Preliminary Evaluation of Dynamics of Subclinical Malaria
Conditions: Malaria
Sponsor: Duke University

Dynamics of subclinical malaria: preliminary study 
 
Version 1.0 
 
12 February 2018 
 ___________________________________________________________________________________________  
1 
 
 
Preliminary Evaluation of Dynamics of Subclinical Malaria 
 
DMID Protocol Number: 
17-0035 
 
Sponsored by: 
National Institute of Allergy and Infectious Diseases (NIAID) 
 
DMID Funding Mechanism: 
NIAID U19AI129386 International Center of Excellence for Malaria Research (ICEMR) 
cooperative agreement “Myanmar Regional Center of Excellence for Malaria Research” 
 
Principal Investigator: 
Myaing M. Nyunt 
 
Coordinating Investigator: 
Christopher V. Plowe 
 
DMID ICEMR Program Officer: 
Malla Rao 
 
Draft or Version Number:  
1.0 
 
Day Month Year 
12 February 2018 
 
 
 

Dynamics of subclinical malaria: preliminary study 
 
Version 1.0 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
1 
Statement of Compliance 
 
The study described in this protocol will be conducted in compliance with the protocol, the 
International Conference on Harmonization Good Clinical Practice E6, the applicable regulatory 
requirements (US 21 CFR Part 50-Protection of Human Subjects and Part 56-Institutional Review 
Boards, US 45 CFR 46), and the applicable rules and regulations of Myanmar, China, and 
Bangladesh. 
The IRBs of the Department of Medical Research (FWA00018816), Defence Services Medical 
Research Centre (FWA00023030), Duke University Health System (FWA00009025), National 
Institute of Parasitic Diseases China CDC (FWA00022996), and icddr,b (FWA00001468) will 
review and approve the protocol prior to study start. Documentation of the approval by these 
bodies will be kept in the PI’s study file and Sponsor’s regulatory files. 
 
 
 

Dynamics of subclinical malaria: preliminary study 
 
Version 1.0 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
2 
SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
Site Investigator: 
Signed: 
Date: 
19 Feb 2018 
 
Myaing Myaing Nyunt MD PhD MPH 
Assistant Professor of Medicine 
Director, Duke Global Health Institute Myanmar 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Dynamics of subclinical malaria: preliminary study 
 
Version 1.0 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
3 
 

Dynamics of subclinical malaria: preliminary study 
 
Version 1.0 
 
12 February 2018  
 ___________________________________________________________________________________________  
 
Table of Contents continued 
 
page 
 ___________________________________________________________________________________________  
4 
Statement of Compliance .............................................................................................................i 
Signature Page ........................................................................................................................... ii 
List of Abbreviations ................................................................................................................... iv 
Protocol Summary .................................................................................................................. …v 
 
1. 
Key Roles ......................................................................................................................... 2 
2. 
Background Information and Scientific Rationale .............................................................. 4 
2.1. 
Background Information .......................................................................................... 4 
2.1.1. Malaria, antimalarial drug resistance, and malaria eradication ........................................ 4 
2.1.2. Myanmar is the frontier of artemisinin resistance and a crucial battleground for malaria 
elimination 4 
2.2. 
Scientific Rationale ................................................................................................. 5 
2.2.1. Risks of Asymptomatic or Subclinical Malaria ................................................................. 5 
2.2.2. Dynamics of Asymptomatic or Subclinical Malaria Identified by Ultrasensitive PCR ....... 7 
2.2.3. Parasite population structure and migration .................................................................... 7 
2.2.4. Serosurveillance: tracking the parasite’s footprints ......................................................... 7 
2.3. 
Hypotheses ............................................................................................................. 8 
2.4. 
Choice of study design ............................................................................................ 8 
2.5. 
Potential Risks and Benefits ................................................................................. 10 
2.5.1. Potential Risks 
10 
2.5.2. Known Potential Benefits .............................................................................................. 10 
3. 
OBJECTIVES AND OUTCOMES ................................................................................... 11 
4. 
Study Design .................................................................................................................. 12 
4.1. 
Overview ............................................................................................................... 12 
4.2. 
Description of the Study Design ............................................................................ 12 
5. 
STUDY POPULATION ................................................................................................... 14 
5.1. 
Selection of the Study Population ......................................................................... 14 
5.2. 
Sampling Strategy ................................................................................................ 14 
5.3. 
Inclusion/Exclusion Criteria ................................................................................... 15 
5.3.1. Inclusion criteria for pilot study ..................................................................................... 15 
5.3.2. Exclusion criteria 15 
6. 
STUDY PROCEDURES/EVALUATIONS ........................................................................ 16 
6.1. 
Study Procedures ................................................................................................. 16 
6.2. 
Laboratory Evaluations ......................................................................................... 19 
6.2.1. Laboratory evaluations/Assays ..................................................................................... 19 
6.2.2. Specimen Collection, Preparation, Handling and Shipping ........................................... 20 
6.2.2.1. 
Instructions for Specimen Preparation, Handling, and Storage .......................................... 20 
6.2.2.2. 
Specimen Shipment ............................................................................................................. 20 
7. 
Statistical Considerations ............................................................................................... 21 
7.1. 
Study Outcome Measures .................................................................................... 21 
7.2. 
Sample Size Considerations ................................................................................. 22 
7.3. 
Participant Enrollment and Follow-Up ................................................................... 22 
7.4. 
Analysis Plan ........................................................................................................ 22 

Dynamics of subclinical malaria: preliminary study 
 
Version 1.0 
 
12 February 2018  
 ___________________________________________________________________________________________  
 
Table of Contents continued 
 
page 
 ___________________________________________________________________________________________  
5 
8. 
Subject Confidentiality .................................................................................................... 24 
8.1. 
Future Use of Stored Specimens .......................................................................... 24 
9. 
Informed Consent Process ............................................................................................. 25 
9.1. 
Informed Consent/Assent Process (in Case of a Minor or Others Unable to 
Consent for Themselves) ...................................................................................... 25 
10. 
Data management .......................................................................................................... 26 
11. 
Literature References ..................................................................................................... 29 
 
 
SUPPLEMENTS/APPENDICES 
 

Dynamics of subclinical malaria: preliminary study 
 
Version 1.0 
 
12 February 2018 
 
 
List of Abbreviations 
 
 ___________________________________________________________________________________________  
6 
ACT 
Artemisinin-based combination 
treatment 
 
ISM 
Independent Safety Monitor 
CDASH 
Clinical Data Acquisition Standards 
Harmonization  
 
LoD 
Limit of detection 
CDISC 
Clinical Data Interchange Standards 
Consortium 
 
MDA 
Mass drug administration 
CFR 
Code of Federal Regulations 
 
MOHS 
Myanmar Ministry of Health & 
Sports 
CIOMS 
Council for International 
Organizations of Medical Sciences 
 
MOP 
Manual of Procedures 
CRF 
Case Report Form 
 
N 
Number of subjects 
DBS 
Dried blood spots 
 
NIAID 
National Institute of Allergy and 
Infectious Diseases, NIH, DHHS 
DGHI 
Duke Global Health Institute 
 
NIH 
National Institutes of Health 
DMID 
Division of Microbiology and 
Infectious Diseases, NIAID, NIH 
 
NIPD 
National Institute of Parasitic 
Diseases, China Center of 
Disease Control (CDC) 
DMR 
Department of Medical Research, 
Myanmar MOHS 
 
NMCP 
National Malaria Control 
Programme 
DSMB 
Data and Safety Monitoring Board 
 
NNT 
Number needed to treat 
DSMRC 
Defence Services Medical 
Research Centre, Myanmar Ministry 
of Defence 
 
OCRA 
Office of Clinical Research Affairs, 
DMID, NIAID, NIH, DHHS 
ERC 
Ethics Review Committee 
 
OHRP 
Office for Human Research 
Protections 
FWA 
Federal-Wide Assurance 
 
ORA 
Office of Regulatory Affairs, 
DMID, NIAID, NIH, DHHS 
GCP 
Good Clinical Practice 
 
PI 
Principal Investigator 
GMS 
Greater Mekong Sub-Region 
 
RDT 
Rapid diagnostic test for malaria 
HIPAA 
Health Insurance Portability and 
Accountability Act 
 
RIP 
Research information portal 

Dynamics of subclinical malaria: preliminary study 
 
Version 1.0 
 
12 February 2018 
 
 
List of Abbreviations 
 
 ___________________________________________________________________________________________  
7 
icddr, b 
International Centre for Diarrhoeal 
Disease Research, Bangladesh 
 
sFTP 
Secure file transfer protocol 
ICF 
Informed Consent Form 
 
SMC 
Safety Monitoring Committee 
ICH 
International Conference on 
Harmonisation 
 
SOP 
Standard Operating Procedure 
IEC 
Independent or Institutional Ethics 
Committee 
 
usPCR 
Ultrasensitive polymerase chain 
reaction 
IRB 
Institutional Review Board 
 
WHO 
World Health Organization 
 
 
 
 
 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ________________________________________________________________________________________  
1 
PROTOCOL SUMMARY 
Title:  
Preliminary Study of Subclinical Malaria  
Population:  
 
Up to 6,000 residents of all ages in villages, refugee camps, military 
bases, and work sites, including children, pregnant women, and the 
elderly; the number of participants screened to achieve enrollment 
ceiling is not known 
Number of Sites:  
 
Up to 6 (each with at least 2 villages, towns, unions, refugee camps 
or plantations, or a single military base)  
Study Duration:  
 
Up to 1.5 years  
 
 
Subject Duration:  
 
Up to four months (military population may be under observation up 
to 4-6 weeks) 
 
 
Ethical Oversight: 
Ethics Review Committee (ERC), Department of Medical Research 
(DMR), Myanmar Ministry of Health and Sports (MoHS) 
Institutional Review Board (IRB), Defence Services Medical 
Research Centre (DSMRC), Myanmar Ministry of Defence 
IRB, International Centre for Diarrhoeal Disease Research (icddr,b), 
Bangladesh 
IRB, National Institute of Parasitic Diseases (NIPD), Chinese Center 
for Disease Control and Prevention (China CDC) 
IRB, Duke University Health System 
 
Study Objectives: 
Primary objective: 
Make a preliminary assessment of the dynamics of subclinical malaria detected by 
ultrasensitive PCR to guide the design of a matched cohort study of subclinical malaria in 
Myanmar and along its borders with China and Bangladesh 
(The objective of the subsequent matched cohort study, to be reviewed as a separate 
protocol, will be to compare the incidence of clinical malaria and malaria transmission 
capacity, in participants with and without subclinical malaria.) 
 
Secondary and exploratory objectives:   
1. Within usPCR+ and usPCR- cohorts, test for association between demographic and 
other risk factors (e.g. occupation, travel) and incidence of clinical malaria and 
transmission capacity 
2. Collect preliminary data to estimate the history of exposure to malaria parasite and 
mosquito vector species by serological profiling 
3. Collect preliminary data to develop a model system to predict future malaria risks using 
geospatial and satellite data on environmental conditions, patterns of parasite 
migration and human mobility, field data on vector abundance and malaria burden over 
time at study sites 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ________________________________________________________________________________________  
2 
1. 
KEY ROLES 
Individuals: DMID Representative: Malla Rao  
 
Investigators:  
Name 
Organization 
Role  
Nyunt, Myaing Myaing 
DGHI 
Principal Investigator 
Plowe, Christopher V. 
DGHI 
Coordinating Investigator 
 
 
 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ________________________________________________________________________________________  
3 
Institutions: 
 
Institutions
Contact information
Duke Global Health Institute, Duke University
310 Trent Drive, Durham NC 27710 
Telephone: 919-681-7760 
Geographical Sciences Department, University of 
Maryland College Park 
7251 Preinkert Dr., Room 2181 
College Park, MD 20742 
Telephone: 301-405-8891 
Department of Medical Research, Myanmar 
Ministry of Health & Sports 
5, Ziwaka Road, Dagon Township 
Yangon 11191, Myanmar 
Telephone: 95 1 375447 
Defence Services Medical Research Centre, 
Directorate of Medical Services, Myanmar Ministry 
of Defence
Nay Pyi Taw, Myanmar 
Telephone: 95 9 860 1604  
icddr,b
GPO Box 128, 
Dhaka 1000, Bangladesh 
Telephone: 88 2 9827001 10 
National Institute of Parasitic Diseases, 
Chinese Center for Disease Control and Prevention
207 Rui Jin Er Road, Shanghai 200025 
People's Republic of China 
Telephone: 86 21 6473805 
 
 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ________________________________________________________________________________________  
4 
2. 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE  
2.1. 
Background Information  
2.1.1. Malaria, antimalarial drug resistance, and malaria eradication 
 
Malaria is caused by a mosquito-borne, protozoan parasite belonging to the genus 
Plasmodium. Of the five species of Plasmodium that infect humans, P. falciparum and P. vivax 
are responsible for the majority of morbidity and death. The malaria burden is greatest in sub-
Saharan Africa, with a high mortality in children under five years of age. In Asia, malaria 
endemicity is lower, but it presents a major public health problem, affecting people of all ages 
due to a lack of the acquired immunity that can be developed through the continuous, heavy 
exposure more typical of Africa. P. falciparum is susceptible to genetic polymorphisms 
associated with antimalarial drug resistance, and Southeast Asia has been the epicenter of 
repeated emergences of drug resistant malaria [1]. Malaria can be cured completely if treated 
with effective drugs. Artemisinin-based combination therapies (ACTs) are the first line malaria 
treatment globally. The widespread implementation of highly efficacious ACTs has contributed 
to large reductions in the malaria burden in many parts of the world, inspiring a renewed hope 
of global malaria eradication [2].   
2.1.2. Myanmar is the frontier of artemisinin resistance and a crucial battleground for 
malaria elimination 
 
Southeast Asia has become a focus in the war against malaria after the emergence of 
artemisinin resistant falciparum malaria. Myanmar, the emerging democracy formerly known 
as Burma, has the heaviest malaria burden in the region, with more cases and deaths than 
the rest of the region combined [3]. Artemisinin resistance first emerged in Cambodia [4-6] 
and has been documented by us and others along the borders of Myanmar with Thailand [7, 
8] and China [9], in central Myanmar, and in southern Laos and southern Vietnam [10]. 
Although artemisinin resistance is manifested chiefly by delayed parasite clearance so long 
as the ACT partner drug is effective, with the new 
emergence of resistance to ACT partner drugs treatment 
failure now occurs in up to 46% of people treated with 
ACTs in western Cambodia [11], raising the specter of 
untreatable malaria, and threatening prospects for 
eradication [12]. The populations currently impacted by 
artemisinin resistance are those living in the six Greater 
Mekong Sub-Region (GMS) countries: Cambodia, 
Vietnam, Laos, Thailand, China’s Yunnan Province, and 
Myanmar (Figure 1). Importantly, Myanmar shares 
borders with Bangladesh and India, two countries with 
no evidence yet of confirmed artemisinin resistance, but 
both with large populations at risk of malaria and travel 
links to Africa.  
 
The initial response to artemisinin resistance was to try 
to contain it by eliminating falciparum malaria from 
western Cambodia and erecting a “firewall” to block its 
spread. The containment strategy, led by the World Health Organization (WHO), the Global 
Figure 1. Map of the Greater Mekong Subregion.

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
5 
Fund to Fight AIDS, Tuberculosis and Malaria, and regional governments, was predicated on 
the assumption—based on past patterns of spreading drug resistance—that a “wave” of 
resistance was spreading westward from Cambodia toward Myanmar [10]. The Myanmar 
Artemisinin Resistance Containment zone was launched along the Thailand-Myanmar border 
where diagnosis, treatment and prevention were scaled up [13] and pilot mass drug 
administration (MDA) schemes were implemented from across the border in Thailand into 
Myanmar’s Kayin [14], as well as in Mon and Tanintharyi States (unpublished).  
 
Using genomic epidemiology approaches, we recently demonstrated that while artemisinin 
resistant parasites have indeed spread between Cambodia and southern Vietnam, the 
resistant parasites initially present in Myanmar emerged de novo there [15]. This unexpected 
finding—which was immediately shared with Myanmar’s National Malaria Control Program 
(NMCP), other country partners, and the WHO—challenged the notion that erecting a firewall 
along Myanmar’s eastern border could stop the spread of resistance: if resistance can pop up 
independently, it will be impossible to contain. An urgent technical expert review [16] led the 
WHO to abandon the containment strategy and to instead launch a regional malaria 
elimination campaign [17, 18]. Myanmar endorsed the goal of malaria elimination by 2030 at 
the East Asia Summit in November 2015, and a costed national malaria elimination plan for 
Myanmar was finalized in 2016.  
 
2.2. Scientific Rationale  
2.2.1. Risks of Asymptomatic or Subclinical Malaria 
 
Myanmar’s NMCP currently stratifies malaria risk at the township level, based on ecological 
risk factors for malaria transmission, reported clinical cases, and presence of or proximity to 
artemisinin resistance. This stratification scheme, though practical, does not account for 
heterogeneous malaria risk or distinguish between locally acquired and imported malaria, 
resulting in inefficient targeting of interventions. Moreover, malaria elimination may require 
eradicating all parasites from all infected persons, not just those with clinical symptoms [19]. 
If clinical malaria represents the “tip of the iceberg”, understanding the rest of the iceberg—
whether it poses risk or even benefit to infected individuals, and how it contributes to 
transmission in different epidemiological settings—will be important for malaria control and 
elimination.  
 
“Asymptomatic”—or more accurately, subclinical malaria—has long been recognized in semi-
immune Africans [20]. New “ultrasensitive” molecular tests [21-23] have recently revealed 
unexpectedly high rates of subclinical malaria in Southeast Asia, where long held dogma had 
it that all infections quickly give rise to symptoms in presumably non-immune people. Some 
argue that “asymptomatic” infections are both harmful and infectious and must be treated [24]. 
The WHO recently endorsed MDA for malaria elimination in Southeast Asia [18], and 
increasingly large scale MDA schemes are being undertaken based on subclinical malaria 
prevalence as measured by ultrasensitive tests [14]. However, no previous studies have 
assessed the clinical risks or transmission potential of the extremely low-density malaria 
infections detected by these tests. 
 
Using an ultrasensitive reverse transcription polymerase chain reaction (usPCR) assay that 
we developed [22], we are finding that the prevalence of malaria at sites within and bordering 
Myanmar is highly heterogeneous. Villages with high prevalence—including some MDA 
sites—are often in close proximity to villages with little or no malaria. While our usPCR test 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
6 
reliably detects P. falciparum and P. vivax at parasite densities below 16 parasites/mL, these 
very low-density infections are difficult to further characterize using currently available 
molecular and in vitro methods, precisely because the 
parasite densities are so low—up to 10,000-fold lower than 
those detected by rapid diagnostic tests (RDTs) and ten to 
100-fold lower than conventional PCR (Figure 2). 
 
Clinical risk or benefit. A recent review concluded that 
“‘asymptomatic malaria’ is a chronic and debilitating condition 
that should be treated” [24]. However, most of the cited 
studies of “asymptomatic” or subclinical malaria infection 
used microscopy or rapid diagnostic tests for malaria (RDTs), 
which have lower limits of detection (LoD) of about 100 
parasites/µL (or 105/mL). More recently studies have used 
standard PCR, with an LoD of about 1000-5000 parasites/mL 
[25]. In contrast, a high-volume intravenous usPCR assay 
has an LoD of 22 parasites/mL [21] and the LoD of a finger-
stick assay that we developed and are using (Figure 2) is <16 
parasites/mL [22]. Although studies are conflicting as to 
whether subclinical malaria at parasite densities detected by 
conventional methods is protective or harmful [26, 27], no 
previous studies have assessed the clinical risks or 
transmission potential of ultralow density subclinical malaria 
infections.  
 
The ability to detect these ultralow parasite densities is 
unprecedented, and for the first time it will be possible to 
study how long these infections last and whether they 
increase the risk of clinical malaria illness in the infected 
individual. Alternatively, ultralow density malaria infection 
could represent a co-adaptation of host and parasite that optimize transmission while 
minimizing symptoms [28]. 
 
Transmission potential. It has been argued that malaria elimination requires eradicating all 
parasites from all infected persons, not just those causing symptoms [19]. Subclinical malaria 
has been known to be a transmission reservoir in Africa since the 19th century [20, 29], and 
40 years ago surveys in Bangladesh found up to 47.3% of people of all ages to be infected 
with falciparum and/or vivax malaria at the end of the transmission season. These studies 
used microscopy to detect much higher parasite densities than those detected along the 
Thailand-Myanmar border by usPCR to guide MDA [14, 23, 28]. Ongoing MDA to eliminate 
ultralow subclinical malaria is predicated on the assumption that extremely low density 
infections contribute meaningfully to transmission. It may seem reasonable to assume that 
any infection is potentially transmissible, but several studies (summarized in [24]) have shown 
that transmissibility falls with declining parasite density.  
 
There is no evidence at present that ultralow subclinical malaria infections contribute 
significantly to transmission. Skeptics about the value of MDA in Southeast Asia rightly point 
out that malaria has been eliminated from many regions without detecting or treating 
submicroscopic infections, much less ultralow density infections. Despite strong advocacy for 
MDA by prominent malaria thought leaders [30], scaling up of this controversial but potentially 
promising intervention is stalled in Southeast Asia as NMCPs await clear answers about the 
Figure 2. Representation of density of 
malaria infections detected by our finger-
stick usPCR (left), standard PCR (center) 
and RDTs (right). Black dots represent 
lower limit of number of parasites 
detected in an equal volume of blood. 
PCR 
RDT 
usPCR 
PCR

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
7 
clinical and transmission risks posed by subclinical—and especially ultralow density—malaria 
infection. This study is designed to provide these clear answers. 
 
Study impact. The results of the longitudinal matched cohort study that this pilot study will 
lead to will influence national and regional malaria control and elimination policies. Whether 
or not the results of the cohort study support scaling up MDA, the development of robust, 
highly sensitive surveillance tools will enhance the ability to map and track malaria, capabilities 
that become increasingly critical as countries approach elimination. Our results may support 
“targeted MDA” based on microstratification at the village level, a new concept just now gaining 
currency. 
2.2.2. Dynamics of Asymptomatic or Subclinical Malaria Identified by Ultrasensitive 
PCR 
 
It is well known that many asymptomatic infections may persist for from weeks to months [31-
33]. Persistent chronic cases of malaria infection in the absence of new exposure has been 
described  [34]. A recent paper extensively reviewed the nature and dynamics of 
asymptomatic malaria and its clinical and transmission impact in different transmission zones 
[35]. However, most of these studies were conducted in areas of high malaria transmission, 
and used standard methods such as microscopy or RDTs LoDs far higher than that of usPCR. 
Studies that used sensitive molecular methods in low transmission regions [36, 37] are limited 
to cross-sectional surveys, and do not assess the dynamics or chronicity of these low-density 
infections. The natural dynamics of low density malaria infections in humans is unknown; 
although it has been suggested that these may be chronic infections, their duration has not 
been described.  
2.2.3. Parasite population structure and migration  
 
There is an urgent push for malaria elimination in the GMS, driven by fear of the spread of 
ACT-resistant malaria. Information on how parasites are migrating, where they come from and 
where they end up (i.e. “sources” and “sinks”), could help to guide elimination interventions. 
Recent studies of P. falciparum whole genome sequences from the GMS identified highly 
structured P. falciparum populations clustering into multiple distinct genetic subpopulations 
[38, 39], some of which are associated with artemisinin resistance. Some P. vivax populations 
show similar patterns of population structure and diversity [40], while others show P. vivax to 
be more diverse and less structured than P. falciparum [41, 42]. Further studies employing 
genome sequencing and genome-wide genotyping linked to ecological, epidemiological and 
geospatial information about risk of exposure to malaria parasite and mosquito vector species 
have the potential to lead to new tools for predicting malaria risk that would aid in elimination 
efforts.  
2.2.4. Serosurveillance: tracking the parasite’s footprints 
 
Serological tools can be used to map malaria risk [43] and assess time-dependent malaria 
exposure [44]. Protein microarrays are used to measure antibody responses to large numbers 
of malaria proteins, including both polymorphic vaccine antigens [45] and variant surface 
antigens associated with immune evasion and pathogenesis [46, 47].  These microarrays may 
prove most useful as surveillance tools to detect recent or remote exposure to specific parasite 
species, life cycle forms, antigenic variants, and even mosquito vectors. Unlike commercially 
available protein microarrays  [44, 48], which measure seroreactivity to hundreds of proteins, 
each derived from the reference genome, our diversity-reflecting arrays contains dozens to 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
8 
hundreds of variants of important polymorphic antigens derived from sequencing many 
hundreds of field isolates. We hypothesize that breadth of recognition of antigen variants will 
provide a simple and robust way to estimate malaria exposure history in a population. 
Moreover, mosquito salivary antigens elicit serological responses in humans [49] that can 
correlate with exposure [50], and we hypothesize that we will be able to estimate exposure to 
different mosquito vectors by measuring antibodies against species-specific variants of 
mosquito proteins. We hope to use this information to target malaria interventions more 
efficiently based on which human populations are exposed to which vectors, e.g. indoor-biting 
versus forest-biting species. 
 
2.2.5. Mapping and forecasting malaria risks using geospatial and satellite data 
 
Understanding malaria risk as it varies over time and space is essential for effective targeting 
of interventions. Changing malaria pattern can be understood using two major transmission 
pathways, namely human and mosquito. Vector ecology likely dominates in determining 
individuals’ risk of exposure at the local scale, because environmental factors largely control 
mosquito population dynamics and distribution. However, human mobility plays a more 
significant role at a regional scale as human flow allows for “import” and “export” of local 
parasite subpopulations, accelerating the dissemination of drug resistance and other heritable 
traits. The data generated through this study will be used to develop spatially explicit and 
temporally dynamic approaches to track, monitor, and forecast malaria risk within Myanmar 
and along its borders with China and Bangladesh, using satellite and geospatial data to aid 
malaria control and elimination. Earth-observing satellite systems acquire data characterizing 
environmental conditions and ecosystem states that are routinely used in habitat and risk 
modeling applications in ecological studies and management applications. At a regional scale, 
studies of movement paths or trajectories of populations are starting to improve our 
understanding of the role of human mobility in spreading infections [51, 52]. We will develop 
highly dynamic depictions of environmental factors in the study areas, including 
meteorological metrics, landscape matrix, and land use drivers derived from satellite imagery 
to model local scale parasite abundance and transmission potential.  
2.3. Hypotheses 
 
This preliminary study is largely descriptive. The subsequent matched cohort study will test 
the hypothesis that individuals harboring persistent chronic subclinical P. falciparum and/or P. 
vivax parasitemias are protected from clinical malaria, but represent a source of malaria 
transmission.  
2.4. Choice of study design 
 
This is a short longitudinal preliminary study that aims to describe the dynamics of low-density 
subclinical malaria to support the final study design of a subsequent matched cohort study. To 
provide the rationale for the current protocol, both studies are described below; a separate 
protocol for the matched cohort study will be finalized based in part on results of this 
preliminary study. 
 
2.4.1. Preliminary study 
 
In this study, a small number of asymptomatic infections detected by ultrasensitive PCR 
(usPCR) will be followed and tested intensively for three months to measure the temporal 
dynamics of these infections. A much larger number of uninfected individuals will be followed 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
9 
for just 2-4 weeks (two visits), providing a comparator group for the infected cohort. The validity 
of our results in a subsequent matched cohort study depends, in part, on our ability to 
accurately classify infection status using a single usPCR result. In other words, do we have 
sufficient confidence in the correct classification of malaria infection positive and negative 
status using usPCR test at the beginning of the study? While there is no gold standard test 
more sensitive than usPCR, repeated usPCR testing offers a surrogate. Presumably, in a low 
transmission setting, someone who is truly uninfected on the first testing should remain 
negative on multiple consecutive tests, but this remains to be confirmed. 
  
If this initial evaluation confirms that usPCR results are relatively stable - i.e., that most 
usPCR+ infections remain positive on most or all repeated testing over the course of three 
months, and that most usPCR- infections remain negative upon re-testing 2-4 weeks later, 
then we can move forward with the cohort study with confidence that our initial classification 
of exposure is a meaningful basis for assessing subsequent outcomes. If, however, a single 
usPCR result is found to be “unstable”, we may need to consider testing at least twice before 
assigning infection status. Similarly, we may wish to increase the sample size if we determine 
that the sensitivity and specificity of a single usPCR test (compared to results of multiple 
testing) is high enough to proceed with single testing at screening and enrollment, but lower 
than ideal (e.g. sensitivity/specificity 50-75%). 
 
2.4.2. Design of the subsequent matched cohort study (to be submitted for ethical 
review later as a separate protocol) 
 
To provide the rationale for this preliminary study, the design of the subsequent matched 
cohort study is described briefly here. The principle design will be a matched cohort study. 
This choice of study design is based on the knowledge of generally low prevalence of malaria 
in most of the proposed study sites. Although a standard cohort study would be appropriate in 
a high transmission setting, it would be difficult, if not impossible, to reach adequate sample 
size in our study sites with such low prevalence. A retrospective case control design is also 
unsuitable, since it will not be possible to measure the exposure of interest (prior subclinical 
malaria) at the time that the primary outcome (RDT+ infection) is detected. Therefore, a 
matched case-cohort design similar those we have previously done in Mali [53] offers a good 
alternative to study a rare exposure.  
 
Matching is not typically used in cohort studies because it precludes testing for association 
between matched variables and outcomes of interest. However, in this situation, independent 
association between clinical protection and transmission risk needs to be understood. 
Because subclinical infection is itself likely to signify higher malaria exposure risk, comparing 
rates of RDT+ infections and transmission capability in unmatched infected and uninfected 
cohorts would likely show that clinical malaria and transmission capacity are more common in 
the infected cohort simply because their initial infection signals greater malaria exposure. 
Therefore, for the subsequent cohort study we will match infected participants (cases) to 
uninfected controls, to control for covariates known and suspected to be associated with 
malaria risk. With this design, we will still be able to test for association with matched variables 
and outcomes of interest within (but not between) infected and control cohorts. 
 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
10 
2.5. Potential Risks and Benefits 
2.5.1. Potential Risks 
 
Effects of finger sticks and venous blood draws. Risks of bruising, bleeding, infection and 
fainting can accompany these procedures. Study-related blood collection for malaria 
diagnostic testing by rapid diagnostic test and for a dried blood spot sample can be 
accomplished by using finger stick or earlobe stick. Venous blood will only be obtained 
routinely from participants with RDT+ infections detected either in screening, enrollment, 
follow-up, or in active or passive surveillance.  
 
DNA sequence information. Samples will be used for genotyping and/or sequencing malaria 
parasite RNA and/or DNA (nucleic acids). Left-over samples will be archived, with consent 
from study subjects, for future investigations—potentially including analysis of human nucleic 
acids or nucleic acids from other infectious organisms—with further appropriate approval. For 
example, future studies may investigate the presence of other causes of fever in participants 
with fever but negative malaria tests, or genomic studies of human migration to complement 
those of parasite migration.  
 
Potential risks to study personnel. The main risks to study personnel are from accidental 
exposure to blood and body fluid borne infections. SOPs for staff safety are used in clinical 
and laboratory areas, including sharps management, hazardous waste management, etc. 
Universal precautions are used for handling all body fluids. 
2.5.2. Known Potential Benefits 
 
Participants may not receive any direct benefit from their participation in the study. However, 
if symptoms of malaria or other illnesses are noted, participants will be referred for prompt 
evaluation and treatment by the appropriate local health provider. By providing evidence-
based strategies for malaria elimination, the information gained from the proposed work is 
beneficial for the study participants in the future, others living with malaria or in malaria-
endemic regions, as well as public health professionals and policymakers in Myanmar and in 
the GMS, who are leading regional malaria elimination efforts. 
  
 
 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
11 
3. 
OBJECTIVES AND OUTCOMES  
Primary objective: 
Make a preliminary assessment of the dynamics of subclinical malaria detected by 
ultrasensitive PCR to guide the design of a matched cohort study of subclinical malaria in 
Myanmar and along its borders with China and Bangladesh 
(The objective of the subsequent matched cohort study, to be reviewed as a separate 
protocol, will be to compare the incidence of clinical malaria and malaria transmission 
capacity, in participants with and without subclinical malaria.) 
Secondary and exploratory objectives:   
1. Within usPCR+ and usPCR- cohorts, test for association between demographic and 
other risk factors (e.g. occupation, travel) and incidence of clinical malaria and 
transmission capacity 
2. Collect preliminary data to estimate the history of exposure to malaria parasite and 
mosquito vector species by serological profiling 
3. Collect preliminary data to develop a model system to predict future malaria risks using 
geospatial and satellite data on environmental conditions, patterns of parasite 
migration and human mobility, field data on vector abundance and malaria burden over 
time at study sites 
Primary outcomes: 
1. Proportion of individuals with initially positive usPCR tests that were followed by 1 or 
more consecutive positive tests on weekly or biweekly testing 
2. Proportion of individuals with initially negative usPCR tests that subsequently had at 
least one usPCR positive test at a second screening 2-4 weeks later. 
Primary endpoints:  
Not applicable 
Secondary and exploratory outcomes: 
Infection outcomes and endpoints 
1. Incidence of falciparum malaria infection by RDT 
2. Incidence of vivax malaria infection by RDT 
3. Incidence of mixed falciparum/vivax malaria infection by RDT 
4. Incidence of falciparum malaria infection by usPCR 
5. Incidence of vivax malaria infection by usPCR 
6. Incidence of mixed falciparum/vivax malaria infection by usPCR 
Serology endpoint 
Seroreactivity to polymorphic malaria/mosquito proteins 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
12 
4. 
STUDY DESIGN 
4.1. Overview  
 
Prospective, multi-site short longitudinal study  
 
Up to six study sites, each with >2 villages, towns, unions, refugee camps or 
plantations, or a single military base. Alternative and additional sites may be added 
to ensure enough infected cases. 
 
Screening for eligibility (age at least 0.5 year; able & willing to strictly follow study 
protocol and to provide written informed consent or assent as appropriate)  
 
Enroll and consent eligible individuals   
 
RDT-positive: one-time enrollment for data and venous blood collection; Refer to 
and ensure appropriate treatment by care providing team; No study-related follow 
up 
 
RDT-negative: Collect data and DBS samples; Return to research clinic in 2-4 
weeks 
 
PCR-negative participants: One follow up visit approximately 2-4 weeks after 
enrollment  
 
PCR-positive participants: Five follow up visits at approximately 4, 6, 8, 10, and 12 
weeks (for those with total 12 weeks follow up) or 2, 3, 4, 5 and 6 weeks (for those 
with total 6 weeks follow up) after enrollment 
 
Total up to 6000 residents in up to six study site of all ages in villages, refugee 
camps, military bases, and work sites are to be enrolled  
 
Finger stick blood sampling with be done for RDT and dried blood spot sampling 
in all participants at each scheduled and unscheduled visit.  
 
Venous sampling will be done for participants with RDT+ infections detected at 
times when study staff trained for venous sampling are present. Blood volumes at 
any time point are limited to 2 mL for children aged < 3 years, 3 mL for age 3-5 
years, and 5 mL for older children and adults. 
 
Estimated duration of study:18 months 
4.2. Description of the Study Design 
This is a multi-site short longitudinal study to evaluate the dynamics of subclinical P. 
falciparum and/or P. vivax malaria. The study areas are chosen to represent a range of 
malaria epidemiologies characteristic of the region, and include Townships in 
Myanmar’s Mandalay and Sagaing Regions and Chin and Rakhine States; Unions in 
the Chittagong Hill Tracts of Bangladesh; and Counties in China’s Yunnan Province 
that border study sites in Shan and Kachin States (Table 1, Figure 3).  

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
13 
A maximum of up to 6,000 participants will be enrolled in the longitudinal study in up to 
six participating sites. Survey participants will provide consent (and assent, if 
appropriate) prior to having blood collected for hemoglobin measurement and for dried 
blood spots used for molecular analyses. A subset of RDT+ participants will also be 
asked to provide venous blood.  
 
 
  
Table 1. Initial study sites 
Township 
County 
or Union* 
Estimated 
population  
P. falciparum 
 
P. vivax 
Location
State 
Region 
Province 
or District 
Prevalence 
by RDT 
% (range) 
Prevalence 
by USqsPCR 
% (range) 
API 
Cases/1000 
(range) 
Prevalence 
by RDT 
% (range) 
Prevalence 
by USqsPCR 
% (range) 
API 
Cases/1000 
(range) 
Central Myanmar  
Mandalay  
Madaya (Singu) 
       22,000  
0.0
(0.0-0.0) 
0.5
(0.0-2.0) 
1.03†
(NA) 
0.0
(0.0-0.0) 
3.5
(0.0-6.0) 
1.03†
(NA) 
Western Myanmar 
Rakhine  
Ann 
(Buthidaung) 
2731  
2.5 
(0.0-8.0) 
11.1 
(4.0-22.0) 
2.09† 
(0.0-4.59) 
0.0 
(0.0-0.0) 
9.0 
(0.0-20.0) 
2.09† 
(0.0-4.59) 
Myanmar-India 
border 
Sagaing 
Monywa/Tamu 
(Paletwa) 
1,200 
troops  
-- 
-- 
1.05† 
(0.0-8.38) 
-- 
-- 
1.05† 
(0.0-8.38) 
China-Myanmar 
Border 
Yunnan  
Waingmaw 
20,000 
refugees  
1.0 
(1.0-1.0) 
0.0 
(0.0-0.0) 
2.99† 
(0.0-26.15) 
0.0 
(0.0-0.0) 
6.0 
(6.0-6.0) 
2.99† 
(0.0-26.15) 
Shan  
Mongmao 
    15,000  
-- 
-- 
0.39 
(NA) 
-- 
-- 
2.02 
(NA) 
Yunnan  
Nabang  
         1,629  
-- 
-- 
0.019 
(NA) 
-- 
-- 
0.22 
(NA) 
Kachin  
Cangyuan 
9,000  
-- 
-- 
0.054 
(NA) 
-- 
-- 
0.22 
(NA) 
Southeastern 
Bangladesh 
Bandarban 
Rajbila 
         8,619  
--
--
3.48
(0.43-14.57) 
--
--
0.67
(0.0-2.73) 
Kuhalong 
       10,361  
--
--
2.03
(1.13-7.16) 
--
--
0.19
(0.16-2.39) 
*Myanmar Townships, Chinese Counties and Bangladeshi Unions are roughly similar administrative units that contain many villages; Chinese counties are much larger in size and population.
Alternative sites that may be substituted in the event that too few cases are found at initial sites are shown in parentheses. 
†All malaria. API data for some townships is reported only for all malaria (any positive RDT). Based on prevalence data, API for Kachin State site is likely to be mostly P. vivax.  
RDT, rapid diagnostic test for malaria; usPCR, ultrasensitive polymerase chain reaction; API, annual parasite index (cases per 1000 population at risk/year); NA, not available—range across 
villages not available for areas only reported by township. 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
14 
5. 
STUDY POPULATION 
5.1. Selection of the 
Study Population 
 
Sites broadly representative of the range of 
malaria epidemiologies seen in the region 
(Table 1, Figure 3) have been identified 
based on available data on the incidence of 
clinical 
malaria 
and 
prevalence 
of 
subclinical malaria (measured by both 
standard testing and by usPCR in surveys 
we have supported). In a staged fashion, 
initial screening will be undertaken at these 
sites, starting with the sites in Myanmar, 
where laboratories proficient in usPCR 
have been established.  
 
To account for the possibility that malaria 
rates have declined since the latest 
available data were collected, alternative 
and additional sites may be identified in 
consultation with our research partners and 
local NMCP officials. The addition or 
substitution of additional sites will be 
reported to the study sponsor and IRBs. 
 
Children will be included because they may 
be at risk for malaria, as well as to measure 
age-specific infection pattern which is 
expected to vary across sites. Adults of all 
ages—including pregnant women and the 
elderly—will be included to address the 
paucity of data on transmission potential in these groups and to detect chronic infections 
persisting even in the absence of recent exposure. 
 
Because technical capacity for molecular diagnosis required for this study is not yet 
established at the Bangladesh and China sites, the study will begin at Myanmar sites following 
review and approval by the Myanmar and U.S.-based IRBs. As laboratory facilities are 
established in Bangladesh and China, the protocol will be reviewed by IRBs in those countries. 
Study activities in each country will only be started after review and approval by the IRB(s) in 
that country. 
5.2. Sampling Strategy 
The primary aim of the screening process is not to obtain an unbiased measurement of malaria 
prevalence, which is already known to be low in many sites. Rather, it is to identify and enroll 
in the study the target number of cases of subclinical malaria infection, which are expected to 
be rare. People and locations suspected or known to harbor malaria will be targeted for 
screening, based on: RDT and usPCR cross-sectional prevalence data from our ongoing 
Figure 3. Study Sites and partners. Department of Medical Research: 
(1) Ann Township, Rakhine State and (2) Madaya Township, 
Mandalay Region; Defence Services Medical Research Centre: (3) 
Monywa Township, Sagaing Region; National Institute of Parasitic 
Diseases, 
Chinese 
CDC: 
(4) 
Waingmaw 
Township, 
Kachin 
State/Nabang, Yingjiang County, China and (5) Mongmao Township, 
Shan State/Canguan County, Yunnan Provice, China; icddr,b: Rajbila 
and Kuhalong Unions, Bandarban District , Bangladesh.  

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
15 
separately-funded malaria surveillance activities; RDT-based malaria incidence data from 
NMCPs and our own previous and ongoing research and surveillance activities; village clinic 
treatment logs; interviews with local health providers and community leaders; and results of 
initial screening.  
As the screening process progresses, household and workplace contacts of RDT+ and 
usPCR+ cases will be traced to find more infected individuals until enrollment targets are 
reached. This targeted screening means that the overall sampling framework will be neither 
random nor fully representative of the larger communities. However, whenever possible, 
comprehensive or representative sampling will be done to provide unbiased prevalence 
estimates of P. falciparum and P. vivax infection for specific locations or populations within the 
larger study areas (e.g. sampling all individuals in a village).  
While sampling strategies will necessarily be locale-specific (e.g. clinic-based, school-based, 
military-based or household-based), at each location the basis for sampling will be carefully 
documented so that we can ascertain when unbiased estimates of prevalence can be made 
for specific locations or populations. Each site has a central location for screening, enrollment 
and follow-up and satellite locations, e.g. clinics or schools, where study activities will occur. 
5.3. Inclusion/Exclusion Criteria 
 
5.3.1. Inclusion criteria for pilot study  
 
Participants must meet all the inclusion criteria to participate in the study: 1) Age 6 
months or older at the time of screening; 2) Written informed consent obtained (from 
the parent/guardian if the subject is less than 18 years old).  
 
5.3.2. Exclusion criteria 
 
Any condition which in the view of the investigator makes participation not in the best 
interests of the prospective participant. Any condition that would interfere with study 
participation or pose risks to participants. Those with clinical malaria infection as 
diagnosed by positive RDT may be invited to return and be re-screened for study 
eligibility after treatment and resolution of their illness. 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
16 
6. 
STUDY PROCEDURES/EVALUATIONS 
6.1. Study Procedures 
 
6.1.1. Screening process 
 
People and locations suspected or known to harbor malaria will be targeted for screening, as 
described above. As screening progresses, household and workplace contacts of RDT+ and 
usPCR+ cases will be traced to find more infected individuals until enrollment targets are 
reached. The sample collection schedule is shown in Table 2. 
 
A very small number of individuals who are found to be RDT+ during the screening process 
will be asked to provide venous blood, dried blood spots, and travel history. Individuals who 
are found to be RDT+ during screening, or at any time during the study, will be referred 
promptly to local health providers for ACT treatment, in compliance with WHO 
recommendations and NMCP policies, which call for treatment of any RDT+ infection.  
 
Table 2a. Study procedures for population with total 12 weeks of follow up 
RDT, rapid diagnostic test; usPCR, ultrasensitive PCR; R, research-related procedure; S, standard care procedure 
 
 
 
 
Procedures 
 
Screening 
Follow-up visits in weeks after 
enrollment 
4 
6 
8 
10 
12 
Eligibility/ RDT testing  
R 
 
 
 
 
 
Informed consent/assent 
R 
 
 
 
 
 
Enrollment 
R 
 
 
 
 
 
Contact & demographic Information; hemoglobin 
testing  
R 
 
 
 
 
 
Travel history 
R 
R 
R 
R 
R 
R 
Dried blood spots (RDT- & usPCR+ at enrollment) 
R 
R 
R 
R 
R 
R 
Dried blood spots (RDT- & usPCR- at enrollment) 
R 
R 
 
 
 
 
Venous blood collection for RDT+ 
R; Any time during the study  
Treatment referral for RDT+ 
S; Any time during the study 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
17 
Table 2b. Study procedures for population with total 6 weeks of follow up 
 
RDT, rapid diagnostic test; usPCR, ultrasensitive PCR; R, research-related procedure; S, standard care procedure 
 
Eligible and consenting individuals will provide dried blood spot (DBS) samples, demographic 
and travel information, and contact information. DBS will be analyzed using usPCR methods 
in designated laboratories. Of those tested at the initial screening visit, usPCR- participants 
will return for just one additional follow up visit at approximately 4 weeks, at which time a 
second DBS and updated travel information will be collected. Individuals who test usPCR+ at 
the initial screening will also return approximately 2-4 weeks after the initial screen, and again 
for four additional visits (weeks ~6, 8, 10, and 12 or weeks ~2, 3, 4, 5 and 6), with DBS and 
travel history update collected at each visit.  
 
 
6.1.2. Enrollment in preliminary study 
 
Eligible and consenting individuals will be enrolled in the study. Eligibility criteria are listed in 
Section 5.3.   
 
6.1.3. Follow-up 
 
All enrolled participants will follow up 2-4 weeks after enrollment. Those who are found to be 
usPCR negative will provide one additional DBS at a second visit at 2-4 weeks and discharged 
from the study. Those who are found to be usPCR positive will provide four additional DBS, 
two weeks apart (weeks 6, 8, 10, and 12), and discharged from the study at Week 12. For the 
military population who are likely able to provide a total of only 6 weeks follow up, they will 
return at 2 weeks after enrollment, and the follow up schedule will be weekly.  
 
At each follow up visit, a brief history (including travel history update) will be taken, and dried 
blood spots will be collected. Those who miss two consecutive follow-up visits will be 
 
Procedures 
 
Screening 
Follow-up visits in weeks after 
enrollment 
2 
3 
4 
5 
6 
Eligibility/ RDT testing  
R 
 
 
 
 
 
Informed consent/assent 
R 
 
 
 
 
 
Enrollment 
R 
 
 
 
 
 
Contact & demographic Information; hemoglobin 
testing
R 
 
 
 
 
 
Travel history 
R 
R 
R 
R 
R 
R 
Dried blood spots (RDT- & usPCR+ at enrollment) 
R 
R 
R 
R 
R 
R 
Dried blood spots (RDT- & usPCR- at enrollment) 
R 
R 
 
 
 
 
Venous blood collection for RDT+ 
R; Any time during the study  
Treatment referral for RDT+ 
S; Any time during the study 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
18 
discharged from the study.  At any time that participants are found to be RDT+, if study 
personnel qualified to collect venous blood are present, RDT+ participants will provide venous 
blood for serology and parasite nucleic acid sequencing. Individuals testing RDT+ at any time 
during the study will be referred for prompt ACT treatment of malaria by local health providers.   
 
Conceptual framework of the study is shown below:  
 
  
 
 
Anticipated duration of screening and enrollment phase: Screening and enrollment will 
begin at each site after obtaining ethical approval from all relevant administrative and ethical 
review committees for that site. Because technical capacity for usPCR is not yet established 
at the China and Bangladesh sites, the study is expected to begin sooner at the Myanmar 
sites. Screening will be continued until enrollment targets are reached.  
 
Duration of follow-up for each subject: Up to 4 months (~2-4 weeks initial assessment plus 
up to 1.5-3 months follow-up).  
 
Total study duration: Up to 1.5 years 
 
Retention: We have achieved retention rates of >95% in longitudinal cohort studies in Madaya 
(unpublished) and Bandarban [54-58] by maintaining strong relationships with the community, 
including regular community meetings; contributing to community welfare through training 
health providers and upgrading facilities; and treating informed consent as an ongoing process 
for the individual and the community [59, 60]. Retention is expected to be lower at sites with 
refugees and military troops, requiring additional screening at those sites, as noted above. 
 
Study termination: Participants will end their participation in the study when they miss two or 
more consecutive follow-up visits. Participants who return after more than two consecutive 
missed visits will be eligible to be re-screened and re-enrolled as a new participant.  
 
Study visit schedule and primary evaluations: Active surveillance will consist of scheduled 
visits every 1-2 weeks for procedures shown in Table 2a and 2b. Passive surveillance will be 
conducted by encouraging participants to see the local health provider when ill. Local 
providers will collect dried blood spot samples whenever RDTs are done and will notify study 
staff of positive tests so that venous blood can be collected. In accord with WHO and NMCP 
policies in all three countries, any positive RDT result, whether testing was done as part of 
active or passive surveillance, will result in referral for ACT treatment, which will be 
administered by local providers as routine care and not as part of the study. 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
19 
6.1.4. Sample Collection 
 
Dried blood spots at all encounters. Our usPCR method preserves both DNA and RNA in 
0.3 mL of blood collected in capillary tubes initially [22], and we have successfully adapted 
this method for DBS on filter paper, achieving nearly the same LoD. At sites and times when 
our dedicated study personnel are present to collect samples, DBS will be collected on two 
filter papers from finger-stick samples. When local health providers collect samples in the 
absence of research staff—e.g. when evaluating fever episodes during unscheduled visits, 
DBS will be collected, following standard procedures that we have successfully trained local 
health providers to perform at dozens of remote sites in Myanmar [22], Bangladesh [54] and 
China [9]. 
 
Whole blood during RDT+ malaria episodes. While DBS and no requirement for cold chain 
is field-friendly and simpler, the ability to sequence parasite genes is impaired by the low 
parasite density. In addition DNA degrades overtime without cold chain. As DNA quantity from 
DBS is not sufficient for whole genome sequencing, venous sampling will be done for RDT+ 
infections detected in screening, follow-up or active surveillance. Blood volumes at any time 
point are limited to 2 mL for children aged < 3 years, 3 mL for age 3-5 years, and 5 mL for 
children aged >5 years and adults. We have extensive experience recovering sera and 
cryopreserved parasites from small blood volumes collected from children [61-65] and have 
developed and used protocols for preserving RNA from field samples in Southeast Asia [22, 
66]. 
 
Quality control and assurance. QC/QA procedures will be followed, including routine 
monitoring and evaluation of study records, using regularly updated study-specific and general 
standard operating procedures, and providing periodic refresher training in Good Clinical 
Practices. 
6.2. Laboratory Evaluations 
6.2.1. Laboratory evaluations/Assays 
Dried blood spots (DBS) on filter paper will be used for usPCR assays and other 
targeted molecular genotyping and serological assays [45-47]. Venous blood samples 
will be used for serology, genotyping [67, 68], targeted and genome-wide sequencing 
[69-71], and related analyses.   
To the greatest extent possible, laboratory analyses, including molecular assays and 
probing of microarrays, will be conducted at malaria research laboratories of 
collaborating institutions in the region (DMR, DSMRC, icddr,b and NIPD), in close 
collaboration and with technical support from the Duke University malaria team. A 
subset of samples will be shipped to Duke University malaria and collaborating 
genomic laboratories for advanced analyses such as whole genome sequencing and 
assembly [70-72] and for quality assurance procedures. 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
20 
6.2.2. Specimen Collection, Preparation, Handling and Shipping 
6.2.2.1. Instructions for Specimen Preparation, Handling, and Storage 
Blood spots on two types of filter paper for study analyses will be collected at 
the time of blood collection for malaria diagnostic testing. DBS specimens will 
be collected using standard methods [73].  
DBS and venous blood samples will be labeled with a participant ID and 
demographic information. After collection, blood spot samples will be air dried 
and stored in zip-lock plastic bags with desiccant at each site in a cool, dry, 
location.  
Venous blood samples obtained from RDT+ participants will be collected into 
tubes with anticoagulant, frozen, and stored in either liquid nitrogen dry 
shippers or -20 ºC or -80 ºC freezers. Frozen samples (either blood or extracted 
DNA) may be shipped to the Duke University in dry ice containers or liquid 
nitrogen dry shippers. 
Future studies of parasite DNA derived from samples may be conducted 
without restriction. Relevant studies of human RNA or DNA, such as human 
polymorphisms associated with risk of or protection from malaria, or genomic 
studies of human migration patterns, may be done on archived samples or 
nucleic acids only if specific permission for additional such studies is obtained 
from the relevant IRBs. Study participants will have the right to withdraw their 
permission for further use of their samples at any time during and after the 
study. 
6.2.2.2. Specimen Shipment 
Samples will be shipped periodically to the malaria laboratories at Duke 
University in Durham NC or other collaborating laboratories for specialized 
genotyping/probing that cannot be done in the region. Frozen venous blood 
samples will be sent in liquid nitrogen dry shippers or on dry ice to Duke 
University. Specimen receipt will be logged into an inventory database.  

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
21 
7. 
STATISTICAL CONSIDERATIONS 
7.1. Study Outcome Measures 
 
Primary objective:   
 
Make a preliminary assessment of the dynamics of subclinical malaria detected by 
ultrasensitive PCR to guide study design of a subsequent longitudinal matched cohort 
study. 
(The objective of the subsequent matched cohort study, to be reviewed as a separate 
protocol, will be to compare the incidence of clinical malaria and malaria transmission 
capacity, in participants with and without subclinical malaria.) 
 
Secondary and exploratory objectives:   
1. Measure the dynamics of subclinical malaria over time and obtain a preliminary 
estimate of the chronicity of infection over a short period of time 
2. Test for association between demographic and other risk factors (e.g. occupation, 
travel) and incidence of clinical malaria and transmission capacity 
3. Collect preliminary data to estimate the history of exposure to malaria parasite and 
mosquito vector species by serological profiling 
4. Collect preliminary data develop a model system to predict future malaria risks using 
geospatial and satellite data on environmental conditions, patterns of parasite 
migration and human mobility, field data on vector abundance and malaria burden over 
time at study sites 
 
Primary outcomes: 
1. Proportion of individuals with initially positive usPCR tests that were followed by 1 or 
more consecutive positive tests on biweekly testing 
2. Proportion of screened individuals with negative usPCR tests that subsequently had 
usPCR positive tests at a second screening test 4 weeks later 
 
Primary endpoints:  
Not applicable 
 
Secondary and exploratory outcomes: 
Infection outcomes and endpoints 
1. Incidence of falciparum malaria infection by RDT 
2. Incidence of vivax malaria infection by RDT 
3. Incidence of mixed falciparum/vivax malaria infection by RDT 
4. Incidence of falciparum malaria infection by usPCR 
5. Incidence of vivax malaria infection by usPCR 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
22 
6. Incidence of mixed falciparum/vivax malaria infection by usPCR 
 
Serology endpoint 
Seroreactivity to polymorphic malaria/mosquito proteins 
7.2. Sample Size Considerations 
 
This preliminary study is designed for better understanding of the dynamics and stability of 
usPCR status. The preliminary data are needed to estimate the required sample size for the 
subsequent matched cohort study, by assuming a McNemar test at α=0.05, two-sided, on the 
observed proportions of usPCR+ and usPCR- participants who develop a positive RDT in 5 
years of follow-up for all sites combined. The results of this preliminary study as well as data 
from other recent and ongoing surveillance and studies will allow us to estimate the average 
annual probability of a positive RDT. We will then be able to be calculate the sample size to 
obtain adequate statistical power to reject the null hypothesis that the true risks in usPCR+ 
and usPCR- individuals are equal if the true relative risk of the estimated 5-year probability of 
a positive RD.  
 
The sample size of this preliminary study (up to 1,000 per site) is planned within logistical and 
financial constraints. A total of 250 usPCR+ is conservatively estimated. Observing usPCR+ 
for 6 or 12 weeks (total number of observation = 6) is feasible, and will provide a meaningful 
preliminary estimate of the stability and dynamics of usPCR+ infections. For every 100 
enrollees, we estimate that at least 75% will be usPCR-. Observation of usPCR-individuals 
(sample size may be as many as 750 per site) for 2-4 weeks (total number of observation = 
2) will also be sufficient and meaningful to judge the stability of usPCR- status.  
 
7.3. Participant Enrollment and Follow-Up 
 
Recruitment will begin in the 2018 May-October rainy season and continue with until 
enrollment targets are reached. The duration of follow-up for each subject will be up to 12 
weeks. The total study duration will be up to 1 year. At each site, enrollment will be paced 
such that at least 100 participants are enrolled in both the rainy and in the dry season, 
permitting us to assess seasonal variation in prevalence of infection. 
7.4. Analysis Plan 
 
Primary analysis will compare the proportion of individuals with initially positive usPCR tests 
that were followed by 1 or more consecutive positive tests on biweekly testing. Using the 
screening results, we will also calculate the proportion of individuals with negative usPCR tests 
that subsequently had usPCR positive tests at the second screening test. 
 
Secondary analyses will include comparisons based on incidence of species-specific RDT+ 
and usPCR+, and, for serological studies, breadth (% seropositivity) and magnitude (mean 
fluorescence intensity) of antibody responses on protein and peptide microarrays.  
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
23 
To understand the dynamics of usPCR positivity, we will construct maximum likelihood models 
using Markov chains to evaluate risk factors that increase or decrease the probability of going 
from state to state between monthly sampling time points.  
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
24 
8. 
SUBJECT CONFIDENTIALITY 
Subject confidentiality is held strictly in trust by the participating investigators and their staff. 
This confidentiality is extended to cover testing of biological samples, in addition to the 
demographic information relating to participating subjects. The study protocol, documentation, 
data and all other information generated will be held in strict confidence. Study records 
including case record forms and consent forms will be stored in locked cabinets in secure 
facilities only accessible to authorized investigators, and/or in password-protected devices and 
computers.  
 
All participants will be assigned a unique study identification (ID) number. This number will be 
used to label all biologic samples and study-related documents except informed 
consent/assent forms which will contain the participant’s identifier and will be stored in secured 
space as described below. All data generated from each study sample will be linked to the 
coded, unique ID and associated study data.  
 
Study data will be stored on password protected computers at the study site under the purview 
of leadership of the DMR, DSMRC, icddr,b and NIPD, with restricted access to specified study 
personnel. Genetic/genomic data generated from malaria parasites isolated from study 
participants may be submitted to publicly available databases, but will not be linked to any 
identifying information from study participants. Participants will not be identified in any 
publications resulting from the study.  
 
The data will be used by the PI and study collaborators to assess parasite gene flow and 
genotype candidate markers of drug resistance as described in this document. The data will 
be stored in secure, password-protected databases at the malaria laboratories at the Duke 
University in Durham NC for up to 20 years, after which the data may be destroyed. 
8.1. 
 Future Use of Stored Specimens 
All study participant will be asked whether we will be permitted to use left-over blood sample 
for future studies 1) related to malaria; 2) related to any infections or 3) related to any diseases. 
Studies of microbial RNA or DNA (chiefly malaria, but also other infectious agents that may 
explain non-malaria fevers) derived from samples may be done without restriction. Studies of 
human RNA or DNA using archived samples or material will be done only if specific permission 
for additional such studies is obtained from the relevant IRBs. Study participants will have the 
right to withdraw their permission for further use of their samples at any time during and after 
the study. Specimens will be maintained in malaria laboratories at the DMR, DSMRC, NIPD, 
icddr,b and/or Duke University. Specimens will be labeled with a coded, unique identifier and 
will not contain identifying information. 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
25 
9. 
INFORMED CONSENT PROCESS 
The principles of informed consent in the current edition of the Declaration of Helsinki will be 
implemented before any protocol-specified procedures are carried out. 
Information about the study will be given to prospective participants in both oral and written 
forms whenever possible. The written consent documents will embody the elements of 
informed consent as described in the current edition of the Declaration of Helsinki, will adhere 
to the ICH Harmonized Tripartite Guideline for Good Clinical Practice and 45CFR46 and 
21CFR50, and will also comply with applicable Myanmar regulations. The oral consent 
process will be consistent with 45CFR46.§46.117, 21CFR50.27 and ICH E6 (R1) Section 4.8. 
Independent witnesses will be used to attest that illiterate potential participants have 
understood the contents of the informed consent document. 
9.1. Informed Consent/Assent Process (in Case of a Minor or 
Others Unable to Consent for Themselves) 
 
We have published detailed descriptions of the processes we use to obtain community 
“permission to enter” and individual informed consent [60]. We consider informed consent to 
be a dynamic, ongoing process, with continuous availability of investigators to answer any 
questions that arise in the course of the study and to ensure that participants and their 
parents/guardians understand study procedures. These same principles will be followed in 
this study. All NMCPs in the GMS routinely conduct multiple annual therapeutic efficacy 
surveys to monitor efficacy of ACT. As a result, communities throughout the GMS have now 
become familiar with the informed consent process, including written, signed consent forms, 
which used for NMCP therapeutic efficacy surveys. The investigators will work with local 
research staff at each site to assure study information and informed consent procedures meet 
requirements of the community and the local IRBs. 
Since the vast majority of study participants’ parents/guardians do not use telephones, fax, or 
mail, contact information is provided in terms of local physicians who can be visited directly 
and who can themselves reach the investigators directly or by telephone or fax.  
Individuals who are at least 18 years old are considered adults and will provide consent for 
themselves; children aged up to 12 years will provide verbal assent in addition to written 
consent provided by their guardians; children aged 13–17 years will provide written assent in 
addition to written consent provided by their guardians. Guardians need not necessarily be 
the child’s parent(s). Any close relative including aunts, uncles, grandparents, adult siblings 
etc. who accompany the child to the clinic may serve as the guardian for the sake of providing 
informed consent. This may include neighbors or village elders in the case of orphans. 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
26 
10. DATA MANAGEMENT  
Source Data, Data Storage, and Access to Data 
Hard copy research records generated for this protocol will be stored in locked rooms at project 
sites. Study data will be entered electronically into a secure, password protected, cloud-based 
research information portal (RIP) developed, managed, and maintained by the Data 
Management and Statistics Core. The data warehouse will be developed using MySQL 
developed by DMB Core staff. The portal will also be built to ensure data security and integrity. 
 
Our primary tool for developing case record forms will be REDCap™ for electronic data 
capture REDCap™ is a free, secure, web-based application for data capture. Developed by a 
multi-institutional consortium initiated by Vanderbilt University, REDCap™ provides user-
friendly, web-based case report forms, real time data entry and validation (e.g., data types, 
range checks), audit trails, and the ability to set up a study-related calendar to schedule and 
track critical study events such as participant visits, reminders, etc. The REDCap consortium 
of users is comprised of 1,837 institutional partners in 99 countries around the world who use 
and support REDCap as a research tool [4,5]. REDCap™ will give us the ability to assign 
different levels of access to members of the research team depending upon their role on the 
project so that end users only have access to forms and data directly related to their role on 
the project. 
 
The REDCap™ system provides tools for users to enter data directly through a web-based 
interface on laptop, Ipad or other tablet device, or enter and save data offline and upload the 
data onto a web server later via internet or satellite service. In addition, CRFs can be saved 
as PDFs for printing in order to allow offline data collection. Any data collected on paper in 
offline mode will require manual entry with double keying to ensure data accuracy. In each 
field site, data will be entered into a REDCap™ CRF on a mobile phone or a tablet, and saved. 
Internet service is reliably available at the local base of operations for each field site, located 
in towns or cities at or near the field site (DMR: Buthidaung and Madaya; DSMRC: Monywa; 
NIPD: Canyuang and Nabang; icddr,b Bandarban), where data will be uploaded daily to the 
central database. 
 
Access to the Duke University REDCap™ system requires university-level credentials. 
Specifically, the tool requires username/password security protection to prevent unauthorized 
access for entry or editing data. Shibboleth [6] middleware is used for identity management 
and will be used for federated identity-based authentication and authorization. A list of security 
level access (full access, edit access, read only access etc.) is documented. REDCap™ 
complies with the Health Insurance Portability and Accountability Act (HIPAA) regulations and 
is 21 CFR Part 11 capable through server configuration. 
 
Data will be stored on a secure, cloud-based server. Connections and access to the RIP will 
be secured with a username and password. Role-based access privileges will be assigned 
and access will be limited to authorized users. PIs will be granted access to all of the data 
from their site, while sub-investigators and other project personnel may only have access to 
data that has been authorized by the study PI. PIs will have final authority for determining who 
is granted access to data and to which data they will have access. 
 
10.2 Data Standardization 
 
To the extent possible, data elements will be developed following the Clinical Data Acquisition 
Standards Harmonization (CDASH) developed by Clinical Data Interchange Standards 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
27 
Consortium (CDISC). Adoption of these standard will facilitate data sharing and harmonization 
with other ICEMRs. 
 
10.3 Audit Trail System  
 
The RIP and tools embedded within the RIP like REDCap™ will support an automatic 
electronic audit trail that permanently tracks and logs every access of data, tools, or reports 
within the RIP. The RIP audit log records the date and time of any access, the RIP element 
that was accessed (e.g., data table, software tool such as REDCap™) and records all changes 
to data stored in the RIP. The system will record and store the username of the individual who 
changes data, the date and time of change, the new and old value of the variable changed 
and the computer used.  
 
10.4 Data Validation and Query Generation  
 
REDCap™ will apply data validation rules at the time data are entered through the REDCap™ 
tool. Other programs within the RIP will identify potentially incorrect data through manual and 
automatic queries. These queries are run after reconciliation procedures, and check for any 
data discrepancies in the system. These queries are documented on a Data Clarification Form 
(DCF). The system has an input mask for incorrect data such as out of range/value or an 
incorrect date. At each in-country partner site (DMR and DSMRC in Myanmar, NIPD/YIPD in 
China, and icddr,b in Bangladesh) a data clerk documents or flags any unclear data or errors 
during the data checking process. The data supervisor generates a query for any discrepant 
data or error and sends it to the field site for resolution. Final quality checks will be done in 
Durham until regional data teams are fully capable of managing the data validation process. 
The resolution of queries by the PI or the designee will be documented and dated. After the 
resolution, the data are updated using the resolved query; the update of this query is then 
signed. This electronic update of data is tracked by the RIS audit trail system. 
 
10.5 Database Lock (Locked Data)  
 
After the cleaning and validation of data, and when data are ready for statistical analyses, the 
data manager for each site will lock the database (Locked data base). This lock is documented 
by the data manager and the Site PI with date/time of locked. Any change after the data lock 
has occurred must be authorized by the PI responsible for the study. 
 
10.6 Quality Assurance and Quality Control 
 
The data managers and data entry clerks involved in the study data management will be 
trained beforehand in all aspects of the specific study data management procedures. Standard 
operating procedures for quality management have been developed and are used to train all 
personnel; these are kept on file with documentation of training. Data are evaluated for 
compliance with protocol and accuracy in relation to source documents. Quality control will be 
also monitored using classical means of the Statistical Process Control (including Control 
Chart of specific indicators). 
 
10.7 Data Integrity and Data Quality 
 
Data integrity will be verified by several means: 1) Comparison of information in the source 
documents vs. those that are in the CRF; 2) comparison of data in the database vs. those in 
the CRF; 3) interviews to search for unacceptable or questionable practices, and review of the 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
28 
analysis procedures; 4) inspection and independent auditing. Furthermore, access to the RIP 
and all data tables will be monitored through regular control of the access log. 
 
10.8 Upload and Download Functions 
 
The Research Information Portal will have data upload and download capability via secure file 
transfer 
protocol 
(sFTP). 
Access 
to 
sFTP 
functionality 
will 
be 
controlled 
by 
username/password strategy, and all uploads and downloads will be monitored via our central 
audit logging tool. 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
29 
11. LITERATURE REFERENCES 
1. 
Plowe CV: The evolution of drug-resistant malaria. Trans R Soc Trop Med Hyg 
2009, 103 Suppl 1:S11-S14. 
2. 
Tanner M, de SD: Malaria eradication back on the table. Bull World Health Organ 
2008, 86(2):82. 
3. 
World Health Organization: World Malaria Report 2015. In. Geneva; 2015: 1-249. 
4. 
Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM: Evidence of 
artemisinin-resistant malaria in western Cambodia. N Engl J Med 2008, 
359(24):2619-2620. 
5. 
Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F, 
Hanpithakpong W, Lee SJ et al: Artemisinin resistance in Plasmodium falciparum 
malaria. N Engl J Med 2009, 361(5):455-467. 
6. 
Dondorp AM, Fairhurst RM, Slutsker L, MacArthur JR, Breman JG, Guerin PJ, 
Wellems TE, Ringwald P, Newman RD, Plowe CV: The threat of artemisinin-
resistant malaria. N Engl J Med 2011, 365(12):1073-1075. 
7. 
Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R, ler MC, Al-Saai 
S, Dondorp AM, Lwin KM et al: Emergence of artemisinin-resistant malaria on the 
western border of Thailand: a longitudinal study. Lancet 2012, 379(9830):1960-
1966. 
8. 
Kyaw MP, Nyunt MH, Chit K, Aye MM, Aye KH, Aye MM, Lindegardh N, Tarning J, 
Imwong M, Jacob CG et al: Reduced susceptibility of Plasmodium falciparum to 
artesunate in southern Myanmar. PLoS ONE 2013, 8(3):e576889. 
9. 
Huang F, Takala-Harrison S, Jacob CG, Liu H, Sun X, Yang H, Nyunt MM, Adams M, 
Zhou S, Xia Z et al: A single mutation in K13 predominates in Southern China and 
is associated with delayed clearance of Plasmodium falciparum following 
artemisinin treatment. J Infect Dis 2015. 
10. 
Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, 
Anderson JM, Mao S, Sam B et al: Spread of artemisinin resistance in Plasmodium 
falciparum malaria. N Engl J Med 2014, 371(5):411-423. 
11. 
Amaratunga C, Lim P, Suon S, Sreng S, Mao S, Sopha C, Sam B, Dek D, Try V, Amato 
R et al: Dihydroartemisinin-piperaquine resistance in Plasmodium falciparum 
malaria in Cambodia: a multisite prospective cohort study. Lancet Infect Dis 2016. 
12. 
Plowe CV: The danger of untreatable malaria is real and present. In. Washington 
DC; 2015. 
13. 
World Health Organization: Strategic Framework for Artemisinin Resistance 
Containment in Myanmar (MARC) 2011‐2015. In.; 2011. 
14. 
Lwin KM, Imwong M, Suangkanarat P, Jeeyapant A, Vihokhern B, Wongsaen K, 
Snounou G, Keereecharoen L, White NJ, Nosten F: Elimination of Plasmodium 
falciparum in an area of multi-drug resistance. Malar J 2015, 14:319. 
15. 
Takala-Harrison S, Jacob CG, Arze C, Cummings MP, Silva JC, Dondorp AM, Fukuda 
MM, Hien TT, Mayxay M, Noedl H et al: Independent emergence of artemisinin 
resistance mutations among Plasmodium falciparum in Southeast Asia. J Infect 
Dis 2015, 211(5):670-679. 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
30 
16. 
World Health Organization: Minutes of the Drug Resistance and Containment 
Technical Expert Group. In. Geneva; 2014. 
17. 
World Health Organization Malaria Policy Advisory Committee: Malaria Policy 
Advisory Committee to the WHO: conclusions and recommendations of sixth 
biannual meeting (September 2014). Malar J 2015, 14:107. 
18. 
World Health Organization: Global technical strategy for malaria 2016-2030. In. 
Geneva; 2015. 
19. 
malERA Consultative Group on Drugs: A research agenda for malaria eradication: 
drugs. PLoS Med 2011, 8(1):e1000402. 
20. 
Stephens JWW, Christophers MB: The Malarial Infection of Native Children. In: 
Reports to the Malaria Committee of The Royal Society. The Royal Society; 1900: 2-
14. 
21. 
Imwong M, Hanchana S, Malleret B, Renia L, Day NP, Dondorp A, Nosten F, Snounou 
G, White NJ: High-throughput ultrasensitive molecular techniques for 
quantifying low-density malaria parasitemias. J Clin Microbiol 2014, 52(9):3303-
3309. 
22. 
Adams M, Joshi SN, Mbambo G, Mu AZ, Roemmich SM, Shrestha B, Strauss KA, 
Johnson NE, Oo KZ, Hlaing TM et al: An ultrasensitive reverse transcription 
polymerase chain reaction assay to detect asymptomatic low-density 
Plasmodium falciparum and Plasmodium vivax infections in small volume blood 
samples. Malar J 2015, 14(1):520. 
23. 
Imwong M, Nguyen TN, Tripura R, Peto TJ, Lee SJ, Lwin KM, Suangkanarat P, 
Jeeyapant A, Vihokhern B, Wongsaen K et al: The epidemiology of subclinical 
malaria infections in South-East Asia: findings from cross-sectional surveys in 
Thailand-Myanmar border areas, Cambodia, and Vietnam. Malar J 2015, 14:381. 
24. 
Chen I, Clarke SE, Gosling R, Hamainza B, Killeen G, Magill A, O'Meara W, Price RN, 
Riley EM: "Asymptomatic" Malaria: A Chronic and Debilitating Infection That 
Should Be Treated. PLoS Med 2016, 13(1):e1001942. 
25. 
Canier L, Khim N, Kim S, Eam R, Khean C, Loch K, Ken M, Pannus P, Bosman P, 
Stassijns J et al: Malaria PCR detection in cambodian low-transmission settings: 
dried blood spots versus venous blood samples. Am J Trop Med Hyg 2015, 
92(3):573-577. 
26. 
Sondén K, Doumbo S, Hammar U: Asymptomatic multiclonal Plasmodium 
falciparum infections carried through the dry season predict protection against 
subsequent clinical malaria. Journal of Infectious … 2015. 
27. 
Liljander A, Bejon P, Mwacharo J, Kai O, Ogada E, Peshu N, Marsh K, Färnert A: 
Clearance of asymptomatic P. falciparum infections interacts with the number 
of clones to predict the risk of subsequent malaria in Kenyan children. PLoS One 
2011, 6(2). 
28. 
Imwong M, Stepniewska K, Tripura R, Peto TJ, Lwin KM, Vihokhern B, Wongsaen K, 
von Seidlein L, Dhorda M, Snounou G et al: Numerical Distributions of Parasite 
Densities During Asymptomatic Malaria. J Infect Dis 2015. 
29. 
Covell G: Relationship between malarial parasitaemia and symptoms of the 
disease: a review of the literature. Bull World Health Organ 1960, 22:605-619. 
30. 
Nosten F: How to Beat Malaria, Once and for All. In: New York Times. New York; 
2014. 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
31 
31. 
Rieckmann KH: Asymptomatic malaria. Lancet 1970, 1(7637):82-83. 
32. 
Felger I, Maire M, Bretscher MT, Falk N, Tiaden A, Sama W, Beck H-P, Owusu-Agyei 
S, Smith TA: The Dynamics of Natural Plasmodium falciparum Infections. PLoS 
ONE 2012, 7(9). 
33. 
Babiker HA, Abdel-Muhsin AM, Ranford-Cartwright LC, Satti G, Walliker D: 
Characteristics of Plasmodium falciparum parasites that survive the lengthy dry 
season in eastern Sudan where malaria transmission is markedly seasonal. Am 
J Trop Med Hyg 1998, 59(4):582-590. 
34. 
Ashley EA, White NJ: The duration of Plasmodium falciparum infections. Malar J 
2014, 13:500. 
35. 
Lindblade KA, Steinhardt L, Samuels A, Kachur SP, Slutsker L: The silent threat: 
asymptomatic parasitemia and malaria transmission. Expert Rev Anti Infect Ther 
2013, 11(6):623-639. 
36. 
Tripura R, Peto TJ, Veugen CC, Nguon C, Davoeung C, James N, Dhorda M, Maude 
RJ, Duanguppama J, Patumrat K et al: Submicroscopic Plasmodium prevalence in 
relation to malaria incidence in 20 villages in western Cambodia. Malar J 2017, 
16(1):56. 
37. 
Zaw MT, Thant M, Hlaing TM, Aung NZ, Thu M, Phumchuea K, Phusri K, Saeseu T, 
Yorsaeng R, Nguitragool W et al: Asymptomatic and sub-microscopic malaria 
infection in Kayah State, eastern Myanmar. Malar J 2017, 16(1):138. 
38. 
Miotto O, Almagro-Garcia J, Manske M, MacInnis B, Campino S, Rockett KA, 
Amaratunga C, Lim P, Suon S, Sreng S et al: Multiple populations of artemisinin-
resistant Plasmodium falciparum in Cambodia. Nat Genet 2013, 45(6):648-655. 
39. 
Miotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, Lim P, 
Mead D, Oyola SO, Dhorda M et al: Genetic architecture of artemisinin-resistant 
Plasmodium falciparum. Nat Genet 2015, 47(3):226-234. 
40. 
Chenet SM, Schneider KA, Villegas L, Escalante AA: Local population structure of 
Plasmodium: impact on malaria control and elimination. Malar J 2012, 11:412. 
41. 
Orjuela-Sanchez P, Sa JM, Brandi MC, Rodrigues PT, Bastos MS, Amaratunga C, 
Duong S, Fairhurst RM, Ferreira MU: Higher microsatellite diversity in Plasmodium 
vivax than in sympatric Plasmodium falciparum populations in Pursat, Western 
Cambodia. Exp Parasitol 2013, 134(3):318-326. 
42. 
Jennison C, Arnott A, Tessier N, Tavul L, Koepfli C, Felger I, Siba PM, Reeder JC, 
Bahlo M, Mueller I et al: Plasmodium vivax populations are more genetically 
diverse and less structured than sympatric Plasmodium falciparum populations. 
PLoS Negl Trop Dis 2015, 9(4):e0003634. 
43. 
Bejon P, Turner L, Lavstsen T, Cham G, Olotu A, Drakeley CJ, Lievens M, Vekemans 
J, Savarese B, Lusingu J et al: Serological evidence of discrete spatial clusters of 
Plasmodium falciparum parasites. PLoS One 2011, 6(6):e21711. 
44. 
Helb DA, Tetteh KK, Felgner PL, Skinner J, Hubbard A, Arinaitwe E, Mayanja-Kizza 
H, Ssewanyana I, Kamya MR, Beeson JG et al: Novel serologic biomarkers provide 
accurate estimates of recent Plasmodium falciparum exposure for individuals 
and communities. Proc Natl Acad Sci U S A 2015, 112(32):E4438-4447. 
45. 
Bailey JA, Pablo J, Niangaly A, Travassos MA, Ouattara A, Coulibaly D, Laurens MB, 
Takala-Harrison SL, Lyke KE, Skinner J et al: Seroreactivity to a large panel of field-
derived Plasmodium falciparum apical membrane antigen 1 and merozoite 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
32 
surface protein 1 variants reflects seasonal and lifetime acquired responses to 
malaria. Am J Trop Med Hyg 2015, 92(1):9-12. 
46. 
Travassos MA, Niangaly A, Bailey JA, Ouattara A, Coulibaly D, Laurens MB, Pablo J, 
Jasinskas A, Nakajima-Sasaki R, Berry AA et al: Seroreactivity to Plasmodium 
falciparum erythrocyte membrane protein 1 intracellular domain in malaria-
exposed children and adults. J Infect Dis 2013, 208(9):1514-1519. 
47. 
Travassos MA, Coulibaly D, Bailey JA, Niangaly A, Adams M, Nyunt MM, Ouattara A, 
Lyke KE, Laurens MB, Pablo J et al: Differential recognition of terminal 
extracellular Plasmodium falciparum VAR2CSA domains by sera from 
multigravid, malaria-exposed Malian women. Am J Trop Med Hyg 2015, 
92(6):1190-1194. 
48. 
King CL, Davies DH, Felgner P, Baum E, Jain A, Randall A, Tetteh K, Drakeley CJ, 
Greenhouse B: Biosignatures of Exposure/Transmission and Immunity. Am J 
Trop Med Hyg 2015, 93(3 Suppl):16-27. 
49. 
Arca B, Lombardo F, Valenzuela JG, Francischetti IM, Marinotti O, Coluzzi M, Ribeiro 
JM: An updated catalogue of salivary gland transcripts in the adult female 
mosquito, Anopheles gambiae. J Exp Biol 2005, 208(Pt 20):3971-3986. 
50. 
Drame PM, Poinsignon A, Dechavanne C, Cottrell G, Farce M, Ladekpo R, 
Massougbodji A, Cornélie S, Courtin D, Migot-Nabias F et al: Specific antibodies to 
Anopheles gSG6-P1 salivary peptide to assess early childhood exposure to 
malaria vector bites. Malaria journal 2015, 14:285. 
51. 
Reiner RC, Le Menach A, Kunene S, Ntshalintshali N, Hsiang MS, Perkins TA, 
Greenhouse B, Tatem AJ, Cohen JM, Smith DL: Mapping residual transmission for 
malaria elimination. Elife 2015, 4. 
52. 
Acevedo MA, Prosper O, Lopiano K, Ruktanonchai N, Caughlin TT, Martcheva M, 
Osenberg CW, Smith DL: Spatial heterogeneity, host movement and mosquito-
borne disease transmission. PLoS One 2015, 10(6):e0127552. 
53. 
Lyke KE, Dicko A, Dabo A, Sangare L, Kone A, Coulibaly D, Guindo A, Traore K, Daou 
M, Diarra I et al: Association of Schistosoma haematobium infection with 
protection against acute Plasmodium falciparum malaria in Malian children. Am 
J Trop Med Hyg 2005, 73(6):1124-1130. 
54. 
Khan WA, Galagan SR, Prue CS, Khyang J, Ahmed S, Ram M, Alam MS, Haq MZ, 
Akter J, Glass G et al: Asymptomatic Plasmodium falciparum malaria in pregnant 
women in the Chittagong Hill Districts of Bangladesh. PLoS One 2014, 
9(5):e98442. 
55. 
Galagan SR, Prue CS, Khyang J, Khan WA, Ahmed S, Ram M, Alam MS, Haq MZ, 
Akter J, Streatfield PK et al: The practice of jhum cultivation and its relationship 
to Plasmodium falciparum infection in the Chittagong Hill Districts of 
Bangladesh. Am J Trop Med Hyg 2014, 91(2):374-383. 
56. 
Ahmed S, Galagan S, Scobie H, Khyang J, Prue CS, Khan WA, Ram M, Alam MS, 
Haq MZ, Akter J et al: Malaria hotspots drive hypoendemic transmission in the 
Chittagong Hill Districts of Bangladesh. PLoS One 2013, 8(8):e69713. 
57. 
Prue CS, Shannon KL, Khyang J, Edwards LJ, Ahmed S, Ram M, Shields T, Hossain 
MS, Glass GE, Nyunt MM et al: Mobile phones improve case detection and 
management of malaria in rural Bangladesh. Malar J 2013, 12:48. 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
33 
58. 
Khan WA, Sack DA, Ahmed S, Prue CS, Alam MS, Haque R, Khyang J, Ram M, Akter 
J, Nyunt MM et al: Mapping hypoendemic, seasonal malaria in rural Bandarban, 
Bangladesh: a prospective surveillance. Malar J 2011, 10:124. 
59. 
Building 
an 
Open 
Burma 
Through 
Medical 
Ethics 
[https://www.opensocietyfoundations.org/voices/building-open-burma-through-
medical-ethics] 
60. 
Diallo DA, Doumbo OK, Plowe CV, Wellems TE, Emanuel EJ, Hurst SA: Community 
permission for medical research in developing countries. Clin Infect Dis 2005, 
41(2):255-259. 
61. 
Lyke KE, Burges R, Cissoko Y, Sangare L, Dao M, Diarra I, Kone A, Harley R, Plowe 
CV, Doumbo OK et al: Serum Levels of the Proinflammatory Cytokines 
Interleukin-1 Beta (IL-1{beta}), IL-6, IL-8, IL-10, Tumor Necrosis Factor Alpha, and 
IL-12(p70) in Malian Children with Severe Plasmodium falciparum Malaria and 
Matched Uncomplicated Malaria or Healthy Controls. Infect Immun 2004, 
72(10):5630-5637. 
62. 
Lyke KE, Burges RB, Cissoko Y, Sangare L, Kone A, Dao M, Diarra I, Fernandez-Vina 
MA, Plowe CV, Doumbo OK et al: HLA-A2 supertype-restricted cell-mediated 
immunity by peripheral blood mononuclear cells derived from Malian children 
with severe or uncomplicated Plasmodium falciparum malaria and healthy 
controls. Infect Immun 2005, 73(9):5799-5808. 
63. 
Deans AM, Lyke KE, Thera MA, Plowe CV, Kone A, Doumbo OK, Kai O, Marsh K, 
Mackinnon MJ, Raza A et al: Low multiplication rates of African Plasmodium 
falciparum isolates and lack of association of multiplication rate and red blood 
cell selectivity with malaria virulence. Am J Trop Med Hyg 2006, 74(4):554-563. 
64. 
Arman M, Raza A, Tempest LJ, Lyke KE, Thera MA, Kone A, Plowe CV, Doumbo OK, 
Rowe JA: Platelet-mediated clumping of Plasmodium falciparum infected 
erythrocytes is associated with high parasitemia but not severe clinical 
manifestations of malaria in African children. Am J Trop Med Hyg 2007, 77(5):943-
946. 
65. 
Lyke KE, Dabo A, Sangare L, Arama C, Daou M, Diarra I, Plowe CV, Doumbo OK, 
Sztein MB: Effects of concomitant Schistosoma haematobium infection on the 
serum cytokine levels elicited by acute Plasmodium falciparum malaria infection 
in Malian children. Infect Immun 2006, 74(10):5718-5724. 
66. 
Takala-Harrison S, Clark TG, Jacob CG, Cummings MP, Miotto O, Dondorp AM, 
Fukuda MM, Nosten F, Noedl H, Imwong M et al: Genetic loci associated with 
delayed clearance of Plasmodium falciparum following artemisinin treatment in 
Southeast Asia. Proc Natl Acad Sci U S A 2013, 110(1):240-245. 
67. 
Jacob CG, Tan JC, Miller BA, Tan A, Takala-Harrison S, Ferdig MT, Plowe CV: A 
microarray platform and novel SNP calling algorithm to evaluate Plasmodium 
falciparum field samples of low DNA quantity. BMC Genomics 2014, 15(1):719. 
68. 
Takala SL, Smith DL, Stine OC, Coulibaly D, Thera MA, Doumbo OK, Plowe CV: A 
high-throughput method for quantifying alleles and haplotypes of the malaria 
vaccine candidate Plasmodium falciparum merozoite surface protein-1 19 kDa. 
Malar J 2006, 5:31. 
69. 
Gandhi K, Thera MA, Coulibaly D, Traore K, Guindo AB, Doumbo OK, Takala-Harrison 
S, Plowe CV: Next generation sequencing to detect variation in the Plasmodium 
falciparum circumsporozoite protein. Am J Trop Med Hyg 2012, 86(5):775-781. 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
34 
70. 
Dara A, Travassos MA, Adams M, Schaffer DeRoo S, Drabek EF, Agrawal S, Laufer 
MK, Plowe CV, Silva JC: A new method for sequencing the hypervariable 
Plasmodium falciparum gene var2csa from clinical samples. Malar J 2017, 
16(1):343. 
71. 
Dara A, Drabek EF, Travassos MA, Moser KA, Delcher AL, Su Q, Hostelley T, 
Coulibaly D, Daou M, Dembele A et al: New var reconstruction algorithm exposes 
high var sequence diversity in a single geographic location in Mali. Genome Med 
2017, 9(1):30. 
72. 
Travassos M, Silva JC, Drabek EF, Delcher AL, Su Q, Dara A, Coulibaly D, Daou M, 
Dembele A, Diarra I et al: An algorithm for ascertaining the var gene repertoire in 
Plasmodium falciparum field samples. American Journal of Tropical Medicine and 
Hygiene 2014, 91:186. 
73. 
Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE: Pyrimethamine and 
proguanil 
resistance-conferring 
mutations 
in 
Plasmodium 
falciparum 
dihydrofolate reductase: polymerase chain reaction methods for surveillance in 
Africa. Am J Trop Med Hyg 1995, 52(6):565-568. 
 
 
 

Dynamics of subclinical malaria: preliminary study 
 
 Version 1.0 
 
 
12 February 2018 
 
 
 
 ___________________________________________________________________________________________  
35 
SUPPLEMENTS/APPENDICES