day 28: ECG 
 
Physical examination and recording of symptoms and adverse events.   
6.6.1 
Time windows  
The time-window for the visit on Day 7 is + 1 day and for the visits on Days 14 - 42 is –1 to +2 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. 
6.6.2 
Additional visits 
Patients presenting to the clinic with a fever or other symptoms on unscheduled days will be as-
sessed 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.  
In the event that a patient becomes pregnant, on study, additional visits will be added at 3 months, 
6 months and 9 months (after birth), to allow documentation of the outcome of the pregnancy. 
6.6.3 
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: 
• 
Repeat parasite count (thick and thin films).  
• 
Dry blood blots (400 microlitres, 4 spots collected on Whatman FTA cards) for:  
o Parasite DNA genotyping for development of genetic surveillance. 
o PCR parasite barcoding (using 24-200 SNPs) to compare genotypes in case of re-
currence of infection. 
 
Full blood count or WBC differential and Hct and Hb if possible (EDTA-anticoagulated) 
• 
Parasite DNA and RNA measurements (Up to 10 ml for adult, up to 5 ml for child) EDTA-
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• 
Validation of qPCR measurements by microscopy results. (2 ml of EDTA-anti-coagulated 
blood for adult and child,). 
• 
DNA/RNA for gametocyte quantification and characterisation (3 ml of Heparinised blood for 
adult, 2.0 ml for child). 
• 
Ex vivo parasite cultivation for drug sensitivity and transcriptomic assays (3 ml adult, 2ml 
child, heparinised blood) 
• 
Parasite cryopreservation (3 ml adult, 2 ml child, heparinised blood) 
In case of a recurrent infection, subjects should still be followed up according to the study protocol 
until day 42. An additional questionnaire on the place of living, work and travel and mobile phone 
use in the period between the initial infection and the recurrent infection will be completed. 
Patients who develop a non-falciparum parasitaemia during follow up will be treated according to 
local guidelines.  
 
6.7. 
Epidemiological data on place of residence, work, travel history and mobile phone 
use 
In order to have a greater understanding of the possible sites of malaria transmission, and to relate 
genetic diversity to geographical location, patients or their guardians will be asked a short set of 
questions on their place of residence, place of work and their history of travel in the last 2 months. 
In addition, basic questions on use of mobile phones will be asked. These questions will help in 
understanding the use of mobile phones in each country in subjects prone to malaria infections. In 
separate studies, we plan to use anonymised, aggregated data on mobile phone use to mode pop-
ulation movement and predict potential routes of spread of malaria and antimalarial drug resistance. 
We will use the mobile phone usage survey to understand how relevant these movement patterns 
are to patients with malaria.  
We will review these travel histories obtained in the hospital and then select a representative sam-
ple of patients for additional in-depth interviews with them and their peers to be conducted in their 
villages. This is to obtain a detailed understanding of the behaviours and risk factors for malaria 
infection. We will GPS the households of all patients and their places of work and places where 
their infection may have occurred, such as forests, farms or plantations. We will collect all availa-
ble local malaria treatment records to describe how the study population compares to the overall 
population who receive treatment for malaria and this will allow us to better understand local ma-
laria epidemiology and transmission patterns. All personal information will be anonymised so that 
no individual can be identified from their treatment records, through interviews, or from mapping 
data.  
 
6.8. 
Rescue treatment 
The indication for rescue treatment is: 
• 
the development of any danger signs or signs of severe malaria at any point. 
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. 
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. 
Patients with persistent asexual parasitaemia on day 7 or who develop a recurrent parasitaemia 
after day 7 with no signs of severity will be treated with either another ACT (Artesunate-mefloquine 
in Cambodia according to National treatment guideline which is now current first line treatment for 
uncomplicated falciparum malaria) 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. 


 
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6.9. 
 Analysis 
6.9.1 
Drug assays 
Partner drug concentrations will be measured in plasma samples. These assays will be performed 
at the Department of Clinical Pharmacology, MORU, Bangkok, Thailand.  
6.9.2 
Analysis of PK data  
Pharmacokinetic analyses will be performed by the Department of Clinical Pharmacology, MORU, 
Bangkok, Thailand at the end of the study period or earlier when requested, for instance by the 
DSMB. 
6.9.3 
Ex vivo drug sensitivity assay 
An ex vivo assay will be performed to measure parasite responses to artemisinin derivates and 
partner drugs according to the latest standards at the time of assessment. Parasites will be also 
be cryopreserved for future studies.  
6.9.4  
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 sequenc-
ing 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. 
6.9.5  
PCR for quantitative parasitaemia 
One of the objectives of this study is to measure assess the parasite clearance dynamics by PCR 
and compare these with clearance rates estimated using this method to microscopy. 
 
6.10.  Discontinuation/ Withdrawal of Participants from the Study  
Each participant has the right to discontinue the study drug or the study at any time. Data accrued 
up until the time of discontinuation will be used in the analysis.  
 
In general, the investigator must make every effort to perform the study procedure until day 42, 
including in the following situations:   
• 
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 inabil-
ity to continue to comply with study procedures 
• 
Development of severe malaria 
• 
Recurrent parasitaemia 
• 
Loss to follow up (every attempt should be made to re-contact the participant) 
• 
Discontinuation of the study drug 
 
However, the investigator may discontinue participation in the study of a participant if he or she 
considers it necessary. 
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The reason for withdrawal or discontinuation, if available, will be recorded in the CRF. If the study 
drug or participation in the study is discontinued due to an adverse event, the investigator will ar-
range for follow-up visits at least 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.  
6.11. 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.  
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 patient number and initials, 
not by name. 
 
7.  
STUDY DRUGS 
7.1 
Storage of Study Drugs 
All efforts will be made to store the study drugs in accordance with the manufacturers' recommen-
dations in a secure area. This may be difficult at some sites where air-conditioned storage rooms 
are not available. The ACTs and TACTs 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 
artemisinin-derivatives and partner drug content of batches of ACTs and TACTs will be retested at 
the end of the study.  
7.2 
Compliance with Study Drugs 
Study drugs will be administered as Directly-Observed-Therapy. If the patient vomits, and is re-
dosed; this will be recorded in the CRF. If vomiting within 1 hour occurs again after retreatment, no 
repeat dosing is allowed. In this case the patient will be treated at the discretion of the Investigator. 
Each patient should be followed up until day 42.  
Once the course of the study drugs is completed, patients will be discharged. All drug doses will be 
recorded in the CRF.    
7.3 
Accountability of the Study Treatment 
All movements of study medication will be recorded. Both study medication of individual patient 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 concomitant medications or treatments deemed 
necessary (e.g. antipyretics or anti-emetics) to provide adequate supportive care except for antibi-
otics with antimalarial activity unless unavoidable (e.g. doxycycline, azithromycin). If these are re-
quired the patients will be kept in the study and this will be noted as a protocol deviation.  Anti-
emetics should not be prescribed as a prophylaxis if no nausea or vomiting is present. If anti-emetics 
are indicated metoclopramide is the preferred anti-emetic as this drug has the least QTc-interval 
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Antimalarials for recurrent infections (see Rescue treatment) and non-falciparum malaria (if applica-
ble) will be prescribed as described above. Any medication, other than the study medication taken 
during the study will be recorded in the CRF.  


 
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8.  
SAFETY REPORTING 
This trial will use drugs that have either been registered or evaluated extensively.    
To allow for comparison of safety and tolerability of both new TACTs compared to the ACTs we will 
record and review all Adverse Events (AEs) and Serious Adverse Events, (SAEs), that occur in the 
study.  
A symptom questionnaire will be performed daily during hospitalisation and at each subsequent visit 
to the health care center, to aid in the identification of adverse events.  An additional questionnaire 
will be administered to patients who report symptoms such as suicide ideation and symptoms that 
might be attributable to a psychosis, to collect further data. 
The investigator is responsible for the detection and documentation of events meeting the criteria 
and definition of an adverse event (AE) or serious adverse event (SAE), as provided in this protocol.   
All SAEs and AEs will be promptly documented from the moment of inclusion in the study to discon-
tinuation of the patient from study participation. Any events occurring between screening and ran-
domization will be considered as baseline, preexisting conditions. 
All adverse events must be recorded in the AE/SAE CRF. To avoid colloquial expressions, the ad-
verse event should be reported in standard medical terminology. Whenever possible, the adverse 
event should be evaluated and reported as a diagnosis rather than as individual signs or symptoms.  
If a definitive diagnosis is not possible, the individual symptoms and signs should be recorded. 
Whenever possible, the aetiology of the abnormal findings will be documented on the CRF.  Any 
additional relevant laboratory results obtained by the Investigator during the course of this study will 
be recorded on the CRF.  
 
If the event meets the criteria for “serious”, the SAE must be reported to the TACT-Cambodia-Vi-
etnam safety team within 24 hours from the time that the event was identified. If further data is 
required, additional documentation can be submitted. All SAEs must be followed until resolution, or 
until the SAE is deemed permanent or leads to death. 
8.1 
Definitions 
8.1.1. Adverse Event (AE) 
An AE is any undesirable event or clinical deterioration that occurs to a study participant during the 
course of the study; that is, from the time of administration of study drugs until study ends (i.e., until 
the follow up visit) whether or not that event is considered related to the study drugs, or to a con-
comitant drug or procedure: e.g.     
• any unfavourable and unintended symptom 
• physical sign 
• abnormal laboratory result 
• an illness 
Any new clinical sign or clinical deterioration that occurs between signing the consent form and the 
administration of study drugs is not an AE. This information will be recorded in the medical records, 
as a pre-existing condition.  
8.1.2 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  
• 
is a congenital anomaly/birth defect 
• 
QTc interval longer than 500 milliseconds 


 
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• 
QTc interval > 60 milliseconds from baseline 
• 
Any other significant medical condition 
All of the above criteria apply to the case as a whole and should not be confused with the outcomes 
of individual reactions/events. More than one of the above criteria can be applicable to the one 
event. Important medical events that may not be immediately life-threatening or result in death or 
hospitalisation may be considered an SAE when, based upon appropriate medical judgment, they 
may jeopardize the patient or require medical or surgical intervention to prevent one of the outcomes 
listed in the definition above.  Examples of such medical events include allergic bronchospasm re-
quiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that 
do not result in hospitalisation, or development of drug dependency or drug abuse. 
8.1.3 QTc Interval  
Electrocardiograms (ECGs) will be recorded at screening, D0H0, D0H4, D1H24, D1H28, D2H48, 
D2H52, D2H60 and D2H64, D7 and D28 in order to measure the QTc-interval. 
 
The screening ECG can be used as the baseline (D0H0) ECG, if the time for administration of the 
D0H0 study drug is within 30 minutes of the time of the screening ECG. If the time between the 
screening ECG and the time for administration of the D0H0 study drug is longer than 30 minutes a 
separate baseline (D0H0) ECG must be performed.  
 
A QTcBazett interval longer than 450 milliseconds at the screening or D0H0 will lead to exclusion 
or discontinuation from the study.  The automated electronic readings at screening or baseline will 
not be confirmed by manual reading. 
 
At the timepoints D0H4, D1H24, D1H28, D2H48 and D2H52, D2H60 and D2H64 an abnormal au-
tomated electronic reading (QTc-interval > 500 milliseconds or QTc > 60 milliseconds from baseline) 
will be confirmed by manual readings. At these timepoints, a QTc-interval > 500 milliseconds or a 
prolongation compared to baseline > 60 milliseconds will be considered a Serious Adverse Event 
and will lead to discontinuation of the study drug but not discontinuation of the patient from the trial. 
A specific SOP will describe procedures such as study drug replacement and follow up of the QTc-
interval until normalization. QTc intervals will be measured every six hours after the first abnormal 
QTc-interval is recorded until the QTc-interval has normalized in two consecutive ECGs. Additional 
blood samples will be taken for pharmacological measurements at the moment of the identification 
of the abnormal QTc-interval and 6 and 12 hours after the first identification of the abnormal QTc-
interval in order to assess the correlation between the prolonged QTc-interval and the drug levels 
that could be leading to the prolonged QTc-interval. 
8.1.4 Biochemical assessments 
In order to assess the safety of the novel TACTs basic biochemical assessments of markers related 
to hepatic and renal toxicity will be performed on day 0 (baseline) day 3, day 7 and 28. If found to 
be abnormal the values will be graded according to the Division of AIDS table mentioned in section 
8.3.1 and followed up according to the local investigators preference and in accordance with section 
8.3.4. In any case, increased ALTs and bilirubin levels found on day 7 and/or day 28 will be followed 
up on day 42. 
 
Screening is performed without assessing biochemistry values. At baseline blood will be drawn for 
biochemistry assessments just minutes before baseline drug administration. Initial treatment at 
baseline will therefore be administered without knowledge of the baseline biochemistry values. If 
baseline biochemistry values turn out to be abnormal the study drugs must be discontinued and 
subjects will be treated according to the standard treatment or artesunate intravenously (if compli-
cated or severe malaria is suspected based on the obtained laboratory values). An abnormal base-
line biochemistry value is defined as falling within grade 3 or 4 of the DAIDS grading table. In any 
case the subject will still be followed up according to the study protocol until day 42).  
 


 
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8.2 
Reporting Procedures for Serious Adverse Events 
All SAEs must be reported by the site investigator to the Sponsor, within one day of his or her 
awareness of the SAE. The CRF documenting the SAE, should be faxed or emailed to the CTSG, 
(Fax No +66 (0) 2 354 9169; email TACTCV@tropmedres.ac).   
Further reports should be submitted, if required, until the SAE is resolved. 
The site investigator must also report the SAEs to the local ethics committee in accordance with 
local requirements. 
8.3 
Evaluating Adverse Events and Serious Adverse Events 
8.3.1  Assessment of Intensity  
Each adverse event will be graded according to the Division of AIDS table for grading the severity 
of ADULT AND PEDIATRIC adverse Events Version 2.0, November 2014 which will be included in 
the safety monitoring SOP. 
If an adverse event is not listed in the Division of AIDS table, the Investigator will assess the severity 
using the following guidelines: 
1 = Mild: awareness of sign or symptom, but easily tolerated 
2 = Moderate: enough discomfort to cause interference with usual activity 
3 = Severe: incapacitating with inability to work or do usual activity 
4 = Life-Threatening 
8.3.2  Clarification of the difference in meaning between ‘severe’ and ‘serious’  
The term “severe” is often used to describe the intensity (severity) of a specific event (as in mild, 
moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor 
medical significance (such as severe headache).  This is not the same as “serious”, which is based 
on the outcome or criteria defined under the serious adverse event definition. An event can be con-
sidered serious without being severe if it conforms to the seriousness criteria, similarly severe events 
that do not conform to the criteria are not necessarily serious.  Seriousness (not severity) serves as 
a guide for defining regulatory reporting obligations. 
8.3.3 Assessment of relatedness 
The investigator is obligated to assess the relationship between study drug and the occurrence of 
each AE/SAE using the following categories of relatedness: 
• Definite: clear-cut temporal association  
• Probable: clear-cut temporal association, with improvement upon drug withdrawal, and not 
reasonably explained by the patient’s known clinical state or other aetiology. 
• Possible: less clear temporal association; other aetiologies are possible. (Other possible aeti-
ologies should be recorded on the CRF). 
• Not related: no temporal association with the study drug; assessed as related to other aetiol-
ogies such as concomitant medications or conditions, or patient’s known clinical state. 
The investigator will provide the assessment of causality as per the AE/SAE data collection tool.  
 
 
8.3.4 Outcome 
The investigator will follow-up the AE and SAE until resolution or until no further medically relevant 
information can be expected. AE and SAE outcome will be classified as follows: 
• 
Continuing/ongoing  
• 
Resolved 
• 
Resolved with sequelae 
• 
Permanent  
• 
Fatal 


 
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9 
STATISTICAL CONSIDERATIONS 
9.1 
Sample size justification  
The sample size calculation is based on the primary endpoint of the 42-day PCR corrected ACPR. 
Our hypothesis is that artemether-lumefantrine+amodiaquine is superior to artemether-lumefantrine. 
Earlier efficacy studies of artemether-lumefantrine found an efficacy of 82.4 and 86.5% [13, 14]. 
Artesunate-mefloquine has not been used for almost a decade in Cambodia (up to 2016, when it 
became the first line treatment again). This has decreased the pfMDR1 copy number counts in the 
Cambodian population [15]. Given the association of pfMDR1 copy number and lumefantrine sensi-
tivity this has potentially increased the efficacy of artemether-lumefantrine, despite increasing levels 
of artemisinin resistance [16]. Therefore, we assume the efficacy of artemether-lumefantrine to be 
90%. Because of the additive therapeutic effect of amodiaquine we assume superiority of arteme-
ther-lumefantrine+amodiaquine with an efficacy of 99%.  
 
A sample size of 100 patients per arm (power 0.80 and alpha=0.05) would allow us to detect this 
superiority. This leads to a total of 200 subjects needed per study site. In Vietnam, the 2 sites will 
recruit a cumulative 200 subjects (the sample size for this country) through competitive-recruitment 
due to the expected low recruitment rates at each site. A total of 600 patient will be recruited for the 
study. 
  
The following Stata command: “sampsi 0.99 0.90, alpha (0.05) power (0.8) nocontinuity” was used 
(Stata 14.0). 
 
9.2 
Statistical Analyses 
Analysis of other endpoints will be described in a Statistical Analysis Plan. A brief overview is given 
below.  
9.2.1 Proportions 
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. 
9.2.2 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 contin-
uous 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 and temporal 
differences.  
9.2.3 Pharmacokinetic data 
The pharmacokinetic parameters of lumefantrine and amodiaquine during the first 24 hours will be 
estimated with a non-compartment analysis and will include standard PK parameters such as Cmax, 
Tmax and AUC. Nonlinear mixed-effects modelling will be employed to integrate all available data 
for an in-depth analysis of the pharmacokinetic properties of the above drugs as well as their rela-
tionship to treatment outcome.  
9.2.4 Safety analysis 
Safety analyses will be based on the whole population that get administered the study drug. Safety 
and tolerability of TACTs versus ACTs will be assessed by comparing the frequency (%) of adverse 
events and serious adverse events, with particular attention to abdominal pain, appetite perturba-
tion, biochemical markers of hepatic and renal toxicity and QT interval prolongation, using the 


 
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Fisher’s exact test.  Safety data will be presented in tabular and/or graphical format and summarized 
descriptively. Any clinically relevant abnormalities or values of potential clinically concern will be 
described. Patients will be analysed according to an intention to treat and a per protocol method 
where appropriate. 
9.2.5 Adverse events 
Adverse events will be graded according to Division of AIDS table for grading the severity of ADULT 
AND PEDIATRIC adverse Events Version 2.0, November 2014 which will be included in the safety 
monitoring SOP. 
All adverse event summaries will refer to treatment emergent adverse events, i.e. adverse events 
that newly started or increased in intensity after the study drug administration. AE summaries will 
be generated for all AEs that occurred after study drug administration, until the end of the study. 
9.2.6 Assessment of relationship between PK parameters and ECG findings. 
In addition to the results of the safety analysis an exploratory analysis of the ECG data will be con-
ducted. The ECG parameters will be compared with the PK properties of the drugs using appropriate 
statistical methods. 
9.2.7 Interim analyses 
Interim analysis reports on the safety of TACTs will be provided by the Project Coordinator in col-
laboration with the Trial Statistician (Dr Mavuto Mukaka, e-mail: Mavuto@tropmedres.ac) after the 
first 60 and 180 patients. To assess the safety of the novel TACTs the DSMB will review the interim 
analysis reports.  
 
 


 
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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.   


 
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11 
QUALITY CONTROL AND QUALITY ASSURANCE PROCEDURES  
The study will be conducted in accordance with the current approved protocol, ICH GCP, any na-
tional 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 
WWARN.  
11.1 
Monitoring 
Study sites may have in place a system for internal monitoring. In addition, regular external moni-
toring 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 re-
quirements. Evaluation of on-site monitoring schemes, such as a reciprocal monitoring scheme, 
may be undertaken at selected sites by CTSG and WWARN. 
11.2 
DSMB   
An independent Data Safety and Monitoring Board (DSMB) will be set up consisting of qualified 
volunteers with the necessary knowledge of clinical trials. The DSMB will receive summary reports, 
prior to each meeting.  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 meet formally at the following timepoints: 
 before the study starts 
 after the first 60 patients have been accrued into the study 
 after the first 180 patients have been accrued into the study 
  At additional time-points before the planned interim analyses, as indicated by the DSMB after 
their review, if deemed necessary 
 At the end of the study (i.e. after the last patient has finished follow up)  
Unscheduled meetings can be held on the initiative of the Medical Monitor (Dr Lorenz Von Seid-
lein, e-mail: Lorenz@tropmedrs.ac), Principal Investigator or Executive committee to consider e.g. 
serious adverse events as they are reported OR if an earlier safety review is thought to be indi-
cated.  


 
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12 
ETHICS 
12.1 
Declaration of Helsinki 
The Investigator will ensure that this study is conducted in compliance with the current revision of 
the Declaration of Helsinki (Fortaleza 2013). 
12.2 
 ICH Guidelines for Good Clinical Practice 
The Investigator will ensure that this study is conducted according to any National Regulations and 
that it will follow the principles of the ICH Guidelines for Good Clinical Practice 1996. 
12.3 
 Approvals 
The study protocol and its associated documents will be submitted to the Oxford Tropical Research 
Ethics Committee (OxTREC) and the appropriate local ethics committees for written approval.   
The Investigator will submit and, where necessary, obtain approval from the above parties for all 
substantial amendments to the original approved documents.    
12.4 
 Risks 
This study will use drugs that have been studied thoroughly and their toxicities are well described. 
In general, they are all well tolerated. 
12.4.1 Risks of artemether-lumefantrine  
Reported A/L side effects have generally been mild. The main side effects are GI upset: anorexia 
(~18%), nausea (~5%), vomiting (~18%), abdominal pain (~5%), and diarrhoea (~10%), headache 
(~10%), dizziness (~4%), fatigue (~1%) and sleep disturbance (~2%). Other symptoms reported 
infrequently include palpitations, myalgia, arthralgia (all of which could be disease related), and rash. 
A/L does not cause prolongation of the QTc interval.  
12.4.2  Risks of amodiaquine 
The main side-effects of amodiaquine are nausea, vomiting and fatigue which are mild to moderate 
in nature. When the drug was used for prophylaxis, rare adverse reactions of agranulocytosis and 
hepatoxicity were observed. 
12.4.3 Risks of new partner drug combinations 
No interactions between lumefantrine and amodiaquine are expected. Based on the relatively safe 
and limited side effect profiles of both drugs, no life-threatening interactions between lumefantrine 
and amodiaquine are expected. In addition, the preliminary results of the TRACII trial indicate that 
the TACT artemether-lumefantrine+amodiaquine has a good safety and tolerability profile.  
12.4.4 Risk of phlebotomy & finger stick  
The primary risks of phlebotomy include local discomfort, occasional bleeding or bruising of the skin 
at the site of needle puncture, and rarely haematoma or infection. 
12.5 
Benefits 
Malaria is a disease that needs to be treated promptly. All patients will benefit from receiving effica-
cious treatment at no cost. They will be followed up closely and will be given rescue treatment if 
clinically indicated.  
12.6 
 Alternatives to Study Participation 
Patients are able to decline freely participation in this study. If so, they will receive standard care for 
their malaria.   
12.7 
 Incentives & Compensation 
Study patients or their guardian in the case of children will be compensated for time lost from work. 
Patients will be reimbursed for the time lost from work as a result of hospitalisation, the cost of local 


 
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transport to attend for the follow up visits and will receive a per diem to cover the costs of meals on 
those days. The amounts in monetary terms will be determined by each site. The study will pay for 
treatment for drug-related SAEs or other research-related injuries. The study cannot pay for long 
term care for disability after hospital discharge resulting from complications of the illness.  
12.8 
Confidentiality 
The trial staff will ensure that the participants’ anonymity is maintained. The participants will be 
identified only by initials and a study number on the CRF and the MACRO EDC database.  All 
documents will be stored securely and be accessible to trial staff and authorised personnel only.  


 
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13 
SAMPLE SHARING AND STORAGE 
Samples collected will be used for the purpose of this study as stated in the protocol and stored for 
future use. Consent will be obtained from patients for sample storage and/or shipment of specific 
samples to collaborating institutions for investigations that cannot be performed locally. Any pro-
posed plans to use samples other than for those investigations detailed in this protocol will be sub-
mitted to the relevant ethics committees prior to any testing. Material transfer agreements will be 
arranged and signed where appropriate/needed. 
 


 
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14 
DATA HANDLING AND RECORD KEEPING 
All study data will be recorded on standard Case Report Forms (CRF), at the study sites. The CRFs 
will be sent to the Clinical Trial Support Group, Data Management team and will be entered on the 
MACRO EDC database in accordance with standard operating procedures. All data management 
activities will be carried out to ensure data quality, in accordance with the data management plan.   
The participants will be identified by a study specific participant number and/or code in any data-
base. The name and any other identifying detail will NOT be included in any study data electronic 
file. Data may be used alone or in combination with data from related studies in secondary analyses.  
The data from this study will be shared with WWARN with the consent of the study sites (The 
WWARN terms of submission are detailed in Appendix 4). The Principal Investigators from each 
site will have the opportunity to review and sign these terms of submission document to indicate 
their agreement.  


 
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15 
SPONSORSHIP AND INSURANCE  
The University of Oxford is the study sponsor and will obtain the necessary insurance.  


 
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16 
PUBLICATION POLICY 
Any data published in the peer-reviewed medical literature will protect the identity of the patients. 
This trial will be registered in a web based protocol registration scheme. All those who have made 
a substantial contribution will be co-authors on publications. The sites have the right to publish their 
data individually and to include members of the sponsor’s team who have made a significant contri-
bution. There will also publications of pooled data which will be coordinated by the MORU group. All 
sites will have the opportunity to contribute to these publications.  
All the research findings from the programme and from relevant research outside the Programme 
will be analysed and integrated, and through the WWARN site and the WHO Global Malaria Pro-
gramme will be disseminated to policy makers, National Malaria Control Programmes (NMCPs) and 
other researchers.  
 


 
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17.  
REFERENCES 
 
 
1. 
Ashley, E.A., et al., Spread of artemisinin resistance in Plasmodium falciparum malaria. N 
Engl J Med, 2014. 371(5): p. 411-23. 
2. 
Saunders, D.L., et al., Dihydroartemisinin-piperaquine failure in Cambodia. N Engl J Med, 
2014. 371(5): p. 484-5. 
3. 
Amaratunga, C., et al., Dihydroartemisinin-piperaquine resistance in Plasmodium falciparum 
malaria in Cambodia: a multisite prospective cohort study. Lancet Infect Dis, 2016. 16(3): p. 
357-65. 
4. 
Phuc, B.Q., et al., Treatment Failure of Dihydroartemisinin/Piperaquine for Plasmodium 
falciparum Malaria, Vietnam. Emerg Infect Dis, 2017. 23(4): p. 715-717. 
5. 
Thanh, N.V., et al., Rapid decline in the susceptibility of Plasmodium falciparum to 
dihydroartemisinin-piperaquine in the south of Vietnam. Malar J, 2017. 16(1): p. 27. 
6. 
Imwong, M., et al., The spread of artemisinin-resistant Plasmodium falciparum in the Greater 
Mekong subregion: a molecular epidemiology observational study. Lancet Infect Dis, 2017. 
17(5): p. 491-497. 
7. 
Smithuis, F., et al., Effectiveness of five artemisinin combination regimens with or without 
primaquine in uncomplicated falciparum malaria: an open-label randomised trial. Lancet 
Infect Dis, 2010. 10(10): p. 673-81. 
8. 
Sidhu, A.B., D. Verdier-Pinard, and D.A. Fidock, Chloroquine resistance in Plasmodium 
falciparum malaria parasites conferred by pfcrt mutations. Science, 2002. 298(5591): p. 210-
3. 
9. 
Sisowath, C., et al., In vivo selection of Plasmodium falciparum pfmdr1 86N coding alleles 
by artemether-lumefantrine (Coartem). J Infect Dis, 2005. 191(6): p. 1014-7. 
10. 
Venkatesan, M., et al., Polymorphisms in Plasmodium falciparum chloroquine resistance 
transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment 
outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-
amodiaquine. Am J Trop Med Hyg, 2014. 91(4): p. 833-43. 
11. 
Boni, M.F., D.L. Smith, and R. Laxminarayan, Benefits of using multiple first-line therapies 
against malaria. Proc Natl Acad Sci U S A, 2008. 105(37): p. 14216-21. 
12. 
Takala-Harrison, S., et al., Genetic loci associated with delayed clearance of Plasmodium 
falciparum following artemisinin treatment in Southeast Asia. Proc Natl Acad Sci U S A, 2013. 
110(1): p. 240-5. 
13. 
Song, J., et al., Randomized trials of artemisinin-piperaquine, dihydroartemisinin-
piperaquine phosphate and artemether-lumefantrine for the treatment of multi-drug resistant 
falciparum malaria in Cambodia-Thailand border area. Malar J, 2011. 10: p. 231. 
14. 
Denis, M.B., et al., Efficacy of artemether-lumefantrine for the treatment of uncomplicated 
falciparum malaria in northwest Cambodia. Trop Med Int Health, 2006. 11(12): p. 1800-7. 
15. 
Lim, P., et al., Decreasing pfmdr1 copy number suggests that Plasmodium falciparum in 
Western Cambodia is regaining in vitro susceptibility to mefloquine. Antimicrob Agents 
Chemother, 2015. 59(5): p. 2934-7. 
16. 
Sidhu, A.B., et al., Decreasing pfmdr1 copy number in plasmodium falciparum malaria 
heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J 
Infect Dis, 2006. 194(4): p. 528-35. 
 


 
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18. 
APPENDIX 1. STUDY SCHEDULES D42 FOLLOW UP 
 
 


 
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19 
APPENDIX 2. DOSING SCHEDULES 
19.1 
Artemether-lumefantrine 
 
Artemether-lumefantrine dosing schedule 
One tablet A/L contains 20mg artemether and 120mg lumefantrine 
 
No. of tablets recommended at approximate timing of dosing 
Weight: Kilo-
gram 
0 h 
8 h 
24 h 
36 h 
48 h 
60 h 
5-14.9 
1 
1 
1 
1 
1 
1 
15-24.9 
2 
2 
2 
2 
2 
2 
25-34.9 
3 
3 
3 
3 
3 
3 
≥35 
4 
4 
4 
4 
4 
4 
19.2 
Amodiaquine 
Amodiaquine dosing schedule 
One tablet contains 150mg Amodiaquine 
 
No. of tablets recommended at approximate timing of dosing 
Weight: Kilo-
gram 
0 h 
8 h 
24 h 
36 h 
48 h 
60 h 
5-14.9 
0.5 
0 
0.5 
0 
0.5 
0 
15-24.9 
0.5 
0.5 
0.5 
0.5 
0.5 
0.5 
25-34.9 
1 
1 
1 
1 
1 
1 
≥35 
1.5 
1.5 
1.5 
1.5 
1.5 
1.5 
 
19.3 
Primaquine 
 
 
 
  
Primaquine dosing schedule 
Weight: Kilogram 
Tab/day (Tab=7.5mg) 
<25 
0.5 tablet on day 1 
25-50 
1 tablet on day 1 
>50 
2 tablets on day 1 


 
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20 
APPENDIX 3.  LIST OF STUDY SITES & PRINCIPAL INVESTIGATORS 
 
CAMBODIA 
CNM:  Dr Huy  Rekol  (Principal investigator) Dr Chea Nguon (Co-Principal Investigator) Dr Lek 
Dysoley (Co-Principal Investigator)  
MORU : Dr. Rupam Tripura (Local investigator), Dr. James Callery (Local investigator), Dr. Tom 
Peto (Local investigator), Professor AM Dondorp (Principal Investigator), MORU, Thailand.  
 
Site 1: Pailin Referral Hospital, Pailin Province,  
Site 2: Stung Treng Referral Hospital/ Siem Pang Health Center, Stung Treng Province:  
 
 
VIETNAM 
 
Country PI:  
Hien Tran Tinh MD, PhD Oxford University Clinical Research Unit (OUCRU), Vietnam, 
Ho Dang Trung Nghia MD, PhD Oxford University Clinical Research Unit (OUCRU), Vietnam. 
  
Site 1: Phuoc Long Hospital, Phuoc Province:  Co-PI: Le Thanh Long, MD, Director 
Site 2: Hospital for Tropical Diseases of Khanh Hoa, Khanh Hoa Province: Co-PI: Nguyen 
Dong MD, Director,  
 


 
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21 
APPENDIX 4: WWARN TERMS OF SUBMISSION 
  
TERMS 
The WWARN project (http://www.wwarn.org) is dependent on the submission of data about individ-
ual patients’ responses to anti-malarial drug treatment. WWARN respects the rights of researchers 
and institutions who may wish to share their data with WWARN. 
These terms of submission (which form part of the WWARN website’s terms of use) provide a frame-
work for sharing data with WWARN, explaining the terms on which you may use the site to submit 
data to WWARN and how WWARN will use that data. Please read these terms carefully before 
submitting data to WWARN: by submitting data to WWARN, you indicate that you accept these 
terms and you agree to abide by them. 
 
WWARN 
WWARN is a project developed by an international collaboration of research institutions and which 
is currently hosted by the University of Oxford. The collaborative group consists of five WWARN 
project teams, each of which will have responsibility for the analysis of submitted data in relation to 
a defined topic (each a WWARN module). 
By submitting data to WWARN you agree that the WWARN project teams will have access to your 
data on substantially the same terms as granted in these terms of submission.  
 
DATA 
In order to ensure compliance with its legal and ethical obligations, WWARN requires that all sub-
mitted data: 
has been obtained in accordance with any laws and ethical approvals applicable in its country of 
origin; 
has been obtained with the knowledge and consent of the individual to which it relates; and 
has been anonymised, so that any individual to which it relates cannot be identified from it. 
You must have the authority to submit the data to be used in accordance with these terms of sub-
mission. 
WWARN reserves the discretionary right to delete any submitted data, in whole or in part, if it con-
tains any information that would identify an individual. 
In order to protect your data and safeguard the rights of the individuals to which it relates, WWARN 
will take appropriate technical and organisational measures against unauthorised or unlawful pro-
cessing of any submitted data and against the accidental loss or destruction of, or damage to, sub-
mitted data. 
 
RIGHTS 
You will continue to own your data. WWARN will not claim any rights of ownership in any data you 
may submit but, in order to allow WWARN to operate, it requires your permission to use the data for 
the purposes set out in these terms of submission. 
By submitting data, you grant to WWARN a right to use, copy, reformat and to make further analyses 
of your data in accordance with these terms of submission. 
You may, at any time and for any reason, withdraw WWARN’s right to use your data, but you accept 
that your data may have been used to prepare and/or create reports and analyses and that these 
reports and analyses cannot be withdrawn once they have been made available. 


 
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In the event that WWARN transfers from the University of Oxford to another entity, the rights you 
grant to WWARN will be transferred to that entity. 
 
USE OF DATA 
Your data will be stored on a secure server and will be accessible only to you, anyone you may 
nominate and WWARN. 
Your data may be further processed by WWARN and used in combination with other data to create 
aggregated data. 
WWARN may use aggregated data to create reports and analyses, including: 
summary reports to define geospatial and temporal trends in anti-malarial drug resistance and drug 
quality; and 
specialised analyses of pooled studies (for example, analyses in relation to a particular drug, patient 
group or time span). 
In the event that WWARN receives a request to contribute data to an external project or collaborative 
analysis, it will not contribute or grant access to your data without your express permission. 
 
PUBLICATION OF DATA 
Your submission of data to WWARN does not affect your right to publish those data, which you may 
do entirely at your own discretion and without consent from or reference to WWARN. 
Data relating to anti-malarial drug resistance is only useful to those involved in the treatment of 
malaria if it is up-to-date and, for that reason, WWARN encourages you to submit your data before 
publication. WWARN will only use your data in an aggregated form, except that where you provide 
your express permission WWARN may include a high-level summary of your research on the 
WWARN Explorer. 
Reports and analyses created by WWARN may be published or otherwise made publicly accessible, 
including through the WWARN website. 
Your contribution to any WWARN publication will be acknowledged in accordance with the guide-
lines of the International Committee of Medical Journal Editors (www.icmje.org/ethical_1au-
thor.html). 
 
YOUR CONCERNS 
If you have any concerns about these terms of submission, please contact info@wwarn.org. 
I accept and agree to abide by these Terms of Submission 
 
Signature………………………………………. 
 
Name…………………………………………… 
Date…………………………………………….. 
 
 

SAP_001.pdf: 
CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
1 
 
 
TACT-CV 
 
 
 
 
STATISTICAL ANALYSIS PLAN 
 
A multi-centre, open-label randomised trial to assess the efficacy, safety and tolerability 
of the Triple ACT  
artemether lumefantrine+amodiaquine (AL+AQ) compared to the ACT artemether-
lumefantrine (AL) 
 in uncomplicated falciparum malaria in Cambodia and Vietnam 
 
 
Version 1.4 
Date 28th June 2020 
 
WRITTEN BY:  
 
MAVUTO MUKAKA 
 
 
 
 
 
 
 
 
DATE:  
 
 
PRINT NAME 
STATISTICIAN 
 
REVIEWED AND APPROVED BY:  
THOMAS PETO
 
 
 
 
 
 
 
 
 
DATE:   
28TH JUNE 2020 
 
PRINT NAME 
PROJECT COORDINATOR 
 
ARJEN DONDORP
 
 
 
 
 
 
 
 
 
DATE:   
28TH JUNE 2020 
 
PRINT NAME 
PRINCIPAL INVESTIGATOR 
28th JUNE 2020


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
2 
 
Table of Contents 
3............................................................................................................................................. 3 
1. 
Analysis Considerations ............................................................................................... 3 
1.1 
General Analysis Approach ...................................................................................... 3 
1.2 
Data integrity ............................................................................................................ 4 
1.3 
Data cleaning and verification ................................................................................... 4 
1.4 
Locking the dataset .................................................................................................. 4 
1.5 
Data format and Analysis logs .................................................................................. 4 
1.6 
Interim analyses ....................................................................................................... 4 
2. 
Introduction .................................................................................................................. 5 
2.1. 
Study objectives and endpoints ................................................................................ 5 
2.1.1. 
Primary objective .................................................................................................. 5 
2.1.2. 
Primary endpoint ................................................................................................... 5 
2.1.3. 
Secondary objectives and endpoints ..................................................................... 5 
2.2. 
Study design ............................................................................................................ 8 
2..2.1 
Determination of sample size ................................................................................ 9 
3. 
Data Analysis ..............................................................................................................10 
3.1 
Trial Profile ..............................................................................................................10 
3.2 
Demographics and other baseline characteristics ....................................................11 
3.3 
Day 42 Efficacy assessments of the ACT (AL) and matching TACT (AL+AQ) .........12 
Primary objective ...............................................................................................................12 
Primary endpoint ................................................................................................................12 
3.4 
Fever clearance and parasite clearance during treatment, by treatment allocation ..16 
3.5 
Safety assessments and tolerability (Incidence of adverse events and serious 
adverse events by study arms within the first 42 days, incidence of adverse events 
concerning markers of hepatic or renal toxicity such as billirubin, ALT, AST, Alkaline 
Phosphatase and creatinine; and QTc Prolongation) .........................................................18 
3.6 
Comparison of changes in the electrocardiogram (such as prolongation of the QTcB-
interval) in patients treated with TACT versus standard ACT .............................................19 
3.7 
Genetic markers of artemisinin (particularly Pfkelch13) and partner drug resistance
 
23 
3.8 
Representability/accuracy of parasite genome sequencing from dry blood spots for 
the genome sequencing results from leukocyte depleted blood samples ...........................24 
3.9 
Differences at the transcriptome level in artemisinin and partner drug sensitive and 
resistant P. falciparum in order to increase the understanding of mechanisms of resistance
 
24 
3.10 
Comparison of clearance dynamics estimated with quantitative PCR measurements 
of parasite loads versus microscopy ..................................................................................24 
3.11 
Measurement and comparison the incidence and duration of gametocyte carriage in 
patients with antimalarial sensitive and resistant malaria before and after treatment with 
primaquine in addition to the ACT (AL) or matching TACT (AL+AQ) ..................................24 
3.12 
DNA and RNA measurement methods for quantification of male and female 
gametocytes. .....................................................................................................................28 
3.13 
ex vivo susceptibility profiles of P. falciparum isolates across geographic regions ...28 
3.14 
Pharmacokinetic profiles of ACT (AL) and Triple ACT (AL+AQ) components ..........29 
3.15 
Change in haematocrit according to geographical location and study arm and 
stratified according to G6PD status ....................................................................................30 
Change in hematocrit on day 1 to 7, 14, 21, 28, 35 and 42 according to geographical 
location and study arm, stratified for G6PD status will be summarized and analyzed. A 
detailed analysis plan will be prepared separately. .............................................................30 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
3 
 
 
 
3.  
1. 
Analysis Considerations 
 
1.1 General Analysis Approach 
The main analytic strategy for the primary outcome will be the intention-to-treat (ITT) principle. 
Patients will be analysed according to the arm of randomisation irrespective of the treatment 
that was actually given. Kaplan Meier 1st event analyses will be the main approach to estimate 
efficacy. In addition, efficacy using proportions will also be presented to allow comparison with 
previous malaria trials. The 95% confidence intervals for efficacy estimates will be presented, 
along with risk differences, and hazard ratios as appropriate. Statistical significance will be 
declared at the 5% level. The analyses will be based on the number of participants that have 
actually been recruited by the trial end or trial closure. 
There is a fortuitous inverse correlation between parasite susceptibility to lumefantrine (which 
is included in the ACT, and hence also in the Triple ACT) and amodiaquine (which is included 
only in the Triple ACT). Amodiaquine was chosen as the third drug partly because of this 
inverse correlation, and partly because its anti-malarial effects persist about as long as those 
of lumefantrine. In patients treated with both these drugs, therefore, neither will persist for 
much longer than the other; this could help avoid or delay the emergence of resistance.  
Hence, in addition to the randomised comparison, a well-documented case series reliably 
treated with Triple ACT (artemether-lumefantrine plus amodiaquine) is of interest. Likewise, a 
well-documented case series assessing the efficacy in various locations in the late 2010s of 
the ACT is also of interest, as artemether-lumefantrine has not previously been widely used 
in areas where artemisinin resistance is common. The planned analyses will assess the 
efficacy of each regimen separately against PCR-corrected recrudescence (particularly for 
artemisinin-resistant Pfkelch13 mutants), in addition to the randomised comparison of the two 
regimens to assess the effects on Pf recrudescence of adding amodiaquine.  
The assessment of Triple ACT against ACT will directly compare the two regimens (to provide 
generalizable evidence on the adverse events of adding the third drug). On the other hand, 
future treatment recommendations depend on the individual efficacy estimates of each 
regimen independently. 
These analyses will be followed by the per protocol (PP) analysis. In this analysis only those 
that adhered to the protocol with respect to the primary outcome will be included for analysis 
of the primary outcome. All inferences will be based on the ITT analyses, but the PP will be 
attached in an appendix to be consistent with reporting guidelines for antimalarial efficacy 
studies (WHO 2009) - as randomization is potentially compromised by PP analysis. 
Key secondary endpoints such as parasite clearance parameters (e.g. half-lives), safety and 
tolerability data will be analysed by ITT approach as for the primary study outcome. In this 
analysis, patients will be analysed according to the arm of randomisation irrespective of the 
treatment that was actually given and all patients will be included in the analyses irrespective 
of their follow-up status as long as they have parasite half-life data. In the event of some 
subjects being withdrawn at baseline and not receiving any does of study drugs will be 
removed from the ITT analyses as there was no intention to treat these patients.  These will 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
4 
 
also be followed by the per protocol analyses. All other secondary analyses will follow the per 
protocol approach. Withdrawals and losses to follow up will not affect the analyses of this data 
as long as the relevant data needed for these analyses is available prior to withdrawal or loss 
to follow up.  
Data analysis will performed using Stata 15 or higher, StataCorp, 4905 Lakeway Drive 
College Station, Texas 77845 USA   
 
1.2 Data integrity 
This study will be conducted in compliance with the protocol, relevant Standard Operating 
Procedures (SOPs), Good Clinical Practice (GCP) and the applicable regulatory 
requirement(s). All the analyses will be performed on clean data only. 
 
1.3 Data cleaning and verification  
All data will be cleaned and verified prior to statistical analysis. The study site will be visited 
by the Monitor periodically at times agreed on with the Investigator. At the time of each 
monitoring visit, the Monitor will review the completed CRFs to ascertain that all items have 
been completed and that the data provided are accurate and obtained in the manner specified 
in the protocol. The Monitor will also check that the data in the CRF are consistent with the 
clinical records (Source Data Verification [SDV]) and that study results are recorded 
completely and correctly. The data manager will ensure that clean data is submitted to the 
statistician for analysis. The statistician will cross-check that the available data for analysis is 
clean. Any data cleaning queries will need to be resolved before statistical analyses. 
 
1.4 Locking the dataset  
After data cleaning and responding to all data queries, the clean data will be locked normally 
in the database that was used for data capturing. The data may also be locked and stored in 
other user-friendly formats such as MS Excel and Stata. The locked data will be stored at an 
identifiable secure place and should be available to the relevant researchers upon request 
following proper request procedures. The data will also be in other robust backup media. 
 
1.5 Data format and Analysis logs  
Prior to dispensing data to the trial statistician, the head of data management will make sure 
that the data to be sent to the trial statistician is clean. This will help the statistician to provide 
the analysis results in a timely manner as there will be a reduced amount of queries if clean 
data is provided to the trial statistician. Data will be given to the Trial Statistician by the head 
of Data Management (or designated person) in a format that is compatible with statistical 
software reading. Statistical analyses will be performed in Stata, version 15 or higher.  
Statistical programs and output logs will be kept for all analysis and made available upon 
request. 
 
1.6 Interim analyses  
We plan to have two interim analyses on clinical, laboratory and electrocardiographic data to 
assess the safety of the novel TACT (AL+AQ). The interim reports will be reviewed by DSMB. 
The interim analyses will be performed after the first 60 patients and after 180 patients. After 
this, safety analysis will be performed based on recommendations by the DSMB. No stopping 
rules both statistical as well as clinical will be specified. The need to stop the trial will be based 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
5 
 
on the perception of the accumulating data by the DSMB. The main strategy of analysis will 
focus on safety data although efficacy data may also be presented.  
 
The Trial coordinator in collaboration with the Trial Statistician will produce the report for the 
DSMB. Only relevant data included in a specific interim report will be made available to the 
DSMB members at the time of sending the report. During a DSMB meeting, the report will be 
presented to the members by the study coordinator in line with the meeting agenda. For a 
normally scheduled DSMB meeting, the report will be sent out to the members at least a week 
before the meeting.  
 
 
2. 
Introduction 
This is a multicentre study with 4 centres in 2 different countries. The study was designed in 
a way that each centre has 80% power to detect a difference in the efficacy outcomes, if they 
exist.  
The principle that multiple drugs with independent mechanisms of action prevent the 
emergence of drug resistance is proven in a range of human diseases. In HIV and tuberculosis 
for example, the occurrence and spread of drug resistance can be prevented by use of a 
combination of three or more antiretroviral or antimycobacterial therapies respectively, but 
until now this was not thought necessary in malaria. In malaria there is a fortuitous inverse 
correlation between susceptibility to amodiaquine and lumefantrine which will be exploited in 
the TACT (AL+AQ). 
 
2.1. 
Study objectives and endpoints 
2.1.1. Primary objective  
To compare the efficacy of the TACT (aretemether-lumefantrine+amodiaquine) versus 
the ACT (artmether-lumefantrine) as defined by the 42-day PCR corrected adequate 
clinical and parasitological response (ACPR). 
 
2.1.2. Primary endpoint 
The Primary endpoint is the 42-day PCR corrected efficacy defined as adequate clinical 
and parasitological response (ACPR) by Kaplan Meier 1st event analyses. 
  
(NB, WHO definition of ACPR: absence of parasitaemia at day 42 irrespective of axillary 
temperature and without previously meeting any of the WHO criteria for early or late 
treatment failure, or late parasitological failure.) 
 
2.1.3. Secondary objectives and endpoints 
 
To compare the efficacy of the TACT (artemether-lumefantrine+amodiaquine) versus ACT 
(artemether-lumefantrine) as defined by the 42-day PCR according to site/geographical 
region 


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STATISTICAL ANALYSIS PLAN 
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o 42-day PCR corrected efficacy defined as adequate clinical and parasitological 
response (ACPR) according to site/geographical region 
 
To assess and compare P. falciparum parasite clearance rates of standard AL and 
matching AL+AQ.  
o Parasite clearance half-life assessed by microscopy as primary parameter to 
determine parasite clearance 
o Additional parameters of parasite clearance dynamics 
 
To assess and compare fever clearance rates of standard AL and matching AL+AQ.  
o Fever clearance time (i.e. the time taken for the tympanic temperature to fall below 
37.5˚C and remain there for at least 24 hours)  
 
To compare the safety and tolerability of AL+AQ versus standard AL 
o Incidence of adverse events and serious adverse events by study arms within the 
first 42 days. 
o Incidence of adverse events concerning markers of hepatic or renal toxicity such 
as billirubin, ALT, AST, Alkaline Phosphatase and creatinine 
o Proportion of patients that reports completing a full course of observed AL+AQ or 
AL without withdrawal of consent or exclusion from study because of drug related 
serious adverse event 
 
To compare changes in the electrocardiogram (such as prolongation of the QTc-interval) 
in patients treated with AL+AQ versus standard AL 
o Incidence of prolongation of the QTc-interval above 500 ms or >60ms above 
baseline values. 
o Prolongation of QTc-interval compared to baseline at timepoint H4, H24, H28, H48, 
H52, H60, H64 and between these timepoints 
 
To assess the spread of genetic markers of artemisinin (such as Kelch13 mutations) and 
partner drug resistance and identify additional genetic determinants of drug resistance.  
o Prevalence of Kelch13 mutations of known functional significance 
o Prevalence/incidence of other genetic markers of antimalarial drug resistance such 
as MDR1 copy number and MDR1 mutations 
 
To assess and increase the representability/accuracy of parasite genome sequencing 
from dry blood spots for the genome sequencing results from leukocyte depleted blood 
samples 
o Correlation between SNPs measured in dry blood spots and whole genome 
sequencing in leukocyte depleted blood samples 
o Genome wide association with in vivo/in vitro sensitivity parasite phenotype 
 
To identify differences at the transcriptome level in artemisinin and partner drug sensitive 
and resistant P. falciparum in order to increase the understanding of mechanisms of 
resistance  
o Transcriptomic patterns at t=0 and t=6h after start of treatment comparing sensitive 
and resistant parasites 


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STATISTICAL ANALYSIS PLAN 
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 
To compare clearance dynamics estimated with quantitative PCR measurements of 
parasite loads versus microscopy  
o Correlation between qPCR based versus microscopy-based assessments of 
parasite clearance dynamics up to day 14 
 
To measure and compare the incidence and duration of gametocyte carriage in patients 
with antimalarial sensitive and resistant malaria before and after treatment with 
TACT(AL+AQ) and matching ACT (AL)  
o Proportion of patients with gametocytemia before, during and after treatment with 
TACT (AL+AQ) or ACT (AL), assessed at admission, up to day 14, stratified by 
presence of gametocytes at enrolment 
 
To develop DNA and RNA measurement methods for quantification of male and female 
gametocytes.  
o Levels of RNA transcription coding for male or female specific gametocytes at 
admission up to day 14, stratified by the presence of gametocytes at enrolment 
 
To compare ex vivo susceptibility profiles of P. falciparum isolates across geographic 
regions  
o In vitro sensitivity of P. falciparum to artemisinins and partner drugs according to 
study sites and genotype 
 
To assess pharmacokinetic and pharmacodynamic interactions between AL and AL+AQ 
o Pharmacokinetic profiles and interactions of artemisinin-derivatives and partner 
drugs (Cmax and AUC) in 20 ACT treated and 20 AL+AQ treated patients of both 
study arms in Vietnam 
o Day 7 drug levels of partner drugs in association with treatment efficacy and 
treatment arm 
 
To obtain additional safety data (in particular incidence and rate of hemolysis) on the 
deployment of single low dose primaquine, stratified according to G6PD status  
o Change in hematocrit on day 1 to 7, 14, 21, 28, 35 and 42 according to 
geographical location and study arm, stratified for G6PD status 
 
To obtain additional data on the effect of the host genotype on the pharmacokinetics and 
pharmacodynamics of antimalarials 
o Correlation between the host genotype and the pharmacokinetics and 
pharmacodynamics of antimalarials 
 
To compare clearance dynamics estimated with plasma HRP2 levels of parasite loads 
versus microscopy 
o Correlation between HRP2 based versus microscopy based assessments of 
parasite clearance dynamics 
 
To obtain data on the place of residence, work, recent travel history and mobile phone 
usage in order to improve the understanding of locations of malaria transmission and 
possible routes spread of malaria and artemisinin resistance. 
o Data on the place of residence, work, recent travel history and mobile phone use 


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STATISTICAL ANALYSIS PLAN 
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 
To obtain data and GPS mapping on a select group of participants and their peers relating 
to their understanding of the behaviours and risk factors associated with malaria infection 
in order to improve understanding of local malaria transmission.   
o Data and GPS mapping for a select group of participants and their peers in relation 
to behaviours and risk factors associated with malaria infection 
 
2.2. 
Study design  
 
Brief Description 
This is an Open-label Randomised Trial comparing standard AL treatment with matching triple 
artemisinin-based combination therapies (AL+AQ), evaluating efficacy in sites experiencing ACT 
failure and safety, tolerability and artemisinin and partner drug resistance in all sites. The study 
sites are shown in figure 1 below. Patients will be randomized to artemether-lumefantrine +/- 
amodiaquine. 
 
Figure 1 Study sites 
 
There are 3 centres of recruitment in 2 different countries (Cambodia and Vietnam). 
200 patients will be recruited from each of three centres: western Cambodia, north-
eastern Cambodia, and from two sites in southern Vietnam (combined as one centre, 
enrolling patients under competitive recruitment). 
 


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STATISTICAL ANALYSIS PLAN 
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2..2.1 
Determination of sample size 
The sample size calculation is based on the primary endpoint of the 42-day PCR corrected ACPR. 
Our hypothesis is that artemether-lumefantrine+amodiaquine is superior to artemether-
lumefantrine. Earlier efficacy studies of artemether-lumefantrine found an efficacy of 82.4 and 
86.5% [13, 14]. Artesunate-mefloquine has not been used for almost a decade in Cambodia (up 
to 2016, when it became the first line treatment again). This has decreased the pfMDR1 copy 
number counts in the Cambodian population [15]. Given the association of pfMDR1 copy number 
and lumefantrine sensitivity this has potentially increased the efficacy of artemether-lumefantrine, 
despite increasing levels of artemisinin resistance [16]. Therefore, we assume the efficacy of 
artemether-lumefantrine to be 90%. Because of the additive therapeutic effect of amodiaquine we 
assume superiority of artemether-lumefantrine+amodiaquine with an efficacy of 99%.  
 
A sample size of 100 patients per arm (power 0.80 and alpha=0.05) would allow us to detect this 
superiority. This leads to a total of 200 subjects needed per site. In Vietnam, the 2 sites will recruit 
a cumulative 200 subjects (the sample size for this country) through competitive-recruitment due 
to the expected low recruitment rates at each site. A total of 600 patient will be recruited for the 
study. 
  
The following Stata command: “sampsi 0.99 0.90, alpha (0.05) power (0.8) nocontinuity” was 
used (Stata 14.0). 
 
 
 


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STATISTICAL ANALYSIS PLAN 
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3. 
Data Analysis 
3.1 
Trial Profile 
The number of patients who will be screened, reasons for non-enrolment, number of patients 
randomized, number of patients lost to follow up and the number of patients assessed for 42-
day endpoint will be summarised in a CONSORT flow diagram, figure 2, below.
 
 
Figure 2 Consort Trial Profile by Arms 
ITT Kaplan-Meier analyses, by follow-up week, of time to Pf recrudescence as a first 
event  (The K-M analyses are to week 6, censored at the first recurrence if it is not a Pf 
recrudescence.)  
 
 
Assessed for eligibility (n=XXXX) 
Excluded (n=XXXX) 
     XX Signs of severe/complicated malaria 
     XX Haematocrit < 25% or Hb < 8 g/dL at 
screening 
     XX Acute illness other than malaria requiring 
treatment 
     XX Pregnancy or breast feeding 
     XX Artemisinin therapy within the previous 7days 
     XX Allergy to artemisinins or AL or AL+AQ 
     XX Previous splenectomy 
     XX QTc-interval>450 ms  
XX Documented history of cardiac conduction
Enrolled (n=XXXX) and randomised   
n=XXX assigned AL 
     Cambodia, site 1 (n=XXX), site 2 (n=XXX) 
     Vietnam, site 1 (n=XXX), site 2 (n=XXX) 
n=XXX assigned AL+AQ 
     Cambodia, site 1 (n=XXX), site 2 (n=XXX) 
     Vietnam, site 1 (n=XXX), site 2 (n=XXX) 
XX Significant non-compliance with treatment 
regimen 
XX An adverse event which requires 
discontinuation 
XX Disease progression which requires 
discontinuation 
XX Development of severe malaria 
XX Recurrent parasitaemia 
XX Loss to follow up 
XX Significant non-compliance with treatment 
regimen 
XX An adverse event which requires 
discontinuation 
XX Disease progression which requires 
discontinuation 
XX Development of severe malaria 
XX Recurrent parasitaemia 
XX Loss to follow up 
AL (n=XX) PP analysis 
Received AL (n=XXX) Safety/ITT analysis 
Received AL+AQ (n=XXX) Safety/ITT analysis 
AL+AQ (n=XX) PP analysis 


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3.2 Demographics and other baseline characteristics 
 
The following baseline characteristics will be described by study arm in table 1 (below). Variables 
such as age, heart rate, respiratory rate will be summarized using median and interquartile rage. 
Continuous variables such as weight, height, systolic and diastolic blood pressure, QTc-intervals, 
haemoglobin will be summarized using mean ± standard deviation. Parasitaemia at baseline will 
be described as geometric mean and range.  Categorical variables such as sex, presence of fever 
and gametocytaemia at baseline will be summarized using frequencies and percentages. 
 
 
 
Table 1. Baseline Characteristics of the Patients 
 
Characteristics 
Artemether-
Lumefantrine 
(N=XXX) 
Artemether-
Lumefantrine 
+Amodiaquine 
(N=XXX) 
Total 
(N=XXX) 
Age (years), med (IQR) 
XX.X (XX.X-XX.X) 
XX.X (XX.X-XX.X) 
XX.X (XX.X-XX.X) 
Male sex, n (%) 
XX (XX.X) 
XX (XX.X) 
XX (XX.X) 
Baseline tympanic 
temperature >37·5°C, n 
(%) 
XX (XX.X) 
XX (XX.X) 
XX (XX.X) 
Weight (kg), mean (SD) 
XX.X (XX.X) 
XX.X (XX.X) 
XX.X (XX.X) 
Height (cm) mean (SD) 
XX.X (XX.X) 
XX.X (XX.X) 
XX.X (XX.X) 
Heart rate 
(beats/minute), med 
(IQR) 
XX.X (XX.X-XX.X) 
XX.X (XX.X-XX.X) 
XX.X (XX.X-XX.X) 
Respiratory rate 
(breaths/minute), med 
(IQR) 
XX.X (XX.X-XX.X) 
XX.X (XX.X-XX.X) 
XX.X (XX.X-XX.X) 
Systolic blood pressure 
(mmHg) , mean (SD) 
XX.X (XX.X) 
XX.X (XX.X) 
XX.X (XX.X) 
Diasystolic blood 
pressure (mmHg) , mean 
(SD) 
XX.X (XX.X) 
XX.X (XX.X) 
XX.X (XX.X) 
QTcB interval 
(miliseconds) , mean 
(SD) 
XX.X (XX.X) 
XX.X (XX.X) 
XX.X (XX.X) 
Hb (g/dL) , mean (SD) 
XX.X (XX.X) 
XX.X (XX.X) 
XX.X (XX.X) 
Parasites/µL,  
geometric mean (range) 
XX.X (XX.X-XX.X) 
XX.X (XX.X-XX.X) 
XX.X (XX.X-XX.X) 
Gametocytemia present 
at baseline, n/N (%) 
XX (XX.X) 
XX (XX.X) 
XX (XX.X) 


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3.3 Day 42 Efficacy assessments of the ACT (AL) and matching TACT (AL+AQ)  
 
Primary objective  
To assess and compare by ITT analyses the efficacies of the ACT and Triple ACT in achieving 
the primary endpoint of durable cure of the original infection. Failure is pre-defined in terms of 
PCR-corrected 42-day adequate clinical and parasitological response. This objective is equivalent 
to avoiding PCR-confirmed P. falciparum recrudescence. 
 
Primary endpoint 
42-day PCR-corrected Pf recrudescence: K-M 1st event analyses, censored at re-infection 
 
Handling of missing data 
In the ITT Kaplan-Meier/survival analysis, participants who are lost to follow up, or who have Pf 
or Pv reinfections or inconclusive PCR correction, will be censored or treated as competing 
risks, as appropriate, from the moment of occurrence of one of these events. Protocol 
deviations/violations (such as those that may switch treatment due to severe malaria, or those 
who may get rescue therapy) that are followed up to the end of the study will be analysed as 
having completed follow up in the ITT, or censored at time of their last follow up. 
In the ITT analyses where efficacy will be presented as a proportions, the outcomes for day 42 
for the patients that had definitive follow up shortly after day 42 will be included according to 
their status (success or recrudescent infection) as described in (Stepniewska & White 2006). 
This is because it is anticipated that some participants will be lost to follow-up (i.e. not seen 
during the 42-day follow-up visit) but that follow up can be performed shortly thereafter. In the 
standard operating procedures (SOPs) it is arranged that, whenever possible, participants will 
be seen as soon as possible after day 42 to ensure they have no malaria parasites. If not seen 
by day 90 no further effort will be made to see these participants. Based on a recommendation 
from Stepniewska and White 2006, these patients who were lost to follow-up but were followed 
up subsequently should not be censored from efficacy assessments if they have remained well 
and are PCR negative, as it is unlikely that a recrudescent malaria infection would have 
occurred during the missed appointment period which then rapidly and symptomless self-cured. 
Thus, such patients will be included as successes and will be clearly identified and documented 
when presenting the results to be easily distinguished from those that were followed up on day 
42 as scheduled. If a participant is seen after day 42 with a recrudescent infection, this will be 
reported along with the interval period in which it emerged (time since last seen to time 
detected) will be reported to estimate the onset of recrudescence.  
In per protocol analyses, for the reporting of proportions, it will be considered that participants 
not seen at day 42 have not completed follow up. Missing follow up visits between D7 and D42 
will not cause participants to be censored if follow up at D42 is complete, and these participants 
will not be removed from per protocol analyses. In the PP analysis in which efficacy will be 


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STATISTICAL ANALYSIS PLAN 
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reported as proportions of outcomes, patients in which study drugs are discontinued and/or 
endpoints are not available due to other reasons (such as withdrawal from the study, loss to 
follow up, Pf or Pv reinfections and inconclusive PCR correction) will be excluded from the 
analysis. 
Similar analyses will be performed for the PCR uncorrected data as well. Where applicable, we 
will also report the cumulative reinfection rate. The treatment efficacy, 95% confidence intervals 
and p-values for the comparison of ACT (AL) with matching TACT (AL+AQ) will be presented as 
outlined in tables 2 and 3 below. 
 
 
 


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Table 2 Comparison of Day 42 PCR corrected Efficacy using survival methods of AL vs 
AL+AQ, overall and by site  
 
PCR corrected ACPR at 
day 42,  
Survival percentage 
(efficacy), % 
Hazard ratio (95% CI) 
p-value 
AL 
(N=XXX) 
AL+AQ 
(N=XXX) 
Overall (all sites - 
pooled) 
 XX.X 
XX.X 
XX.X (XX.X - XX.X) 
X.XXX  
By sites 
 
 
 
  
     Cambodia, west 
XX.X 
XX.X 
XX.X (XX.X - XX.X) 
X.XXX 
     Cambodia, east 
XX.X 
XX.X 
XX.X (XX.X - XX.X) 
X.XXX 
     Vietnam 
XX.X 
XX.X 
XX.X (XX.X - XX.X) 
X.XXX 
 
Table 3 Comparison of Day 42 PCR corrected efficacy as proportions of AL vs AL+AQ, 
overall, and by site  
 
PCR corrected ACPR at day 42,  
n/N, (%)  
Risk difference (95% CI) p-value 
AL 
(N=XXX) 
AL+AQ 
(N=XXX) 
Overall 
XX/XXX 
(XX.X) 
XX/XXX  
(XX.X) 
XX.X (XX.X - XX.X) 
X.XXX  
By sites 
 
 
  
 
     Cambodia, 
west 
XX/XXX 
(XX.X) 
XX/XXX  
(XX.X) 
XX.X (XX.X - XX.X) 
X.XXX 
     Cambodia, 
east 
XX/XXX 
(XX.X) 
XX/XXX  
(XX.X) 
XX.X (XX.X - XX.X) 
X.XXX 
     Vietnam 
XX/XXX 
(XX.X) 
XX/XXX  
(XX.X) 
XX.X (XX.X - XX.X) 
X.XXX 
 
 
 
 


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Table 3b PCR-confirmed weekly numbers with Pf recrudescence, and with new malarial 
re-infection without Pf recrudescence 
 
 
Recrudescence 
of P. falciparum 
Re-infection by a 
new P. falciparum 
Re-infection by 
P. vivax only 
Day * 
tested 
ACT 
(AL) 
Triple ACT 
(AL+AQ) 
ACT 
(AL) 
Triple ACT 
(AL+AQ) 
ACT 
(AL) 
Triple ACT 
(AL+AQ) 
Day 7 
x 
x 
x 
x 
x 
x 
Day 14 
x 
x 
x 
x 
x 
x 
Day 21 
x 
x 
x 
x 
x 
x 
Day 28 
x 
x 
x 
x 
x 
x 
Day 35 
x 
x 
x 
x 
x 
x 
Day 42 
x 
x 
x 
x 
x 
x 
Total  
x 
x 
x 
x 
x 
x 
* Rounded to nearest multiple of 7, with tests just after Day 42 rounded to Day 42 
 
Note to be added to state falciparum infections not due to artemisinin-resistant Pf  
 
 
 
 


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STATISTICAL ANALYSIS PLAN 
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3.4 Fever clearance and parasite clearance during treatment, by treatment allocation 
 
Early parasite clearance rates should be dominated by the effects of the artemisinin rather than 
the partner drugs. Hence, little effect on them is expected from the random allocation to Triple 
ACT rather than ACT. Similar analyses, split by Pfkelch13 mutation status (determined for all 
trial participants) rather than trial treatment, will be given as Table 5 and plotted as Figure 3. 
 
 Table 4 Fever clearance and parasite clearance by allocated treatment 
 
   
ACT 
(AL) 
N=XXX 
  Triple ACT 
(AL+AQ) 
N=XXX 
Both  
groups 
N=XXX 
P- 
value 
(2-sided) 
Hours to fever clearance, mean (SD) 
(baseline to start of first 24h period <37.5C;  
temperature was recorded every 6 hours)  
     Western Cambodia 
XX.X (X.X) 
XX.X (X.X) 
XX.X (X.X) 
X.XXX 
     Eastern Cambodia 
XX.X (X.X) 
XX.X (X.X) 
XX.X (X.X) 
X.XXX 
     Vietnam 
XX.X (X.X) 
XX.X (X.X) 
XX.X (X.X) 
X.XXX 
All 3 study sites 
XX.X (X.X) 
XX.X (X.X) 
XX.X (X.X) 
X.XXX 
Day 3 blood smear still positive, n/N (%) 
(taking prior clearance or discharge as negative) 
     Western Cambodia 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
     Eastern Cambodia 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
     Vietnam 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
All 3 study sites 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
Parasite clearance half-life longer than 5 hours, n/N (%) 
(excluding patients with initial count insufficient to estimate half-life) 
     Western Cambodia 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
     Eastern Cambodia 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
     Vietnam 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
All 3 study sites 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
Parasite clearance half-life in hours, mean (SD) 
(excluding patients with initial count insufficient to estimate half-life) 
     Western Cambodia 
X.X (X.X) 
X.X (X.X) 
X.X (X.X) 
X.XXX 
     Eastern Cambodia 
X.X (X.X) 
X.X (X.X) 
X.X (X.X) 
X.XXX 
     Vietnam 
X.X (X.X) 
X.X (X.X) 
X.X (X.X) 
X.XXX 
All 3 study sites 
X.X (X.X) 
X.X (X.X) 
X.X (X.X) 
X.XXX 


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Table 5 Fever clearance and parasite clearance by Pfkelch13 status 
 
 
 
 
 
Relevant Pfkelch13 mutation detected? 
 
   
Yes 
(N=XXX) 
No (wild type) 
(N=XXX) 
Difference, 
Yes minus No 
P-value 
(2-sided) 
Hours to fever clearance, mean (SD) 
(baseline to start of first 24h period <37.5C;  
temperature was recorded every 6 hours) 
     Western Cambodia 
XX.X (X.X) 
XX.X (X.X) 
XX.X (X.X) 
X.XXX 
     Eastern Cambodia 
XX.X (X.X) 
XX.X (X.X) 
XX.X (X.X) 
X.XXX 
     Vietnam 
XX.X (X.X) 
XX.X (X.X) 
XX.X (X.X) 
X.XXX 
All 3 study sites 
XX.X (X.X) 
XX.X (X.X) 
XX.X (X.X) 
X.XXX 
Day 3 blood-smear still positive, n/N (%) 
(taking prior clearance or discharge as negative) 
     Western Cambodia 
X.X (X.X) 
X.X (X.X) 
X.X (X.X) 
X.XXX 
     Eastern Cambodia 
X.X (X.X) 
X.X (X.X) 
X.X (X.X) 
X.XXX 
     Vietnam 
X.X (X.X) 
X.X (X.X) 
X.X (X.X) 
X.XXX 
All 3 study sites 
X.X (X.X) 
X.X (X.X) 
X.X (X.X) 
X.XXX 
Parasite clearance half-life longer than 5 hours, n/N (%) 
(excluding patients with initial count insufficient to estimate half-life) 
     Western Cambodia 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
     Eastern Cambodia 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
     Vietnam 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
All 3 study sites 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
Parasite clearance half-life in hours, mean (SD) 
(excluding patients with initial count insufficient to estimate half-life) 
     Western Cambodia 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
     Eastern Cambodia 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
     Vietnam 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 
All 3 study sites 
XX/XXX (XX) 
XX/XXX (XX) 
XX/XXX (XX) 
X.XXX 


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Figure 3 Parasite clearance half-life by PfKelch13 mutation status and treatment  
 
 
3.5 Safety assessments and tolerability (Incidence of adverse events and serious adverse 
events by study arms within the first 42 days, incidence of adverse events concerning 
markers of hepatic or renal toxicity such as billirubin, ALT, AST, Alkaline Phosphatase 
and creatinine; and QTc Prolongation) 
Safety analyses will be based on the whole population that get administered the study drug. That 
is, the safety and tolerability data will be pooled from all the sites that received the same 
antimalarial treatment.  
The safety and tolerability of AL+AQ versus AL will be assessed by comparing the frequency (%) 
of adverse events and serious adverse events, with particular attention to abdominal pain, appetite 
perturbation, biochemical markers of renal toxicity (creatinine) and hepatic toxicity (such as 
billirubin, ALT, AST, Alkaline Phosphatase) and markers of bone marrow toxicity (Haemoglobin, 
White blood cell count and differentiation, platelet counts), using the Fisher’s exact test. 
Safety data will be presented in tabular and/or graphical format and summarized descriptively. Any 
clinically relevant abnormalities or values of potential clinically concern will be described. Patients 
will be analysed following the intention to treat analysis. All adverse event summaries will refer to 
treatment emergent adverse events, i.e. adverse events that newly started or increased in intensity 
after the study drug administration. Adverse events will be graded according to Division of AIDS 
table for grading the severity of ADULT AND PEDIATRIC adverse Events Version 2.0, November, 
2014. The relevant DAIDS table will be appended to this document. The safety and tolerability 
summaries will be presented in table 5. A previous randomised comparison of these two regimens 
(R van der Pluijm et al, 2020 https://doi.org/10.1016/S0140-6736(20)30560-2 ), which involved a 


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STATISTICAL ANALYSIS PLAN 
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total of 575 patients, reported an increased incidence of bradycardia and possible increases in 
vomiting and in creatinine. 
 
3.6 Comparison of changes in the electrocardiogram (such as prolongation of the QTcB-
interval) in patients treated with TACT versus standard ACT 
Incidence of prolongations of the QTc-interval>60 miliseconds compared to baseline or >500 
miliseconds and bradycardia (heart rate<54 beats/minute) at least once will be summarized 
in table 5. Absolute changes in the QTc-interval will be summarized as in tables 7 and 8, and will 
be graphically displayed by arm as in figure 5 (only QTcB). Comparisons on the changes in the 
QTcB-interval will be made using the unpaired t-test for each specific time point.  
 
Table 5 Incidence of adverse events within first 42 days by arm 
AL 
AL+AQ 
p-
value 
Number of subjects 
XX 
XX 
 
Vomiting/number of treatments, n/N, (%) 
X/XX (X.X) 
X/XX (X.X) 
X.XXX 
Serious adverse events (SAEs) , n/N, (%) 
XX 
XX 
X.XXX 
Possible, probable or definite drug related 
SAEs, n/N, (%)  
X/XX (X.X) 
X/XX (X.X) 
X.XXX 
QTc >60ms above baseline (H64) 
X/XX (X.X) 
X/XX (X.X) 
X.XXX 
QTc >500ms (H64) 
X/XX (X.X) 
X/XX (X.X) 
X.XXX 
Bradycardia (<=54 bpm) (H64) 
X/XX (X.X) 
X/XX (X.X) 
X.XXX 
Grading of adverse events, n/N, (%) 
1-2 
3-4 
1-2 
3-4 
 
Symptoms 
  
  
  
  
 
Headache 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Fatigue  
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Abdominal pain 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Loss of appetite 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Nausea 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Vomiting 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Diarrhea 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Itching  
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Dizziness  
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Blurred vision 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Sleeping disturbance 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
20 
 
Total 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Laboratory abnormalities 
  
  
  
  
 
Creatinine 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Total bilirubin 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Alkaline phosphatase 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Alanyl transfarase (ALT) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Aspartate transfarase (AST) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Hemoglobin decrease 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Leukocytopenia 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Neutrocytopenia 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Lymphocytopenia 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
Thrombocytopenia 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X/XX 
(X.X) 
X.XXX 
QTc: QT corrected using Bazett's formula. Adverse event grading: grade1: mild; grade 2: 
moderate; grade 3: severe; grade 4: potentially life-threatening 
Incidence of QTc abnormalities (>60 miliseconds above baseline or >500 miliseconds) and 
bradycardia is defined as a subjects experience these abnormalities at one or more timepoints. 
 
 
The frequency and proportions of patients that report a full course of observed TACT or ACT 
without withdrawal of consent or exclusion from the study because of drug related serious 
adverse events will be summarized for the whole study population. The 95% confidence 
intervals will be reported for the proportions. The proportions will be compared between 
matching drugs. The analyses will be summarised in table 6. 
 
Table 6 Proportions of patients that report a full course of observed TACT or ACT 
AL,  
n/N (%, 95% CI) 
 A+AQ, 
n/N (%, 95% CI) 
Completing full course of study drug  
 
 
 
 
 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
21 
 
Table 7 QTcB interval and change in it since study entry, by time and allocated treatment 
 
ACT (AL) 
Triple ACT (AL+AQ) 
QTcB 
(ms) 
Change  
in QTcB 
p-value 
QTcB 
(ms) 
Change  
in QTcB 
p-value 
Hour   0 
XXX (XX) 
- 
- 
XXX (XX) 
- 
- 
Hour   4 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
Hour 24 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
Hour 28 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
Hour 48 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
Hour 52 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
Hour 60 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
Hour 64 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
 Day   7 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
 Day 28 
XXX (XX) 
XX.X (X.X) 
X.XXX 
XXX (XX) 
XX.X (X.X) 
X.XXX 
 
 
 
Figure 5 Changes in QTc-intervals at hour 64 from baseline by treatment arm 
 
 
 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
22 
 
Table 8: Heart rate and change in it since study entry, by time and allocated treatment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ACT (AL) 
Triple ACT (AL+AQ) 
Heart rate 
(bpm) 
Change in 
heart rate 
p-value 
Heart rate 
(bpm) 
Change in 
heart rate 
p-value 
Hour   0 
XX (XX) 
- 
- 
XX (XX) 
- 
- 
Hour   4 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 
Hour 24 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 
Hour 28 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 
Hour 48 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 
Hour 52 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 
Hour 60 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 
Hour 64 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 
 Day   7 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 
 Day 28 
XX (XX) 
X.X (X.X) 
X.XXX 
XX (XX) 
X.X (X.X) 
X.XXX 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
23 
 
3.7 
Genetic markers of artemisinin (particularly Pfkelch13) and partner drug 
resistance   
All parasite samples collected at baseline and at the times of any recurrences within the first 6 
weeks after recruitment will be genotyped for known markers of resistance to artemisins and to 
the other study drugs. This information will be used to help interpret parasite clearance rates, 
and to help investigate the reasons for any recurrences during the first 6 weeks after baseline. 
 
Table 8 Molecular resistance markers in baseline P. falciparum specimens, by study site 
 
 
Number 
studied, 
N 
Wild-type 
Kelch13 
n (%) 
C580Y 
Kelch13 
n (%) 
Other 
Kelch13  
n (%) 
Plasmepsin 
2/3 amplified 
n (%) 
MDR1 
amplified 
n (%) 
Western 
Cambodia 
XXX 
xxx (xx) 
xxx (xx) 
xxx (xx) 
xxx (xx) 
xxx (xx) 
Eastern 
Cambodia 
XXX 
xxx (xx) 
xxx (xx) 
xxx (xx) 
xxx (xx) 
xxx (xx) 
Vietnam 
XXX 
xxx (xx) 
xxx (xx) 
xxx (xx) 
xxx (xx) 
xxx (xx) 
All three  
study sites 
XXX 
xxx (xx) 
xxx (xx) 
xxx (xx) 
xxx (xx) 
xxx (xx) 
 
 
 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
24 
 
 
3.8 
Representability/accuracy of parasite genome sequencing from dry blood spots 
for the genome sequencing results from leukocyte depleted blood samples 
 
A detailed analysis plan will be prepared separately. 
 
 
3.9 
Differences at the transcriptome level in artemisinin and partner drug sensitive 
and resistant P. falciparum in order to increase the understanding of mechanisms 
of resistance  
A detailed analysis plan for the analysis of the transcriptome will be prepared separately. 
 
3.10 
Comparison of clearance dynamics estimated with quantitative PCR 
measurements of parasite loads versus microscopy 
At each time point the level of agreement (if absolute values will be of interest) or 
correlation (if relative relationship will be of interest) will be assessed between qPCR 
and microscopy parasite load measurements. If the agreement or correlation (whichever 
will be appropriate) will show a consistent relationship/agreement at all time points, the 
data may be pooled across all time points. If it will be deemed appropriate/ feasible, 
half-lives achieved by the two methods will be compared. A detailed analysis plan will be 
prepared separately. 
 
3.11 
Measurement and comparison the incidence and duration of gametocyte carriage 
in patients with antimalarial sensitive and resistant malaria before and after 
treatment with primaquine in addition to the ACT (AL) or matching TACT (AL+AQ)  
The baseline proportion of participants with patent gametocytaemia (gametocyte densities above 
the microscopy level of detection) will be summarized and reported by site. To measure and 
compare the incidence and duration of gametocyte carriage in patients with antimalarial sensitive 
and resistant malaria before and after treatment with primaquine in addition to TACT (AL+AQ) or 
matching ACT (AL). Proportion of patients with gametocytaemia before, during and after 
treatment with primaquine in addition to TACT (AL+AQ) or matching ACT (AL), assessed at 
admission, up to day 14. The patients will be categorized into four gametocytaemia status and 
summarized as presented in the tables 10a and 10b below. McNemar’s test will be used to assess 
whether there will be a significant difference in proportions of gametocytaemia between admission 
and either day 7 or day 14. 
 
 
 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
25 
 
Table 10a Proportion of patients with gametocytaemia at admission and day 7 
Country, site 
AL 
AL+AQ 
 
Admission  Day 
7 
n (%) 
p-
value 
Admission  Day 
7 
n (%) 
p-
value 
Cambodia, 
east 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
 
Yes 
No 
XX 
(XX.X) 
Yes 
No 
XX 
(XX.X) 
 
No 
Yes 
XX 
(XX.X) 
No 
Yes 
XX 
(XX.X) 
 
No 
No 
XX 
(XX.X) 
No 
No 
XX 
(XX.X) 
Cambodia, 
west 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
 
Yes 
No 
XX 
(XX.X) 
Yes 
No 
XX 
(XX.X) 
 
No 
Yes 
XX 
(XX.X) 
No 
Yes 
XX 
(XX.X) 
 
No 
No 
XX 
(XX.X) 
No 
No 
XX 
(XX.X) 
Vietnam 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
 
Yes 
No 
XX 
(XX.X) 
Yes 
No 
XX 
(XX.X) 
 
No 
Yes 
XX 
(XX.X) 
No 
Yes 
XX 
(XX.X) 
 
No 
No 
XX 
(XX.X) 
No 
No 
XX 
(XX.X) 
 
 
 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
26 
 
Table 10b Proportion of patients with gametocytaemia at admission and day 14 
Country, site 
AL 
AL+AQ 
 
Admission  Day 
14 
n (%) 
p-
value 
Admission  Day 
14 
n (%) 
p-
value 
Cambodia, 
east 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
 
Yes 
No 
XX 
(XX.X) 
Yes 
No 
XX 
(XX.X) 
 
No 
Yes 
XX 
(XX.X) 
No 
Yes 
XX 
(XX.X) 
 
No 
No 
XX 
(XX.X) 
No 
No 
XX 
(XX.X) 
Cambodia, 
west 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
 
Yes 
No 
XX 
(XX.X) 
Yes 
No 
XX 
(XX.X) 
 
No 
Yes 
XX 
(XX.X) 
No 
Yes 
XX 
(XX.X) 
 
No 
No 
XX 
(XX.X) 
No 
No 
XX 
(XX.X) 
Vietnam 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
Yes 
Yes 
XX 
(XX.X) 
X.XXX 
 
Yes 
No 
XX 
(XX.X) 
Yes 
No 
XX 
(XX.X) 
 
No 
Yes 
XX 
(XX.X) 
No 
Yes 
XX 
(XX.X) 
 
No 
No 
XX 
(XX.X) 
No 
No 
XX 
(XX.X) 
 
Time to gametocyte appearance will be summarised in a Kaplan-Meier Plot for patients who will 
have no gametocytaemina at admission but develop gametocytemia during follow-up. Similarly, 
time to gametocyte disappearance will be summarised in a Kaplan-Meier Plot for patients who will 
have gametocytaemina either at admission or during follow-up.   
 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
27 
 
 
Figure 6 Kaplan-Meier Plot on gametocyte appearance in patients who have no 
gametocytaemina at admission 
 
 
Figure 7 Kaplan-Meier Plot on gametocyte disappearance in patients who have 
gametocytaemina at admission 
 
 
For those with gametoctytaemia at admission, the duration of patent gametocytemia will be 
summarized using median and interquartilerange (IQR) and/or range in table 10. Mann-Whitney U 
test will be used to test whether the median duration of patent gametocytemia differ according to 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
28 
 
treatment given in each site. We will also assess whether there is correlation between the duration 
of patent gametocytemia and the parasite clearance half-life.  
 
 
 
 
Table 11 Duration of gametocyte carriage in patients with gametoctytaemia at admission 
Country, site 
Duration of gametocyte carriage 
p-value 
 
AL 
AL+AQ 
Cambodia, east 
XX.X (XX.X – XX.X) XX.X (XX.X – XX.X) X.XXX 
Cambodia, west XX.X (XX.X – XX.X) XX.X (XX.X – XX.X) X.XXX 
Vietnam 
XX.X (XX.X – XX.X) XX.X (XX.X – XX.X) X.XXX 
 
The proportion and duration of gametocyte carriage in patients with antimalarial sensitive and 
resistant malaria before and after treatment with primaquine will be summarized. 
 
3.12 
DNA and RNA measurement methods for quantification of male and female 
gametocytes.  
Samples will be obtained at baseline, hour 24 and hour 48. A detailed analysis plan for the 
analysis of the transcriptome and genome will be prepared separately. 
 
3.13 
ex vivo susceptibility profiles of P. falciparum isolates across geographic regions  
Parasite half-lives (IC50 or other related parameters) of the drugs will be used to assess drug 
susceptibility. The drug susceptibility half-life cut-off points will be determined from a statistical 
model that will be developed based on previous data. A statistical approach will be developed 
from previous drug susceptibility data to infer the characteristics of the component distributions 
and their relative contribution to the composite mixture. The model may be either unimodal, 
bimodal or multimodal depending on prevalence of resistance from the different areas. The 
geometric mean (IQR) parasite half-lives consistent with the sub-distributions will be reported 
together with the 95% confidence intervals to determine the cut-off point from the model. The half-
lives of parasites in the individual data will be used to obtain an estimate of the likelihood of a 
resistant infection depending of an individual which will depend on the population prevalence of 
resistance in that area as determined by the model 
 
 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
29 
 
 
3.14 
Pharmacokinetic profiles of ACT (AL) and Triple ACT (AL+AQ) components 
A sub-study of multiple blood samples from each of about 40 patients in Vietnam, half given Triple 
ACT and half given standard ACT will be undertaken. Standard pharmacokinetic parameters of 
lumefantrine and amodiaquine during the first 24 hours will be estimated from a non-compartment 
analysis, including as Cmax, Tmax, T1/2 and area under the curve (AUC). Results will be presented 
as below, to check whether amodiaquine affects artemether or lumefantrine pharmacokinetics. 
 
Table 12 Pharmacokinetic parameters of the individual drugs artemether and lumefantrine when 
given in combination with amodiaquine (Triple ACT) and when given without amodiaquine   
 
 
ACT (AL) 
Triple ACT (AL+AQ) 
p-value 
Artemether  
(geometric mean, 95%CI) 
Cmax (ng/ml)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
Tmax (hours, h)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
AUC (h × ng/ml)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
Lumefantrine 
(geometric mean, 95%CI) 
Cmax (ng/ml)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
Tmax (hours, h)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
AUC (h × ng/ml)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
Amodiaquine 
(geometric mean, 95%CI) 
Cmax (ng/ml)  
NA 
x.xx (xx.x - xx.x) 
NA 
Tmax (hours, h)  
NA 
x.xx (xx.x - xx.x) 
NA 
AUC (h × ng/ml)  
NA 
x.xx (xx.x - xx.x) 
NA 
 
 
 
 


CTSG 
 
 
STATISTICAL ANALYSIS PLAN 
30 
 
Table 13 Pharmacokinetic parameters of lumefantrine on day 7 when given in combination with 
amodiaquine (Triple ACT) and when given without amodiaquine in all patients from Cambodia 
and Vietnam. Also amodiaquine levels in all patients allocated to amodiaquine (Triple ACT). 
 
 
ACT (AL) 
Triple ACT (AL+AQ) 
p-value 
Lumefantrine 
(geometric mean, 95%CI) 
Cmax (ng/ml)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
Tmax (hours, h)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
AUC (h × ng/ml)  
x.xx (xx.x - xx.x) 
x.xx (xx.x - xx.x) 
x.xxx 
Amodiaquine 
(geometric mean, 95%CI) 
Cmax (ng/ml)  
NA 
x.xx (xx.x - xx.x) 
NA 
Tmax (hours, h)  
NA 
x.xx (xx.x - xx.x) 
NA 
AUC (h × ng/ml)  
NA 
x.xx (xx.x - xx.x) 
NA 
 
 
3.15 
Change in haematocrit according to geographical location and study arm and 
stratified according to G6PD status  
Change in hematocrit on day 1 to 7, 14, 21, 28, 35 and 42 according to geographical location and 
study arm, stratified for G6PD status will be summarized and analyzed. A detailed analysis plan 
will be prepared separately.