A Five-arm Trial Comparing Artesunate-amodiaquine and Artemether-lumefantrine-amodiaquine With or Without Single-dose Primaquine to Reduce P. Falciparum Transmission in Mali
Phase: PHASE2
Conditions: Malaria,Falciparum
Sponsor: London School of Hygiene and Tropical Medicine

NECTAR4: A five-arm trial comparing artesunate-amodiaquine and 
artemether-lumefantrine-amodiaquine with or without single-dose 
primaquine to reduce P. falciparum transmission in Mali  
 
Principal Investigators 
Prof Alassane Dicko 
Faculty of Pharmacy and Faculty of Medicine 
and 
Dentistry, 
University 
of 
Sciences 
Techniques and Technologies of Bamako, 
Mali 
Prof. Dr. Chris Drakeley 
London School of Hygiene & Tropical 
Medicine, United Kingdom 
 
 
Co-investigators: Mali 
Dr. Halimatou Diawara 
Dr. Almahamoudou Mahamar 
Dr. Harouna Soumare 
Dr. Djibrilla Issiaka 
Prof. Cheick Traore 
Malaria Research and Training Centre, 
Faculty of Pharmacy and Faculty of Medicine 
and 
Dentistry, 
University 
of 
Sciences 
Techniques and Technologies of Bamako, 
Mali 
 
Co-investigators: International 
Prof. Teun Bousema 
Drs. Merel Smit 
Radboud University Medical Center, Nijmegen, 
The Netherlands 
 
Dr. William Stone 
Dr. Leen Vanheer 
London School of Hygiene & Tropical Medicine, 
United Kingdom 
 
 
London School of Hygiene & Tropical Medicine is the main research sponsor for this study.  
For further information regarding the sponsorship conditions, please contact the Research 
Governance and Integrity Office: 
 
London School of Hygiene & Tropical Medicine 
Keppel Street 
London WC1E 7HT 
Tel: +44 207 927 2626 
Email: RGIO@lshtm.ac.uk 
 
Planned start date:     September 2022 
Planned end date:      July 2023 
 

 
2 
TABLE OF CONTENTS 
1. 
LIST OF ABBREVIATIONS ......................................................................................... 4 
2. 
STUDY SYNOPSIS .................................................................................................... 4 
3. 
STUDY BACKGROUND AND RATIONALE ................................................................... 5 
3.1. 
Background .......................................................................................................................... 5 
3.2. 
Rationale .............................................................................................................................. 6 
4. 
OBJECTIVES............................................................................................................. 7 
4.1. 
Primary specific objective .................................................................................................... 7 
4.2. 
Secondary specific objectives .............................................................................................. 8 
4.3. 
Exploratory objectives .......................................................................................................... 8 
5. 
STUDY DESIGN ........................................................................................................ 8 
5.1. 
Study site ............................................................................................................................. 9 
5.2. 
Study population ................................................................................................................ 10 
5.3. 
Intervention ........................................................................................................................ 10 
5.4. 
Sample size calculation ..................................................................................................... 10 
5.5. 
Inclusion criteria ................................................................................................................. 10 
5.6. 
Exclusion criteria ................................................................................................................ 11 
6. 
STUDY DRUGS ....................................................................................................... 11 
6.1. 
Artesunate-Amodiaquine (ASAQ) ...................................................................................... 11 
6.2. 
Primaquine (PQ) ................................................................................................................ 12 
6.3. 
Artemether-Lumefantrine (AL) ........................................................................................... 12 
6.4. 
Amodiaquine (AQ) ............................................................................................................. 12 
7. 
OUTCOME MEASURES ........................................................................................... 13 
7.1. 
Primary outcome measure ................................................................................................. 13 
7.2. 
Secondary outcome measures .......................................................................................... 13 
7.3. 
Exploratory outcomes ........................................................................................................ 14 
8. 
PROCEDURES ........................................................................................................ 14 
8.1. 
Informed consent ............................................................................................................... 14 
8.2. 
Study procedures ............................................................................................................... 15 
8.3. 
Randomization procedure .................................................................................................. 16 
8.4. 
Blinding .............................................................................................................................. 16 
9. 
SAFETY EVALUATION ............................................................................................. 16 
9.1. 
Definitions .......................................................................................................................... 17 
9.1.1. 
Adverse events .................................................................................................................. 17 
9.1.2. 
Serious adverse events ..................................................................................................... 17 
9.1.3. 
Unexpected adverse events .............................................................................................. 17 
9.1.4. 
Serious and Unexpected Suspected Adverse Reaction (SUSAR) .................................... 17 
9.1.5. 
Serious Unanticipated Problem (UP) ................................................................................. 17 
9.1.6. 
Unanticipated Problem that is not an Adverse Event (UPnonAE) ..................................... 18 
9.1.7. 
Confirmatory pregnancy test at the end of follow-up ......................................................... 18 
9.2. 
Adverse Event Data Collection .......................................................................................... 18 
9.3. 
Haematological severe AE................................................................................................. 19 
9.4. 
Assessment of causality .................................................................................................... 19 
9.5. 
Assessment of outcome..................................................................................................... 20 
9.6. 
Data and Safety Monitoring Committee (DSMB) ............................................................... 20 
9.7. 
Treatment failure criteria .................................................................................................... 21 
10. LABORATORY PROCEDURES ................................................................................. 21 
10.1. 
Blood film ........................................................................................................................... 21 
10.2. 
Gametocyte and asexual stage density measurement ..................................................... 22 
10.3. 
Serology and biomarkers ................................................................................................... 22 
10.4. 
Mosquito infectivity assay .................................................................................................. 22 

 
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10.5. 
Magnetic-activated cell sorting .......................................................................................... 22 
10.6. 
Haemoglobin concentrations ............................................................................................. 22 
10.7. 
Complete blood count ........................................................................................................ 23 
10.8. 
Biochemistry....................................................................................................................... 23 
11. DATA MANAGEMENT AND ANALYSIS...................................................................... 23 
11.1. 
Analysis plan ...................................................................................................................... 23 
11.2. 
Publication/Dissemination .................................................................................................. 24 
12. ETHICAL CONSIDERATIONS ................................................................................... 24 
12.1. 
Confidentiality..................................................................................................................... 24 
12.2. 
Risk .................................................................................................................................... 25 
12.2.1. 
Risk of artesunate-amodiaquine ........................................................................................ 25 
12.2.2. 
Risk of primaquine ............................................................................................................. 25 
12.2.3. 
Risk of artemether-lumefantrine ........................................................................................ 25 
12.2.4. 
Risk of amodiaquine .......................................................................................................... 26 
12.2.5. 
Risk of artemether-lumefantrine-amodiaquine .................................................................. 26 
12.3. 
Benefits .............................................................................................................................. 26 
12.4. 
Compensation .................................................................................................................... 26 
13. USE AND STORAGE OF STUDY SAMPLES .............................................................. 26 
14. SPONSOR INFORMATION ....................................................................................... 27 
14.1. 
Indemnity ............................................................................................................................ 27 
14.2. 
Sponsor .............................................................................................................................. 27 
14.3. 
Audits and Inspections ....................................................................................................... 27 
15. REFERENCES......................................................................................................... 27 
 
 
 
 

 
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1. LIST OF ABBREVIATIONS 
ACT 
 
Artemisinin-based combination therapy 
AE 
 
Adverse event 
AL 
 
Artemether-Lumefantrine 
AQ 
 
Amodiaquine 
AS 
 
Artesunate 
ASAQ  
Artesunate-Amodiaquine 
AUC 
 
Area under the curve 
CBC 
 
Complete Blood Count 
CRF 
 
Case report form 
DNA 
 
Deoxyribonucleic acid 
DP 
 
Dihydroartemisinin-Piperaquine 
DSMB  
Data and Safety Monitoring Board 
EC 
 
Ethics Committee 
EDTA  
Ethylenediaminetetraacetic acid  
G6PD  
Glucose-6-phosphatase dehydrogenase 
GCP 
 
Good clinical practice 
Hb 
 
Haemoglobin 
Hct 
 
Haematocrit 
LSHTM 
London School of Hygiene and Tropical Medicine 
MACS  
Magnetic-activated cell sorting  
MFA 
 
Membrane feeding assay 
MRTC  
Malaria Research and Training Centre (Bamako, Mali) 
P. falciparum Plasmodium falciparum 
PA 
 
Pyronaridine-Artesunate 
PCR 
 
Polymerase chain reaction 
PQ  
 
Primaquine 
RBCs  
Red blood cells 
RT-qPCR 
Quantitative reverse transcriptase polymerase chain reaction 
SAE 
 
Serious Adverse Event 
SLD 
 
Single low dose 
SOP 
 
Standard operating procedure 
SUSAR 
Serious and Unexpected Suspected Adverse Reaction 
TACT  
Triple Artemisinin-based Combination Therapy 
UP 
 
Unanticipated Problem 
UPnonAE 
Unanticipated Problem that is not an Adverse Event 
WHO  
World Health Organisation 
2. STUDY SYNOPSIS 
Artemisinin Combination Therapies (ACT) are the first-line treatment for uncomplicated 
Plasmodium falciparum malaria. ACTs rapidly clear asexual P. falciparum parasites, 
responsible for clinical symptoms, but have limited activity against mature gametocytes, which 
are the only life stages capable of transmitting to mosquitoes. To reduce P. falciparum 
transmission, the World Health Organisation (WHO) recommends the addition of a single low 
dose of primaquine (0.25 mg/kg), a potent and fast-acting gametocytocidal drug, to ACT [1]. 
Although Artesunate-Amodiaquine (ASAQ) is a commonly used ACT, limited available data 

 
5 
suggest poor activity against gametocytes [2] and the added benefit of SLD PQ in combination 
with ASAQ remains unknown. We previously showed that Artemether-Lumefantrine (AL) is a 
more potent ACT in terms of gametocyte clearance and transmission reduction efficacy 
compared to Dihydroartemisinin-Piperaquine (DP) [3], [4] [NECTAR3, in preparation]. 
Recently, triple artemisinin-based combination therapies (TACT) such as Artemether-
Lumefantrine plus Amodiaquine (ALAQ) have been proposed to delay the emergence of drug 
resistance and to provide efficacious treatment for multi-drug resistance P. falciparum 
infections [5], [6]. The transmission reducing efficacy of ALAQ or ALAQ-PQ has not yet been 
tested. 
In this study we aim to determine transmission reducing efficacy of Artesunate-Amodiaquine 
(ASAQ) with and without a single low dose (0.25mg/kg) of Primaquine (SLD PQ) and 
Artemether-Lumefantrine (AL) with and without Amodiaquine (AQ) and Primaquine (PQ). To 
achieve this, we propose to conduct a five-arm, single-blind, randomised control trial to test 
the gametocytocidal and transmission-blocking efficacy of SLD PQ in combination with 
Artesunate-Amodiaquine and Artemether-Lumefantrine-Amodiaquine. Individuals will be 
recruited to an AL-only arm as an internal control for the ALAQ arm, and for comparison to 
recent trials at the same site [NECTAR3, in preparation]. Individuals who meet inclusion 
criteria will be randomised in a 1:1:1:1:1 ratio to: ASAQ, ASAQ-PQ, AL, ALAQ, ALAQ-PQ. 
For participation in the trial, individuals between 10-50 years will be recruited from villages 
around Ouelessebougou, near Bamako in Mali. Written, informed consent will be obtained. 
Inclusion criteria include presence of ≥16 gametocytes/µL, absence of other non-P. falciparum 
species on blood film, absence of symptomatic falciparum malaria, haemoglobin ≥ 10 g/dL, 
and no evidence of acute severe or chronic disease or pregnancy. Recruitment will continue 
until 100 individuals who meet the inclusion criteria are recruited and randomised. G6PD 
enzyme status will not be tested. 
Participants will be followed for 28 days. Blood samples will be taken pre-treatment, and at 
days 2, 7, 14, 21 and 28 post-treatment for parasitology, genetic, haematology and 
biochemistry analysis. MFA will be conducted pre-treatment, and at days 2, 7 and 14 post-
treatment. MFA will also be conducted at day 21 for any individuals infectious at day 7 or 14; 
and at day 28 for any individuals infectious at day 14 or 21. Magnetic-activated cell sorting 
(MACS) to enrich for gametocytes will be done at two timepoints per individual to determine 
per-gametocyte infectivity and confirm whether PQ sterilizes gametocytes, followed by MFA. 
The primary outcome will be the reduction in mosquito infection rate within each arm at day 2 
post-treatment. Secondary outcomes include within and between arm comparisons in other 
mosquito infection, gametocyte, safety, and biochemical parameters. 
3. STUDY BACKGROUND AND RATIONALE 
3.1. Background 
Worldwide malaria morbidity and mortality remains unacceptably high, with an estimated 241 
million cases in 2020, a substantial increase from 214 million in 2015 [7]. In addition to this, 
the emergence and spread of resistance to first-line drugs is threatening to further increase 
this rise in malaria cases and deaths. Therefore, there is a clear need for interventions that 
reduce malaria transmission. 

 
6 
Artemisinin combination-based therapies (ACTs) are the first-line treatment for uncomplicated 
P. falciparum malaria; however, they have very limited activity against mature gametocytes, 
the stages responsible for transmission to the mosquito. The 8-aminoquinolone primaquine is 
a potent and fast-acting gametocytocidal drug and is currently the only drug recommended for 
blocking malaria transmission. To reduce P. falciparum transmission, the World Health 
Organisation (WHO) recommends the addition of a single low dose of primaquine (SLD PQ) 
(0.25 mg/kg), to ACT. The benefit of adding SLD PQ has been assessed with 
Dihydroartemisinin-Piperaquine (DP), Pyronaridine-Artesunate (PA) and Artemether-
Lumefantrine (AL), concluding effective blockage of transmission within 48 hours  [3], [8] 
[NECTAR3, in preparation]. However, the added benefit of combining PQ with Artesunate-
Amodiaquine (ASAQ) has not yet been tested, despite ASAQ being the first-line ACT in many 
countries [7]. Importantly, an individual-level meta-analysis of gametocyte data before and 
after ACT treatment suggested that ASAQ may be inefficient in clearing gametocytes with a 
fourfold higher odds of gametocyte carriage compared to the most widely used ACT that is AL 
[2].  
The stagnation of progress toward reducing global malaria morbidity and mortality may be 
compounded by the emergence of artemisinin resistance. Artemisinin resistance, 
characterized by slow parasite clearance [9], [10] because of reduced susceptibility of ring-
stage parasites [11], first emerged in Southeast Asia, a region that has been the epicentre for 
resistance development against all the major anti-malaria drugs in the past. In many areas 
with artemisinin resistance, resistance to the partner drug has now emerged as well, leading 
to a rapid rise in treatment failures following ACT [12], [13].  
Artemisinin resistance is mediated by mutations in the propeller region of the Kelch 13 (K13) 
gene. Recently, K13 mutations and associated delayed clearance of parasites have been 
found in East-Africa as well [14], [15]. Wider spread of artemisinin resistance across the 
African continent, which bears the vast majority of the global malaria burden, could be 
disastrous. In order to avert a public health emergency, there is an urgent need for alternative 
treatments with combinations of existing drugs that could be deployed rapidly. Triple 
Artemisinin-based Combination Therapies (TACT), which combine an existing ACT with a 
second partner drug that is slowly eliminated, could be part of such an approach.  
The rationale for TACT is similar to the one underlying ACT. By combining drugs with different 
treatment targets and/or resistance mechanisms, the probability that resistance will emerge to 
the combination is very low (i.e., the product of the probabilities for each individual drug). TACT 
could counteract resistance emergence and provide efficacious treatment against drug 
resistant infections. Artemether-Lumefantrine with Amodiaquine (ALAQ) is one such TACT 
and has been proven a safe, well-tolerated and efficacious treatment for uncomplicated P. 
falciparum malaria, including in areas with artemisinin and partner-drug resistance [5], [6]. The 
effect of ALAQ on mature gametocytes and infectivity is unknown, as well as the added benefit 
of combining PQ with ALAQ. Given that a major rationale for using TACT is a reduction in the 
transmission of artemisinin resistance, it is pressing to understand the impact of TACT on 
gametocyte production and infectivity.  
3.2. Rationale 
Artesunate-Amodiaquine (ASAQ) is a commonly used ACT, however, its transmission 
reducing efficacy remains largely unknown. Studies microscopically assessing gametocyte 
carriage post-ASAQ treatment reached very different conclusions, ranging from increased 

 
7 
gametocyte carriage until day 3 [16] or day 7 [17] post-treatment to an immediate decrease in 
gametocytaemia [18]. Compared to AL, ASAQ appears to be less efficient in preventing post-
treatment gametocyte carriage [2]. In addition, it is important to note that gametocyte carriage 
is a poor measurement for human-to-mosquito transmission, as antimalarial drugs can 
sterilise gametocytes before removing them from the blood stream [19] and transmissible 
gametocytes can persist at submicroscopic densities [20]. Membrane feeding assays (MFA) 
are therefore the most reliable method to assess transmission. MFA can be supported by 
magnetic enrichment of gametocytes that can allow for assessments of infectivity of increased 
gametocyte densities [21] and differentiate between lack of infectivity because of gametocyte 
densities that are too low to permit transmission and gametocyte sterilization [22].This is 
relevant since uncertainties remain about the exact working mechanism of transmission-
blocking antimalarial drugs that may clear gametocytes, distort their sex-ratio or sterilize 
gametocytes without clearance from circulation [19]. Understanding the mode by which 
transmission is prevented can support drug development. 
Only one study has performed MFA after ASAQ treatment, reporting an increase in 
transmission [17]. If there is indeed substantial transmission after treatment with ASAQ, the 
addition of a single low dose PQ may be considered. To date, the added benefit of PQ when 
used in combination with ASAQ is unknown.  
We previously showed that Artemether-Lumefantrine (AL) is a more potent ACT in terms of 
gametocyte clearance and transmission reduction efficacy compared to Dihydroartemisinin-
Piperaquine (DP) [3], [4], [NECTAR3, in preparation]. It is currently unclear what the reason is 
for this marked difference and whether gametocyte-sterilization plays a role or the effect is 
driven by gametocyte-clearance. Recently, triple artemisinin-based combination therapies 
(TACT) such as Artemether-Lumefantrine plus Amodiaquine (ALAQ), which combine an 
existing ACT with a second partner drug that is slowly eliminated, have been proposed to 
provide efficacious treatment for multidrug-resistant P. falciparum infections and to delay the 
emergence of drug resistance [5], [6]. The transmission reducing efficacy of ALAQ or ALAQ 
combined with a single dose of PQ has not yet been tested.  
Therefore, we propose to conduct a five-arm, phase 2, single-blind, randomised control trial 
to test the gametocytocidal and transmission-blocking efficacy of SLD PQ in combination with 
Artesunate-Amodiaquine and Artemether-Lumefantrine-Amodiaquine. Individuals will be 
recruited to an AL only arm as an internal control for the ALAQ arm, and for comparison to 
recent trial [NECTAR3, in prep]. Individuals who meet inclusion criteria will be randomised in 
a 1:1:1:1:1 ratio to: ASAQ, ASAQ-PQ, AL, ALAQ, ALAQ-PQ. 
4. OBJECTIVES 
4.1. Primary specific objective 
To assess the within-person reduction of infectivity of gametocytes following administration of 
ASAQ alone or with single-dose PQ and AL alone or with AQ and ALAQ alone or with single-
dose PQ in children and adults at day 2 post-treatment compared to pre-treatment (day 0). 

 
8 
4.2. Secondary specific objectives 
• To assess differences in other mosquito infectivity parameters (mosquito infection rate, 
change in infection rate, infectivity to mosquitoes, and oocyst density) following treatment 
with ASAQ, ASAQ-PQ, AL, ALAQ and ALAQ-PQ in children and adults at all feeding 
timepoints compared to pre-treatment (day 0) and between treatment matched arms 
(ASAQ vs ASAQ-PQ, AL vs ALAQ, ALAQ vs ALAQ-PQ). 
• To assess differences in gametocyte parameters (prevalence, density, circulation time, 
area-under-the-curve, sex-ratio) following treatment with ASAQ, ASAQ-PQ, AL, ALAQ and 
ALAQ-PQ in children and adults at all feeding timepoints compared to pre-treatment (day 
0) and between treatment matched arms (ASAQ vs ASAQ-PQ, AL vs ALAQ, ALAQ vs 
ALAQ-PQ). 
• To assess differences in safety parameters (AE frequency, Hb density, median drop in Hb) 
following treatment with ASAQ, ASAQ-PQ, AL, ALAQ and ALAQ-PQ in children and adults 
at all feeding timepoints compared to pre-treatment (day 0) and between treatment 
matched arms (ASAQ vs ASAQ-PQ, AL vs ALAQ, ALAQ vs ALAQ-PQ). 
• To assess differences in biochemical parameters (ALT, CBC, Creatinine) following 
treatment with ASAQ, ASAQ-PQ, AL, ALAQ and ALAQ-PQ in children and adults at all 
feeding timepoints compared to pre-treatment (day 0) and between treatment matched 
arms (ASAQ vs ASAQ-PQ, AL vs ALAQ, ALAQ vs ALAQ-PQ). 
4.3. Exploratory objectives 
• To assess parasite genomic and transcriptomic variation at baseline and at select post-
treatment timepoints. 
• To assess human genomic variation (i.e., HBB type) and association with parasite 
measures. 
• To assess the association of parasite and plasma biomarkers on malaria transmission 
efficiency. 
• To assess the efficiency of Plasmodium transmission to mosquitoes before and after 
enrichment of gametocytes by MACS. 
• To determine the association between gametocyte density and mosquito infection rates, 
before and after MACS enrichment, to estimate possible loss in per-gametocyte infectivity 
following treatment. 
5. STUDY DESIGN 
This is a five arm, single-blinded, randomized clinical trial. The study will be conducted 
according to the following steps for each individual: 
Individuals aged 10-50 years will be invited to participate. They will be asked to provide 
informed consent and assent (for those under 18 years of age). After obtaining the informed 
consent and assent (if applicable), participants will undergo a screening including clinical 
evaluation and for the presence of microscopically detectable P. falciparum infection. 
Individuals with ≥16 gametocytes/µL will be recruited and go through additional screening 
including haemoglobin measurement, CBC, assessment of renal and liver functions, and 
pregnancy tests. Recruitment will continue until 100 infected individuals with microscopically 
detectable P. falciparum gametocytes (≥16/µL) are recruited and randomised. 

 
9 
Individuals who meet the inclusion and exclusion criteria will be randomised in a 1:1:1:1:1 ratio 
into five groups (arms); ASAQ, ASAQ-PQ (0.25mg/kg), AL, ALAQ, ALAQ-PQ (Table 1). 
Randomisation will be performed by the study pharmacist. All other staff will be blinded to the 
treatment allocation. 
Table 1. Treatment arms 
Arm ACT 
PQ (mg/kg) 
Sample size 
1 
ASAQ  
20 
2 
ASAQ 0.25  
20 
3 
AL 
 
20 
4 
ALAQ 
 
20 
5 
ALAQ 
0.25 
20 
 
Each participant will be followed for 28 days. Participants will receive a full clinical and 
parasitological examination on days 2, 7, 14, 21, 28 after receiving the first dose of the study 
drugs. Blood samples will be taken at all visits for parasitology (microscopy, and whole blood 
for qRT-PCR) and serology with additional samples for haematology and biochemistry 
analysis at days 0, 2 and 14. A clinical and haematological examination will also be performed 
on day 1, at which point no other samples will be collected.  
Infectivity to locally reared mosquitoes will be assessed with membrane feeding assays at 
baseline, on the final day of treatment (day 2) and at days 7 and 14 for all participants. Feeds 
will then be performed at day 21 for any individuals infectious at day 7 or 14; and at day 28 for 
any individuals infectious at day 14 or 21. Magnetic-activated cell sorting (MACS) to enrich for 
gametocytes will be done at two timepoints per individual, followed by MFA. 
In addition to this passive monitoring, participants will be prompted to contact the study 
coordinators if they feel sick at any point during follow up. A full clinical examination will be 
performed, and treatment will be provided according to national treatment guidelines. 
5.1. Study site 
The Malaria Research and Training Centre (MRTC) in Bamako, Mali will use the field site of 
Ouelessebougou for recruitment. The MRTC has the unique position of being one of very few 
sites in Africa that can study malaria transmission endpoints. Since 2013 the MRTC 
successfully carried out similar trials at this site with great success, in collaboration with the 
University of California San Francisco, the London School of Hygiene and Tropical Medicine 
and the Radboud University Medical Center, Nijmegen, the Netherlands. In the 2013-2014 
study, the MRTC demonstrated the efficacy of SLD PQ of doses of 0.125mg/kg or greater in 
combination with dihydroartemisinin-piperaquine for blocking P. falciparum transmission 
safely in G6PD normal Malian males from the age of 10-50 years [8]. MRTC has already 
established all the assays required for the study described in this protocol.  

 
10 
The MRTC is experienced in conducting Good Clinical Practice (GCP) compliant clinical trials 
including phase 1, 2, 3 and 4 of malaria vaccine and drug studies. Ouelessebougou village 
has been conducting ongoing clinical research since 2008 and is endemic for malaria with 
marked seasonality and a high burden of malaria in both children and adult populations. In 
recent years the prevalence of P. falciparum malaria in children under 5 years of age has 
ranged between 14% and 54% during the transmission season. The frequency of G6PD 
deficiency is in the range of 10 to 15%. 
5.2. Study population 
The study population will be derived from individuals aged 10-50 years with asymptomatic P. 
falciparum malaria infection who agree to be screened for malaria infection. 
For the randomised trial, we will aim to recruit 100 individuals. Eligible individuals will have 
microscopically detectable P. falciparum gametocyte densities of ≥16 gametocytes/µL.   
5.3. Intervention 
Individuals who meet the inclusion criteria in the current study will be randomised in a 1:1:1:1:1 
ratio into five groups (arms); ASAQ, ASAQ-PQ (0.25 mg/kg), AL, ALAQ, ALAQ-PQ (0.25 
mg/kg). Randomisation will be performed by the study pharmacist. All staff other than the 
study pharmacist and clinician will be blinded to the treatment allocation. Participants will be 
told prior to enrolment that they can ask at any time which treatment arm they are in. The 
clinician or pharmacist will discuss this with them. 
5.4. Sample size calculation 
For sample size considerations, we estimated the infectivity for participants in previous trials 
in the same study setting [3], [4], [8], [23] using a mixed effects logistic regression model that 
accounted for correlation between mosquito observations from the same participant. Based 
on a mixed effects logistic regression we looked at 100 simulations with an expected reduction 
in infectivity of 90%, an expected baseline proportion of infectivity of 17% for the average 
participant, and an expected intra-cluster correlation of 0.5. We estimated 88% empirical 
power to detect >80% reduction in infectivity with a one-tailed test with a 0.025 level of 
significance when including 20 participants and dissecting 50 mosquitos per participant at 
each timepoint. 
5.5. Inclusion criteria 
• Age ≥ 10 years and ≤ 50 years 
• Absence of symptomatic falciparum malaria, defined by fever on enrolment  
• Presence of P. falciparum gametocytes on thick blood film at a density >16 gametocytes/µL 
(i.e. ≥ gametocytes recorded in the thick film against 500 white blood cells) 
• Absence of other non-P. falciparum species on blood film 
• Haemoglobin ≥ 10 g/dL 
• Individuals weighing < = 80 kg 
• No evidence of acute severe or chronic disease  
• Written, informed consent 

 
11 
5.6. Exclusion criteria 
• Women who are pregnant or lactating (tested at baseline). Urine and/or serum pregnancy 
testing (β-hCG) will be used. 
• Detection of a non-P. falciparum species by microscopy 
• Previous reaction to study drugs / known allergy to study drugs, such as sudden high 
fevers, shaking or severe sore throat or ulcers in the mouth during treatment with 
Amodiaquine  
• Current eye disease with retinal damage 
• Signs of severe malaria, including hyperparasitaemia (defined as asexual parasitaemia > 
100,000 parasites / µL) 
• Signs of acute or chronic illness, including hepatitis 
• The use of other medication (except for paracetamol and/or aspirin), including antacids, 
other medicines used to treat malaria, abnormal heart rhythm, depression or mental illness 
or HIV/AIDS, and medicines that have antibiotic/antifungal properties 
• Use of antimalarial drugs over the past 7 days (as reported by the participant) 
• Clinically significant illness (intercurrent illness e.g., pneumonia, pre-existing condition e.g., 
renal disease or HIV/AIDS, malignancy or conditions that may affect absorption of study 
medication e.g., severe diarrhoea or any signs of malnutrition as defined clinically) 
• Signs of hepatic injury (such as nausea and/or abdominal pain associated with jaundice) 
or known severe liver disease (i.e., decompensated cirrhosis, Child Pugh stage B or C) 
• Signs, symptoms or known renal impairment 
• Clinically significant abnormal laboratory values as determined by history, physical 
examination, or routine blood chemistries and haematology values (laboratory guideline 
values for exclusion are haemoglobin < 10 g/dL, platelets < 50,000/μl, White Blood Cell 
count (WBC) < 2000/μl, serum creatinine >2.0mg/dL, or ALT more than 3 times the upper 
limit of normal for age. 
• Blood transfusion in the last 90 days. 
• Known Electrocardiogram (ECG) corrected QT interval of more than 450 ms 
• Documented or self-reported history of cardiac conduction problems 
• Documented or self-reported history of epileptic seizures 
6. STUDY DRUGS 
6.1. Artesunate-Amodiaquine (ASAQ) 
Participants in the ASAQ arm will receive a fixed-dose combination in tablets containing 
50mg/135 mg or 100mg/270 mg of artesunate/amodiaquine (Fosun Pharma, Shanghai, 
China). Tablets will be administered according to manufacturer guidelines (Table 2). 
Table 2. ASAQ dosing 
Weight 
Tablets 
D0 
D1 
D2 
9 to < 18 kg 
50 mg AS/135 mg AQ base 
1 tab 
1 tab 
1 tab 
18 to < 36 kg 
100 mg AS/270 mg AQ base 
1 tab 
1 tab 
1 tab 
blister pack of 3 tab 
≥ 36 kg 
100 mg AS/270 mg AQ base 
2 tab 
2 tab 
2 tab 

 
12 
blister pack of 6 tab 
 
6.2. Primaquine (PQ) 
Participants in the ASAQ-PQ arm will receive PQ (ACE Pharmaceuticals, Zeewolde, The 
Netherlands) at a single low dose of 0.25mg/kg as is currently recommended by the World 
Health Organization. The single dose of PQ will be given on day 0 together with ASAQ, 
administered in an aqueous solution, according to a standard operating procedure (SOP) 
provided by the manufacturer as previously done at the study site when PQ was combined 
with DP, PA, AL [3], [8], [23]. 
6.3. Artemether-Lumefantrine (AL) 
Participants in the AL arm will be treated with standard doses of AL (Fosun Pharma, Shanghai, 
China). Tablets containing 20 mg artemether and 120 mg lumefantrine will be administered 
per manufacturer guidelines (Table 3) 
Table 3. AL dosing 
Bodyweight (kg) 
20/120 mg tablet 
 
D0 
D1 
D2 
5 to < 15 kg 
1 disp tab x 2 
1 disp tab x 2 
1 disp tab x 2 
15 to < 25 kg 
2 disp tab x 2 
2 disp tab x 2 
2 disp tab x 2 
25 to < 35 kg 
3 tab x 2 
3 tab x 2 
3 tab x 2 
≥ 35 kg 
4 tab x 2 
4 tab x 2 
4 tab x 2 
6.4. Amodiaquine (AQ) 
Participants in the ALAQ and ALAQ-PQ arms will receive AQ as tablets of 153 mg. The weight-
based treatment schedule in table 4 aims for a dosage of approximately 10 mg (7.7-
15.3mg)/kg/day, given once or twice daily (together with artemether–lumefantrine) for three 
days. 
Table 4. AQ dosing 
Bodyweight (kg) 
153 mg tablet 
D0  
(0hr) 
D0  
(8hr) 
D1 
(24hr) 
D1 
(36hr) 
D2 
(48hr) 
D2 
(60hr) 
10 to 19.9 
1 tab 
0 tab 
1 tab 
0 tab 
1 tab 
0 tab 
20 to 29.9 
1 tab 
1 tab 
1 tab 
1 tab 
1 tab 
1 tab 
30 to 54.9 
2 tab 
1 tab 
2 tab 
1 tab 
2 tab 
1 tab 
55 to 80 
3 tab 
2 tab 
3 tab 
2 tab 
3 tab 
2 tab 
 

 
13 
7. OUTCOME MEASURES  
7.1. Primary outcome measure 
Median within person percent change (presented as percent reduction) in mosquito infection 
rate in infectious individuals from baseline (day 0, pre-treatment) to day 2 post treatment in 
the ASAQ, ASAQ-PQ, AL, ALAQ and ALAQ-PQ arms. 
7.2. Secondary outcome measures 
• Median within person percent change (presented as percent reduction) in mosquito 
infection rate from baseline to all feeding time-points, with comparison within and between 
arms. 
• Median mosquito infection rate at all feeding time-points, with comparison within treatment 
arms compared to baseline, and between arms. 
• Infectivity to mosquitoes at all feeding time-points, with comparison within treatment arms 
compared to baseline, and between arms. 
• Mean oocyst intensity (in all/all infected mosquitoes) at all feeding time-points, with 
comparison within treatment arms compared to baseline, and between arms. 
• Male and female gametocyte prevalence at all time-points, determined by microscopy or 
molecular assays, with comparison within treatment arms compared to baseline, and 
between arms. 
• Male and female gametocyte density at all time-points, determined by microscopy or 
molecular assays, with comparison within treatment arms compared to baseline, and 
between arms. 
• Male and female gametocyte sex ratio (proportion male) at all time-points, determined by 
microscopy or molecular assays, with comparison within treatment arms compared to 
baseline, and between arms. 
• Gametocyte circulation time (cumulative), determined by microscopy or molecular assays, 
compared between treatment arms. 
• Gametocyte area under the curve (cumulative), determined by microscopy or molecular 
assays, compared between treatment arms. 
• Asexual and total parasite prevalence at all time-points, determined by microscopy or 
molecular assays, with comparison within treatment arms compared to baseline, and 
between arms. 
• Asexual and total parasite density at all time-points, determined by microscopy or molecular 
assays, with comparison within treatment arms compared to baseline, and between arms. 
• Haemoglobin density at all time-points, with comparison within treatment arms compared 
to baseline, and between arms. 
• Median within person percent change (presented as percent reduction) in haemoglobin 
density from baseline to all time-points, with comparison within and between arms. 
• The frequency and prevalence of adverse events (all AE’s, treatment related AE’s, and 
haematological AE’s) observed up to and including day 2, 7, and 14 post-treatment, and at 
all timepoints. 

 
14 
7.3. Exploratory outcomes 
• Parasite genotype and transcriptional analysis at baseline and at post-treatment timepoints, 
both for blood stage parasites and oocysts. 
• Plasma biomarkers (antibodies and parasite protein) at baseline and at post-treatment 
timepoints. 
• Association between gametocyte density and mosquito infection rates per study drug and 
time-point, to examine evidence for loss in gametocyte fitness/sterilization. 
• Human genotype analysis at baseline (G6PD, CYP2D6, HBB) 
• ALT/Creatine density at all time-points with comparison within treatment arms compared to 
baseline, and between arms. 
• Gametocyte density, mosquito infection rate and mean oocyst intensity before and after 
MACS, for within treatment arm comparison to baseline and comparison between treatment 
arms. 
 
Table 5. Outcome measures 
Outcome measure 
Day 0 
Day 1 
Day 2 
Day 7 
Day 14 
Day 21 
Day 28 
Mosquito infectivity 
* 
 
* 
* 
* 
** 
** 
Asexual parasite prevalence & 
density 
* 
 
* 
* 
* 
* 
* 
Gametocyte prevalence, 
density, sex ratio, genotype 
* 
 
* 
* 
* 
* 
* 
Biochemistry (ALT/Creatinine) 
* 
 
* 
 
* 
 
 
CBC 
* 
 
* 
 
* 
 
 
Haemoglobin  
* 
* 
* 
* 
* 
* 
* 
Safety assessment: signs of 
haemolysis 
* 
* 
* 
* 
* 
* 
* 
Safety assessment: Adverse 
Events 
* 
* 
* 
* 
* 
* 
* 
Plasma biomarkers 
* 
 
* 
* 
* 
* 
* 
Mosquito infectivity after 
MACS 
* 
 
* 
 
 
 
 
** Mosquito infectivity assays will be performed at day 21 for any individuals infectious at day 7 or 14; 
and at day 28 for any individuals infectious at day 14 or 21. 
8. PROCEDURES 
8.1. Informed consent 
Consenting procedures will vary based on age of the potential participant. Participants aged 
18 years and above will provide informed consent. For participants under 18 years of age we 
will seek parental consent. In addition to parental consent, assent will be sought for children 
aged 10-17 years. The Ethics committee in Mali does not require that participants under the 
age of 10 provide assent. The written, informed consent procedure will be conducted in French 
or in a local language understood by subject as described in the information and consent form.  

 
15 
The informed consent document will be used to explain the risks and benefits of study 
participation to the participant and in the case of participants < 18 years, the participant’s 
parent in simple terms before the subject is enrolled in the study. The informed consent 
document contains a statement that the consent is freely given, that the subject is aware of 
the risks and benefits of entering the study, and that the subject is free to withdraw from the 
study at any time. Written consent must be given by the participant or the participant’s parent, 
after the receipt of detailed information on the study. The informed consent form will be signed 
and personally dated by the participant or the participant’s parent and the person who 
conducts the informed consent discussion. The original signed informed consent form will be 
retained in the participant’s chart and another copy will be provided to him. A participant who 
is unable to read or write will place an imprint of his finger in place of a signature; in addition, 
an independent witness will sign the consent form to attest that the information in the consent 
form was orally conveyed to the participant. Specific consent will be provided for sharing 
samples and anonymized data to collaborating institutes outside Mali and for sharing data in 
anonymous form on a digital repository. 
8.2. Study procedures 
Participants who agree to participate and provide written, informed consent will be assessed 
for the presence of sexual blood stage parasite with a thick blood film. Individuals with ≥16 
gametocytes/µL will go through additional screening for eligibility including haemoglobin 
measurement, CBC, assessment of the renal and liver functions and pregnancy tests. 
Recruitment will continue until 100 infected individuals with microscopically detectable P. 
falciparum gametocytes (≥16/µL) are recruited and randomised. Individuals with symptomatic 
malaria will not be enrolled in the study and will be treated with artemether + lumefantrine, the 
standard treatment for uncomplicated malaria as per the Ministry of Health policy in Mali.  
Individuals screened found with other acute diseases will also be treated according to the 
standard of care in Mali. Those with chronic disease will be referred to the district hospital for 
further evaluation and management.  
Eligible patients will then be randomized (see below under randomization procedure) to one 
of the treatment groups, and drugs will be administered. In case of vomiting within 30 minutes 
after drug intake, the drugs will be re-administered once. 
The participants will be compensated for any travel costs and for work loss income. 
Participants will be followed for 28 days as described in Table 6 (below). Single venous draws 
or finger pricks will be used.  
Any individuals determined to have an adverse event (haemolytic or otherwise) will be treated 
in accordance with national guidelines and best practice and will be withdrawn from the study 
at the discretion of the study clinician.  
 
Table 6. Sampling framework for study participants 
Required 
vacutainer 
Time of sampling 
(d = day) 
Required 
volume 
(mL) 
Day 0  
Day 1 
Day 2 
Day 7 
Day 14 
Day 21 
Day 28 
Type of sampling$ 
V 
C 
V 
V 
V 
V 
V 
Heparin (2-5mL) 
Mosquito infectivity  
2 
* 
 
* 
* 
* 
** 
** 

 
16 
MACS 
3 
* 
 
* 
 
 
 
 
EDTA (0.05-
3.4mL) 
Blood in RNA protect for 
RNA analysis/parasite 
genotyping 
0.7 
* 
 
* 
* 
* 
* 
* 
Blood smear for asexual 
parasite and gametocyte 
density  
0.1 
* 
 
* 
* 
* 
* 
* 
Filter paper blood spots 
0.05 
* 
 
* 
* 
* 
* 
* 
Haemoglobin    
0.05 
* 
* 
* 
* 
* 
* 
* 
Plasma  
1 
* 
 
* 
* 
* 
* 
* 
CBC 
0.5 
* 
 
* 
* 
* 
 
 
Human genotyping  
1 
* 
 
 
 
 
 
 
SST (0.8 mL) 
Biochemistry 
0.8 
* 
 
* 
* 
* 
 
 
 
Maximum total Volume 
of blood sampled (mL) 
38.65 
 9.2 
0.05 
9.2 
6.2 
6.2 
3.9 
3.9 
$ V denotes venous blood sampling and C denotes capillary sampling from a finger prick.  
** Denotes volume taken for any individuals infectious at day 7 or 14 (day 21) and for any 
individuals infectious at day 14 or 21 (day 28) . 
8.3. Randomization procedure 
Eligible participants will be assigned the next sequentially numbered study ID number based 
on a pre-printed Study ID List created by a study investigator. Randomization will be computer 
generated using a 1:1:1:1:1 ratio. The randomisation codes will be provided in opaque sealed 
envelope to the study pharmacist. The study pharmacist in Mali will open the corresponding 
sealed, opaque envelope and provide the intervention to study participants. Randomization 
will be performed in blocks of varying sizes. 
8.4. Blinding 
This is a single-blind randomised controlled trial. The treating physician and staff involved with 
assessing all laboratory outcomes of the study are blinded, but no placebo will be used. The 
study pharmacist will be unblinded and responsible for randomisation and treatment 
administration. Entomology staff involved in the mosquito feeding assays will be blinded for 
the parasitology results. 
9. SAFETY EVALUATION 
The major safety endpoint is haemolysis. For this reason, haemoglobin will be measured 
before treatment, at days 1, 2, 7, 14, 21, and 28. Previous studies have shown that highest 
haemoglobin fall related to drug induced haemolysis can be detected within 7 days after 
treatment. In addition at 24 hours, and subsequent days of follow up, a questionnaire 

 
17 
assessing adverse events (AE) will be carried out. All participants will have access to contact 
study medical staff 24 hours a day and medical facilities that can give safe blood transfusions. 
Participants who require blood transfusions will be hospitalised at the district hospital in 
Ouelessebougou or referred to the University hospitals in Bamako for proper management. 
Additional safety precautions due to the small risk of transient haemolysis in this population 
will include the performance of clinical examinations and monitoring for adverse events (AE) 
at all sampling time points, carefree of charge to all participants throughout the duration of 
follow-up.  
9.1. 
Definitions 
9.1.1. Adverse events 
Adverse events are defined as any undesirable experience occurring to a subject during the 
study, whether or not considered related to trial. An AE can therefore be any unfavourable and 
unintended sign (including an abnormal laboratory finding), symptom, or disease (new or 
exacerbated) temporally associated with a study intervention. AEs may include events that 
occur as a result of protocol-mandated procedures (i.e. invasive procedures). All AEs reported 
spontaneously by the subject or observed by the trial clinicians, or their staff will be recorded. 
Abnormal laboratory findings or other abnormal assessments that are judged by the trial 
clinicians to be clinically significant will be recorded as AEs or serious adverse events (SAEs) 
if they meet the definition. The investigators and trial clinicians will exercise their medical and 
scientific judgement in deciding whether an abnormal laboratory finding, or other abnormal 
assessment is clinically significant. 
9.1.2. Serious adverse events 
A serious adverse event is any untoward medical occurrence or effect that at any dose:  
• 
Results in death 
• 
Is life threatening (at the time of the event) 
• 
Requires hospitalisation  
• 
Results in persistent or significant disability or incapacity 
• 
Congenital anomaly/birth defect 
• 
Medically important event 
All SAEs will be reported by the principal investigators to the Ethics Committees and DSMB 
within 48 hours of awareness of the PI.   
9.1.3. Unexpected adverse events 
An AE is considered unexpected if it is not listed in the Investigator’s Brochure (IB) or Package 
Insert (for marketed products) or is not listed at the specificity or severity that has been 
observed.  
9.1.4. Serious and Unexpected Suspected Adverse Reaction (SUSAR) 
A SUSAR is any AE that is assessed as being serious, is suspected of having a causal 
relationship to the trial treatment and is unexpected (as per 3.2.4), will require expedited 
reporting to the Sponsor, RA and IRB/IEC which have approved the clinical trial and all other 

 
18 
organisations as required under the terms of the individual contracts (e.g. relevant 
pharmaceutical companies, NHS Trusts etc.).  
Fatal or life-threatening SUSARs must be reported within 7 days of receipt of the report and 
all other SUSARs within 15 days (see Appendix 3 for reporting procedure for SUSARs for 
LSHTM sponsored trials). 
9.1.5. Serious Unanticipated Problem (UP) 
An UP is any event, incident, experience, or outcome that is 
1. 
unexpected in terms of nature, severity, or frequency in relation to 
a. 
the research risks that are described in the approved research protocol and 
informed consent document, IB, or other study documents; and 
b. 
the characteristics of the subject population being studied; and 
2. 
possibly, probably, or definitely related to participation in the research; and 
3. 
places subjects or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized.  
9.1.6. Unanticipated Problem that is not an Adverse Event (UPnonAE) 
An UPnonAE is an UP that does not fit the definition of an adverse event, but which may, in 
the opinion of the investigator, involve risk to the subject, affect others in the research study, 
or significantly impact the integrity of research data. Such events would be considered a non-
serious UP. For example, we will report occurrences of breaches of confidentiality, accidental 
destruction of study records, or unaccounted-for study drug. 
All UPs that are also adverse events will be reported to the sponsor no later than 7 calendar 
days of site awareness of the event. UPs that are not AEs will also be reported to the sponsor. 
9.1.7. Confirmatory pregnancy test at the end of follow-up 
Urine and/or serum pregnancy test (β-hCG) will be repeated on the final day of follow-up (day 
28) for participants who received a pregnancy test at enrolment. The results will be reported 
in the communication to the DSMB. 
9.2. Adverse Event Data Collection 
Safety assessments will be performed and recorded by the trial clinicians. All AEs/reactions, 
observed by the trial clinicians or by the subject, will be accurately documented in the case 
report form. For each event/reaction the following details will be recorded:  
1. 
Description of the event(s)/reaction(s)  
2. 
Date and time of occurrence  
3. 
Duration 
4. 
Intensity 
5. 
Relationship with the intervention 
6. 
Action taken, including treatment 

 
19 
7. 
Outcome 
In addition, symptoms will be ranked as (1) mild, (2) moderate, or (3) severe, depending on 
their intensity. All AEs except fever will be judged for their intensity according to the following 
scale: 
• 
Mild (grade 1): awareness of symptoms that are easily tolerated and do not interfere 
with usual daily activity  
• 
Moderate (grade 2): discomfort that interferes with or limits usual daily activity 
• 
Severe (grade 3): disabling, with subsequent inability to perform usual daily activity, 
resulting in absence or required bed rest 
For fever, the following scale will be used: 
• 
Mild (grade 1): 37.5 - 38.0°C 
• 
Moderate (grade 2): > 38.0 to 39.0°C  
• 
Severe (grade 3): > 39.0°C 
When an AE/SAE occurs, it is the responsibility of the principal investigators and trial clinicians 
to review all documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) 
related to the event. The principal investigators and trial clinicians will then record all relevant 
information regarding an AE/SAE on the Case Report Form (CRF) or SAE Report Form, 
respectively. Furthermore, the trial clinicians will attempt to establish a diagnosis of the event 
based on signs, symptoms, and/or other clinical information. In such cases, the diagnosis 
should be documented as the AE/SAE and not the individual signs/symptoms. 
9.3. Haematological severe AE 
A haematological severe AE is defined as a drop in Hb density of >40% from baseline. These 
will be reported separately in the data safety and monitoring reports and in any resulting 
publications, due to their potential linkage with PQ treatment. 
9.4. Assessment of causality 
The principal investigators and trial clinicians are obligated to assess the relationship between 
study procedures and the occurrence of each AE/SAE. The clinicians will use clinical judgment 
to determine the relationship. Alternative causes, such as natural history of the underlying 
diseases, concomitant therapy, other risk factors and the temporal relationship of the event to 
the study procedures will be considered and investigated. The relationship of the AE with the 
study procedures will be assessed considering the factors listed under the following 
categories: 
 
Table 7. Causality 
Definitely Related 
 
• 
reasonable temporal relationship 
• 
follows a known response pattern 
• 
clear evidence to suggest a causal relationship 
• 
there is no alternative aetiology 

 
20 
Probably Related 
 
• 
reasonable temporal relationship 
• 
follows a suspected response pattern (based on similar agents) 
• 
no evidence of a more likely alternative aetiology 
Possibly Related 
 
• 
reasonable temporal relationship 
• 
little evidence for a more likely alternative aetiology 
Unlikely Related 
 
• 
does not have a reasonable temporal relationship  
OR 
• 
good evidence for a more likely alternative aetiology 
Not Related 
 
• 
does not have a temporal relationship 
OR 
• 
definitely due to an alternative aetiology 
Note: Other factors will also be considered for each causality category when appropriate. 
Causality assessment is based on available information at the time of the assessment of the 
AE. The investigator may revise the causality assessment as additional information becomes 
available. 
9.5. Assessment of outcome 
Assessment of each AE must be assessed according to the following classification: 
Table 8. Outcome 
Completely recovered 
The subject has fully recovered with no observable residual effects 
Not yet completely recovered 
The subject condition has improved, but still has some residual effects 
Deterioration 
The subject’s overall condition has worsened 
Permanent damage 
The AE has resulted in a permanent impairment 
Death 
The subject died due to the AE 
Ongoing 
The AE remains the same as at onset 
Unknown 
The outcome of the AE is not known because of lost to follow-up 
All adverse events will be recorded on individual CRF with the following information: the 
severity grade (mild, moderate, severe), its relationship to the study drug(s) (related/not 
related), its duration (start and end dates or if continuing at final exam), actions taken, 
outcome, whether it constitutes a serious adverse event (SAE). 
9.6. Data and Safety Monitoring Committee (DSMB) 
A Data and Safety Monitoring Committee (DSMB) will be established and convened before 
the onset of the trial; an independent local safety monitor (LSM) will be appointed in Mali and 
form part of the DSMB. The DSMB will convene meetings in person or by telephone 

 
21 
conference prior to the study initiation, after the first 50 subjects have been enrolled and upon 
completion of the study. In addition to these three scheduled meetings, ad hoc meetings may 
be organised based on monthly progress reports that are provided to the DSMB. These 
monthly reports include an update on the number of participants who were enrolled, who 
completed the study and who discontinued the study (with reason provided). In addition, key 
safety data are provided: AEs and up to day 14 haemoglobin concentrations. Any SAEs or 
Suspected Unexpected Serious Adverse Reactions (SUSARs) are first reported and 
discussed among the research team and LSM and subsequently reported within 48 hours to 
the complete DSMB. If an SAE or SUSAR is deemed possibly, probably, or definitely related 
to the study medication by the DSMB, the trial will be stopped until the DSMB has decided 
how to proceed or to stop the study. 
9.7. Treatment failure criteria 
In case a patient develops symptomatic malaria (denoted as fever and the presence of asexual 
parasitaemia denoted by microscopy) at any time during follow-up, they will be treated with a 
full course of Dihydroartemisinin-Piperaquine. DP treatment tablets contain 40 mg 
dihydroartemisinin/320 mg piperaquine (Eurartesim, Sigma Tau) and will be administered per 
manufacturer guidelines shown in Table 5. 
Table 9. DP dosing 
Body weight (kg) 
Daily dose (mg)  
Tablet strength and number of tablets 
 
Piperaquine 
DHA 
 
5 to <7 
80 
10 
½ x 160mg / 20mg tablet 
7 to <13 
160 
20 
1 x 160mg / 20mg tablet 
13 to <24 
320 
40 
1 x 320mg / 40mg tablet 
24 to <36 
640 
80 
2 x 320mg / 40mg tablet 
36 to <75 
960 
120 
3 x 320mg / 40mg tablet 
75 to 80 
1,280 
160 
4 x 320mg / 40mg tablet 
>80 
Not eligible 
 
10. LABORATORY PROCEDURES  
10.1. 
Blood film 
Thick and/or thin blood films for parasite counts will be obtained and examined at screening 
to confirm P. falciparum monoinfection. Giemsa-stained thick and/or thin blood films will be 
examined at a magnification of 100. The blood smear will be considered negative if no 
parasites are seen after examining 100 high powered fields. 

 
22 
10.2. 
Gametocyte and asexual stage density measurement 
Blood slides stained with Giemsa will be double read over 500 fields for quantification of 
gametocytes and asexual stages. EDTA samples of blood (200 µL) will be tested for molecular 
quantification of gametocytes and asexual parasites. Parasite DNA and RNA will be extracted 
from whole blood samples in EDTA tubes and tested using quantitative real-time polymerase 
chain reaction (qPCR) with a detection limit of 0.02-0.1 parasites/µL of blood and highly 
precise parasite quantification [24]. Quantification of gametocytes will be based on sex specific 
female (Pfs25/CCP4) and male (PfMGET) mRNA detection and quantification by reverse 
transcriptase-PCR [25]. Markers of asexual stage parasites will include 18s Ribosmal and 
SBP1 mRNA. Markers of sexual commitment will also be assessed (e.g. Ap2-g). 
Further molecular analysis of samples from individuals who have undergone feeding assays 
(either from whole blood samples or from mosquito midguts) will include parasite genotyping 
(e.g. to assess clonal complexity or determine the presence of specific deletions e.g. HRP2) 
and human genotyping for specific genes (HBB, CYP2D6, G6PD).  
10.3. 
Serology and biomarkers 
Serological analysis of these samples will include assessment of antibody responses to 
gametocyte and asexual stage proteins in the microarray platform [26]. Non antibody protein 
biomarker assessment will be conducted using bead-based assays to assess for correlates of 
infection and infectivity (i.e. proteins associated with inflammation), or parasite density 
(HRP2), or parasite proteins linked with gamete activation in the mosquito midgut (i.e Pfg377). 
If plasma remains after mosquito feeding, some may be stored and used in standard 
membrane feeding assays in Radboud University, Nijmegen, the Netherlands, for the 
assessment of functional transmission reducing immunity [27]. 
10.4. 
Mosquito infectivity assay 
For each assessment of infectivity 2 ml of heparinized blood will be drawn from the study 
participant and stored at 37oC and transported to the insectary. At the insectary, using 
standard procedures, ~70 A. gambiae will be fed on the subjects’ blood for 15-20 minutes (this 
figure is based on a previous study where 61 fed mosquitoes allowed for an average of 50 
mosquitoes surviving until day 7 after the feeding experiment and being available for 
dissection and examination for oocysts). All of these mosquitoes will be dissected on the 7th 
day after the feeding assay for prevalence of mosquitoes with oocysts and quantification of 
oocysts. Infected guts will be stored for later PCR confirmation of oocysts. 
10.5. 
Magnetic-activated cell sorting  
Blood samples will be enriched for gametocytes using MACS LS columns as previously 
described [28].  
10.6. 
Haemoglobin concentrations 
Haemoglobin concentrations will be also measured regularly throughout the study using the 
HemoCue system (Hemocue AB, Angelholm, Sweden). 

 
23 
10.7. 
Complete blood count 
Using standard techniques, the clinical laboratory will perform the complete blood count (CBC) 
and platelet count. The following CBC parameters will be evaluated for exclusion criteria at 
baseline and for safety assessment during the study on days 2, 7 and 14: white blood cells, 
haemoglobin, haematocrit, platelets. 
10.8. 
Biochemistry 
Blood chemistries values will be also measured throughout the study. The following 
parameters will be evaluated for exclusion criteria at baseline and for safety assessment 
during the study on days 2, 7 and 14: serum creatinine and ALT. 
11. DATA MANAGEMENT AND ANALYSIS 
All data will be stored on secure password protected databases. Data that are collected on 
paper forms will be double entered. Data collected through handheld devices or directly 
produced by laboratory equipment will be examined for quality assurance and fed into the 
database using double data entry. Handheld devices used for data input and temporary 
storage will be password protected and data will be encrypted. 
11.1. 
Analysis plan 
Mosquito infectivity will be assessed at three levels: the mean number of oocysts in a sample 
of mosquitoes (i.e. oocyst intensity), the proportion of mosquitoes infected with any number of 
oocysts (i.e. mosquito infection rate), and the infectivity of the study participant to any number 
of mosquitoes (i.e. infectivity to mosquitoes). The primary outcome measure will be the percent 
change in mosquito infection rate within each arm by day 2 compared to baseline, with other 
time-points of infectivity assessments as secondary outcomes. Percent change will be 
reported as percent reduction (with 100% as total reduction of transmission, and negative 
values as enhanced transmission). Other secondary transmission outcomes are as described 
in the ‘Objectives’ section above. Some exploratory objectives may be published separately 
from the main primary/secondary outcomes. 
Statistical analysis will be conducted using the most recent STATA and SAS versions (16.0 
and SAS version 9.4 at time of writing). Mosquito infection data will be analysed at time-points 
after baseline only for those individuals who are infectious at baseline, although the data and 
analyses from all enrolled individuals will be published in the appendix for completeness. 
Individuals will be classified as infectious to mosquitoes if they infect at least 1 mosquito, with 
any number of oocysts. Infectivity to mosquitoes and parasite/gametocyte prevalence will be 
compared within and between treatment arms using generalised linear models (family: 
binomial, z-score, co-efficient with 95% CI) or fishers exact tests. Absolute haemoglobin 
density and percent change in haemoglobin density (relative to baseline) will be compared 
using paired t-tests (t-score for difference compared to day 0) and two-way t-tests (t-score for 
difference between ACT matched treatment groups at each time-point). The proportion of 
gametocytes that are male will be calculated for all values with total gametocyte densities of 
0.2/µL and over. Gametocyte circulation time will be calculated to determine the average 
period (in days) that a mature gametocyte circulates in the blood prior to clearance, using a 
deterministic compartmental model that assumes a constant rate of clearance and has a 

 
24 
random effect to account for repeated measures on individuals, as described previously [29]; 
circulation time will be analysed using t-tests (t score for difference between ACT matched 
treatment groups) or linear regression analyses. Area under the curve (AUC) of gametocyte 
density per participant over time will be calculated using the linear trapezoid method [30] using 
the first 28 days of observation only, and analysed by fitting linear regression models to the 
log10 adjusted AUC values, with and without adjustment for baseline gametocyte density (t-
score, coefficient with 95% CI). All other analyses of quantitative data will be performed using 
Wilcoxon sign rank tests (z-score for difference compared to matched values at day 0) and 
Wilcoxon rank-sum tests (z-score for difference between ACT matched treatment arms at 
each time-point). The nonparametric van Elteren’s test, an extension of the Wilcoxon rank 
sum test, will be used to compare the infectivity between treatment arms after adjusting for 
gametocyte density, as previously described [19]. This will compare infectivity between groups 
after stratifying into categories based on total gametocytes per microliter. Within these bins 
differences in infectivity between arms will be tested, thereby investigating sterilizing effects 
of gametocytocidal drugs. For all analyses, the threshold for statistical significance will be set 
at p<0.05. 
11.2. 
Publication/Dissemination 
Findings will be analysed and presented to local researchers and stakeholders in Mali and at 
other research meetings and symposium. Data will be submitted for publication in peer 
reviewed journals in due course. Anonymized data will be shared in public repositories upon 
publication. 
12. ETHICAL CONSIDERATIONS 
This research will be conducted in compliance with the protocol, Good Clinical Practice (GCP) 
guidelines, and all applicable regulatory requirements. A copy of the protocol, informed 
consent forms, and any other documents given to study participants will be submitted to the 
ethics committees (EC) of all the institutions involved. Written approval will be obtained for all 
subsequent amendments to the protocol, informed consent documents, and other study 
documentation referenced above. The investigator will notify the ECs and DSMB of violations 
of the protocol and serious adverse events. Ethical approval will be sought from the Ethics 
Committee of the Faculty of Medicine, Pharmacy, and Dentistry of the University of Science, 
Techniques, and Technologies of Bamako (Bamako, Mali) and the Research Ethics 
Committee of the London School of Hygiene & Tropical Medicine (London, UK). No activities 
will start before approval has been received by both committees. 
12.1. 
Confidentiality 
The investigator will ensure that the subject’s anonymity is maintained. Participants will not be 
identified in any publicly released reports of this study. All records will be kept confidential to 
the extent provided by laws and regulations. The study monitors and other authorized 
representatives of the regulatory authorities may inspect all documents and records required 
to be maintained by the Investigator.  
All laboratory specimens, evaluation forms, reports, and other records that leave the site will 
be identified only by a coded number to maintain subject confidentiality.  

 
25 
Fully anonymized data will be uploaded to publicly available data repositories (e.g. 
https://datadryad.org/, https://www.biorxiv.org/) if requested as a pre-requisite for publication 
of this research. This will be explained in the participant information and during the consenting 
procedure. 
The 
upload 
of 
anonymized 
data 
will 
only 
be 
permitted 
if 
the 
participants/participants parents provide explicit approval during consent (a tick box in addition 
to the signature required for trial participation). 
12.2. 
Risk 
This study only involves the use of antimalarial drugs at doses equal or lower than the 
recommended dosing and schedules. All drugs have been studied thoroughly and their 
toxicities are well described. 
Venipuncture can be associated with pain and bruising at the site of the prick, and rarely 
infection. Risks will be minimized by the study sites by ensuring adequate training of staff in 
all procedures and supply of consumables. The study site adheres to the standards of GCP 
and will be monitored.  
12.2.1. Risk of artesunate-amodiaquine 
About 30% of treated patients experience adverse reactions. Most of the reported adverse 
reactions are similar to symptoms usually seen during a malaria attack. Common side effects 
of artesunate-amodiaquine include anorexia, abdominal pain, nausea, asthenia, somnolence, 
insomnia and cough. More serious adverse reactions, although infrequently, are anaemia and 
vertigo. 
12.2.2. Risk of primaquine 
Primaquine is known to cause dose-dependent haemolysis among individuals who are 
deficient in glucose-6-phosphate dehydrogenase (G6PD), an enzyme involved in glucose 
metabolism prevalent in malaria endemic geographies [31]. Haemolysis in G6PD-deficient 
individuals is caused by oxidative stress induced by primaquine within red blood cells (RBCs); 
the extent of haemolysis depends on several factors including the dose of primaquine, the 
type of G6PD deficiency, the functional G6PD assessment at the time of dosing, the 
concurrent use of other oxidant drugs, and whether the person is hemizygous (homozygous) 
or heterozygous for G6PD-deficiency [31]. In 2010 the WHO recommended the addition of a 
single low dose of primaquine (0.25 mg/kg) to standard ACT treatment, without mandatory 
G6PD testing to block P. falciparum transmission in areas threatened by artemisinin resistance 
or approaching malaria elimination [1], [32]. The safety of single low dose PQ has 
subsequently been confirmed in clinical trials where PQ was combined with either DP or AL 
[33]–[35]; two of these studies were specifically designed to assess safety in G6PD deficient 
individuals and found no clinically relevant reductions in haemoglobin concentrations [33], 
[34]. PQ use is now widespread throughout the world, and many countries have adopted the 
0.25 mg/kg WHO recommendation for single low dose PQ without prior G6PD testing [36]. 
Common side effects include heartburn, nausea and abdominal cramps. 
12.2.3. Risk of artemether-lumefantrine 
Reported AL side effects have generally been mild. The main side effects are GI upset: 
anorexia (~18%), nausea (~5%), vomiting (~18%), abdominal pain (~5%), and diarrhoea 

 
26 
(~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.  
12.2.4. Risk 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 hepatotoxicity were observed. 
12.2.5. Risk of artemether-lumefantrine-amodiaquine 
In two recent studies [5], [6], artemether-lumefantrine-amodiaquine was found to be generally 
well tolerated and safe, although mild adverse events (mainly gastrointestinal) were more 
frequent compared to artemether-lumefantrine alone. The addition of amodiaquine was also 
observed to cause a small prolongation of the electrocardiogram corrected QT interval 
compared to artemether-lumefantrine alone (mean increase at 52 h of 8.8 ms versus 0.9 ms), 
but not to the extent of cardiac arrhythmias. In addition, amodiaquine use was associated with 
a bigger decrease in heart rate compared to artemether-lumefantrine alone and a higher 
incidence of bradycardia (<54 beats/minute), both in adults and children. 
12.3. 
Benefits 
The benefits of the participation include free treatment of malaria and other acute diseases 
that may occur during the follow-up period. Participants who are in the groups that receive 
primaquine may be less infectious to mosquitoes and reduce the chance of having mosquitoes 
in their environment becoming infectious. Findings will help in the treatment of future patients 
and communities with malaria and further optimize the design of community intervention 
programmes. 
12.4. 
Compensation 
Participants will receive compensation for the time and travel for protocol specified visits. This 
compensation will be carefully evaluated and provided upon agreement of the local ethics 
committee. In Mali, the estimated cost of time and travel expense per visit in Ouelessebougou 
for residents of Ouelessebougou is estimated to be 1500 CFA (~3 US $). The compensation 
for enrolment visit will be 3000 CFA (~6.0 US $) and 1500 CFA (~3 US $) for each additional 
visit. 
13. USE AND STORAGE OF STUDY SAMPLES 
Samples collected will be stored at MRTC in Bamako, Mali and some will also be shipped to 
collaborating research centres such as the London School of Hygiene & Tropical Medicine 
and/or Radboud University medical centre to perform specific tests as described above. 
Samples may be kept for a maximum of 10 years. As described in the patient consent forms 
secondary use of patient samples may be shared with collaborating institutes, if ethical 
approval and appropriate material transfer agreements are in place. 

 
27 
14. SPONSOR INFORMATION 
14.1. 
Indemnity 
London School of Hygiene & Tropical Medicine holds Public Liability ("negligent harm") and 
Clinical Trial ("non-negligent harm") insurance policies which apply to this trial. 
14.2. 
Sponsor 
London School of Hygiene & Tropical Medicine will act as the main sponsor for this study. 
Delegated responsibilities will be assigned locally. 
14.3. 
Audits and Inspections 
The study may be subject audit by the London School of Hygiene & Tropical Medicine under 
their remit as sponsor, the Study Coordination Centre, and other regulatory bodies to ensure 
adherence to GCP. 
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