Serological Screen and Treat Trial for P. Vivax: a Proof-of-concept Trial in Western Indonesia
Phase: NA
Conditions: Malaria, Vivax
Sponsor: Indonesia University

SEROLOGICAL SCREEN AND TREAT TRIAL FOR PLASMODIUM VIVAX: A 
PROOF-OF-CONCEPT TRIAL IN EASTERN INDONESIA 
Sponsorship: 
Walter and Eliza Hall Institute 
1 G Royal Parade, Parkville Victoria 3052 
Principal Investigator 
Prof. DR. lnge Sutanto, MPhil 
OXTREC 57-19/SSAT PROTOCOL_v1 210819

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
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TABLE OF CONTENTS 
1. TITLE OF RESEARCH PROTOCOL .................................................................... 3
2. INVESTIGATORS AND INSTITUTIONS .............................................................. 3
3. STUDY SYNOPSIS .............................................................................................. 4
4. BACKGROUND AND RATIONALE ...................................................................... 8
5. OBJECTIVES AND ENDPOINTS ....................................................................... 10
6. METHODOLOGY ................................................................................................ 12
7. LABORATORY METHOD .................................................................................... 19
8. DATA MANAGEMENT AND ANALYSIS .............................................................. 23
9. ETHICAL CONSIDERATION ............................................................................... 26
10. EXPECTED APPLICATION OF RESULT OF THE STUDY ............................... 26
11. STUDY INSURANCE ........................................................................................ 27
12. REFERENCES .................................................................................................. 27
13. ROLE OF INVESTIGATORS ............................................................................. 29
14. APPENDICES ................................................................................................... 32

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1. TITLE OF RESEARCH PROTOCOL
Serological screen and treat trial for P. vivax: a proof-of-concept trial in eastern 
Indonesia   
2. INVESTIGATORS AND INSTITUTIONS
Principal Investigator 
Inge Sutanto1 
Co-Investigators 
Rintis Noviyanti2 
Ari Satyagraha2 
Ayleen Alicia Kosasih3 
Lenny Ekawati3 
Iqbal RF. Elyazar3 
Decy Subekti3 
Mulya Rahma Karyanti4 
Erni Juwita Nelwan5 
Instiaty6 
Jeanne Rini Poespoprodjo7 
J. Kevin Baird3 
Shazia Ruybal Pesántez8
Leanne J. Robinson8 
Ivo Mueller8 
1. Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta,
Indonesia
2. Eijkman Institute for Molecular Biology, Jakarta, Indonesia
3. Eijkman-Oxford Clinical Research Unit, Jakarta, Indonesia
4. Department of Pediatrics, Faculty of Medicine, University of Indonesia, Jakarta,
Indonesia

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5. Department of Internal Medicine, Faculty of Medicine, University of Indonesia,
Jakarta, Indonesia
6. Department of Pharmacology and Theurapeutics, Faculty of Medicine, University
of Indonesia, Jakarta, Indonesia
7. Rumah Sakit Umum Daerah Kabupaten Mimika, Papua, Indonesia
8. Walter and Eliza Hall Institute, Melbourne, Australia
Funding: Australian Government National Health and Medical Research Council 
3. Study Synopsis
Study title 
SEROLOGICAL SCREEN AND TREAT TRIAL FOR P. VIVAX: A 
PROOF-OF-CONCEPT TRIAL IN EASTERN INDONESIA  
Regulatory Status 
Investigational – individually randomised phase IIb proof-of-concept 
trial 
Investigational 
Product and 
Route 
Multi-antigen sero-diagnostic test for measurement of P. vivax 
antibodies in plasma from finger stick as a means to detect 
hypnozoite carriers for treatment 
Study Objectives 
Primary objective: 
To determine the efficacy of a Serological Screening and Treatment 
(SSAT) intervention for the prevention of recurrent P. vivax malaria.  
Secondary objectives: 
(I) 
To evaluate the performance of a point-of-care (POC) anti-
P. vivax antibody detection test relative to Luminex 
(MagPix). 
(II) 
To evaluate safety of high dose PQ administration in 
schoolchildren. 
(III) 
To evaluate the accuracy of Sahli’s method and Standard 
G6PD (SD Biosensor Inc., Suwon-si, Gyeonggi-do, 
Republic of Korea) relative to HemoCue for haemoglobin 
measurement. 
(IV) 
To estimate the sensitivity and specificity of a microscopic 
diagnosis of malaria by blood sample from skin (ankle) 
relative to standard finger stick microscopic and PCR. 
(V) 
To measure duration of gametocyte in the blood samples 
of the skin of the ankle and finger prick. 
(VI) 
To estimate the sensitivity and specificity of magneto-
optical hemozoin detection by Gazelle® (Hemex Health, 
USA) relative to standard finger stick microscopic and 

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PCR.  
(VII) 
To analyse hemoglobinopathies in schoolchildren using 
HemeChip by Gazelle (Hemex Health, USA) 
Primary study endpoint: the proportional reduction of P. vivax 
incidence by PCR in children serologically screened relative to those 
receiving routine care during 9 months of active follow-up. 
Secondary endpoints: 
(I) 
Difference in the time-to recur of P. vivax by PCR in SSAT 
and control arms. 
(II) 
Difference in the number of recurrent P. vivax by PCR in 
SSAT vs. control arms. 
(III) 
Difference in the incidence of recurrent symptomatic P. 
vivax by LMF in SSAT vs control arms. 
(IV) 
Seroconversion rate before and after intervention in SSAT 
and control arms. 
(V) 
Sensitivity and specificity of POC antibody detection test 
vs. gold standard Luminex assay. 
(VI) 
Adverse event (AE) and Severe Adverse Event (SAE) of 
high dose PQ in schoolchildren.   
(VII) 
Hb level in Sahli’s method, Standard G6PD (SD Biosensor 
Inc., ROK), and Gazelle® (Hemex Health, USA), in 
comparison with HemoCue (HemoCue AB, Angelholm, 
Sweden).  
(VIII) Sensitivity and specificity of microscopic examination to 
detect parasitemia from the shallow skin vasculature of the 
ankle (light microscopy-skin/LMS) compared to standard 
microscopic (light microscopy-finger/LMF) and PCR. 
(IX) 
Mean duration time of gametocyte in LMS and LMF 
(X) 
Sensitivity and specificity of magneto-optical hemozoin 
detection (MOD) compared to standard malaria detection 
and PCR.  
(XI) 
Hemoglobinopathy rate in schoolchildren 
Study Site 
Malaka regency, East Nusatenggara province, Indonesia. 
Study Duration 
January 2020 to January 2021 
Number of 
Participants, 
Randomisation, 
and Treatment 
A total of 800 elementary schoolchildren and their households living 
at Malaka, West Timor.  
Randomization and Treatment: 

Participants will be randomized into two groups: (1)
intervention group (SSAT; n=400) and (2) routine care group
(n=400). All participants will be tested for presence of
antibodies to a validated panel of P. vivax antigens and

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standard finger stick microscopic. 

In the SSAT arm at enrollment, only children who are Pv
seropositive and/or LMF Pv/Po positive receive supervised
high-dose primaquine (1mg/kg for 7 days) regardless of their
symptoms. All blood slides will be processed and read directly
after blood collection. Then treatment will follow if positive.
Sero-positive children, on the other hand, will commence
treatment after the result is available (approximately 1 week).
Symptomatic or asymptomatic LMF positive for Pf/Pm will be
treated according to current national guidelines.

In the control arm at enrollment, only children who are
symptomatic and LMF Pv/Po positive will receive supervised
high-dose primaquine (1mg/kg for 7 days).

In both arms at every other timepoint, symptomatic LMF
positive children will receive treatment in accordance with
national guidelines (unsupervised).

In both arms at the final timepoint, LMF will be conducted on
all children and if positive will be treated according to national
guidelines.

As blood schizontocides, DHA-PP for 3 days will be given
according to body weight (national guidelines).
Inclusion and 
Exclusion Criteria 
Inclusion: 

resident of study area and attending selected elementary
school in Grade 1-5

no evidence of health condition that would interefere with
study participation

assent of child and documented parental informed consent
Exclusion: 

G6PD deficiency as determined by SD Biosensor quantitative
determination of <70% G6PD activity (male: <4 U/g Hb,
female: <6 U/g Hb).

Haemoglobin < 9 g/dL
Analysis plan and 
sample size 
calculation 
The study is designed to show a superiority of SSAT vs. routine care 
for the prevention of recurrent P. vivax infections. The primary study 
endpoint will be the proportion of children with recurrent P. vivax by 
PCR during 9 months of follow-up. Secondary endpoints include 
time-to-first P. vivax recurrence as well as incidence of symptomatic 

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P. vivax.  
SSAT intervention is expected to reduce incidence of P. vivax in 
comparison to standard care. With the previous prevalence rate of 
20%, we will have a power of >90% to detect a significant difference 
with the sample size of 350 children per group. We will anticipate the 
exclusion of 15% by recruiting 400 children per group. 
Lay Summary 
This study evaluates an experimental serological diagnostic 
technique intended to identify people at high risk of having dormant 
malaria parasites in their liver. The findings will assess the ability of 
this sero test to identify those carriers.   
800 children living in eastern Indonesia will be individually 
randomised to the experimental serologic test or routine care.  
Children in the serological diagnosis arm will be screened for the 
presence of antibodies to a previously validated panel of malaria 
antigens optimized for sensitivity to infection during the prior 9 
months. Furthermore, they also will be screened by microscopy. If 
positive by either test, they will be treated for that malaria infection.  
Children assigned to routine care will be screened by microscopic 
examination and treated when they show or have history of 
symptoms in the last 3 days. 
After initial screening and treating according to diagnostic technique, 
all children will be actively followed for 9 months with PCR detection 
every 4 weeks. 
During 9 month follow up, all children becoming acutely ill and 
proven to be positive for malaria by microscopy will be treated, in 
addition, all asymptomatic children will be treated at the final time 
point. 
POC serology test will be performed at baseline and final timepoint 
on all children in both arms. The results from this POC serology test 
will not be used to inform diagnosis or treatment in any way at either 
of the timepoints. 
None of these blood samples will be collected by venopuncture but 
instead minimally invasive lancet devices will be used.    
Sero-diagnostic 
test 
Quantitative measurement of antibody titres to a panel of 8 specially 
selected P. vivax antigens in study subject plasma samples using a 
Luminex MAGPIX® bead array system. This testing will be done at 
the Eijkman Institute in Jakarta. The results will be generated in no 

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more than 7 days.  
4. Background and Rationale  
In order to eliminate P. vivax within proposed timeframes (i.e. by 2030) it is 
essential to directly target the ‘silent’ hypnozoite reservoir sustaining P. vivax 
transmission. Modelling studies show that population mass-screening and treatment 
(MSAT) of parasite positive individuals will not have a persistent effect on P. vivax 
transmission even if screening is done by PCR (1). Cohort studies conducted in 
Thailand and Solomon Islands demonstrate that qPCR at a single timepoint detects 
only 25-30% of all individuals who subsequently have recurrent P. vivax infections in 
the following 10-12 months.  
While MDA including an anti-hypnozoitocidal drug such a primaquine is 
predicted to be highly effective against P. vivax, this is logistically challenging: G6PD 
testing is required to prevent the potentially severe haemolysis associated with 8-
aminoquinoline treatment in G6PD-deficient people and directly observed treatment 
(DOTs) is needed to assure treatment success. However, between 70 – 90% of 
people living in low- transmission and pre-elimination settings do not experience any 
P. vivax infections over a 12 months period and are thus highly unlikely to carry 
hypnozoites and would be exposed to the risks of primaquine treatment, without any 
of its benefits. In short, whilst MSAT misses up to 75% of all individuals requiring 
treatment (2), MDA “over-treats” up to 90% of the population.  
Effective screening with a P. vivax sero-diagnostic test may address this gap. 
As all non-temperate P. vivax strains cause a primary infection followed by a primary 
relapse within 4 weeks to 9 months, a validated panel of serological exposure 
markers (SEMs) are expected to identify most individuals that potentially harbour 
hypnozoites (3). SSAT with our novel antigens is therefore predicted to be superior to 
routine diagnosis and treatment in preventing recurrent P. vivax infections and thus 
reducing P. vivax transmission. 
Further to the idea of identifying silent and latent/subpatent infections, this 
project explores the possibility of the shallow vasculature of human skin sequesters 
and sustains infectious gametocytes of the plasmodia. Gametocytes are the only 
stage able to continue living inside the mosquito body to undergo a sexual 
reproduction. Therefore, anopheline feeding time is a critical phase for survival of 

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this species. However, a typical anopheline has a proboscis about 300 microns in 
length, in which its protrudable feeding parts are housed. Its feeding depth would 
certainly not exceed that reach and would probably be considerably shorter. 
Gametocytes must be near to the surface of the skin in order to infect anopheline 
mosquitoes (i.e., survive to reproduce). We know that microscopically sub-patent 
volunteers very often infect anophelines allowed to feed on their skin while being 5-
times less likely to infect anophelines simultaneously allowed to membrane feed on a 
sample of venous blood (4). Other studies have reported very similar findings (5). 
Those studies corroborate earlier reports of sexual stage gametocytes in skin 
vasculature occurring at 3-fold higher numbers relative to peripheral blood (finger 
stick) (6). We hypothesize that gametocytes observed in finger stick samples of 
vasculature well below 300 microns (typically about 600 microns) rapidly transit that 
compartment on their way to longer-term sequestration in the microvasculature of 
the uppermost dermis, just below the epidermis. That is precisely where they need to 
be, and a protracted residence in that compartment may explain their virtually 
dormant metabolic state (7), much like that of latent hypnozoites in hepatic cells (8). 
Human hosts experiencing exceptional and fleeting states of infection like 
symptomatic patency may go on to host silent, stable, and durable infections of the 
shallow skin microvasculature by sexual plasmodia. Moreover, these sexual stage 
should be distributed in such a way to increase the odds of being taken up by the 
mosquito, i.e. feet and ankle, where anopheles shows the strong preference (9). 
There is also possibility they have circardian rhythm (10). 
The current standard for the diagnosis of malaria in ill patients or healthy 
residents of endemic areas perform poorly at parasite counts below 50 per microliter 
blood (11,12). Nucleic acid-based diagnostic techniques offer relatively much greater 
sensitivity (i.e., from approx. 1/uL to 1/mL), but these assays cannot be done at 
point-of-care (a sophisticated laboratory and equipment are required), takes days or 
weeks to complete, requires highly specialized training, and imposes prohibitive 
direct costs (13). Malaria research leaders and communities identified and 
acknowledged the need for a point-of-care diagnostic tool with PCR-like sensitivity 
as a critical need (14,15). The simple, practical, and inexpensive approach to a 
bedside diagnosis within an hour would effectively empower mass screening and 
treatment as a means of interrupting malaria transmission in endemic communities, 
whereas today that practice is futile due to the weakness of diagnostic technology 

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and the epidemiological dominance of clinically silent sub-patent parasitemias (5).  In 
search of point-of-care diagnostic test, potential of hemozoin as detection tool will 
also be explored. Previous studies employing magnetic field to capture hemozoin 
have demonstrated strong preference for gametocytes and increased sensitivity for 
detection of P. vivax asexual stages (16,17). This project will investigate the 
performance of a point-of-care diagnostic tool employing magneto-optical detection 
in ankle/feet area which can provide further evidence for the skin reservoir 
hypothesis.  
Moreover, 
previous 
studies 
have 
demonstrated 
a 
higher 
rate 
of 
hemoglobinopathy in malarious areas due to evolutionary pressure (18).  Apart from 
its controversial benefit against malaria, this trait brings substantial health problem to 
individuals carrying it. This project will investigate the rate of hemoglobinopathy in 
schoolchildren.         
Lastly, radical cure of P. vivax is crucial part to stop malaria transmission, 
therefore compliance and safety monitoring of currently available hypnozoitocidal PQ 
is essential (19). This project will contribute evidence towards the development of 
guidelines for high dose PQ in Primary Health Center to establish its use in national 
scale. The utilisation of cheap and widely used Sahli’s Hb measurement for such 
purpose will be evaluated as well.        
5. Objectives and Endpoints 
5.1. Primary objective: to determine the efficacy of a Serological Screening and 
Treatment (SSAT) intervention for the prevention of recurrent P. vivax malaria. 
5.2. Secondary objectives:  
1. To evaluate the performance of a point-of-care (POC) anti-P. vivax antibody 
detection test relative to Luminex (MagPix). 
2. To evaluate safety of high dose PQ administration in schoolchildren. 
3. To evaluate the accuracy of Sahli’s method and Standard G6PD (SD 
Biosensor Inc., Deogyeong-daero, Korea) relative to HemoCue for 
haemoglobin measurement. 

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4. To estimate the sensitivity and specificity of a microscopic diagnosis of blood 
by sample from skin (ankle) relative to standard finger stick microscopic and 
PCR. 
5. To measure duration of gametocyte in the blood samples of the skin of the 
ankle and finger prick. 
6. To estimate the sensitivity and specificity of magneto-optical hemozoin 
detection by Gazelle® (Hemex Health, USA) relative to standard finger stick 
microscopic and PCR. To analyse hemoglobinopathies in schoolchildren using 
HemeChip by Gazelle (Hemex Health, USA) 
 
 
Primary endpoint: the proportional reduction of P. vivax incidence by PCR in children 
serologically screened relative to those receiving routine care during 9 months of 
active follow-up. 
Secondary endpoints:  
(I) 
Difference in the time-to recur of P. vivax by PCR in SSAT and control 
arms. 
(II) 
Difference in the number of recurrent P. vivax by PCR in SSAT vs. control 
arms. 
(III) 
Difference in the incidence of recurrent symptomatic P. vivax by LMF in 
SSAT vs control arms. 
(IV) 
Seroconversion rate before and after intervention in SSAT and control 
arms. 
(V) 
Sensitivity and specificity of POC antibody detection test vs. gold standard 
Luminex assay. 
(VI) 
Adverse event (AE) and Severe Adverse Event (SAE) of high dose PQ in 
schoolchildren.   
(VII) 
Hb level in Sahli’s method, Standard G6PD (SD Biosensor Inc., 
Deogyeong-daero, Korea), and Gazelle® (Hemex Health, USA), in 
comparison with HemoCue (HemoCue AB, Angelholm, Sweden).  
(VIII) Sensitivity and specificity of microscopic examination to detect parasitemia 
from the shallow skin vasculature of the ankle (light microscopy-skin/LMS) 
compared to standard microscopic (light microscopy-finger/LMF) and 
PCR. 

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(IX) 
Mean duration time of gametocyte in LMS and LMF 
(X) 
Sensitivity and specificity of magneto-optical hemozoin detection (MOD) 
compared to standard malaria detection and PCR.   
(XI) 
Sensitivity and specificity of Standard G6PD (SD Biosensor, Inc., 
Deogyeong-daero, Korea Korea) compared to CareStart G6PD RDT 
(Access Bio. Inc., New Jersey, USA).  
(XII) 
Hemoglobinopathy rate in schoolchildren 
  
6. Methodology 
6.1. Study site and Participants 
 
The study will be conducted at mesoendemic area of Malaka regency (124º 
38’ 32.17” – 125º 5’ 21.38” E and 9º 18’ 7.19” – 9º 47’26.68” S), West Timor, 
Indonesia. Formerly known as part of Belu regency, Malaka regency was established 
in 2012. At north it is adjacent to Belu regency, at south Timor sea, at east 
Democratic Republic of Timor Leste, and at west South Central Timor and North 
Central Timor.  This area has 20 Primary Health Center (Puskesmas) and one 
hospital. Most of these Puskesmas (14) reside in malaria endemic areas. The 
highest Annual Parasite Incidence (API) were reported from Puskesmas Alkani 
(21.43), Weoe (4.79), and Tunabesi (4.46) in 2018. Working area of Puskesmas 
Weoe includes 15,652 residents and 16 elementary schools, in contrast with 
Puskesmas Alkani which only has 6,111 residents and 4 elementary schools. 
Therefore, we propose to conduct the study at working area of Puskesmas Weoe. 
 
6.2. Schools selection and demographic census 
Before the start of the study, a serological survey will be conducted to select 
the schools. Blood samples will be collected from  20 randomly selected children 
from each school in Weoe for serological screening with Luminex. Only schools with 
sero-prevalence of 30-70% will be eligible for enrollment. Maximal number of 
selected schools will be 6-7, where 800 children will be individually randomized. 
Demographic census will be conducted during which name, date of birth/age, 
gender, village, hamlet, parents/guardian will be recorded (census form). Photograph 
will be taken from each participants to ensure the correct identification for treatment 
administration and follow up. 

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6.3. Study design and study overview 
The study design is a randomized controlled trial with serological screening 
and treatment as the intervention. Selected schoolchildren will be allocated into two 
study arms: (1) serological screening and treatment (SSAT), and (2) routine care as 
control arm.  
At enrolment, in the SSAT arm, individuals found P. vivax positive by either 
serological (Luminex) or Giemsa stained microscopic examination regardless of the 
symptoms will receive supervised treatment (direct observation) with high dose PQ 
(daily 1 mg/kg BW) for 7 days (Po=Pv). In the control arm, only symptomatic 
individuals found P. vivax positive by Giemsa stained microscopic examination will 
receive the same regimen (Po=Pv). In addition, in the treated children of both arms, 
dyhidroartemisinin-piperaquine (DHA-PP) will be given as blood schizontocide 
according to body weight.  All other species (P. falciparum, P. malariae) found by 
Giemsa stained microscopic examination will be treated according to national 
guideline. Serological examination will be performed by Luminex (done at Eijkman, 
Jakarta) and point-of-care (POC) antibody detection using rapid test on site 
Follow up will be perfomed monthly for 9 months. During this period, 400 µL of 
finger pricked blood samples will be collected from all children in both arms. These 
samples will be tested for malaria presence by PCR which will be done at EOCRU, 
Jakarta. However, children with clinical symptoms (body temperature of >37.5oC 
and/or history of fever within 3 days), will be examined by Giemsa stained 
microscopy on site, and treated if positive (any species) according to national 
guideline (unsupervised).  
At the last follow up, a mass blood survey will be conducted to both arms for 
Pv serological test, LMF, and PCR. Those found positive by LMF for any species will 
be treated regardless of their symptoms.  
6.4. Inclusion and exclusion criteria 
Inclusion: 

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 resident of study area and attending selected elementary  school in Grade 1-5 
 no evidence of health condition that would interefere with study participation 
 assent of child and documented parental informed consent  
Exclusion:  
 G6PD deficiency as determined by SD Biosensor quantitative determination 
of <70% G6PD activity (male: <4 U/g Hb, female: <6 U/g Hb). 
 Haemoglobin < 9 g/dL as determined by HemoCue. 
These subjects will be referred to PHC for treatment. 
Figure 1. Study Framework  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomized: 6-7 schools  randomised 
800 elementary schoolchildren  
16 elementary schools: 
20 children from each school will be 
screened with serology  
Eligible schools: 
seroprevalence of 30-70%  
SSAT group (n=400) 
Control group (n=400) 
MBS: Pv serology, POC Pv antibody  
           rapid test, LMF, PCR, Hb, G6PD,  
           and other substudies (Hemex,  
           LMS). 
Treatment: Pv serology (+) and/or  
                     microscopic (+) 
MBS: Pv serology, POC Pv antibody  
           rapid test, LMF, PCR, Hb, G6PD,  
           and other substudies (Hemex,  
            LMS). 
Treatment: microscopic (+) in      
                     symptomatic 
Monthly follow up (month 1-8): 
MBS: PCR & LMS  
           LMF in symptomatic, and if (+)  
 
            Screen for family members  
            (LMF, LMS, PCR)       
            Treat all LMF (+) 
Monthly follow up (month 1-8): 
MBS: PCR & LMS  
           LMF in symptomatic, and if (+) 
 
            Screen for family members  
            (LMF, LMS, PCR) 
            Treat all LMF (+) 

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6.5. Study Socialization, Informed Consent, and Training 
Staff of Primary Health Center (PHC) of Weoe will be gathered and explained 
about the study plan. They will be asked to assist our team in the field activity. Then, 
head and teachers of selected schools will be gathered and explained about the 
study plan. Finally, meetings which invite parents/ legal guardians of the selected 
children will be organized 2-3 weeks prior to the study to obtain informed consent 
from every child. During these meetings the study will be explained to parents/legal 
guardian of the schoolchildren. At the end of activity, there will be a discussion 
session. The explanation is arranged in a seminar form by the trained investigator 
team with ample time of question and answer session. At the end of discussion, 
informed consent form will be distributed for each parent. They have 1 week to 
consider whether they will participate in our study or not. The signed informed 
consent form will be collected by the class teacher and field team.  
Trainings will be given for the personnel of PHC that involved in the study, 
school teachers and health care volunteers. Explanation will be given in details with 
standard operating procedure (SOP) provided on their duty of each group to facilitate 
the process of data and sample collection.     
 
6.6. Enrolment  
During enrolment, finger pricked blood samples will be collected from all 
participants. At this activity, body temperature will be recorded using infrared 
thermometers, body weight will be scaled, history of fever within 3 days and taking 
antimalarial drugs within last 2 weeks will be recorded. Finger pricked blood samples 
will be made into malaria blood smear and tested by POC Pv antibody rapid 
detection. A total of 400 µL will be collected into microtainer tube containing EDTA to 
be sent to field laboratory, and separated for various tests, e.g. serology (Luminex), 
PCR, Hb, G6PD, and Hemex.  
All participants’ blood samples will be tested for the presence of antibodies to 
a validated panel of P. vivax antigens (using a gold-standard laboratory test) at the 
Eijkman Institute for Molecular Biology, Jakarta. Blood samples will be shipped every 
2 days, and the result will be reported to the field team within no more than 1 week.  
End of study (month 9):  
MBS: Pv serology, LMF, PCR & LMS 
Treatment: all LMF (+)  
End of study (month 9):  
MBS: Pv serology, LMF, PCR, & LMS 
Treatment: all LMF (+) 

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On site, blood slides will be Giemsa stained (see 7.3) and assessed by 
microscopy with 1000x magnification. Screening for G6PD deficiency will be 
performed quantitatively by Standard G6PD (SD Biosensor, ROK) with the cut off of 
4 U/g Hb (male) or 6 U/g Hb (female) on the same day of blood collection. Hb level 
will be measured using HemoCue and Hb Sahli. Other substudies (LMS, hemozoin 
detection, hemoglobinopathy) will also be conducted (see 6.10) 
 
6.7. Follow up and end of study  
First follow up will be conducted one month after the last dose of PQ 
treatment administration. Every visit, blood samples from finger prick and skin (LMS) 
will be taken. Unlike in the 16ecurrent process, malaria blood smear will be made 
only from participants with clinical symptoms suggestive for malaria. If positive, they 
will be treated.  All treatment during follow up period will be provided by PHC staff 
according to national guideline (Appendix B table 1 & 3).  
At the end of study (month-9 of the follow up), mass blood survey will be 
conducted to collect finger prick blood (LMF, Pv serological test, qPCR) and LMS. 
Seroconversion will be assessed. All LMF (+) will be treated as above. 
All study subjects will be followed until the end of study (month 9) although 
they cannot fulfil the complete schedule.  
6.8. Antimalarial treatment 
Treatment will be provided based on body weight (Appendix B). Blood 
schizontocide for any species is 3-day DHA-PP (daily dose: 6-10 kg=1/2 tab, 11-17 
kg=1 tab, 18-30 kg=1 ½ tab, 31-40 kg=2 tab). For P. vivax subjects, the 
administration of high dose primaquine will be started at the same day of DHA-PP 
treatment (see Table 2 appendix B for PQ dosage). Meal should be given prior to PQ 
administration daily. The total dose of PQ should be completed within 10 days as the 
window period. Subjects who does not complete the dose will be recorded, and still 
be followed until end of study. Monitoring of Hb level and hemoglobinuria for treated 
subjects will be done daily before and during treatment by Hb measurement using 
HemoCue (HemoCue AB, Angelholm, Sweden) and urine test (inspection and 
urinalysis) in order to detect hemolysis. Primaquine will be stopped if hemolysis 

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occur, the subject will be observed for daily vital sign, Hb level, and urine until 
recovery. If worsen, the subject will be referred to the hospital for further 
management, and given blood transfusion if necessary (Appendix E).  
Primaquine for P. falciparum will be given as single dose 0.25 mg/kgBW (6-17 
kg=1/4 tab, 18-30 kg=1/2 tab, 31-40 kg=3/4 tab). P. ovale will be treated as P. vivax. 
Mixed infection with Pv will be treated as Pv. 
 
6.9. Adverse Event (AE) and Serious Adverse Event (SAE) 
Adverse events (AE) are defined as any untoward and unintended reaction 
during the study period. AE will be identified and recorded in CRF by field team in 
collaboration with the teachers and primary health center attendants. These subjects 
will be given symptomatic treatment as necessary. Specific AE are black urine, 
gastro intestinal disturbances, blue lips and breathless.  
Serious adverse events (SAE) is an AE which becomes serious and could 
endanger the life of subjects, cause hospitalization/prolong hospitalization, and 
disability to conduct normal life. The SAE will be recorded in SAE form and reported 
to PI, clinician coinvestigators, Ethic Committee, and DSMB within 24 hours. SAE’ 
subjects will be treated in RSPP/RS Atambua until recover completely. Complete 
SAE report will be distributed within one month after the occurrence of SAE.  
 
The observation of AE and SAE will be performed routinely according to the 
schedule of drug administration (7 days).  
If subject is allergic to DHA-PP, second-line treatment (7 days quinine) will be 
provided by the team in close collaboration with local primary health center (11-17 
kg: 3x1, 18-33 kg: 3x1½, 34-40 kg: 3x2).  All treatments for AE/SAE which will be 
given either at the PHC or in hospital, should be reported to the investigator team 
and being documented in the database.   
Treatment will be stopped immediately if any of these following occurs: 1) 
allergy to the antimalarial regimen being given, 2) Hb drop of more than 20%, 3) 
active hemolysis which is proven by urine test and Hb drop, 4) Hb drop with warning 
sign of MetHb (breathless, blue lip/cyanosis, drowsiness, lethargy, palpitation), 5) 
Any possible SAE related to antimalarial treatment. Hospitalisation is indicated for 2) 
to 5).  
 
6.10. Gametocyte study 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
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At enrollment, blood samples will also be taken from shallow vasculature of 
the skin from the ankle of all participants from both arms for microscopic examination 
(light microscopic-skin/LMS) (figure 2). Malaria status by LMS will be assessed post-
hoc and will not indicate any treatment. 
To determine longevity of these skin gametocytes, LMS samples will be taken 
at monthly follow up. Moreover, to explore any diurnal variation of this stage, we 
should obtain blood samples at night and during the day. Therefore, during follow up 
activity, house visit will be made to the malaria infected children with symptoms for 
collecting two times blood samples, i.e. 7-10 AM and 7-10 PM.  In addition, 
household members will be asked to contribute their blood samples (LMF, LMS, and 
PCR). For the household members, LMF will be read on site to indicate treatment.  
Figure 2. Location of ankle blood sampling 
 
6.11. Other substudies 
During enrollment, several diagnostic tests will be performed to all 
schoolchildren as substudies:  magneto-optical detection of hemozoin/MOD (finger 
prick and skin), hemoglobinopathy screening by HemeChip (finger prick), and Hb 
Sahli.  
 
6.12. Ethical clearance 
The study protocol will be submitted to Ethic Committee Faculty of Medicine, 
University of Indonesia, the Oxford Tropical Diseases Research Ethics Committee 
(OXTREC), and Walter and Eliza Hall Institute Human Research Ethics Committee 
to obtain ethical clearance. Upon above approval, the protocol will be sent to the 
local government of Malaka regency to obtain the local permit. 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
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7. 
Laboratory Methods 
7.1.  Blood Collection and Transportation  
7.1.1. Finger-prick Blood Sample Collection 
Blood samples will be drawn from fingertip using BD lancet. During enrolment, 
malaria blood smear, POC rapid Pv antibody detection, hemozoin detection, and 
hemoglobinopathy screening will be done directly on site. Then 400 µL blood will be 
collected into microtainer tube containing EDTA. The microtainer tubes will be placed 
into the ice box, and delivered in no more than 4 hours to the field laboratory for 
further process.  
 
During follow up (month 1-month 8), only 400 µL of finger pricked blood will be 
collected into the microtainer. At month-9, malaria blood smear and POC Pv 
antibody detection will again be done on site, as an addition to the 400 µL 
microtainer blood.  
 
7.1.2. Skin Blood Samples  
To collect blood from skin of the ankle, a vacuum-assisted lancet (Genteel®) 
will be used with penetration depth of 600 µm. The selected location is shown in the 
Figure 2. An alternative site will be dorsal surface of the foot between second and 
middle toe. Thin blood smear will be done directly from the skin blood using 
microcapillary tube or micropippette. This almost completely painless procedure may 
repeated one or more times (up to 3 times) to sufficient blood volume.  
 
7.2. Separation of Whole Blood Specimen and Shipment 
This step will be done in the field laboratory. During enrolment, blood collected 
in the microtainer will be separated for quantitative G6PD screening test by Standard 
G6PD (SD Biosensor, ROK), Hb measurement by HemoCue and Sahli, serological 
test, and qPCR.  
Ten microliters will be used for Standard G6PD (SD Biosensor), 30 µL for 
hemoglobin measurement (10 µL for HemoCue and 20 µL for Sahli). The remaining 

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blood will be centrifuged at 5000 RPM for 10 minutes at room temperature. Plasma 
(supernatant, yellow transparent top layer) will be transfered into new screwed 
cryogenic tubes. Tubes containing plasma for serological test and pellet for qPCR 
will be immediately stored in the -20oC freezer. Every 2 days, tubes containing 
plasma will be shipped to Jakarta for serological assay using cold-chain, and 
monitored for temperature during transportation. During follow up, blood will be 
separated into plasma (for serological analysis when needed) and pellet for qPCR to 
detect recurrence. 
 
7.3. Blood smear Giemsa staining and microscopic reading  
Blood films will be dried in room temperature overnight, and fixed with 
methanol (thin smear only) before it is stained using 3% Giemsa made by mixing the 
stock solution with buffer. The stain will be gently poured into the slides until they are 
totally covered. After 45-60 minutes, slides will be rinsed with water and allowed to 
dry and read under binocular microscope with 1,000 magnification (20).  
WHO certified microscopist will read the slides in the field, and cross check 
will be done at Jakarta. Asexual stages and gametocytes will be counted separately. 
For LMF, parasites will be counted against leucocytes in the thick smear. Density will 
be recorded as the number of parasites found per 200 leucocytes. A conversion to 
number of parasites in microliter of blood will conducted under the assumption of one 
microliter of blood contains 8,000 leucocytes. 
For LMS, parasites will be counted against RBC found in 20 HPF. Density will 
be recorded as number of parasites per 1 microliter. A conversion to number of 
parasites in microliter of blood will conducted under the assumption of one microliter 
of blood contains 5,000,000 RBC. 
 
7.4. Serological Test  
7.4.1. Gold-standard Luminex   
This test will measure IgG antibodies titres to a panel of 8 specially selected 
P. vivax antigens in plasma samples and will be performed using a Luminex 
MAGPIX® bead array system. This test will be run in plate format containing blank 
(in duplicate), positive (singlicate), and negative (singlicate) controls. Blank contains 
only PBT(1x PBS, 1% BSA, 0.05% Tween-20 (Sterile filter the PBS-BSA prior to 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
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adding Tween-20). A 2-fold serial dilution from 1/50 to 1/25,600 of positive controls 
will be run in singlicate. A 1/100 dilution of plasma sample will be run in each well.  
 
7.4.2. Point-of-Care anti-P. vivax antibody detection test 
This test will be done on site by finger prick. This laminar flow rapid diagnostic 
test will measure IgG antibodies titres to a fragment of the P. vivax RBP2b 
(reticulocyte binding protein 2b) antigen in whole samples. This test will require 
placing 15µL of blood and a buffer into a laminar flow cartridge. The test can be read 
after 10-15 minutes either by eye or with a specific reader (if quantification is 
desired).  
 
7.5. Glucose-6-Phosphate Dehydrogenase (G-6-PDH) Screening Test  
Quantitative G6PD screening test will be performed to all schoolchildren. 
Standard G6PD (SD Biosensor, ROK) will detect amount of G6PD enzyme in U/gHb. 
The result is shown as number of U/gHb. G6PD normal is defined as G6PD level >4 
U/g Hb for male or >6 U/gHb for female.  
 
7.6. 
Hemoglobin Level Measurement  
Hemoglobin level will be measured in the field lab using HemoCue® 
(HemoCue AB, Angelholm, Sweden) as golden standard. After mix properly,10 µL 
blood will be transferred into the Hemocue microcuvette. Blood filled microcuvette 
will be inserted into the Hemocue Hb 301+ automated machine system (HemoCue 
AB, Angelholm, Sweden). The haemoglobin result will be then displayed after about 
30 to 45 seconds in g/dL units. Result will be saved, and transferred to PC. Result 
will also be recorded in the Case Report Form manually. Additionally, Sahli’s 
hemoglobinometer and Standard G6PD (SD Biosensor Inc., Deogyeong-daero, 
Korea) will be evaluated.  
For Sahli’s method, 20 µL blood will be transferred by micropipette into the 
tube containing 2 g% of HCl. The solution is left for 10 minutes, then diluted with 
distilled water drop by drop until the colour changes to matched the comparator in 
the Sahli’s hemoglobinometer. 
  
7.7. 
Gazelle® (Hemex Health, USA) test 

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This point-of-care test will be done on site during enrolment. It will detect the 
presence of malaria paramagnetic byproduct hemozoin using magneto-optical 
detection (MOD) principle. A pipette is used to drop 10uL from skin vasculature or 
15uL of whole blood from finger prick into the cartridge bottom, and mixed with 85uL 
of Gazelle Diluent (mostly water). The cartridge is then manually inserted into the 
chamber in the Reader. Inside the cartridge, blood sample is sonicated, causing it to 
lyse the blood and release hemozoin. The magnetic field is used to align the 
hemozoin. The alignment will block the transmission of light which is proportional to 
the quantity of hemozoin in the sample. The malaria positive/negative (qualitative) 
test result is displayed on the Reader within approximately one minute. The result 
will be stored in the Reader’s memory which can be printed or transferred to a laptop 
or printer.  
The machine will also identify hemoglobin type by electrophoresis on 
microchip. Twenty microliter of finger pricked blood sample will be inserted into the 
cartridge and read within 10 minutes.  
7.8. 
qPCR Malaria Speciation  
 
This step will be performed at Universities of Indonesia and Oxford Clinical 
Research Laboratory (UIOCRL), Jakarta. DNA will be extracted from 200 µL of red 
cell pellet using a commercially available kit. All blood samples will be screened for 
the presence of malaria parasites using Real-Time PCR targeting a genus-specific, 
conserved region of 18S rRNA.  For all positive samples, speciation and 
quantification will be conducted using probe-based Real-Time PCR, which will 
amplify the multicopy gene 18S rRNA (21).   
 
7.9.  Genotyping  
All samples from schoolchildren found to be qPCR-positive for Plasmodium 
falciparum or Plasmodium vivax will undergo genotyping using a targeted amplicon 
deep sequencing approach to genotype clones within individual infections, measure 
genetic relatedness, and discriminate new and existing infections. Each sample will 
undergo PCR-amplification of highly polymorphic genomic regions followed by next-
generation sequencing (22,23).  
 
7.10. Storage of the Specimens 

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Malaria stained blood smears will be stored in the UIOCRL, Jakarta. Blood 
samples will be stored in the -20oC freezer at UIOCRL, Jakarta until further process.  
 
 
 
 
8. 
Data management and analysis 
8.1. 
Data management 
Qualitative and quantitative data will be collected during study period. 
Research team will visit selected elementary schools to conduct demographic 
census of study participants with the assistance of class teacher and cadres.  
Body weight and temperature, history of fever, results of malaria examination 
and other substudies, anti-malarial treatment, AE, SAE (if any) will be recorded in 
Case Report Forms. CRF will only contain ID number of the subjects instead of their 
full name. Labelling of specimen will be given by adding activity number to the 
subject ID, e.g. QQxxx-0 (enrollment), QQxxx-1 (follow up 1), etc. (QQ=school initial, 
xxx=subject number) 
After end of study, hard copy files will be stored for at least 5 years at 
UIOCRL. All CRFs will be transferred into electronic database. Only authorized 
individuals using secure, authorized computers may access this data management 
system. 
8.2. Sample size consideration 
Sample size is calculated based on the difference in the incidence of P. vivax 
infection between the intervention and control arm. The impact of the SSAT 
intervention is expected to reduce incidence of P. vivax in schoolchildren compared 
to control group. Our study in 2013 found Pv prevalence of ~20% by PCR (2). 
Different effect and sample sizes were simulated using a specific mathematical 
transmission model (24) and statistical power calculated. With the proposed sample 
size of 350 children per arm, we will have a power of >90% to detect a significant 
difference (Figure 3). Assumming 15% exclusion rate, we will recruite 400 children 
per arm. 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
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Figure 3. Simulation of various sample size (n=50-500) to detect significant 
difference in the proportion of infected subjects during follow up period.    
 
  
 
8.3. Data analysis  
Plasmodium vivax incidence is defined as the total number of new genetically 
distinct P. vivax clones per child per detected during the follow up period. The 
analysis will use the comparison of two incidence rates between SSAT group and 
control group. For comparison, data will be entered as the number of recurrent Pv 
(numerators) and the total length of time people contributed in the cohort (person-
time) as denominators. We will calculate the exact probabilities (Fisher's and Mid-P), 
the rate ratio with the exact confidence intervals and the measures of the impact of 
intervention. We will report the 95% of confidence interval for this comparison. 
Measures of the impact of intervention are computed for the prevented fractions in 
the exposed and in the population and the preventable fraction in the population.  
As secondary endpoints, we will use Kaplan-Meier method to detect 
differences in the time-to-first recurrence of Pv by PCR in SSAT and control 
arms. The log-rank test is used to detect any difference between the two groups. Cox 
proportion hazard will be used to measure the effect size.  
Multiple recurrences of P. vivax infections will be calculated during follow up. 
Number of events will be calculated, divided by the total length of time people 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
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contributed in the cohort (person-time) as denominators. We will calculate the exact 
probabilities, rate ratio with the exact confidence intervals, and the measures of the 
impact of intervention using same tests as above.  
Symptomatic recurrent P. vivax is defined as LMF positive with fever 
(>=37.5oC) or history of fever in the last 3 days during follow up. The same statistical 
test will be performed to compare both groups.  
Difference of seropositivity between the two arms before and after the 
intervention will be calculated using chi-square test with p value < 0.05 as limit of 
significance.   
Sensitivity, specificity, positive predictive value, and negative predictive value 
of POC Pv antibody rapid test will be reported with Luminex as the golden standard.  
Type and number of AE and SAE will recorded, and reported with total length 
of time people contributed in the cohort (person-time) as denominators. 
 Plasmodium gametocytes prevalence in LMF and LMS will be calculated as 
the number positive for gametocytes by each diagnostic method divided by the 
number of subject examined. Diagnostic performance will be derived using 2 × 2 
contingency to determine sensitivity, specificity, positive predictive value (PPV), 
negative predictive value (NPV). Results of qPCR detection will be used as the gold 
standard for diagnostic method evaluation. All statistics will be reported as absolute 
values with their 95% confidence interval (95% CI). To assess the significance of 
different results of LMF and LMS methods, we will apply McNemar’s chi-square test 
on 2x2 contingency tables derived from the results of each detection method.  
We will evaluate durability analysis by measuring population half-life of the 
existence of Plasmodium gametocyte detected by LMS samples. Gametocyte 
prevalence-based approach will use data on the appearance and disappearance of 
any circulating gametocytes according to the prevalence at month 0 and the 
prevalence at the time when they are fully cleared from skin vasculature.  
Sensitivity, specificity, positive predictive value, and negative predictive value 
of hemozoin detection will be reported using microscopic Giemsa examination as 
golden standard.  
Hemoglobin measurement using Sahli method and Standard G6PD will be 
compared to HemoCue by t test. 
Hemoglobinopathy rate will be reported as descriptive value.  
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
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9. 
Ethical consideration   
9.1. 
The subject risks  
Blood samples collection will only bring minimal risk to the subjects. Only 
small volume of blood samples (~0.5 ml) will be drawn from the fingertip and using 
modern equipment which only cause small lesion on the incision site where the 
lancet or microtainer is introduced. A lancing device of the latest technology will be 
used to collect blood samples from the skin to collect sufficient blood volume with 
minimal pain. Blood sample collection will only be performed by trained staff of our 
investigator team to minimize the possibility of infection or other unlikely adverse 
events.   
The risk of hemolysis in G6PD deficient subjects has been minimized by 
conducting G6PD screening to all schoolchildren and daily monitoring for hemolysis 
before and during PQ administration.   
 
9.2. 
Benefit for study subjects 
All positive individuals will receive standard antimalarial treatment by the PHC 
staff. A G6PD screening and Hb measurement will be performed to maximize the 
safety of subjects receving primaquine. 
 
10. 
Expected application of results of the study 
The study is expected to provide an improved screen and treat strategy to 
clear hypnozoites by the use of highly sensitive detection tools. The reduction of 
hypnozoites in the liver will lower the reservoirs of Plasmodium vivax which hopefully 
will bring down the transmission.  
Moreover, this study may offer a highly sensitive and practical diagnostic 
procedure that detects these carriers allows treatment, effectively empowering an 
efficacious approach to mass screen and treat (MSAT) interventions. The malaria 
research community engaged in discovery of targets of chemotherapy or 
immunoprevention would be offered a whole new universe of possibilities with 
sequestered gametocytes in the upper vasculature of the dermis.    
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
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11.  
Study Insurance 
 
The research will be appropriately covered by the Oxford University's 
insurances. 
12. References 
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Bousema T, Dinglasan RR, Morlais I, Gouagna LC, van Warmerdam T, Awono-
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Chardome M, Janssen P. Enquetê sur l’incidence malarienne par la method 
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Krotoski WA. Discovery of the hypnozoite and a new theory of malarial 
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Braack L, Hunt R, Koekemoer LL, Gericke A, Munhenga G, Haddow AD, et al. 
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Hemingway J, Shretta R, Wells TNC, Bell D, Djimdé AA, Achee N, et al. Tools 
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12.  Roles of Investigators 
 
Inge Sutanto is Principal Investigator of this study. She is professor at Faculty of 
Medicine, University of Indonesia. She will provide scientific support, coordination 
and supervision of the study, and will participate in manuscript preparation. 
Rintis Noviyanti is senior researcher at Eijkman Institute for Molecular Biology. She 
will be responsible for molecular diagnostic of Plasmodium. She will provide scientific 
support and will participate in manuscript preparation. 

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Ari Satyagraha is a senior researcher at Eijkman Institute for Molecular Biology. She 
will be responsible for G6PD analysis. She will provide scientific support and will 
participate in manuscript preparation 
Ayleen Alicia Kosasih is the study coordinator of the study. She will assist with 
supervision of the study, scientific support, and will participate in manuscript 
preparation. 
Lenny Ekawati is an public health scientist of Eijkman-Oxford Clinical Research Unit 
who will provide regular support in study design, data analysis, and will participate in 
manuscript preparation. 
Iqbal Elyazar is statistician & epidemiologist of Eijkman-Oxford Clinical Research 
Unit who will provide regular support in study design, data analysis, and will 
participate in manuscript preparation. 
Decy Subekti is laboratory manager of Eijkman-Oxford Clinical Research Unit who 
will provide regular support in laboratory, molecular work, and data analysis, and will 
participate in manuscript preparation. 
Mulya Rahma Karyanti is academic staff at Department of Pediatrics, Faculty of 
Medicine, University of Indonesia. She will provide assistance in developing safety 
protocol, andShe will support the clinical aspect of this study, particularly in terms of 
safety.   
Erni Juwita Nelwan is academic staff at Department of Internal Medicine, Faculty of 
Medicine, University of Indonesia. She will provide assistance in developing safety 
protocol, and support the clinical aspect of this study, particularly in terms of safety.   
Instiaty is is academic staff at Department of Pharmacology and Theuraptics, 
Faculty of Medicine, University of Indonesia. She will provide assistance in 
developing treatment and safety protocol. She will also support the clinical aspect of 
this study, particularly in terms of safety. 
Jeanne Rini Poespoprodjo is staff at Rumah Sakit Umum Daerah Kabupaten 
Mimika, Papua. She will provide scientific insight for the protocol and safety guideline 
of the participants.  
J. Kevin Baird, head of Eijkman-Oxford Clinical Research Unit. He will provide 
scientific support and support in study design, data analysis, and will participate in 
manuscript preparation. 
Shazia Ruybal Pesántez is post-doctoral researcher in the Population Health & 
Immunity Division, Walter & Eliza Hall Institute. She will provide assistance in data 

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management, analysis of the molecular epidemiology, and will participate in 
manuscript preparation. 
Leanne J. Robinson is Laboratory Head in the Population Health & Immunity 
Division, Walter & Eliza Hall Institute, Group Head of Vector Borne Diseases and 
Tropical Public Health, Burnet Institute, Adjunct Principal Research Fellow,  Vector 
Borne Disease Unit, Papua New Guinea Institute of Medical Research. She will 
provide scientific support and support in study design, data analysis, and will 
participate in manuscript preparation. 
Ivo Mueller is Laboratory Head and Co-Head of Population Health & Immunity 
Division, Walter & Eliza Hall Institute. He will provide scientific support and support in 
study design, data analysis, and will participate in manuscript preparation. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
32 
 
 
 
 
 
 
 
APPENDIXES 
 
APPENDIX A. MAP OF STUDY SITE 
 
 
 
 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
33 
 
 
 
 
 
APPENDIX B. DRUG DOSAGE 
Table 1. DHA-PP  
DHA-PP will be given for any species. Same dosage will be given at the enrollment 
and during follow up. 
Day 
Drug 
Daily dosage based on body weight  
< 5 kg 6-10 
kg 
11-17 
kg 
18-30 
kg 
31-40 
kg 
41-59 
kg 
> 60 
kg 
1-3 
DHA-PP 
¼ 
½ 
1 
1 ½  
2 
3 
4 
 
Table 2. High dose Primaquine (PQ)  
Body weight (kg) 
mg/tab 
Tablet 
given/day 
Actual dose 
given mg/kg 
BW 
Total dose 
received (mg) 
12 
15 
0,75 
0,9375 
6,5625 
13 
15 
1 
1,153846 
8,076923 
14 
15 
1 
1,071429 
7,5 
15 
15 
1 
1 
7 
16 
15 
1 
0,9375 
6,5625 
17 
15 
1,25 
1,102941 
7,720588 
18 
15 
1,25 
1,041667 
7,291667 
19 
15 
1,25 
0,986842 
6,907895 
20 
15 
1,5 
1,125 
7,875 
21 
15 
1,5 
1,071429 
7,5 
22 
15 
1,5 
1,022727 
7,159091 
23 
15 
2 
1,304348 
9,130435 
24 
15 
2 
1,25 
8,75 
25 
15 
2 
1,2 
8,4 
26 
15 
2 
1,153846 
8,076923 
27 
15 
2 
1,111111 
7,777778 
28 
15 
2 
1,071429 
7,5 
29 
15 
2 
1,034483 
7,241379 
30 
15 
2 
1 
7 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
34 
 
31 
15 
2 
0,967742 
6,774194 
32 
15 
2 
0,9375 
6,5625 
33 
15 
2 
0,909091 
6,363636 
34 
15 
2 
0,882353 
6,176471 
35 
15 
3 
1,285714 
9 
36 
15 
3 
1,25 
8,75 
37 
15 
3 
1,216216 
8,513514 
38 
15 
3 
1,184211 
8,289474 
39 
15 
3 
1,153846 
8,076923 
40 
15 
3 
1,125 
7,875 
41 
15 
3 
1,097561 
7,682927 
42 
15 
3 
1,071429 
7,5 
43 
15 
3 
1,046512 
7,325581 
44 
15 
3 
1,022727 
7,159091 
45 
15 
3 
1 
7 
 
 
Table 3. Low dose Primaquine 
Single 0.25 mg/kg BW dose PQ will be given for P. falciparum or 14 days 0.25 mg/kg 
BW dose PQ for P. vivax/P. ovale infection any time during the study period. 
Day 
Species 
Daily dosage based on body weight 
< 5 kg  6-17 
kg 
18-30 
kg 
31-40 
kg 
41-59 
kg 
> 60 
kg 
1 
P. 
falciparum 
- 
¼  
½    
¾   
1  
1 
1-14 
P. vivax/P. 
ovale 
- 
¼  
½  
¾  
1 
1 
 
Table 4 Quinine dose 
Day 
Drug 
Daily dosage based on body weight 
< 5 
kg  
6-10 
kg 
11-17 
kg 
18-33 
kg 
34-40 
kg 
41-60 
kg 
1-7 
Quinine 
3 x 10 
mg/kg 
BW 
3 x ½   
3 x 1    3 x 1½  3 x 2  
3 x 
2½  

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
35 
 
1 (1-14) 
Primaquine - 
¼  
½  
¾  
1 
1 
 
 

APPENDIX C. TIME FRAME 
 
Activity 
2019 
2020 
2021 
  
V 
VI 
VII 
VIII 
IX 
X 
XI 
XII 
I 
II 
III 
IV 
V 
VI 
VII 
VIII 
IX 
X 
XI 
XII 
I 
II 
III 
IV 
V 
VI 
VII 
VIII 
IX 
X 
Protocol  
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ethical review 
 
 
 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Investigator meeting & 
trainings 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study approval 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Site establishment 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Socialization  
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sero-positivity screening 
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
School selection and 
randomization  
 
 
 
 
 
 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Socialization, Informed 
Consent, and Training 
 
 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enrolment 
 
 
 
 
 
 
 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Monthly sampling 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
Family screening 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
PCR & genotyping 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
Data analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
 
 
 
 
 
 
Publish findings 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
x 
x 
x 
x 
x 
x 

APPENDIX  D.  SAMPLE HANDLING WORKFLOW DURING INITIAL SCREENING   
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
MagPix 
qPCR
400 µL whole blood 
in microtainer tube 
15 µL 
MOD  
10 µL  thick 
and thin 
smear 
10 uL 
HemoCue 
Finger pricked 
blood 
35 µL 
Gazelle 
10 µL POC 
antibody 
detection  
20 µL 
HemeChip  
Genotyping 
Finger pricked 
blood 
400 µL whole blood 
in microtainer tube 
10 µL  thick 
and thin 
smear 
35 µL 
Gazelle 
10 µL POC 
antibody 
detection  
310 µL 
15 µL 
MOD  
10 µL 
G6PD 
Standard  
20 uL 
Sahli 
20 µL 
HemeChip  
Plasma 
Pellet 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
38 
 
APPENDIX E. MANAGEMENT OF ACUTE HEMOLYSIS 
Any clinical concern of acute hemolysis (sign of anaemia e.g. breathless, fatigue, 
pale, dark urine, clinical jaundice) or falling haemoglobin during 7 days of treatment 
should trigger the following: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Indication to stop PQ: 
Hb<7 with clinical decompensation 
(breathless, exercise intolerant, tachycardy, 
abdominal pain, tachypneu, bibasilar fine 
crackles, leg pit edema) 
Refered to Atambua hospital for blood 
transfusion 
Continue monitoring of the subject and 
coordinate with clinical Jakarta team for 
further management until resolved 
Continue the monthly follow up 
Indication to withhold PQ: 
1.
Hb <7 but daily activities unaffected 
2.
Macroscopic hemoglobinuria 
(Hillmen >=5) 
3.
Fractional haemoglobin fall >=20% 
from baseline 
4.
Other significant clinical concern of 
anaemia or hemolysis  
In patient care at PHC for close 
monitoring of clinical (vital sign) and 
Hb for 24 hours 
Hb drop 
continuously  
Hb stable 
Redose if Hb 
stable and >=9 
Continue 
monitoring as 
outpatient for 2x24 
hours  
1.
History taking and vital sign measurement 
2.
Repeat of haemoglobin measurement by HemoCue 
3.
Repeat of Hillmen urine colour estimation for hemoglobinuria 
4.
Repeat of G6PD test (SD Biosensor) 
5.
Contact clinical team in Jakarta (dr. Erni JN, dr. RM Karyanti, dr. Instiaty) 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
39 
 
APPENDIX F. MANAGEMENT OF METHEMOGLOBINEMIA 
Any sign of metHb (dyspnea, headache, fatigue, weakness, dizziness, syncope) 
without sign of hemolysis should be followed by metHb measurement. 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
metHb >3-20 
with clinical sign 
metHb >20 
Withold PQ, 
give O2 
Withold PQ, 
give O2 
Monitoring of 
metHb every 6 
hours 
Redose PQ  
metHb has 
returned to 
baseline with 
resolved clinical 
symptom  
Worsen clinically 
or laboratory 
Refer to 
hospital 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
40 
 
APPENDIX G. PENETRATION DEPTH OF GENTEEL® LANCING DEVICE 
Contact Tip Color                     Penetration Depth in mm  
 
Blue                                                      0.025 
Yellow                                                   0.66 
Green                                                    1.30 
Clear                                                      2.00 
Orange                                                   2.57 
Violet                                                      3.20 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
41 
 
APPENDIX H. SCHEDULE OF PROCEDURES 
  
Day 
Month 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
2 
3 
4 
5 
6 
7 
8 
9 
10 
History of fever 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Temperature taking 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Body weight measurement 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
Finger prick samples & 
microtainer up to 400 µL 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
LMF 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
POC antibody detection test 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
Hemex test for hemozoin 
detection & 
hemoglobinopathy 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
SD Biosensor G6PD screening 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
HemoCue 
x 
  
  
  
  
  
  
  
d0 
d1 
d2 
d3 
d4  
 d5 
d6 
d7 
Sahli 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
MagPix 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
qPCR 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Genotyping 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Drug treatment 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
DHA-PPQ1 
  
  
  
  
  
  
  
  
D1 
D2 
D3 
  
  
  
  
 
PQ1 
  
  
  
  
  
  
  
  
D1 
D2 
D3 
D4 
D5 
D6 
D7 
 
  
  
  
  
  
  
  
  
  
Ankle prick 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 x 
 x 
x  
x  
x  
x  
x  
x  
x  
LMS 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
 x 
x  
x  
x  
x  
x  
x  
x  
x  
Hemex test for hemozoin 
detection 
x 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
Urine color inspection1 
  
  
  
  
  
  
  
  
d0 
d1 
d2 
d3 
d4  
 d5 
d6 
d7 
  
  
  
  
  
  
  
  
  
Urinalysis1,2  
 
 
 
 
 
 
 
 
d0 
d1 
d2 
d3 
d4  
 d5 
d6 
d7 
 
 
 
 
 
 
 
 
 
1For microscopic positive subjects, treatment should be given promptly according to malaria species  
2Macroscopic, chemistry (protein), microscopic (RBC)   
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
42 
 
APPENDIX I. RETREATMENT SCHEDULE ON ENROLMENT 
LMF 
Antimalarial drugs 
Pv Serology 
Positive 
Negative 
Pf 
3d DHP 
7d PQ (1 mg/kgBW) 
1d PQ (0.25 mg/kg 
BW) 
Pv 
3d DHP + 7d PQ 
No further treatment 
No further treatment 
Pm 
3d DHP 
7d PQ (1 mg/kgBW) 
No further treatment 
Po 
3d DHP + 7d PQ 
No further treatment 
No further treatment 
Mixed Pf&Pv 
3d DHP + 7d PQ 
No further treatment 
No further treatment 
Mixed Pf&Pm 
3d DHP 
7d PQ (1 mg/kgBW) 
1d PQ (0.25 mg/kg 
BW) 
Mixed Pv&Pm 
3d DHP + 7d PQ 
No further treatment 
No further treatment 
  
 
 
 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
43 
 
APPENDIX J. FORMS 
 
CENSUS FORM 
 
SEKOLAH 
: 
 
 
 
 
 
 KELAS 
: 
TANGGAL 
: 
No 
Nama 
L/P Tgl lahir / 
Usia 
Nama Bapak 
Nama Ibu 
Desa 
Dusun 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
44 
 
FORM SCREENING 
SEKOLAH 
: 
 
 
 
 
KELAS 
: 
 
TANGGAL 
: 
No 
ID  
Nama 
L/P 
Usia 
BB 
Suhu 
Demam 
seminggu 
terakhir  
Serologi 
 
Mikroskopik 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
 
 
 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
45 
 
FORM FOLLOW UP 
SEKOLAH 
: 
ID 
:  
 
 
 
KELAS: 
 
 
USIA: 
NAMA: 
 
 
 
L/P 
 
 
 
Kohort 
Tgl 
BB 
Suhu 
Demam 
seminggu 
terakhir 
(Y/T) 
LMF 
LMS 
 
qPCR 
1 
  
  
  
  
 
 
 
2 
  
  
  
  
  
  
 
3 
  
  
  
  
  
  
 
4 
  
  
  
  
  
  
 
5 
  
  
  
  
  
  
 
6 
  
  
  
  
  
  
 
7 
 
 
 
 
 
 
 
8 
 
 
 
 
 
 
 
9 
 
 
 
 
 
 
 
Di luar jadwal 
  
  
  
  
  
  
 
Di luar jadwal 
  
  
  
  
  
  
 
Di luar jadwal 
  
  
  
  
  
  
 
 
 
 
 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
46 
 
FAMILY SCREENING 
ID SUBJEK :  
 
 
 
 
NAMA SUBJEK: 
 
 
 
 
 
 
 
No 
 
Nama anggota 
keluarga 
ID 
BB 
Suhu 
Demam 
seminggu 
terakhir (Y/T) 
LMF 
LMS 
 
Filter paper 
1 
  
 
  
  
  
Malam: 
Malam: 
Malam: 
Siang: 
Siang: 
Siang: 
2 
  
 
  
  
  
Malam: 
Malam: 
Malam: 
Siang: 
Siang: 
Siang: 
3 
  
 
  
  
  
Malam: 
Malam: 
Malam: 
Siang: 
Siang: 
Siang: 
4 
  
 
  
  
  
Malam: 
Malam: 
Malam: 
Siang: 
Siang: 
Siang: 
5 
  
 
  
  
  
Malam: 
Malam: 
Malam: 
Siang: 
Siang: 
Siang: 
 
 
 
 
 
 
 
 
 
 
 

OXTREC 57-19/SSAT PROTOCOL_v1 210819 
47 
 
CASE REPORT FORM 
ID 
: 
 
 
 
 
NAMA: 
 
L/P 
 
 
FAMILY SCREENING/SEKOLAH: 
G6PD: 
 
HASIL: P. FALCIPARUM / P.VIVAX / P. MALARIAE / P. OVALE 
 
Hari 
D-1 
D-2 
D-3 
D-4 
D-5 
D-6 
D-7 
D-8 
D-9 
D-10 
D-11 
D-12 
D-13 
D-14 
Tanggal 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
DHP (jml tablet) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Primakuin (jml tablet) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hb (D-1 sebelum minum obat & D-3)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urin (D-1 sebelum minum obat) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Keluhan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Alergi 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pusing 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sakit kepala 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Bibir kebiruan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Keluhan di dada 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Mual 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Muntah 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Gangguan napsu makan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Gangguan Buang Air Besar (BAB) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Gangguan Buang Air Kecil (BAK) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Gangguan tidur 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tanda tangan pasien 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tanda Tangan Petugas Pengawas Minum 
Obat 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tanda Tangan Manajer Site/Wakil