Targeting High-risk Populations With Enhanced Reactive Case Detection: a Study to Assess the Effectiveness and Feasibility for Reducing Plasmodium Falciparum and P. Vivax Malaria in Southern Lao Peoples Democratic Republic
Phase: NA
Conditions: Plasmodium Falciparum Malaria, Plasmodium Vivax Malaria
Sponsor: University of California, San Francisco

Lao PDR protocol 
 
 
Protocol 
 
 
 
 
 
Targeting high-risk populations with enhanced reactive case 
detection: a study to assess the effectiveness and feasibility 
for reducing Plasmodium falciparum and P. vivax malaria in 
Southern Lao PDR 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Version Number: 6 
July 12, 2019 
 
 
 
 
 
 
 
 
 
 
 

Lao PDR protocol 
 
 
2 
Study Personnel and Institutions ......................................................................................... 4 
1 
Protocol summary ......................................................................................................... 6 
2 
Background and introduction ........................................................................................ 8 
2.1. 
Rationale ........................................................................................................................... 9 
2.2. 
Significance ........................................................................................................................ 9 
2.3. 
Primary aim ....................................................................................................................... 9 
2.4. 
Primary research questions and hypotheses ..................................................................... 9 
2.5. 
Study overview ................................................................................................................ 10 
2.6. 
Summary of ethical issues ............................................................................................... 10 
3 
Methodology ............................................................................................................... 11 
3.1. 
Study sites ....................................................................................................................... 11 
3.2. 
Study population ............................................................................................................. 12 
3.3. 
Interventions ................................................................................................................... 13 
3.3.1. 
Reactive case detection (RACD) ............................................................................................. 13 
3.3.2. 
Adherence and safety monitoring ......................................................................................... 16 
3.4. 
Study evaluation methods ............................................................................................... 17 
3.4.1. 
Overview of evaluation activities by study objectives, outcomes, and data collection activities
 
17 
3.4.2. 
Impact evaluation of enhanced RACD .................................................................................... 19 
3.4.2.1. 
Study design ......................................................................................................................... 19 
3.4.2.2. 
Randomization ...................................................................................................................... 19 
3.4.2.3. 
Primary outcome measures .................................................................................................. 19 
3.4.2.4. 
Sample size ........................................................................................................................... 19 
3.4.2.4.1. 
Confirmed P. falciparum and P. vivax malaria parasite incidence ....................................... 19 
3.4.2.4.2. 
Population-level P. falciparum and P. vivax malaria parasite prevalence ............................ 20 
3.4.2.4.3. 
HS-RDT/RDT test positivity during RACD intervention ........................................................ 20 
3.4.2.5. 
Data collection ...................................................................................................................... 20 
3.4.2.5.1. 
Confirmed malaria case incidence ..................................................................................... 20 
3.4.2.5.2. 
Cross-sectional surveys ..................................................................................................... 20 
3.4.2.5.3. 
Intervention implementation data .................................................................................... 21 
3.4.2.6. 
Data management and analysis ............................................................................................. 21 
3.4.2.7. 
Laboratory analysis ............................................................................................................... 22 
3.4.3. 
Assessment of the feasibility, acceptability, and safety .......................................................... 24 
3.4.3.1. 
Implementation procedures .................................................................................................. 24 
3.4.3.2. 
Primary outcomes measures ................................................................................................. 24 
3.4.3.3. 
Sample size ........................................................................................................................... 24 
3.4.3.4. 
Data collection ...................................................................................................................... 25 
3.4.3.5. 
Analytical plan ...................................................................................................................... 27 
3.4.4. 
Assessment of the risk of malaria vector biting in forest and village settings .......................... 28 
4 
Ethical issues related to human subjects research ...................................................... 28 
4.1. 
Adequacy of protection against risks ............................................................................... 28 
4.2. 
Protection against risks associated with administration of PQ ........................................ 29 
4.3. 
Potential benefits of the proposed research to the participants and others ................... 30 
4.4. 
Alternatives to participation ............................................................................................ 30 

Lao PDR protocol 
 
 
3 
4.5. 
Data and safety monitoring plan ..................................................................................... 30 
4.6. 
Collection of specimens ................................................................................................... 31 
4.6.1. 
Racial and ethnic origin ......................................................................................................... 31 
4.6.2. 
Inclusion of vulnerable subjects – children and pregnant women .......................................... 31 
4.7. 
Participant consent/assent .............................................................................................. 31 
5 
Potential risks, limitations, data quality assurance, and dissemination plan .............. 32 
5.1. 
Potential risks from participation .................................................................................... 32 
5.2. 
Limitations ....................................................................................................................... 32 
5.3. 
Data quality assurance plan ............................................................................................. 33 
5.4. 
Dissemination Plan .......................................................................................................... 33 
6 
Timeline ...................................................................................................................... 34 
7 
References................................................................................................................... 35 
8 Appendices ...................................................................................................................... 36 
 
 

Lao PDR protocol 
 
 
4 
Study Personnel and Institutions 
Principal Investigators 
 
 
Adam Bennett, PhD  
Assistant Professor 
 
MEI Programmatic Lead, Malaria 
Elimination Initiative, Global Health 
Group 
University 
of 
California, 
San 
Francisco 
Phone: 1-415-476-5590 
Adam.Bennett@ucsf.edu 
 
Viengxay Vanisaveth, MD 
Director            
 
Center 
of 
Malariology, 
Parasitology, Entomology (CMPE) 
Vientiane, Lao PDR 
v.viengxay@gmail.com 
 
 
Co-investigators and Key Personnel 
 
 
Jimee Hwang MD, MPH 
Medical Epidemiologist 
 
US 
Center 
for 
Disease 
Control 
&
Prevention 
Center for Global Health/DPDM 
Malaria Branch/US President’s Malaria
Initiative, and 
Malaria Elimination Initiative,  
Global 
Health 
Group 
University of California, San Francisco 
Jimee.Hwang@ucsf.edu 
 
Timothy Finn, PhD MPH 
Senior Research Manager and 
Study Coordinator 
 
 
Lao PDR Malaria Elimination 
Research Partnership 
Global Health Group / UCSF Global 
Health Sciences 
Vientiane, Lao PDR  
timothy.finn@ucsf.edu  
Valerie Scott, MPH 
Program Manager 
 
Malaria Elimination Initiative, Global 
Health Group 
University of California, San Francisco 
Valerie.Scott@ucsf.edu   
Chris Cotter, MPH 
Senior Research Manager 
 
Malaria Elimination Initiative, 
Global Health Group 
University 
of 
California, 
San 
Francisco 
Phone: +1-415-476-5657 
Chris.cotter@ucsf.edu  
Roly Gosling, MD, PhD 
Associate Professor 
 
Co-Director, Malaria Elimination 
Initiative, Global Health Group 
University 
of 
California, 
San 
Francisco 
Roly.gosling@ucsf.edu 
 
Michelle Hsiang, MD, MSc 
Assistant Professor 
 
Malaria Elimination Initiative,  
Global 
Health 
Group 
University of California, San Francisco 
Dept of Pediatrics 
University of Texas Southwestern 
michelle.hsiang@ucsf.edu 
michelle.hsiang@utsouthwestern.edu  
Francois Rerolle, MS 
Geospatial specialist; PhD student 
 
 
Malaria Elimination Initiative,  
Global 
Health 
Group 
University of California, San  
 Jenny Smith, MSc, PhD 
Assistant Professor  
 
 
Senior Research Scientist, Malaria
Elimination Initiative 
Global Health Group 
Henry Ntuku, MD, PhD 
High Risk Populations Surveillance 
Specialist 
 
Malaria Elimination Initiative,  
Global 
Health 
Group 
University of California, San  

Lao PDR protocol 
 
 
5 
Francisco 
Francois.Rerolle@ucsf.edu 
University 
of 
California, 
San
Francisco 
Jennifer.smith@ucsf.edu   
Francisco 
henry.ntuku@ucglobalprograms.org 
  
Andrew Lover MPH, PhD 
Assistant Professor 
 
Dept of Biostatistics and Epidemiology 
School  of Public Health and Health
Sciences 
University of Massachusetts – Amherst 
alover@umass.edu  
 
Jerry Jacobson, PhD 
Independent consultant 
 
jerryojacobson@gmail.com  
Joshua Yukich, PhD 
Assistant Professor 
 
Tulane University School of Public 
Health and Tropical Medicine 
jyukich@tulane.edu 
Bryan Greenhouse, MD, MA  
Assistant Professor 
 
Dept of Medicine / Div. of Infectious 
Diseases 
University of California, San Francisco 
bryan.greenhouse@ucsf.edu 
 
 
 
 
 
 

Lao PDR protocol 
 
 
6 
1 
 Protocol summary 
 
Aim 
To determine the effectiveness and feasibility of enhanced reactive case 
detection (RACD) targeting high-risk villages and forest workers for reducing 
Plasmodium falciparum and Plasmodium vivax transmission in southern Lao 
PDR 
Primary research 
question 
and 
hypothesis 
1. Null hypothesis:  Compared to standard of care, which includes case 
management and RACD with conventional RDTs and is directed at 
villagers, there is no additional benefit of enhanced community-based 
RACD, which is conducted by village malaria workers (VMWs), uses HS-
RDTs, and targets forest workers in addition to villagers, for reducing 
P. falciparum and P. vivax confirmed case incidence and parasite 
prevalence over an 18-month period in Lao PDR. 
 
Research hypothesis: Enhanced community-based RACD will be more 
effective than standard of care case management and RACD at 
reducing P. falciparum and P. vivax confirmed case incidence and 
parasite prevalence over an 18-month period in Lao PDR. 
 
Research 
objectives 
Primary Objective: To evaluate the effectiveness of reactive case detection 
(RACD) using HS-RDTs, targeting both village and forest working populations, 
compared to control for reducing the health center catchment-level incidence 
and prevalence of P. falciparum and P. vivax within two provinces in Lao PDR. 
 
Secondary Objectives:  
1. To determine the operational feasibility, cost-effectiveness, and 
acceptability of targeting high-risk populations with community-based 
reactive case detection strategies aimed at reducing P. falciparum and 
P. vivax transmission among village and forest working populations. 
2. To determine the operational feasibility and safety of conducting 
G6PD testing and PQ administration and follow-up at the community 
level. 
3. To determine factors associated with G6PD testing after referral to 
district or provincial-level facilities for persons with P. vivax positive 
RDT results, and subsequent adherence to Primaquine treatment.  
4. To assess malaria vector species composition and bionomics in both 
village and forest settings 
 
Study site and 
target 
populations 
Total population of approximately 120,000 in 32 health center catchment 
areas of two Provinces in Lao PDR (Champasak and Saravan Province) 
 
Study design 
A cluster randomized controlled trial design will be used to evaluate the 
enhanced RACD intervention against standard of care 
 
Primary outcome 
measures 
for 
assessing impact 
1. Cumulative incidence over the study period at the health facility level  
2. Prevalence of infection (P. falciparum and/or P. vivax) using qPCR 
3. HS-RDT and RDT test positivity rate for RACD 
Data 
collection 
and 
sampling 
period 
18 months from start date (June 2019 – November 2020) 

Lao PDR protocol 
 
 
7 
Sample size 
32 health center catchment areas in Lao PDR 
Estimated 
enrollment 
1) Confirmed case incidence across 32 health center catchment areas: 
population of approximately 145,000 
2) Village-based reactive case detection: estimated 14,400 
3) Forest worker reactive case detection: estimated 2,880 
4) Endline household survey: 14,080 persons at end-line 
5) Qualitative surveys: 6 Focus group discussions (FGDs) and 18 Key informant 
interviews (KIIs) at baseline, 9 FGDs and 18 KIIs at end-line.  
6) Competency checklists with 32 VMWs at baseline, midline, and endline 
7) Entomological human landing catches: 20 collectors over three nights at 
baseline 
Statistical 
and 
analytical plan 
Rapid reporting of microscopy or RDT and HS-RDT positivity will be analyzed 
as interventions are implemented (or shortly thereafter) to provide 
preliminary assessment of interventions. Negative binomial (for incidence) 
and generalized linear mixed effects (for prevalence) models will be used to 
test the effect of each intervention independently (main effects) relative to 
control (standard of care). For the enhanced RACD vs. control comparison, 
both country-specific and pooled analyses will be conducted.  
 
 
 
 

Lao PDR protocol 
 
 
8 
2 
 Background and introduction 
A standard and widely practiced method to identify infections at the community level is reactive case 
detection (RACD), whereby household and community members of a passively identified index case 
are tested with either an RDT or by microscopy 1–3. However, traditional household-based RACD 
methods using current diagnostics have shown limited effectiveness for identifying additional 
infections, especially in settings of forest malaria transmission such as the GMS. Results from a 
recently conducted UCSF-MEI study of RACD in two Thailand border areas (Chanthaburi and 
Kanchanaburi Provinces) identified only 0.2% parasite prevalence by PCR in household and community 
members tested residing around passively identified index cases (n=27); all additional secondary 
infections identified were P. vivax (n=4). Furthermore, a higher risk of malaria infection was associated 
with being male, aged 31 years and older, and having worked in or near the forest fringe. While there 
have been several published small-scale observational studies examining the yield of household-based 
RACD using conventional RDTs in the GMS 4, none have examined its impact on transmission. Recent 
pilot studies in Cambodia suggest targeting high risk groups (using PCR) will substantially increase the 
infections found and cleared 5. Preliminary results of an observational study of RACD targeted to high-
risk forest workers (using LAMP) in Aceh Province, Indonesia carried out by UCSF-MEI suggest that 
substantially more secondary infections will be identified when RACD is targeted to high-risk groups 
compared to standard RACD in the neighborhood of cases. None of these studies have used the newly 
available HS-RDTs for P. falciparum 6; evidence suggests these will increase the sensitivity of active 
case detection over standard RDTs and have greater utility for community-based approaches where 
PCR or LAMP are not likely to be feasible, but rigorous testing is needed in routine operational settings, 
especially in P. vivax co-endemic settings. 
 
Recent results from an observational trial in Myanmar demonstrate the effectiveness of early 
diagnosis and treatment through village-based malaria posts and mass drug administration for P 
falciparum in hotspot villages 7. In 2018, the UCSF-MEI conducted a community randomized control 
trial of mass test and treat (MTAT) in villages combined with focal test and treat (FTAT) in forest 
workers led by forest-working peer navigators using HS-RDTs in Champasak, Lao PDR, which helped 
inform community-based approaches for targeting RACD to high-risk forest workers in this proposal. 
Additionally, the UCSF-MEI is conducting formative ethnographic and epidemiological research to 
understand forest worker and other high-risk populations in several provinces in southern Lao PDR, 
working closely with Health Poverty Action (HPA) within their Malaria Post/village malaria worker 
(VMW) networks. These studies have highlighted the importance of involving the community, 
especially high-risk individuals, as part of community-based active surveillance and response 
strategies, and further operational research is essential to assess feasibility and challenges associated 
with scaling up such community-based approaches. 
 
Safe and effective management of P. vivax cases requires addressing glucose-6-phosphate 
dehydrogenase (G6PD) deficiency and ensuring patients with G6PD normal function complete a 14-
day treatment regimen with primaquine. The heterogeneity and severity of G6PD deficiencies in SE 
Asia and associated risk of severe hemolysis with oxidative drugs such as primaquine is an especially 
serious public health issue 8. The majority of P. vivax cases in most transmission settings are due to 
relapses 9, making primaquine (and tafenoquine when available) a critical component of elimination 
activities. However, treatment adherence for full 14-day primaquine is generally sub-optimal, and 
standard of care use of primaquine in health systems has shown minimal effectiveness in preventing 
relapses 10. There are limited data on primaquine use in Lao PDR; a study conducted by WHO and 
CMPE in 2015 enrolled 1,577 P. vivax cases (363 women) for G6PD testing of which 67 (4.2%; 95% CI: 
3.3 to 5.3%) were classified as deficient.11  A total of 1,225 G6PD non-deficient individuals with P. vivax 
infection were enrolled for PQ administration, of which 889 (72.5%) completed follow-up hematocrit 
testing, and only one individual experienced a serious drop in hematocrit (below 25%). However, 
treatment adherence and parasite clearance were not measured in the study. This study and others 

Lao PDR protocol 
 
 
9 
suggest that there remain key gaps in knowledge to inform safe and effective programmatic use of 
primaquine drug regimens to target the hypnozoite reservoir, especially with regards to ensuring 
adequate follow-up and safety monitoring at all levels of the health system including the community 
level. 
2.1.  Rationale 
The Center for Malariology, Parasitology, and Entomology (CMPE) in Lao PDR over the past two years 
has completed the National Strategic Plan 2016-202012, and new National Surveillance Guidelines that 
includes case-based surveillance and focal response including RACD. After some setbacks from 2011-
2015, there has been major progress and incidence has recently fallen dramatically in several southern 
provinces, making foci-based active surveillance strategies now feasible in these areas.  
 
Under the current national and regional artemisinin initiative 2 elimination (RAI2E) funding, VMWs 
and Malaria Posts will be expanded to improve coverage across these provinces. Household-level 
RACD has yet to be operationalized in southern Lao PDR, but is planned for roll-out over the next year 
by teams from national, provincial, and district levels. In order to maximize sustainability and impact, 
this strategy will need to be community-based and target the highest risk populations. 
 
The current management of P. vivax cases and G6PD deficiency varies by country; in Laos, current 
policy states that all P. vivax cases are to be tested for G6PD deficiency and only those without 
deficiency receive a 14-day course of primaquine. Rigorous follow-up of cases to monitor safety and 
adherence is not conducted, and the use of G6PD testing and primaquine treatment has not yet been 
widely operationalized.  
 
The research proposed here will evaluate the effectiveness and feasibility of a community-based 
reactive case detection strategy when combined with the use of HS-RDTs, targeting high-risk villages 
and forest populations, and P. vivax testing treatment adherence after referral to district or provincial-
level facilities. Results of this research will have direct implications for continued roll out and 
sustainability of community-based foci management, and provide practical guidance that other 
malaria programs can utilize. 
2.2.  Significance 
We propose to conduct this operational research in a large number of the remaining active foci across 
in two of the remaining higher burden provinces in southern Lao PDR. The findings will help Laos 
assess the benefit and potential challenges of community-based approaches to active foci 
management including RACD for P. falciparum and P. vivax, and the effectiveness of RACD using HS-
RDTs in this context, which will provide a rigorous evidence base for other countries in the GMS 
applying these approaches. Importantly, this trial will incorporate community-based approaches for 
targeting high-risk forest workers, which are a uniquely prevalent high-risk population in the GMS. 
2.3.  Primary aim 
The primary aim of this project is to determine the effectiveness and feasibility of enhanced 
community-based reactive case detection (RACD) in Lao PDR, targeting high-risk villages and forest 
workers, for reducing P. falciparum and P. vivax transmission. 
2.4.  Primary research questions and hypotheses 
This project aims to answer the following research question and hypotheses: 
Is community-based RACD using HS-RDTs targeting both village and forest working populations more 
effective than the standard of care (community case management, and RACD conducted by district 
surveillance teams) in reducing P. falciparum and P. vivax confirmed case incidence and parasite 
prevalence over an 18-month intervention period? 
 

Lao PDR protocol 
 
 
10 
Null hypothesis 1:  Compared to standard of care, which includes case management and RACD with 
conventional RDTs and is directed at villagers, there is no additional benefit of enhanced community-
based RACD, which is conducted by VMWs, uses HS-RDTs, and targets forest workers in addition to 
villagers, for reducing P. falciparum and P. vivax confirmed case incidence and parasite prevalence 
over an 18-month period in Lao PDR. 
 
Research hypothesis 1: Enhanced community-based RACD will be more effective than standard of care 
case management and RACD at reducing P. falciparum and P. vivax confirmed case incidence and 
parasite prevalence over an 18-month period in Lao PDR. 
2.5. Study overview 
To test this hypothesis, this study will employ a cluster randomized controlled trial design with two 
comparison arms:  
 
1. Control: standard of care - passive case management provided through community-based VMWs 
and existing health facilities; includes village-based RACD with conventional RDTs conducted by 
district surveillance teams. 
2. Enhanced community-based RACD: RACD conducted by community-based VMWs using both HS-
RDTs and conventional RDTs within villages and among forest workers. 
 
The primary outcome measures to assess effectiveness include P. falciparum and P. vivax confirmed 
case incidence over the study period; PCR-based P. falciparum and P. vivax prevalence at endline; and 
HS-RDT test positivity rate in village and forest worker RACD. Secondary outcomes measures will 
examine the operational feasibility, safety, and acceptability of VMW-led reactive approaches and 
G6PD testing, referral to district or provincial-level facilities, safety and treatment adherence for P. 
vivax cases. The trial will be implemented between June 2019 and December 2020 in Lao PDR. The 
primary evaluation will be conducted following an end-line cross-sectional survey in 
November/December 2020.  
2.6. Summary of ethical issues 
Prior to implementation, the protocol and all related project activities will be reviewed and approved 
by the institutional review boards in Lao PDR and at UCSF. For other partnering institutions (i.e., 
University of Texas Southwestern, University of Massachusetts, Tulane University), requests will be 
made for reliance on UCSF IRB. The protocol will be submitted to CDC Human Subjects for non-
engaged review. Participation in either the research or intervention is voluntary. Individuals will be 
included in the study only if they or their parents/guardians provide written informed consent. The 
consent process will be conducted in appropriate local languages at the start of every new contact 
with an individual. For individuals under 18 years of age, informed consent will be obtained from a 
parent or guardian. Oral assent for adolescents 12 to <18 years of age will be obtained in addition to 
consent from a parent or guardian. As part of the informed consent, specific consent for the pregnancy 
test will also be required for female participants of child-bearing age. 
 
The safety risks associated with participation in this trial are expected to be minimal. The study drugs, 
which are first-line treatment regimens for the respective areas in Lao PDR, artemether-lumefantrine 
(AL) and PQ in G6PD non-deficient individuals, are well tolerated and safe.  Prior to drug 
administration, participants will be asked about known contraindications, and if such 
contraindications are reported, participants will be restricted from taking the relevant medication. PQ 
administered as a single low dose has been found to be safe in individuals with any of the G6PD 
variants and is recommended by WHO without G6PD testing 13. The 14-day course of PQ will only be 
given to G6PD non-deficient individuals after G6PD testing with a qualitative RDT. Should a subject be 
misclassified as G6PD non-deficient when in actual fact they are G6PD deficient and had begun their 
14-day PQ regimen, participants will be asked to stop treatment with primaquine immediately and a 

Lao PDR protocol 
 
 
11 
robust follow-up and severe adverse event management system will pick up early cases of hemolysis, 
stop treatment with primaquine and if necessary refer for further clinical management. 
 
The risks associated with loss of privacy in this study are likely to be low. To ensure confidentiality is 
maintained, all information will be treated as private by study personnel, and records kept securely in 
locked filing cabinets and offices. Electronic records will be kept on a secure, firewall- and password-
protected server. For all data collected as part of the study, participants will be assigned a unique 
identification number. No personal identification information such as names will be used in any 
reports arising out of this research. All project staff will be trained on procedures for maintaining 
confidentiality. 
Study participants with malaria infections identified by RDTs/HS RDTs will benefit directly from 
treatment with recommended first line treatment. 
 
Participants will not be paid to take part in this study. Most assessments will be conducted at 
households or working sites, which will eliminate the need for participant travel and minimize 
opportunity costs for the participants. Any diagnosis and treatment associated with the study will be 
provided free of charge. 
3 
Methodology 
3.1.  Study sites 
The selected study sites include health facility catchment areas in two of the highest burden provinces 
in Southern Laos. All selected health facilities had at least 3 malaria cases in the previous year. The 
districts included in Laos are shown in Table 1 and Figure 1. In addition to the number of health 
facilities included in the study, the total number of villages and population in those selected areas, 
and the P. falciparum and P. vivax annual parasite index (API) for Oct 2017 – Sep 2018 is shown. The 
anticipated period for the study is June 2019 – December 2020. 
 
Table 1. Overview of incidence and population within selected health facility catchment areas in Lao PDR 
District 
Province 
Total 
health 
facilities 
Total 
villages 
Total 
populatio
n 
Pf 2017-
18 (API) 
Pv 2017-
18 
(API) 
Total 
cases 
2017-18 
(API) 
Khong 
Champasak 
4 
32 
22764 
3.43 
2.81 
6.24 
Mounlapamok 
Champasak 
4 
23 
18005 
0.56 
1.94 
2.5 
Pathoumphon 
Champasak 
3 
34 
22975 
3 
4.61 
7.62 
Sukhuma 
Champasak 
1 
8 
10300 
0.58 
1.65 
2.23 
Samouay 
Saravan 
3 
28 
6177 
0.65 
13.11 
13.76 
Saravan 
Saravan 
3 
35 
17877 
1.51 
1.29 
2.8 
Taoi 
Saravan 
5 
57 
12713 
16.6 
1.65 
18.25 
Toumlan 
Saravan 
6 
46 
18366 
6.81 
1.47 
8.28 
Vapy 
Saravan 
3 
37 
18621 
3.11 
2.09 
5.21 
Total 
 
32 
300 
147,798 
3.98 
2.79 
6.77 
 
Figure 1. Selected study districts in Lao PDR (highlighted in orange) 

Lao PDR protocol 
 
 
12 
 
 
3.2. Study population 
The study population includes all villagers and forest workers in the selected health facility 
catchments. Inclusion and exclusion criteria for the interventions are described below. 
 
Inclusion and exclusion criteria 
Inclusion criteria 
Subjects must fulfill the following inclusion criteria to be eligible for enhanced RACD: 
• 
Index cases: 
o Presented as a confirmed malaria case to an intervention health facility or village 
malaria worker, and lives in a village within a selected intervention health facility 
catchment area, or worked or spent at least one night at a forest or forest-fringe site 
in the past 30 days located within an intervention health facility catchment area 
• 
Village residents: 
o Lives in a village within a selected intervention health facility catchment area and in 
one of the five households closest to the residence of an index case of malaria 
• 
Co-worker/traveler referral: 
o Worked or traveled and spent at least one night in forest in past 30 days in same 
location within an intervention health facility catchment area as an index case of 
malaria 
• 
All participants: 
o Willing and available to participate in the study 
• 
Informed consent for participant under the age of 18 will be provided by the parent or 
guardian. 
 
Exclusion criteria: 
• 
All RACD referrals: 
o Previous participation in the study as a result of any RACD event in the past 30 days. 

Lao PDR protocol 
 
 
13 
o Individuals with severe disease or drug contra-indications will be excluded from the 
treatment component only (detailed below) 
3.3. Interventions 
Each intervention cluster (i.e., health facility catchment area) will be randomly assigned to either the 
RACD intervention or control arm (in a 1:1 ratio) before the study begins. The randomization process 
is described in more detail in section 3.4.2.2 below. Interventions (RACD) will be conducted by VMWs 
and/or community volunteers in response to an index case (presenting to VMW, malaria post, health 
facility, or other testing facilities) occurring anywhere within a selected intervention health facility 
catchment area. At presentation, all index cases will initially be asked about recent forest exposure. 
The intervention response for cases with no history of forest exposure in the past two months will be 
conducted only at the village level, while the response for cases with history of recent forest exposure 
will include both village and forest-worker populations. Forest-worker RACD will be conducted at 
recent work sites if accessible and located within the health facility catchment and/or amongst peer 
forest-goers referred by the index case. VMWs in intervention villages will maintain a register of forest 
workers and locations to assist them with tracking. In Lao PDR, health center staff will assist with RACD 
during the first six months of the study only, after which point VMWs will operate independently. 
 
Index case capture  
Interventions will be initiated after an individual from a cluster assigned to an intervention is 
diagnosed with malaria and notified to the study team. Suspected malaria cases attending local health 
services (hospital, health center, health post, or VMW) will be tested with an RDT or microscopy 
according to routine practice. All cases parasitologically confirmed by RDT will be treated according to 
national guidelines and asked for informed consent to participate in the study. If the patient gives 
informed consent, the VMW or health staff will promptly notify the study team using mobile phone. 
A short screening questionnaire will be administered to determine location of residence and recent 
forest or other travel. Additionally, finger prick blood will be collected on DBS from the index case for 
subsequent analyses.   
3.3.1. Reactive case detection (RACD) 
Household RACD 
Within 7 days of the index case notification, the VMW will conduct the investigation visit at the 
residence of the index, and the index case will be interviewed using a standard case investigation form. 
All members of the index case’s household will then be invited to participate in the study. A GPS point 
will be captured for the index house, and the nearest five households around the index case’s 
household will be identified by the VMW. In the neighboring five households, a GPS point will be 
captured and all household members, including temporary visitors, will be invited to participate in the 
study. Household members and parents/guardians of children will be verbally informed of the general 
purpose of the intervention and the study, and the possible risks and potential benefits associated 
with participation. Individual written consent will be obtained at each household and for each blood 
sample collected for testing. For children <6 to 12 years old, consent will be obtained from the parent 
or guardian. For children 12 to <18 years of age, the child’s oral assent will also be required in addition 
to parents/guardians’ consent.  All informed consent procedures will be conducted in the appropriate 
local language. All consenting participants will be interviewed using the participants’ questionnaire to 
assess history of malaria in previous year, travel information, and utilization of malaria prevention 
measures. 
A finger stick blood sample will be collected for each consenting individual for testing with the HS-RDT 
for P. falciparum, a standard combination RDT, and four blood spots on filter paper. Individuals will 
be told of their test result and a positive test result on either RDT will prompt treatment as per the 
national treatment guidelines. See tables 2 and 3 below for treatment guidelines. 

Lao PDR protocol 
 
 
14 
In the event that household members are not present at the time of testing, the VMW will ask if any 
absent household member has been ill with fever during the previous two weeks. If any absent 
household members are reported as having recent fever history or if any other household members 
test positive for malaria, the VMW will schedule a time to revisit the household. At this time, the VMW 
will administer the informed consent form to any previously absent household members and then test 
these individuals (and treat positive cases). Febrile individuals testing negative by RDT(s) will be 
referred to the nearest health center. 
 
RACD of cases’ co-workers/co-travelers 
Index cases will be screened by the VMW at their households at the time of case investigation to 
determine if they have traveled or worked in a forest or forest-fringe area within the past 30 days. If 
eligible, the case will trigger two reactive recruitment strategies to screen and treat others who 
recently traveled or worked with the case in a forest or forest-fringe location:  
 
1. Peer-referral RACD (PR-RACD): In this strategy, the case will identify specific co-travelers or 
co-workers resident in their village who had spent the night with the case at a forest or forest-
fringe area and spent the night there in the past 30 days.  
2. Venue-based RACD (VB-RACD): In this strategy, co-workers will be recruited directly from 
(accessible) forest or forest-fringe work sites where the index case worked and spent at least 
one night in the past 30 days.  
 
Participating index cases who meet eligibility criteria will be asked to provide a list of all people who 
have worked or traveled in the forest or forest-fringe with him/her in the past 30 days between dusk 
and dawn and their contact information (names, phone numbers, home addresses) (peer-referrals). 
The questionnaire will elicit from index case all forest and forest-fringe work sites where the case has 
spent the night in the past 30 days, as well as contact information for any respective employers. 
 
For PR-RACD, VMWs will contact peer-referrals by phone, at their home, or through village leaders 
and invite them to participate in a survey interview and malaria testing at a time and place that is 
most convenient. This may be the referral’s place of residence, a health facility, or another location in 
the village that provides sufficient confidentiality, such as a coffee shop.   
a. Peer referrals will be screened for eligibility by the VMW and, if eligible, informed 
consent will be conducted in the appropriate local language. 
b. Consenting referrals will provide a fingerstick blood sample for testing with the HS-
RDT, standard combination RDT and four blood spots on filter paper. If infection is 
present according to any RDT, they will receive treatment following the guidelines in 
the following section.  
c. All consenting referrals will complete an interviewer-administered survey 
questionnaire. 
For VB-RACD, if available, employers will be contacted by the VMW to coordinate a potential visit to 
identified work sites.  
a. Work sites considered for VB-RACD will meet the following criteria: 
 
o Case reports that the work site is currently active 
o Case reports there are at least five other workers who work or sleep there (the 
minimum threshold ensures that screening there is worthwhile, given logistic 
constraints, and will be specified in SOP) 
o The work site is safe to visit and accessible 
o The nearest VMW has necessary permissions to travel to site 

Lao PDR protocol 
 
 
15 
b. The nearest VMW will travel to any such work sites identified 
c. All workers present at the site(s) will be screened for eligibility and, if eligible, 
informed consent will be conducted. 
d. Consenting referrals will provide a finger stick blood sample for testing with the HS-
RDT, standard combination RDT, and four blood spots on filter paper. If infection is 
present according to any RDT they will receive treatment following the guidelines in 
the following section.  
e. All consenting referrals will complete an interviewer-administered survey 
questionnaire. 
All participants will receive a small token of participation such as a bar of soap or phone credit.  
 
Forest workers may be difficult to reach at their places of residence as they are often away from the 
household.  When following-up with PR-RACD referrals, VMWs will attempt to contact them directly 
by phone multiple times and visit the household if the address was provided or they are familiar with 
the location. When referrals are unreachable by phone or the residential location is unknown, VMWs 
will elicit the help of the village leader to identify where they live or how best to contact them. VMWs 
will record each contact into a register of all individuals in their community known to travel to forest, 
the location of their residence, and location of work-site if possible.  
 
Case Management and Follow-up 
All individuals who test positive by either HS-RDT or Standard RDT will be told of their results and 
treated on site per national guidelines: 
• 
Individuals with P. falciparum infection will be treated with an age-appropriate course of 
artemether-lumefantrine (AL) and a single low dose of primaquine (SLD-PQ). Weight-based 
dosing is described in Tables 2 and 3 below. 
• 
At all study sites in Lao PDR, patients with a P. vivax infection identified by RDT (both febrile 
and asymptomatic) will be given a unique coded and signed informational letter directing 
them to the nearest district hospital (or other testing facility) for G6PD deficiency testing and 
possible radical cure administration depending on results. Study contact information will also 
be included if the participants present at other health facilities. At the health facility, G6PD 
normal individuals will be treated with AL and a 14-day course of PQ, whereas G6PD deficient 
individuals will receive AL alone as per the national guidelines and referred to a hospital for 
further primaquine management decisions. The study staff will meet with district hospital and 
health facility staff monthly from all study areas and nearby district hospitals and health 
facilities to collect these forms, with small incentives for health staff to report receiving a 
study card (phone top up card, etc.) to maximize data completeness. After six months of 
implementation, a subset of VMWs will begin conducting qualitative G6PD testing and 
treatment administration at the community-level with the assistance of a health facility staff 
member. 
 
Table 2. AL weight-based dosing 
Body 
weight 
(kg) 
Tablet strength (mg) 
Tablets/ 
dose 
Mg of drug per dose 
Tablets/day 
Artemether 
Lumefantrine 
Artemether 
Lumefantrine 
5 to 14  
20 
120 
1 
20 
120 
1 tablet given twice* per day 
for 3 consecutive days 

Lao PDR protocol 
 
 
16 
15 to 24 
20 
120 
2 
40 
240 
2 tablets given twice* per 
day for 3 consecutive days 
25 to 34 
20 
120 
3 
60 
360 
3 tablets given twice* per 
day for 3 consecutive days 
35 
or 
greater 
20 
120 
4 
80 
480 
4 tablets given twice* per 
day for 3 consecutive days  
*Approx. 8 hours between doses 1 and 2. 
Approx. 12 hours between all other consecutive doses. 
 
Table 3. Weight-based dosing of SLD- Primaquine (.25 mg/kg) for P. falciparum 
Body weight (kg)  
SLD-PQ dose  
(7.5 mg tablet)  
(as base) 
Tablets 
10 to < 25 
3.75 
1/2 
25 to < 50 
7.5 
1 
50 to 100 
15 
2 
*Given as SLD in addition to AL for P falciparum positive  
 
Table 4. Age-based dosing of Primaquine for P. vivax  
Items 
Dose 
No. 
of 
tablet/day 
Frequency 
Duration 
G6PD Normal 
Children 
 
4-8 years old 
5 mg/day 
1/3 
Once per day 
after meal 
14 days 
9-14 years old 
10 mg/ day 
2/3 
>14 years old (adults) 
15 mg/ day 
1 
 
Per national guidelines in Laos, AL will not be given to women who are pregnant or may be in their 
first trimester. All women of reproductive age with a last menstrual period greater than four weeks 
positive HS RDT or Standard RDT will be offered a pregnancy test. Pregnancy testing and consent will 
be embedded in the participant questionnaire. Any woman who acknowledges pregnancy in the first 
trimester or confirmed by a positive pregnancy test or refuses to take a pregnancy test will be excluded 
from receiving AL and will be referred to the nearest health facilities for treatment. 
Contraindications to primaquine include pregnancy (any trimester), age less than 12 months, infants 
weighing less than 10 kilograms, women in the first 12 months of breastfeeding, and prior allergic 
reaction to primaquine. Participants with contraindications to primaquine will not be excluded from 
the study and will receive first-line treatment alone, provided they are eligible. 
 
All individuals with suspected severe malaria or other severe illness (including those with symptoms 
of severe anemia, prostration, impaired consciousness, respiratory distress, convulsions, circulatory 
collapse, abnormal bleeding, jaundice or passing dark urine) will be referred to the nearest health 
facility for clinical assessment and treatment. 
3.3.2. Adherence and safety monitoring 
 
To ensure participants’ adherence to their medication and monitor safety, participants will be 
informed on how to take their medications, about the side effects of the treatment and the 
importance of compliance to the treatment. In addition to DOT on day 0, The VMW will conduct a day 

Lao PDR protocol 
 
 
17 
3, day 7, and day 14 adherence and safety monitoring visit for all individuals treated with primaquine 
for P. vivax, and the VMW will ask a series of questions to determine adherence with each dose, 
number of remaining tablets, potential adverse events, and reasons for non-adherence or treatment 
refusal. Participants with phone number will be sent a daily text message to remind them to adhere 
to their treatment and remind them about any symptoms suggesting serious adverse events. All 
participants and their family members will be provided a chart to record and track their adherence as 
well as symptoms that may indicate an adverse event.  
 
The safety risks associated with participation in this trial are expected to be minimal. The study drugs, 
AL and PQ, are well tolerated first-line treatments and safe for G6PD non-deficient individuals. Prior 
to drug administration, participants will be asked about known contraindications, and if such 
contraindications are reported, participants will be restricted from taking the medication.  
 
Drug 
Contraindications 
AL 
Pregnancy, infants < 5kg, prior allergic reaction, any 
severe disease 
PQ 
Pregnancy, age < 12months, infants < 10kg, women in 
first 12 months of breastfeeding, prior allergic reaction, 
any severe disease, G6PD deficient 
 
The common adverse events reported for each drug are as below: 
§ 
AL: headache, dizziness, loss of appetite, generalized weakness, fever, chills, arthralgia, 
myalgia, nausea, vomiting, and abdominal pain 
§ 
PQ: nausea, headache, fatigue, dizziness, hives, chest pain, painless darkening of urine, 
jaundice of skin and vomiting. 
 
The Primaquine Roll Out Pharmacovigilance Tool Data Collection Forms (PROMPT) will be used to track 
the status of individuals treated with PQ. Identification of serious adverse events (SAEs) will occur 
both passively and actively. As part of the consent process, participants will be instructed to call or 
visit VMW to report any adverse events that occur between VMW visits. During adherence and follow 
up visits, the VMW will also ask participants for specific adverse events related to hemolysis. SAEs as 
well as serious unexpected serious adverse reactions (SUSAR) will be reported to the study 
management and PI who will report to the data safety monitoring board (DSMB). At each study site, 
a local physician will be recruited (Local Safety Monitor, LSM) to determine causality and grade of the 
SAE. All SAEs will be reported to the Pharmacovigilance center of the Ministry of Health (Lao PDR) and 
the respective IRBs. All potential AEs or SAEs will be referred by VMW to the nearest health center or 
district hospital for management and documented as per 4.2 below, and the study will reimburse all 
patient travel and hospitalization resulting from study drug administration.  
 
3.4. Study evaluation methods 
3.4.1. Overview of evaluation activities by study objectives, outcomes, and data 
collection activities 
 
Table 4. Overview of evaluation activities 
Study activity 
Research objective 
Primary outcome measures 
Data collection activity 
Activity 1: Impact 
evaluation 
of 
enhanced RACD 
1. 
Evaluate 
the 
relative 
effectiveness 
of 
enhanced 
community-
based RACD using HS-
Total confirmed outpatient (OPD) 
P. falciparum and P. vivax malaria 
case incidence among all ages by 
health facility catchment 
Routine malaria data from 
all reporting VMWs, health 
centers 
and 
district 
hospitals 

Lao PDR protocol 
 
 
18 
RDTs in villages and 
forest-based 
HRPs, 
compared to control 
P. 
falciparum 
and 
P. 
vivax 
prevalence in all individuals 
Cross-sectional 
parasite 
survey 
November-
December 2020 
HS-RDT and RDT test positivity 
rate 
HS-RDT and RDT positivity 
data 
from 
RACD 
intervention data 
Activity 
2: 
Assessment of the 
feasibility 
and 
acceptability 
of 
community-based 
RACD  
1. 
Assess the operational 
feasibility 
of 
community-based RACD  
Population coverage of RACD and 
interventions 
RACD intervention data 
Proportion of VMWs who rate 
conducting 
the 
RACD 
interventions 
as 
very 
easy, 
somewhat 
easy, 
somewhat 
difficult, and very difficult, and 
changes in proportions over time 
 
Entrance, mid-intervention, 
and 
exit 
competency 
checklists 
with 
VMWs; 
focus group discussion and 
interviews with VMWs at 
end-line 
Costs of RACD as unit and total 
costs; cost-effectiveness will be 
assessed as an incremental cost 
effectiveness ratio (ICER) 
 
Program data on costs 
combined with estimates of 
program effectiveness 
2. 
Assess the acceptability 
of 
community-based 
RACD  
Proportion of targeted individuals 
refusing RACD intervention 
RACD intervention data 
Proportion of survey respondents 
who strongly disagree, disagree, 
are 
ambivalent, 
agree 
and 
strongly agree on the importance 
and acceptability of community-
based RACD 
Cross-sectional survey and 
qualitative 
studies 
at 
endline 
with 
villagers, 
forest workers, VMWs and 
other health sector staff 
Activity 
3: 
Assessment of the 
operational 
feasibility and safety 
of 
conducting 
G6PDd 
testing, 
referral, 
and 
treatment 
adherence 
among 
positive P. vivax RDT 
cases 
1. 
Assess the operational 
feasibility 
of 
G6PD 
referral at community 
level 
Proportion of VMWs who rate 
conducting G6PD referral as very 
easy, somewhat easy, somewhat 
difficult, and very difficult, and 
changes in proportions over time 
Entrance, mid-intervention, 
and 
exit 
competency 
checklists 
with 
VMWs; 
focus group discussion and 
interviews with VMWs at 
end-line 
Proportion of P. vivax cases with 
valid recorded G6PD result 
VMW and health facility 
records 
2. 
Determine risk factors 
for non-attendance at 
hospitals 
or 
health 
centers after referral for 
G6PD testing 
Proportion of referred cases 
presenting at hospitals or health 
centers for G6PDd testing. 
Cross-sectional survey and 
qualitative 
studies 
at 
endline 
with 
villagers, 
forest workers, VMWs 
3. 
Assess 
treatment 
adherence 
among 
positive P. vivax RDT 
cases 
Proportion 
of 
patients 
with 
physical 
evidence 
of 
full 
adherence with the prescribed 
drug regimen 
VMW follow-up visits on 
day 3, 7, and 14 
Proportion of P. vivax cases who 
relapsed in the six months 
following treatment 
VMW and health facility 
records 
4. 
Assess the safety of 
community-level G6PD 
testing, referral, and P. 
vivax treatment follow-
up 
Adverse event rate per treated 
individual 
RACD intervention data and 
VMW 
follow-up 
visits; 
health facility adverse event 
reporting forms 
Activity 4:  
Assessment of the 
risk 
of 
malaria 
vector 
biting 
in 
forest and village 
settings 
1.     Describe local vector   
species 
composition 
and 
bionomics 
in forest and 
village settings  
Vector occurrence and density by 
species; vector biting behaviors; 
human behavioral observations in 
relation to human biting rate 
(HBR) 
Human Landing Catches 
(HLCs) vector sampling at 
baseline 
 

Lao PDR protocol 
 
 
19 
3.4.2. Impact evaluation of enhanced RACD 
3.4.2.1. 
Study design 
This study will employ a two-arm cluster-randomized control trial design with randomization of 
clusters into either RACD or control.  
3.4.2.2. 
Randomization 
A total of 32 health center catchment areas (HCCAs) in Laos will be selected for inclusion based upon 
incidence per 1000 population between October 2017 and September 2018. HCCAs with higher 
incidence will be prioritized to improve power; where possible, directly neighboring HCCAs will not 
both be included to reduce contamination. Restricted randomization of the HCCAs into either RACD 
or control arms will be conducted, whereby sets of randomizations (with 16 HCCAs in each arm) will 
be generated that achieve balance across arms on incidence per 1,000 over the prior year, population 
size, and amount of forest cover. From these sets of potential randomizations, a single randomization 
will be randomly selected to assign 16 HCCAs each to the RACD or control group. Restricted 
randomization in this way will ensure balance across intervention and control groups on potential 
confounding factors. The ‘sample’ command in Stata v14 (StataCorp, College Station, TX) will be used 
to implement randomization. 
 
Table 5. Study arms 
Study arm 
Description 
Lao PDR 
RACD 
Reactive case detection led by VMWs in response to cases in study 
area HCCA, with follow up testing with HS-RDTs/RDTs in both 
villages and forest workers; referrals for qualitative G6PD testing for 
P. vivax cases and 14-day PQ for G6PD non-deficient  
16 
Control 
Standard of care including case management through health 
facilities and malaria posts/VMWs; village-based RACD conducted 
by district staff in some areas 
16 
 
3.4.2.3. 
Primary outcome measures 
1. Confirmed P. falciparum and P. vivax malaria parasite incidence: defined as the number of 
OPD malaria confirmed and suspected cases per person per year for each HCCA, as 
ascertained from the health facility registers, utilizing administrative catchment population 
size estimates for the exposure denominator. 
2. PCR-based P. falciparum and P. vivax parasite prevalence in sampled HCCAs: defined as the 
proportion of individuals ≥18 months old with P. falciparum or P. vivax infection (detected by 
PCR) out of all individuals ≥18 months tested within the endline survey (2020). 
3. HS-RDT/RDT -based test positivity rate in village and forest-based reactive case detection: 
defined as the proportion of all individuals tested by HS-RDT/RDT in response to an index 
cases, with a positive HS-RDT/RDT, among the population older than 18 months. 
3.4.2.4. 
Sample size 
3.4.2.4.1. 
Confirmed P. falciparum and P. vivax malaria parasite incidence 
Assuming an average baseline transmission of 2.5 per 1000 population per year (all species, from 2017 
health system data across study sites and a 50% reduction), an average population of 4000 per health 
facility catchment, and coefficient of variation of 0.6 (based upon 2017 health system data), in order 
to detect a 50% reduction in cumulative infection incidence (as measured by routine health system 
surveillance data over an 18-month period) in a random effects Poisson or negative binomial model, 
a minimum of 16 randomization units will be required in each study arm. A total of 32 health facility 
catchment areas will be included in Lao PDR, with 16 allocated each to enhanced RACD and control. 

Lao PDR protocol 
 
 
20 
Only health facility catchment areas with at least 3 malaria cases in the past year will be considered 
for inclusion.  
3.4.2.4.2. 
Population-level P. falciparum and P. vivax malaria parasite prevalence 
Assuming 16 health catchment areas per arm, a baseline control prevalence by qPCR (all species) of 
2.5%, and a coefficient of variation of 0.5, a minimum of 400 individuals per catchment area will be 
required in order to detect a 50% reduction in prevalence at endline. Assuming a 10% refusal rate, a 
total of 14,080 individuals will be required across both arms at the end-line survey. Assuming 4 
individuals per household, this will result in a total of 3,520 households sampled, or 110 per health 
facility catchment area. 
3.4.2.4.3. 
HS-RDT/RDT test positivity during RACD intervention 
Test positivity by HS-RDT and RDT will be monitored throughout the intervention period. There is no 
formal sample size calculation for this outcome, but based upon an estimated 720 cases in Lao PDR 
over the study period, an average of 5 households per response event, 4 people per household, and 
4 forest-workers per case, we estimate a total of 17,280 individuals will participate in testing during 
RACD in Laos.  
3.4.2.5. 
Data collection 
3.4.2.5.1. 
Confirmed malaria case incidence 
The confirmed parasite incidence from all reporting units (including HCs, district hospitals, VMWs with 
RDTs, and PPM sites) will be captured throughout the study with support from study or health center 
staff. In the months prior to the start of study activities, all villages within each health facility 
catchment area will be mapped, and trainings conducted with health facility staff to systematize the 
collection of village names at health facilities for confirmed malaria cases. Routine supervision of 
health facility staff will be conducted by study staff to ensure accurate recording of village names in 
health facility registers throughout the trial. Forest sites visited by malaria cases will be recorded and 
mapped where possible. 
3.4.2.5.2. 
Cross-sectional surveys 
An endline survey will be conducted in November/December 2020 to obtain an unbiased estimate of 
P. falciparum and P. vivax malaria parasite prevalence in each study arm, as well as assess intervention 
coverage, treatment seeking, and mobility and forest going activities. The endline survey will include 
testing with HS-RDTs/RDTs and collection of DBS for qPCR-based testing. 
 
Sampling frame and sampling strategy 
Within each of the HCCAs selected for study inclusion, survey staff will work with village authorities 
to update household ledgers, and then all households will be enumerated and a GPS point captured. 
All households will be given a study ID card and household sticker with a unique barcode, which will 
be used throughout the study to identify repeat visits at each household, as well as for individuals to 
present at health facilities if they report for care. 
 
In order to reduce potential contamination due to proximity to intervention areas (for the control 
arm), households within 2 km of a neighboring study village will be removed from the sampling frame. 
Households will be selected via simple random sampling from the remaining HH lists.    
 
Questionnaires and human specimen collection 
During the endline survey, selected households will be visited and the head of household interviewed 
by a study staff member using a tablet or paper form. To capture any household members not present 
at time of survey, study staff will plan for an overnight stay whenever feasible to schedule visits for 
early morning or late evening, but will have a maximum of four visits to each HH. The survey 
questionnaire used for interviewing will be developed in English with input from local health staff. This 

Lao PDR protocol 
 
 
21 
will then be translated to Lao, and back-translated by a fluent bilingual health expert prior to field 
testing.  
 
The survey questionnaire will capture household-level demographics, and assess potential risk factors 
for malaria infection. Information collected will include age, gender, pregnancy, nationality, ethnicity, 
occupation, socioeconomic status, travel history, history of malaria, treatment seeking for fever in the 
past two weeks, individual and household use of vector control measures, housing structure type, 
proximity to forest and forest-fringe, and frequency of overnight sleeping in forest or forest-fringe 
areas.  
 
At the endline survey, all household members (residents and temporary visitors) aged 18 months and 
older will be invited to participate in an RDT and blood collection component. Informed consent will 
be obtained from all participants, including parental consent for any participant younger than 18 years 
of age. After consenting, the study team will capture axillary temperature, and test each individual 
using a standard combination RDT and an HS-RDT (SD Bioline Malaria Ag P.f High Sensitive Cat# 
05FK140), and Hemocue kit, followed by collection of four DBS on filter paper. 
  
If found positive by RDT or HS-RDT, treatment will be administered as in Section 3.1.1. Testing results 
from the collected DBSs will not be provided to the participants.  
3.4.2.5.3. 
Intervention implementation data 
Data collection during RACD activities is described below.  
 
Questionnaires and human specimen collection: reactive case detection (RACD) 
For all household members and co-worker referrals from forest workers in RACD intervention areas, 
after obtaining informed consent, a short demographic and malaria risk factor survey will be 
conducted to obtain information on individuals’ occupations, recent forest work or travel, ITN usage, 
and recent care-seeking behavior for fever. Household members and referrals will be linked to the 
index case through unique codes in order to facilitate follow-up and allow for statistical adjustments, 
such as clustering by index case.  
 
In the intervention areas, all patients with a P. vivax positive RDT will be given a coded and signed 
informational letter directing them to the nearest health center of hospital for G6PD testing and 
follow-up treatment. All P. vivax patients will be recorded into a registry and given an identification 
card for presentation and treatment and follow-up visits. 
 
Sites where interviews occur will be geo-located if all parties agree. VMWs may also obtain contact 
information from consenting persons to allow for follow-up on positive cases and potential creation 
of support networks for other HRPs. 
3.4.2.6. 
Data management and analysis 
Data will be collected via ODK-based tablet application with internal range checks or paper-based with 
subsequent double-entry, and will be stored in Microsoft Excel or Access. The number of malaria 
cases, based on RDT and PCR results, will be mapped by village or sub-village and compared between 
surveys.  
 
Primary outcomes 
 
Outcome 1: Community-level confirmed P. falciparum and P. vivax malaria parasite incidence 
Data pertaining to this outcome will be analyzed on an intention-to-treat basis at the level of health 
facility catchment area (randomization unit). Cumulative counts of confirmed malaria cases from the 
health facility registers over the study period and the previous year will be analyzed in a Poisson or 

Lao PDR protocol 
 
 
22 
negative binomial regression model with random intercepts at the health center catchment level and 
an offset for the estimated population size of the catchment area (from administrative data). The 
models will include a fixed effect for each study arm and a fixed effect for time period (pre- and post-
intervention start). The interaction between these two terms will be the primary effect measure (also 
known as the difference-in-differences estimator). Pre/post-intervention will be determined as all 
time periods before the start date of the intervention in the areas considered as being pre-
intervention and all time periods after (and including the start date of the intervention in the area 
considered as being post-treatment).  
 
Outcome 2: Community-level PCR-based P. falciparum and P. vivax parasite prevalence in sampled 
villages 
The effectiveness of the interventions will be assessed as P. falciparum and P. vivax prevalence via 
PCR at end-line (post only) using generalized linear mixed effects models with separate random 
intercepts to allow for clustering within health center catchments. The binomial distribution will be 
used to analyze prevalence outcomes (logistic regression). All main analyses will be analyzed as 
intention-to-treat, and all survey clusters will be analyzed within the intervention group assigned at 
randomization, regardless of adherence. The primary effect estimate will be evaluated using the fixed 
effects for RACD. Secondary analyses will include adjustment for age, sex, and other potential 
confounders, and a per-protocol analysis of the primary effect estimate.  
 
Outcome 3: HS-RDT and RDT-based test positivity rate in village-based and forest-based samples 
The HS-RDT and RDT test positivity rate will be estimated for the RACD areas during each intervention. 
This will be done as soon as data on HS-RDT results are available. Differences in test positivity 
measures over time and between village and forest base samples will be assessed using a χ2 test, as 
well as logistic regression models to account for potential confounding factors.  
3.4.2.7. 
Laboratory analysis 
The laboratory procedures described below will be followed for all laboratory-based activities 
conducted during this project. The laboratories performing these tests include the Pasteur Institutes 
in Phnom Penh and Paris, and UCSF. 
 
Rapid diagnostics tests 
Standard combination RDT, HS-RDTs and G6PD Carestart RDT will be used during field activities. 
Standard combination RDT and HS-RDTs will be used to determine malaria infection status and will be 
performed on participants every time a blood sample is collected. The standard combination RDT can 
detect P falciparum and P vivax malaria infection while HS-RDT can detect whether P falciparum 
infection is present or not and is estimated to be several times more sensitive than standard RDTs; 
new HS-RDTs including detection of P. vivax will be used if they become available over the study 
period. The VMW will use a finger prick blood sample to run the Standard SD Bioline and HS RDTs in 
parallel with results available within 20 minutes and recorded by study staff. The G6PD Carestart RDT 
will be used by health center and hospital staff to determine the G6PD status of all individuals positive 
for P vivax by standard RDT. All RDTs will be used according to the manufacturer's instructions. The 
results of these tests will be provided to the participant. For quality assurance of qualitative G6PD 
tests, quality assurance processes including user training, proficiency testing, and regular quality 
control of tests using control reagents will be introduced in intervention facilities. Images of G6PD 
RDTs conducted over the first three months of the study will be reviewed by laboratory technicians 
at the district hospital. 
 
Filter paper sample collection 
Dried blood spots (DBS) will be collected onto filter paper for future molecular analysis including 
parasite and human (i.e., G6PD) genotyping for research purposes only. These results will not be 
provided to the participants. Filter paper (Whatman 3MM) will be pre-cut into individual squares and 

Lao PDR protocol 
 
 
23 
stapled to a thick card that will serve as its cover. Blood spots will be collected onto the filter paper in 
volumes of approximately 25 µl aliquots per blood spot (4 blood spots per card). Filter paper samples 
labelled with the individual’s barcodes or ID number on the covering cardboard and will be allowed to 
dry at ambient temperature and relative humidity before closing the card over the filter paper. Filter 
paper samples will be transported from the field in a Ziploc bag then placed in a stock card filter paper 
box with desiccant and humidity indicator card and stored at 4˚C within one week, and at -20˚C within 
one month. Dried blood spots (DBS) will be regularly transported to the district or provincial offices 
for refrigerated storage prior to bulk transport and shipment to designated laboratories.  
 
Serology 
Serology, a test of past infection as assessed by the presence of antimalarial antibodies, will be used 
to improve the identification of hotspots and estimate current and historical transmission 
intensities14. Using DBS, ELISA assays will be performed using previously described methods. Briefly, 
antibodies will be eluted from DBS and assayed to detect antibodies against the P. falciparum blood 
stage antigens including merozoite surface protein-1 (MSP-1) and apical membrane antigen-1 (AMA-
1), both biomarkers of P. falciparum exposure15. Markers for P. vivax exposure will include Pvmsp-1 
and Pvcsp 16. Other antigens that are sensitive and specific for recent exposure (currently undergoing 
evaluation) for P. falciparum and P. vivax may also be used. ELISA assays will be performed in duplicate 
and optical densities recorded with an ELISA reader. Other serological and antigenic platforms (bead 
array, protein microarray) may be used to analyze responses to multiple antigens/antibodies, if 
available. 
 
Genotyping 
Genotyping of P. falciparum infections to assess transmission networks will consist of a panel of 
microsatellites located throughout the genome. Briefly, DNA samples will be amplified in a multiplex 
pre-amplification step followed by amplification of microsatellites in individual reactions using 
fluorescently tagged primers and sized using denaturing capillary electrophoresis. Multilocus 
genotypes from mixed infections will be reconstructed, where possible, by quantifying alleles at each 
locus. Genotyping of additional loci including for HRP2 deletion will be performed as needed. 
 
Individual microsatellite amplifications will be undertaken using single round or nested PCR assays 
with fluorescently labeled primers, and the amplicons sized by denaturing capillary electrophoresis 
with internal size standards. Allele-calling will be undertaken with the aid of the GeneMapper v4.0 
software. The potential for effective multilocus haplotype reconstruction in polyclonal infections will 
be explored. Additional informative SNP markers identified in whole genome sequencing efforts may 
also be genotyped as necessary to improve sample fingerprinting. 
 
Genotyping for markers of drug resistance will include PCR and/or sequencing to identify markers of 
resistance to artemisinin-based combination therapies (ACTs), in particular artemether-lumefantrine, 
artesunate-amodiaquine, dihydroartemisinin-piperaquine (including K13 and plasmepsin), and the 
antifolate combination sulfadoxine-pyrimethamine (SP). 
 
Human DNA may be used in the future to screen for genetic risk factors for malaria, such as inherited 
blood disorders and drug metabolism e.g., G6PD variant, CYP2D6 variant. In subjects who experience 
an SAE and where hemolysis is suspected, G6PD genotype will be examined as well as a number of 
control subjects of people who were given primaquine but did not hemolyse or have an SAE. A random 
sample from the cross-sectional surveys may be examined for G6PD genotype in order to assess the 
prevalence of G6PD deficiency in study areas. No additional human genetic testing unrelated to 
malaria will be performed.   
 

Lao PDR protocol 
 
 
24 
3.4.3. Assessment of the feasibility, acceptability, and safety  
Qualitative key informant interviews (KII) and focus group discussions (FGDs) will be conducted at 
baseline and endline in Laos PDR. 
3.4.3.1. 
Implementation procedures 
The feasibility, acceptability and safety evaluations will be conducted using a mix of quantitative data 
collection and key informant (KI) interviews and focus group discussions (FGDs) of key study and 
health sector personnel including provincial, district, and health facility staff, VMWs, forest 
workers/employers and villagers/community members. In addition, participatory research methods 
including a process-mapping workshop at a subset of health facilities and a workshop based on the 
co-design of training and counselling messages for G6PD deficiency and risk factors for radical cure 
treatment will be conducted at baseline.     
3.4.3.2. 
Primary outcomes measures 
 
1. Population coverage of enhanced RACD interventions: this indicator will be measured in two 
ways. Operational program coverage will be defined as the proportion of individuals ≥18 
months old and households visited and offered the RACD interventions within the target areas 
per time period. Effective program coverage is defined as the proportion of individuals (≥18 
months old) that agreed to participate in the RACD intervention among all individuals ≥18 
months old eligible to participate in the intervention in the target population per time period.  
2. Feasibility of conducting enhanced RACD intervention at the community level: feasibility will 
be determined based upon a combination of population coverage data, responses of 
provincial, district, and health staff, VMWs, and community members to KIIs and FGDs at 
baseline and endline, VMW competency checklists at baseline, midline, and endline, and cost 
data. 
3. Acceptability of community-based RACD approaches: acceptability will be determined based 
upon refusal rates during interventions and responses of community members and VMWs to 
endline questionnaire, interviews, and focus groups. 
4. Safety of community-based RACD and treatment follow-up: safety measures will include the 
adverse event rate amongst treated individuals and hemoglobin measurement pre and post 
treatment for individuals receiving PQ. 
5. Operational feasibility of G6PD referral: operational feasibility of G6PD referral will be 
determined by the results of the process-mapping and co-design workshops at baseline, 
responses of health staff and VMWs to interviews and focus groups at baseline and endline 
and competency checklists at baseline, midline, and endline, the proportion of referred P. 
vivax cases presenting at a health facility for G6PD testing, and the proportion of P. vivax cases 
with a valid recorded G6PD result. 
6. Assessment of P. vivax treatment adherence: treatment adherence will be determined by the 
proportion of P. vivax cases with physical evidence of adherence through pill count, the P. 
vivax relapse rate across study arms, and the proportion of P. vivax cases with successful 
parasite clearance on day 14. 
3.4.3.3. 
Sample size 
Population coverage data for RACD interventions will be collected routinely and estimated numbers 
of individuals included are described in section 3.4.2.4.3 above. Focus group discussion (FGD) and key 
informant interview (KII) activities for feasibility and acceptability outcomes will aim to reach 
saturation and be comprehensive. At baseline and endline there will be a total of 1 FGD and 2 KIIs per 
district with provincial, district or study staff (36 KIIs total), VMWs, and recent malaria cases reporting 
forest travel. Supervisors will observe and complete a competency checklist on a random subset of 32 
VMWs at three different time points: within the first month of intervention launch, at the midpoint of 
the intervention, and within the final month of the intervention. Sample sizes for quantitative (Likert 

Lao PDR protocol 
 
 
25 
scale) acceptability data derived from cross-sectional surveys will be based on end-line sample size of 
3,520 households. 
3.4.3.4. 
Data collection 
Baseline and endline KIIs with province and district authorities and FGDs with health center staff, 
VMWs, and high-risk populations (HRPs) will be conducted to qualitatively assess the feasibility, 
acceptability and safety of community-based RACD practices, including G6PD deficiency and 
primaquine knowledge, attitudes, and practices. The outputs of a process-mapping workshop and co-
design workshop at two purposively selected health facilities will be documented and used to inform 
downstream training materials and counselling messages. 
 
Standardized tools including interview/discussion guides and a competency checklist will be 
developed using best practices in qualitative research, and translated and back-translated prior to 
implementation. Study staff and an independent FGD/KII moderator will be trained on the appropriate 
tools, and all interviews will be conducted in the local language. The end-line FGDs and KIIs will explore 
constraints and any issues related to all the topics identified from the quantitative survey, field 
experience implementing the RACD intervention, and barriers to implementation of malaria control 
efforts. Informed consent will be obtained from all FGD, KII, and HRP participants in the local language 
prior to any data collection. FGDs and KIIs will be audio-recorded, transcribed, and the transcripts 
translated into English. 
 
Themes to be explored during the KIIs and FGDs include the following: Demographics of HRP groups;  
Occupations of HRP groups, including seasonality of work; Forest-going behaviors; Migratory patterns; 
Health-seeking patterns and behaviors; Social networks and congregation/frequented sites (locations 
and days/times visited); Ideal locations and times to access HRPs for case management and 
surveillance activities; Operational feasibility and attitudes towards VMW-led active case detection; 
Capacity to conduct active case detection in forest workers; Knowledge, attitudes, and practices 
related to P. vivax infection, G6PDd testing, and primaquine. 
 
FGDs and KIIs will be grouped as follows:  
 
1) High risk populations (HRPs), including recent malaria cases who have worked or traveled at 
least one night at a forest or forest-fringe site located outside of a permanent settlement in 
the past 30 days 
2) Health facility-based staff involved in malaria diagnosis and treatment 
3) Village Malaria Workers and other community members 
4) Provincial and district health officers 
5) Other health sector and ministry personnel 
6)  Process-mapping and co-design workshops. Participation will include a cross-section of 
supervisors, health center staff, VMWs, and community members at 2 health centers. The 
health centers will be purposively selected based on malaria case volume. 
 
At endline, FGDs and KIIs will be conducted with health center staff, VMWs, and HRPs. Themes to be 
explored during KIIs and FGDs include: Forest-going behaviors; Migratory patterns; Health-seeking 
patterns and behaviors; Operational feasibility and attitudes towards VMW-led active case detection; 
Capacity to conduct active case detection in forest workers; Knowledge, attitudes, and practices 
related to P. vivax infection, G6PDd testing, and primaquine; Facilitating factors and challenges to 
scale-up of the interventions.  
 
These KIIs, FGDs and participatory research methods will focus on assessing the operational feasibility 
and acceptability of community-based active case detection in both villages and forest workers, 
G6PDd referral, and treatment follow-up of radical cure for P. vivax infection using 14-day primaquine. 

Lao PDR protocol 
 
 
26 
 
The costs of RACD will be collected. Total costs as a unit as well as cost-effectiveness will be assessed 
as an incremental cost effectiveness ratio (ICER). The types and potential sources of expenditure data 
are shown in Table 6.  
 
Table 6. Types and potential sources of expenditures 
 
 
Focus groups will be facilitated by two members of the research staff:  1 moderator and 1 note-taker. 
A locally-appropriate location will be used to hold the focus group discussions (i.e., community center, 
health facility, etc). After a brief introduction, the moderator will obtain informed consent separately 
from each participant. All sessions will be audio recorded. During the sessions, the research staff will 
generate notes as the discussion unfolds to help formulate follow-up questions and probes, with no 
identifying information. 
 
For interviews and focus groups, the note taker and moderator will discuss interview 
notes/similarities, and responses will be recorded/finalized at the end of each interview. The 
moderator for each group will transcribe the recording and once all focus groups have been conducted 
and data transcribed, and results disseminated, all audio recordings will be destroyed. A professional 
translator will translate the transcript to English. The local study coordinator will supervise and review 
the local language and English transcripts to ensure accuracy. Each focus group discussion will take 
approximately 1-2 hours total and occur at an appropriate time for the study respondents (i.e., 
weekday vs weekend; morning vs afternoon). 
 
Data from the interviews and focus groups will be entered into Dedoose or a similar program for 
simple thematic analysis and stored on a password protected device. All data will be stored on a 
password-protected database and will only be available to the study personnel. All data from 
participants will be coded using a study identification number in place of the individual’s name. Data 
will be analyzed to provide preliminary descriptive statistics on the study population and recurrent 
themes within the assessment. Data will only be used for the purposes of this study. 
 
All participants will receive locally-appropriate compensation for participating in the focus group or 
interview to compensate for travel costs and time, and will be determined following discussions with 
Expenditure category 
Types of expenditure needed 
Potential sources of information 
Personnel 
All human resource expenditures and 
time contributions 
• 
Salary/wage payments 
• 
Value of benefits 
• 
Volunteer labor 
• 
Annual program budgets 
for salaries and benefits 
• 
Work logs or reports 
from volunteers 
 
Commodities and Services 
All supplies and services used toward 
RACD activities 
• 
In-kind donations 
• 
Purchased 
commodities 
(including acquisition costs) 
• 
Utilities 
• 
Travel/transit costs: fuel, transit 
fees and services, airfare 
• 
Reproduction costs, postage 
• 
Staff 
trainings: 
per 
diem 
payments, 
trainer 
fees, 
consultants 
• 
Current rental value of property 
• 
Printing 
and 
postage 
receipts 
• 
Training 
budgets 
and 
receipts 
• 
Vehicle travel logs, fuel 
receipts 
• 
Utility bills 
• 
Consultant invoices 
 

Lao PDR protocol 
 
 
27 
local partners. Light snacks and non-alcoholic refreshments will be provided during the focus group 
session. 
3.4.3.5. 
Analytical plan 
Outcome 1: Village-based population coverage of test and treat interventions 
The operational coverage will be estimated at the individual and household level as the percent of the 
population that received a visit from the intervention teams to offer the RACD intervention, among 
those eligible for inclusion. This will be obtained from a combination of RACD program data and 
enumeration data. Additionally, the proportion of individuals accepting the RACD interventions, 
among those eligible for inclusion in the intervention, will be estimated, providing an estimate of the 
effect coverage of each program. Data for the denominator of individuals and households targeted 
for the intervention will be ascertained from the household enumeration for the sampling frame. 
Additionally, to validate the enumeration, attempts will be made to use remote sensing data, and/or 
Google Earth, to enumerate household structures. To the extent possible, individual, household and 
community level factors associated with coverage will be assessed using mixed effects logistic 
regression.  
 
Outcome 2: Feasibility of conducting RACD interventions  
The operational feasibility of VMWs to conduct RACD interventions will be estimated at the individual 
level as the percent of VMWs and health center staff who rate the intervention activities as very easy, 
somewhat easy, somewhat difficult, and very difficult. Supervisors will conduct a competency 
checklist on 2 VMWs per intervention area (32 total) at baseline, midline, and end-line; the capacity 
of following case investigation, recording, management, and follow-up SOPs will be assessed over 
time. Qualitative surveys will be conducted with VMWs and health center staff at endline. The costs 
of RACD and cost-effectiveness will be assessed as an incremental cost effectiveness ratio (ICER). 
Program data on costs/expenditure for RACD will be collected and combined with estimates of 
program effectiveness to determine ICER.  
 
Outcome 3: Acceptability of community-based RACD approaches 
The acceptability of a community-based approach for RACD will be assessed as the proportion of 
targeted individuals refusing RACD intervention, among those eligible for inclusion. Qualitative data 
collection methods such as focus groups and key informant interviews with villagers, forest workers, 
VMWs and other health sector staff will be implemented. 
 
Outcome 4: Safety of community-based RACD and treatment follow-up 
The adverse event rate for RDT positive participants will be assessed using intervention data and 
health facility adverse event reporting forms. Study staff and VMWs will be trained on detection of 
adverse events for positive cases receiving treatment. 
 
Outcome 5: Operational feasibility of G6PD referral, testing and non-adherence 
The operational feasibility of G6PD referral, testing and non-adherence at the community and facility 
levels will be evaluated. For testing, interview data at baseline and endline will be collected from 
district health staff and community members, as well as focus group discussions with both groups at 
end-line. Risk factors for non-adherence to G6PD testing after referral will be determined through 
comparison of factors captured at enrollment for individuals who were tested at a facility versus those 
who were not. 
 
Outcome 6: Assessment of P. vivax treatment adherence  
The treatment adherence of P. vivax positive study participants will be assessed through physical 
evidence of adherence during VMW follow-up visits on day 3, 7 and 14 and comparison of relapse 
rates across study arms. 
 

Lao PDR protocol 
 
 
28 
3.4.4. Assessment of the risk of malaria vector biting in forest and village settings 
A baseline entomological investigation will be conducted along with the baseline qualitative activities 
in Aug/Sep 2019. The investigation will involve collection of vector occurrence and density data (by 
species), human landing catches (HLC) to assess vector biting behaviors (indoor vs outdoor and forest 
sites), and household structure type, and human behavioral observations. All collections will take 
place over a one-week period. HLC collections will occur in 4-5 sleeping structures each in the forest 
and village, with 2 HLC collectors by structure. At the end of each HLC collection hour, collectors will 
also record human behavior (sleeping (or awake) under net/sleeping without net/awake), both 
indoors and outdoors. Field collectors will be community volunteers that will have been properly 
informed about the sampling methods and their implications, and who have consented to 
participating by signing consent forms. Prior to the start of collections, volunteers will also need to be 
tested for malaria. Depending on national policy, they may also be required to take malaria 
prophylaxis. HLC data collection forms are found in the Appendix.  
4 
Ethical issues related to human subjects research 
4.1. Adequacy of protection against risks 
We will administer an informed consent form both verbally and in writing to all participants in the 
local language for participation in all study activities. These forms will be read or will be given to 
participants to read themselves and will include a full description of voluntary participation, the right 
to withdraw from the study at any time, and the right to not answer any question or participate in 
any component of the research.  
 
These forms will also address the risks, benefits, and purpose of the study and what we hope to 
learn, with a specific focus on the potential risks associated with the administration of SLD-PQ. We 
will train all interviewers extensively on the consent procedure, and each form will be co-signed by 
a team member to ensure all participants have consented. Checks in the field by the PI and project 
leaders will further ensure the consent process is followed in all cases. Data collection team 
members will provide the contact information for study coordinators who can be contacted for any 
further information on the topics brought up in the interview, or for additional treatment if 
necessary. The confidentiality procedures are designed to meet all contingencies to ensure the 
confidentiality of participant data and the privacy of the participants is preserved. 
 
Our proposed strategies to reduce risks to privacy or of disclosure of confidential information 
include: 
 
1. Identifying information will be recorded only in secure database software on password 
protected computers, and data collectors will only have access to the data that they 
themselves directly collect which will be cleared from their devices after all follow-up visits 
are completed. All data will be stored only in password-protected files on password-
protected computers in locked offices.   
2. Prior to analysis, data will be de-identified with the exception of geo-location codes, which 
are necessary for specific per-protocol analyses. The absence of individual identifying 
information will protect subject confidentiality.  
3. All paper records will be stored in a locked location. 
The potential risks of drawing blood from a finger-prick include temporary discomfort, pain, transient 
bleeding, bruising, skin infection, and fainting. The volumes of blood taken will be too small to produce 
any adverse physiologic effects from blood loss anemia and overall the aforementioned risks 
associated with blood draws are likely to be low. Study staff will be trained in the proper conduct of a 
finger-prick according to standard operating procedures to minimize the risk of discomfort and 
infection.  

Lao PDR protocol 
 
 
29 
4.2. Protection against risks associated with administration of PQ 
The main safety concern for primaquine administration is the risk of acute hemolytic anemia in 
individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency17. In a context where G6PD 
testing is not available, a dose of 0.25mg/kg of PQ has been found to be safe in individuals with any 
of the G6PD variants and is recommended by WHO 13. All individuals RDT+ for P. vivax will be referred 
to and tested at a health facility using a G6PD Carestart RDT and a 14-day course of primaquine will 
only be given to G6PD non-deficient individuals. After the first six months, a subset of VMWs will 
conduct G6PD testing under supervision by a study staff member for P. vivax cases diagnosed in the 
community. Primaquine is typically well tolerated but possible side effects include nausea, headache, 
fatigue, dizziness, hives, chest pain, painless darkening of urine, jaundice of skin, vomiting and syncope 
(fainting). In line with national guidelines, PQ will not be administered to pregnant women in any 
trimester or to women unable to undergo pregnancy testing, children less than 6 months of age, 
infants weighing less than 10 kg, women in the first 6 months of breastfeeding, or individuals with 
prior allergic reaction to PQ or related drugs. 
The key to managing side effects and any potential adverse events (AEs) or serious AEs (SAEs) is 
thorough training of VMWs/VHVs, health facility staff and supervisors, and intensive sensitization of 
the community. HC workers and VMWs/VHVs will passively monitor their respective communities 
for AEs and refer any potential SAEs to designated health facilities. Health workers at these facilities 
will be informed of the project and potential side effects, and undergo training on completion of AE 
reporting forms (8.14 and 8.15). Cases that cannot be treated at health centers will be referred to 
district or provincial hospitals.  
 
All participants receiving study drug will be provided with an informational sheet listing potential 
side effects and instruction to seek care at the nearest health center should they experience any of 
the defined symptoms or other adverse events. Community sensitization events and targeted 
IEC/BCC materials will also help increase community awareness of potential adverse events and 
encourage early care-seeking if events arise.  
 
A passive case detection system will be employed in both intervention and control villages to help 
detect and refer any potential SAEs and active follow-ups will be conducted by VMWs on days 3, 7, 
and 14; VMWs/VHVs will be oriented on potential drug side effects and asked in addition to passively 
monitor their communities and refer any potentially serious or unexpected AEs. All public health 
facilities in the nine target districts will be trained on AE reporting procedures prior to 
implementation of any study activities. A data safety monitoring board (DSMB) will be established 
to oversee and report on any SAEs that are potentially linked to the administration of AL or SLD-PQ, 
as outlined in further detail in Section 4.5. The following steps will be taken for all AEs: 
 
The health facility will complete an AE reporting form (Appendix 8.14). The completed form will then 
be submitted to the Local Safety Monitor (a medical doctor), who with the support of the UCSF 
project manager will assess if the event is an SAE or unexpected AE based upon specific criteria 
determined by the DSMB. If determined to be an SAE or unexpected AE, the UCSF project manager 
will submit the AE reporting form and any accompanying documentation (clinical records, laboratory 
reports) to the DSMB and the UCSF PI within 48 hours from the identification of the potential SAE.  
 
Upon receipt of the AE reporting form, the DSMB will complete an AE investigation form (Appendix 
8.14) within 24 hours to a) confirm if the event is an SAE, and 2) to determine if the SAE was caused 
by the administration of AL with SLD-PQ as part of this study. If the event is confirmed to meet the 
criteria of an SAE, the DSMB will submit the completed AE investigation form to the PI, who will 
submit to the research ethics committees (RECs) at UCSF and NIOPH. The respective RECs will be 
notified within 24 hours of the DSMB’s completion of all potential SAE investigation forms, and 
within 48 hours of DSMB notification if deemed an AE, but not fulfilling criteria for an SAE.     

Lao PDR protocol 
 
 
30 
 
Travel and hospitalization costs for any SAEs resulting from PQ administration will be covered by the 
study.  
4.3. Potential benefits of the proposed research to the participants and others 
The proposed research may benefit patients in direct and indirect ways. Participants will directly 
benefit from detection of low-level parasitemia, and the curative effects of AL administration on 
existing malaria infections in the RACD intervention arm. Furthermore, patients may directly benefit 
due to community-wide reductions in malaria transmission that are expected to occur after the 
application of the interventions. Finally, entire target village populations will benefit from SLD-PQ, 
which targets the parasite sexual stages thereby decreasing overall transmission. 
 
Participants may also indirectly benefit, as the information gained from this research will be used to 
help establish the safety and efficacy of new malaria control interventions in both countries. The 
research will benefit the scientific and malaria control communities more generally by expanding the 
evidence base on RACD targeting high risk populations.   
4.4. Alternatives to participation 
Participation in the research study is voluntary. Individuals electing not to participate in the research 
study may still receive testing and treatment as part of the routine malaria case management. 
Individuals who do not wish to receive testing and treatment or presumptive treatment during the 
intervention campaigns may visit local health facilities for malaria testing and treatment.   
4.5. Data and safety monitoring plan 
The project will follow US National Institutes of Health (NIH) guidelines for establishing a data safety 
monitoring board (DSMB). The DSMB will be established prior to any data collection as part of this 
study. The members of the DSMB will serve in an individual capacity and provide their expertise and 
recommendations. The primary responsibilities of the DSMB will be to periodically review and 
evaluate the accumulated study data for participant safety, study conduct and progress, and, when 
appropriate make recommendations to investigators concerning the continuation, modification, or 
termination of the trial. DSMB will consider study-specific data as well as relevant background 
knowledge about malaria, adverse events from drugs used in the study, drug resistance, and the 
participant population under study. 
 
Membership of the DSMB will consist of five independent experts in malaria control, diagnosis, case 
management and epidemiology, and one member of the research team to advise and clarify study 
activities for the independent experts. No independent member of the DSMB shall have any conflict 
of interest with the study team, the organizations funding or conducting the research, or the results 
of the study. The DSMB will be comprised of experts in the following areas, with an emphasis on 
local Lao expert participation: 
 
• 
The study population in Lao PDR (Lao representatives) 
• 
Malaria diagnosis and case management 
• 
Malaria epidemiology 
• 
Biostatistics 
• 
Conduct of clinical trials 
• 
Malaria drug resistance 
 
No data on futility or benefit of the intervention will be estimable during the course of the trial as 
the timing of outcome data collection precludes developing stopping rules based on outcome data 
collected during implementation. Safety concerns associated with the wide-scale use of AL + SLD-
PQ, although unexpected, will form the basis of development of a stopping rule. The stopping rules 

Lao PDR protocol 
 
 
31 
for this trial will be based on detection of a significantly higher rate of mortality, hospitalization for 
possible drug-related events, or any other SAE in the intervention villages versus rates recorded at 
health facilities for the control villages. Further, any investigated SAE that results in death that is 
found to be due to the administration of one of the study drugs will be grounds for stoppage. 
 
The Local Safety Monitor (physician) will review all SAEs, hospital admissions and deaths in the study 
areas, give an opinion on causality, and give regular feedback to the DSMB. 
4.6. Collection of specimens 
RDT kits for malaria using finger prick blood samples will be collected during the research and 
intervention. These samples and their byproducts will be collected for disposal according to local 
disposal standards for biohazard and sharps waste at the nearest health facility (generally 
incineration). DBS will also be collected for PCR analysis of malaria parasite infection. These blood 
spots will be disposed of after the research and not stored for future use. The research period will 
be defined by the approval dates from the local research ethics committee, the UCSF, and ministry 
of health, and blood samples will not be stored for any longer than three years past the date the 
research study end. The informed consent documents will specify the uses and storage plans for 
dried blood specimens. Patient identifying information will be replaced with an unrelated unique 
identifier on each filter paper. All appropriate universal and site-specific safety precautions will be 
used in handling sharps, RDTs, and filter paper blood spots. All survey workers will be trained in the 
proper storage and handling of blood samples prior to fieldwork. 
4.6.1. Racial and ethnic origin 
A diverse range of ethnic groups will be included in the study, including several marginalized ethnic 
minorities. Our study will be conducted outside of the U.S., and no racial/ethnic group will be 
excluded. We do not expect to find race/ethnicity differences in the intervention effect, but refusal 
rates may differ. 
4.6.2. Inclusion of vulnerable subjects – children and pregnant women 
All age groups will be included in this study, except for those < 18 months. All children that 
participate must have the consent of the parent or guardian. Children older than 6 years and less 
than 18 must also provide oral assent before participation. All women who are pregnant or believe 
they may be pregnant will be assessed appropriately before treatment. 
4.7. Participant consent/assent 
Written informed consent will be obtained from the household members, neighbors, and co-workers 
eligible for the study. For the field procedures (survey, blood testing, treatment and follow-up visits), 
people will be asked to consent each time they are eligible to be part of the study. Consent will not be 
required for collection of de-identified, used RDTs from health facilities inside and outside of the study 
areas, if this collection is deemed feasible.  
Consent will be conducted in the participant’s household or in a private area prior to study activities. 
Consents may be collected from all members of the household and/or co-workers once. Parents will 
be able to sign one form to consent for themselves and all of their participating children (under 18 
years old) at once. Each additional adult member of the house will sign separate consent forms. Each 
minor (12-<18 years old) will sign a separate assent form.  
Informed consent will be conducted in local languages. If the participant is unable to read or write, an 
X will substitute for a signature.  
As part of the informed consent process, study personnel will assess participants’ understanding of 
the study procedures that were explained by using a checklist comprised of key components of the 
study. Participants who pass will be allowed to sign the written consent form. If the participant does 
not pass, the consent discussion will be repeated, before asking for a signature. In this case, the 
consent form will be read again, focusing on areas where understanding was limited, and encouraging 

Lao PDR protocol 
 
 
32 
the subject to ask questions. Up to five attempts will be permitted per participant. If, despite five 
consecutive attempts (each incorrect one followed by the team’s correction with explanation), the 
participant still has not answer a minimum number of questions correctly, then he/she will not be 
allowed to take part in the study.  
5 
Potential risks, limitations, data quality assurance, and dissemination plan 
5.1. Potential risks from participation 
Detailed discussion of potential AEs related to drug regimens is discussed above in section 4.2. Finger 
pricks for RDTs and DBS are associated with small risks of bleeding, hematoma, and infection. To 
minimize these risks, the skin will be cleaned with alcohol prior to puncture, and sterile unused lancets 
will always be used, and pressure will be placed on the puncture site after removal of the lancet using 
sterile gauze. Although the quantity of blood drawn would not lead to any ill effects on the 
participants’ health, some adults and rarely children feel faint from the blood during the finger prick. 
The risks will be minimized by having trained health staff perform all procedures, and all untoward 
effects will be evaluated by health center staff. 
 
For those subjects with P. vivax diagnosis that are tested for G6PD deficiency, there is a risk of 
misclassification and wrongly ascribed to being G6PD non-deficient and then being treated with 14 
days of PQ. In this study, the team will ensure a high-quality of training for all those using the G6PD 
RDTs, in addition subjects will be given information on what to do if they experience adverse events, 
and on days 3 and 14 all subjects given PQ will be actively followed up. If hemolysis is suspected PQ 
treatment will be stopped and the subject monitored to at least day 14. Should the hemolysis event 
require further clinical management, the study team will ensure access to appropriate health services. 
5.2. Limitations 
Several limitations have the potential to compromise study outcomes. 
 
Malaria declines 
There may be large-scale changes in malaria incidence and prevalence throughout the study area 
over the course of the 18-month implementation period, which could compromise study power.  
 
Malaria increases 
Any large increases in malaria burden or other diseases (e.g., dengue) in target districts could 
increase overall caseloads at health centers, potentially impacting availability of VMWs or other staff 
to support test and treat campaigns, especially in areas with ethnic minorities where their expertise 
is crucial. 
 
Safety of PQ 
Accidental administration of PQ to G6PD deficient individuals could result in an SAE and study 
stoppage. 
 
Adherence to treatment 
Poor study outcomes could result from poor adherence to the 14-day PQ course. 
 
Widespread HRP2/3 deletions 
There have been no surveys for P. falciparum HRP2/3 deletions in Lao PDR, and only limited data 
from other settings in the GMS: in China and Myanmar deletions were detected in 4/87 samples 
(unpublished data WHO MPAC, 2017). If these deletions are common in the study sites, the impact 
of HRP2-based test and treat could be severely compromised. P. falciparum positive DBS from the 
baseline cross-sectional survey will be rapidly analyzed to assess the level of HRP2/3 deletions in 
Southern Lao PDR. 

Lao PDR protocol 
 
 
33 
 
Changes in forest-going activity patterns 
The Lao government decree (May 2016) is believed to have had a major impact on the total number 
of illegal and semi-legal forest-goers. However, the future status is unknown; if there are changes to 
the decree itself or to its enforcement, the VMWs could potentially be overwhelmed with interviews 
or sampling sites to target. 
 
Mobility of target populations 
The fluxional nature of HRPs targeted in this intervention and the mobility inherent in forest-based 
economic activities has the potential to contaminate the HCCA-level randomization. While HRPs may 
freely transit between intervention and non-intervention arms, and thereby ‘carry-over’ community-
based interventions, the use of unique IDs will allow the impact of these movements to be assessed 
and adjusted for in exploratory analyses. 
 
Other partners’ interventions impact study outcomes 
The implementation of diverse programs by other partners across the study area has the potential 
to impact study outcomes if implementation is differential across the study arms. The nature of the 
randomization should minimize biases from partner activities, and a detailed matrix of other project 
activities will be created, and used to assess and adjust outcomes for exploratory analyses.  
5.3. Data quality assurance plan 
Data quality and management 
Data collection will occur in multiple locations, and differences in data collection systems may exist 
at different locations, which could potentially bias results. However, study teams and regular health 
staff will receive comprehensive training, as well as ongoing evaluation, supervision, and 
supplementary capacity building as necessary to ensure data quality and completeness. 
 
Procedures to minimize biases 
A survey instrument based on the formative work (carried out in December 2016) will be developed 
in English with input from collaborators at CMPE. This will then be translated to Lao and back-
translated by a fluent bilingual health expert before field testing. Any ethnic minority language 
interviews will be conducted in the appropriate language, and translated into Lao. Formative work 
will assess the feasibility of using tablets for data entry versus paper questionnaires. A pilot study 
will test the utility of the survey instruments; these data will then be discarded if significant changes 
are made to the survey instrument. Study coordinators will be responsible for monitoring data 
quality to ensure that questionnaires are completed and entered correctly. 
 
Potential changes to this protocol based on the piloting of tools and methods  
The organization and supervision of the peer navigators may be changed based on initial feasibility 
studies during field-testing of survey instruments. 
5.4. Dissemination Plan 
The finalized protocol will be filed at http://www.clinicaltrials.gov/ prior to implementation, and will 
conform to CONSORT recommendations for cluster randomized trials18. 
 
The results of the baseline survey, as well ‘hotspots’ identified during community-based RACD 
activities will be shared on a real-time basis with national, provincial, district and village-level 
partners as well as other key stakeholders (i.e., WHO, PMI, CHAI, PSI) throughout implementation to 
ensure that the most up-to-date information about malaria is available in the target districts.  
 

Lao PDR protocol 
 
 
34 
6 
Timeline 
Activities  
2019 
2020 
2021 
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
Q1 
Study Preparation 
  
  
  
  
  
  
  
  
  
Finalize protocol  
  
  
  
  
  
  
  
  
  
• Consent forms, questionnaires, SOPs, training 
materials 
      
  
  
  
  
  
  
  
  
IRB submissions  
  
  
  
  
  
  
  
  
  
  
  
Partner engagement  
  
  
  
  
  
  
  
  
  
Develop subcontracts with local institutions 
  
  
  
  
  
  
  
  
  
Hire Staff 
  
  
  
  
  
  
  
  
  
Procurement of materials  
  
  
  
  
  
  
  
  
  
•  RDTs, HS-RDTs, G6PD RDTs, AL, PQ, laboratory 
supplies, field supplies 
  
  
  
  
  
  
  
  
  
Electronic data collection tool development 
  
  
  
  
  
  
  
  
  
Meeting with provincial and district staff 
  
      
  
  
  
  
  
  
  
Pilot & Training Staff  
  
  
  
  
  
  
  
  
  
Revisions electronic data collection tool 
  
  
  
  
  
  
  
  
  
Baseline qualitative data collection, mapping, and 
surveillance data collection 
  
  
  
  
  
  
  
  
  
Data collection (Intervention) 
  
  
  
  
  
  
  
  
  
Midline review and competency checks 
  
  
  
  
      
  
  
  
  
Data collection (Endline survey) 
  
  
  
  
  
  
  
  
  
Laboratory analysis 
  
  
  
  
  
  
  
  
  
Data analysis 
  
  
  
  
  
  
  
  
  
Prepare final manuscripts and reports 
  
  
  
  
  
  
  
  
  
 

Lao PDR protocol 
 
 
35 
7 
References 
1. Bousema, T. et al. Hitting hotspots: Spatial targeting of malaria for control and 
elimination. PLoS Medicine 9, 1–7 (2012). 
2. Cotter, C. et al. The changing epidemiology of malaria elimination : new strategies for new 
challenges. The Lancet 382, 900–911 (2013). 
3. Moonen, B. et al. Operational strategies to achieve and maintain malaria elimination. The 
Lancet 376, 1592–1603 (2010). 
4. Hustedt, J. et al. Reactive case-detection of malaria in Pailin Province, Western Cambodia: 
Lessons from a year-long evaluation in a pre-elimination setting. Malaria Journal 15, 1–
10 (2016). 
5. Rossi, G. et al. Adapting Reactive Case Detection Strategies for falciparum Malaria in a 
Low-Transmission Area in Cambodia. Clinical Infectious Diseases 66, 296–298 (2018). 
6. Das, S. et al. Performance of an ultra - sensitive Plasmodium falciparum HRP2 - based 
rapid diagnostic test with recombinant HRP2 , culture parasites , and archived whole 
blood samples. Malaria Journal 17, 1–7 (2018). 
7. Landier, J. et al. Effect of generalised access to early diagnosis and treatment and targeted 
mass drug administration on Plasmodium falciparum malaria in Eastern Myanmar: an 
observational study of a regional elimination programme. The Lancet 391, 1916–1926 
(2018). 
8. Howes, R. E. et al. Spatial distribution of G6PD deficiency variants across malaria-endemic 
regions. Malaria Journal 12, 1–15 (2013). 
9. White, M. T. et al. Modelling the contribution of the hypnozoite reservoir to Plasmodium 
vivax transmission. eLife 3, 1–19 (2014). 
10. Douglas, N. M. et al. Unsupervised primaquine for the treatment of Plasmodium vivax 
malaria relapses in southern Papua: A hospital-based cohort study. PLoS Medicine 14, 1–
19 (2017). 
11. Ministry of Health Lao PDR. The Evaluation of G6PD Rapid Tests and the Use of Primaquine 
for the Treatment of Plasmodium Vivax Infections in Luangprabang, Savannakhet and 
Champasak Provinces (Apr-Nov 2015). (2015). 
12. Center for Malariology Parasitology and Entomology Lao PDR. Lao PDR Malaria National 
Strategic Plan 2016-2020. (2015). 
13. WHO. Updated WHO policy recommendation: Single dose primaquine as a 
gametocytocide in Plasmodium falciparum malaria. World Health Organisation (2012). 
14. Baum, E. et al. Submicroscopic and asymptomatic Plasmodium falciparum and 
Plasmodium vivax infections are common in western Thailand - Molecular and serological 
evidence. Malaria Journal 14, 1–11 (2015). 
15. Corran, P., Coleman, P., Riley, E. & Drakeley, C. Serology: a robust indicator of malaria 
transmission intensity? Trends in Parasitology 23, 575–582 (2007). 
16. Chan, C. W. et al. Plasmodium vivax and Plasmodium falciparum at the crossroads of 
exchange among Islands in Vanuatu: Implications for malaria elimination strategies. PLoS 
ONE 10, 1–13 (2015). 
17. Beutler, E. & Duparc, S. Glucose-6-phosphate dehydrogenase deficiency and antimalarial 
drug development. The American journal of tropical medicine and hygiene 77, 779–789 
(2007). 
18. Campbell, M. K., Piaggio, G., Elbourne, D. R. & Altman, D. G. Consort 2010 statement: 
Extension to cluster randomised trials. BMJ (Online) 345, 1–21 (2012). 

Lao PDR protocol 
 
 
36 
8 Appendices 
 
8.1 Schematic of study design 
8.2 Household survey informed consent 
8.3 Household survey questionnaire 
8.4 Blood collection informed consent 
8.5 Blood collection results  
8.6 RACD household informed consent 
8.7 RACD HH and Forest Questionnaire 
8.8 RACD household blood collection informed consent 
8.9 RACD forest informed consent 
8.10 VMW forest registry form 
8.11 VMW G6PD referral form 
8.12 HF G6PD register form 
8.13 HF Case investigation form 
8.14 Adverse event reporting form 
8.15 Severe adverse event investigation form 
8.16 FGD/KII informed consent form 
8.17 VMW FGD guide 
8.18 VMW interview guide 
8.19 VMW competency checklist 
8.20 HF FGD guide 
8.21 PAMS and DAMS KII guide 
8.22 District and supervisor KII guide 
8.23 HRP interview guide 
8.24 Treatment adherence follow-up form 
8.25 PROMPT Primaquine treatment data collection form 
8.26 Entomological data collection form 
8.27 Human Behavior Observations form 
8.28 Informed Consent Form for Persons Conducting Human Landing Catches 
8.29 Informed Consent Form for Heads of Household 
8.30 RACD Household Survey Oral Assent