Evaluation of the Performance of a Highly-sensitive Rapid Diagnostic Test (RDT) Versus Conventional RDT, Compared With PCR as the Gold Standard, in Reactive Case Detection of Malaria Infections in Rakhine State, Myanmar
Phase: NA
Conditions: Malaria Diagnosis, Malaria,Falciparum
Sponsor: University Research Co, LLC

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hsRDT reactive case detection
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$EEUHYLDWLRQV
ACT
Artemisinin-based Combination Therapy
CNP
Copy Number Polymorphism
DBS
Dried Blood Spot
DMR
Department of Malaria Research
DNA
Deoxyribonucleic acid
ERC
Ethics Review Committee
GCP
Good Clinical Practice
GMS
Greater Mekong Sub-region
GWAS
Genome-Wide Association Study
ICH
International Conference on Harmonization
ID
Identification
N
Number (typically refers to subjects)
NMCP
National Malaria Control Program
PCR
Polymerase Chain Reaction
PI
Principal Investigator
PMI
U.S. President’s Malaria Initiative
SOP
Standard Operating Procedure
UMB
University of Maryland, Baltimore
URC
University Research Co.
USA
United States of America
USAID
United States Agency for International Development
VBDC
Vector Borne Disease Control
WHO
World Health Organization
WWARN
Worldwide Antimalarial Resistance Network



hsRDT reactive case detection
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3URWRFRO6XPPDU\
7LWOH:
Evaluation of the performance of a highly-sensitive rapid diagnostic
test (hsRDT) YHUVXV conventional RDT (cRDT), compared with
polymerase chain reaction (PCR) as the gold standard, in reactive case
detection of malaria infections in Rakhine State, Myanmar
3RSXODWLRQ:
Adults and children at least 6 months of age in a specified study site
1XPEHURI6LWHV:
One
6WXG\'XUDWLRQ:
One year

6XEMHFW'XUDWLRQ:
One encounter lasting no more than 30 min

6WXG\REMHFWLYHV:
*HQHUDOREMHFWLYH: To evaluate the performance of the highly-sensitive rapid diagnostic test
(hsRDT), developed by SD Bioline, YHUVXV conventional RDT (cRDT), compared with PCR
as the gold standard, in reactive case detection to identify additional malaria infections in
Rakhine State, Myanmar
6SHFLILFREMHFWLYHV
1. To evaluate the prevalence of malaria identified by the new hsRDT in comparison
with that by cRDT and PCR
Outcomes: test positivity rate by cRDT, hsRDT and PCR, respectively
2. To assess the diagnostic performance characteristics of hsRDT YHUVXV cRDT, using
PCR as gold standard, in the detection of 3IDOFLSDUXP infections
Outcomes: Sensitivity, specificity, positive and negative predictive value of hsRDT and
cRDT
3. To evaluate correlation of detection capability between cRDT and hsRDT
Outcomes: Correlation of test positivity rate by the three different study tests
4. To identify risk factors associated with malaria infection, including but not limited to,
socio-demographic factors and travel history related with malaria index cases
Outcomes: Relative risk of malaria in association with different risk factors identified


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4

,QYHVWLJDWRUVLQVWLWXWLRQDODIILOLDWLRQVDQGUROH

3ULQFLSDO,QYHVWLJDWRU

San Kyawt Khine
Assistant Director
Vector Borne Disease Control (VBDC) Rakhine State
Department of Public Health
Myanmar Ministry of Health and Sports (MoHS)
&RLQYHVWLJDWRUV

Dr. Aung Thi
Program Manager, National Malaria Control Program (NMCP),
Myanmar
Dr. Aye Nyein
Team Leader, Vector Borne Disease Control (VBDC) Team,
Rakhine State
Dr. Nay Yi Yi Linn
Assistant Director, NMCP
Dr. Zay Min Tun
Medical Officer, NMCP
Dr. Saw Lwin
Deputy Chief of Party &Technical Advisor, Defeat Malaria,
University Research Co. (URC)
Dr. Feliciano Monti
Senior Malaria Advisor, U.S. President’s Malaria Initiative
(PMI), U.S. Agency for International Development (USAID)
Dr. Jimee Hwang
Medical Epidemiologist, U.S. Center for Disease Control &
Prevention (CDC)
Dr. Nelli Westercamp
Epidemiologist, U.S. Center for Disease Control & Prevention
(CDC)
Dr. Khin Than Win
Senior Technical Director, Defeat Malaria, URC
Dr. Kyaw Myint Tun
Monitoring & Evaluation (M&E) Technical Advisor, Defeat
Malaria, URC
Dr. Kay Thwe Han
Deputy Director, Parasitology Division, Department of Medical
Research, Myanmar MoHS
Dr. Myaing M. Nyunt
Director, Institute for Global Health (IGH) Myanmar,
University of Maryland Baltimore (UMB)
Dr. Janie Zuber
Infectious Disease Fellow, IGH, UMB
Dr. Christopher Plowe
Director of IGH & Professor of Molecular Epidemiology &
Infectious Diseases, UMB
Dr. Yuanyuan Liang
Professor of Biostatistics, IGH, UMB
Matt Adams
Lab supervisor, UMB

6SRQVRUU.S. President’s Malaria Initiative


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For questions regarding this protocol, contact:
,QGLYLGXDOV:
6SRQVRUUHSUHVHQWDWLYH

Dr.Feliciano Monti
U.S. President’s Malaria Initiative
U.S. Embassy, Yangon
Tel. +95-(0)9-5419651
Email: fmonti@usaid.gov

3ULQFLSDO,QYHVWLJDWRUV:
Dr. San Kyawt Khine
Assistant Director
Vector Borne Disease Control (VBDC) Program, Rakhine State
General Hospital Compound, Sittwe, Rakhine State
Tel. +95-(0)9-450542076
Email: skkhine.khine75@gmail.com

6HQLRU7HFKQLFDO$GYLVRU

Dr. Saw Lwin
Deputy Chief of Party
University Research Co. (URC), Defeat Malaria
Room 602/701, Shwe Than Lwin Condominium, New University Avenue
Road, Bahan Township, Yangon]
Tel. +95-(0)9-5014887
Email: slwin@URC-CHS.COM
3DUWLFLSDWLQJ,QVWLWXWLRQV:


9HFWRU%RUQH'LVHDVH&RQWURO3URJUDP
Diseases Control Office,
Zabu Thiri Township
Nay Pyi Taw, Myanamar]
Tel. 067-421204
Fax 067-421204

8QLYHUVLW\5HVHDUFK&R0\DQPDU
Room 602/701, Shwe Than Lwin
Condominium,
New University Avenue Road,
Bahan Township, Yangon
Tel. +95-1-543353
Fax: 01-1-220658

,QVWLWXWHIRU*OREDO+HDOWK
University of Maryland School of Medicine
485 West Baltimore Street, HSFII-480
Baltimore, Maryland 21201 USA
Tel. +1-410-706-2491
Fax. +1-410-706-1204

'HSDUWPHQWRI0HGLFDO5HVHDUFK
Ministry of Health and Sports
5, Ziwaka Road, Dagon P.O.
Yangon 11191, Myanmar
Tel. +95-1-380129
Fax. +95-1-251514
86&HQWHUVIRU'LVHDVH&RQWURODQG
3UHYHQWLRQ
1600 Clifton Rd, Atlanta, GA, 30333, USA
Tel. +1-770-488-7100
Fax +1-770-488-4206


hsRDT reactive case detection
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&RLQYHVWLJDWRUVDQGWKHLUUROH
Name
Institution
Role
Brief description
Dr. San Kyawt
Khaing
VBDC-
Rakhine
Principle
Investigator
Responsible for the overall technical, regulatory,
financial and ethical adequacy of the study; provide
necessary administrative and technical support for
study team; Respond timely and adequately for the
needs from study team; Preparation of study
protocol and related documents; Ensure accuracy
and completion of all study related documents, and
study samples and data timely.
Dr. Saw Lwin
URC
Defeat
Malaria
Co-investigator
Dr. Aung Thi
NMCP
Co-investigator
Overall administrative and technical leadership;
Guidance for study conduct and decision on
interpretation and implication of study findings
Dr. Aye Nyein
VBDC-
Rakhine
Team leader
Responsible for leading and managing daily
activities of study team; Guide and coordinate
issues related to local circumstances and overall
organization of implementation of the study and
responsible for all communications with the
authorities concerning the study.
Dr. Nay Yi Yi Linn
NMCP
Assistant
Director
Assist Program Manager of NMCP in critically
reviewing the paper
Dr. Zay Min Tun
NMCP
Medical
Officer
Assist Program Manager of NMCP
Dr. Feliciano Monti
PMI
Senior Malaria
Advisor
Provide guidance for administrative and regulatory
adequacy of study conduct; Preparation of study
protocol and related documents
Dr. Jimee Hwang
CDC
Medical
Epidemiologist
Provide guidance for scientific and technical
adequacy of study design, preparation and conduct,
and for study data analysis and interpretation
Dr. Nelli
Westercamp
CDC
Epidemiologist

hsRDT reactive case detection
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Dr. Khin Than Win
URC
Defeat
Malaria
Co-investigator
Oversee and ensure technical adequacy of the study
team and responsible for overall accomplishment of
the study by advocacy to responsible persons at all
level, seeking approval from Department of Public
Health, engage the research team with community,
arrangement of specimen for proper storage and
transport.
Dr. Kyaw Myint
Tun
URC
Defeat
Malaria
Co-investigator
Support successful submission of protocol to Ethics
Review Committee, recruit and organize survey
team, plan and facilitate necessary trainings for
implementation
of
operational
research,
site
selection
for
study
villages,
monitoring
and
supervision of survey activities, ensure data quality
for analysis, and data validation.
Dr. Kay Thwe Han
DMR
Co-investigator
Oversees and lead all aspects of lab work including
sample management; Co-train with UMB the study
teams; Provide research and lab support for study
team
Dr. Myaing Myaing
Nyunt
IGH-UMB
Co-investigator
Responsible for all communication and
coordination between UMB, URC and DMR;
Design and prepare study protocol and related
documents; Assist NMCP and URC for admin and
ethical approval; Ensure timely and adequate
preparation and completion of laboratory work; data
analysis, interpretation and manuscript preparation;
Assist NMCP and URC for study related matter,
data dissemination.
Dr. Chris Plowe
IGH-UMB
Supervising
investigator/
Molecular
Epidemiologist
Provide overall administrative and technical expert
opinion; support for study epidemiologic design,
molecular aspect of study conduct and analyses;
statistical consideration, lab analysis, data
interpretation and dissemination
Matt Adams
IGH-UMB
Lab supervisor
Responsible for readiness of all field and lab
supplies and equipment & Quality control &
assurance; Train lab team; Oversees lab analysis;
problem solves and act according to results and
needs; Train, manage and supervise lab data
analysis; Ensure timely completion of lab work and
data dissemination

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Dr. Janie Zuber
IGH-UMB
Infectious
Disease
Research
fellow
Assist protocol development, study planning and
conduct, data organization, analysis and
interpretation; Manuscript preparation; Work under
Nyunt’s supervision
Dr. Yuanyuan Liang
IGH-UMB
Statistician
Responsible for sampling and statistical procedures,
analysis, interpretation of results and formulation of
recommendations. Advice on study design, data
analysis and interpretation; manuscript preparation


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
%DFNJURXQGLQIRUPDWLRQDQGVFLHQWLILFUDWLRQDOH


%DFNJURXQGDQGLQWURGXFWLRQ
Myanmar, along with several other countries in the Grater Mekong Sub-region (GMS), is
committed to eliminate malaria by 2030.
In the National Plan for Malaria Elimination in
Myanmar (2016–2030), the NMCP recommended that standard operating procedures (SOPs)
for malaria active case detection (ACD) should be developed, and that the role of rapid
diagnostic tests (RDTs), microscopy, and PCR-based techniques in ACD at different levels of
the transmission-reduction and elimination phases be evaluated.
Recent epidemiological data indicates that 30-50% of all malaria infections exist in a form of
asymptomatic infection (also known as “chronic malaria”) where the parasite density is
generally below the limit of detection of all point-of-care diagnostic tests [1]. The requirement
for more sensitive diagnostic tests for malaria is predicated on the idea that malaria elimination
may require eradicating all parasites from all infected persons, not just those with clinical
symptoms [2]. “Asymptomatic”—or more accurately, VXEFOLQLFDO malaria—has long been
recognized in semi-immune Africans [3], but not in Southeast Asia where malaria transmission
is generally lower. Many believed that subclinical infections are unlikely in low transmission
settings, partly due to a long held dogma that partial immunity does not develop to allow for
such low grade infections, and partly to the lack of detection methods adequately sensitive to
identify very low parasite density typically found in the GMS [4-6].
New “ultrasensitive” PCR-based tests, with a lower limit of detection 16–22 parasites/mL [4,
7-9], have recently found unexpectedly high rates of subclinical malaria in Southeast Asia.
These infections are missed by standard diagnostics such as microscopy or conventional RDT
which have a lower limit of detection about 100 parasites/uL. Though the PCR is adequately
sensitive to detect these low-density infections, its utility is quite severely limited by its
complexity, requirement for advanced skills and infrastructure, cost, and by the fact that it is
not a point-of-care test. Today’s malaria elimination toolkit essentially consists of prompt
diagnosis and treatment using rapid diagnostic tests (RDTs) and artemisinin-based
combination treatments (ACTs), vector control methods, and surveillance using RDTs and/or
microscopy. The available diagnostic tests are inadequate to detect these low-density malaria
infections, and new tools are urgently needed.
Three studies, a survey, and a systematic review assessing the role, appropriateness and
benefits of the ACD strategy, both proactive and reactive, in low transmission settings have
been recently conducted in Cambodia, Indonesia, Thailand, India and other Asian-Pacific
countries [10-12]. The data clearly indicated that the ACD strategy needs to be optimized based
on the local context, and on evidence for the adoption of improved methods. The need for
better, cheaper and more accurate diagnostic tools, with an increased capacity to detect low
density malaria infections, was highlighted in recent literature.
To fill this critical gap, a new hsRDT that has a potential to be at least 10- to 20-fold more
sensitive for detecting low density infections than standard cRDT was developed (Standard
Diagnostics Bioline Inc. Gyeonggi-do, Republic of Korea), and is now under field evaluation
in several malaria endemic countries. This test is similar in design to cRDT, using a handheld
point-of-care laminar flow test to detect low levels of the 3IDOFLSDUXPantigen Histidine Rich
Protein 2 (HRP2), which is also one of the detection targets of cRDT. Initial prototype testing
of the new hsRDT designed to detect the 3IDOFLSDUXP-specific antigen +53 in a format

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similar to the current cRDT resulted in a limit of detection (LOD) of +53 of about 80 pg/mL
compared to cRDTs with a LOD of 800–1000 pg/mL (personal communication, S. Allauzen,
Diagnostics & Global Health, Bill & Melinda Gates Foundation).
We propose to evaluate the role of this new hsRDT in the setting of reactive case detection
(RCD) in specified townships where malaria prevalence has been steadily declining. Our
primary goal is to assess whether or not this new hsRDT performs significantly better than
cRDT in identifying subclinical submicroscopic infection, and to subsequently evaluate if
hsRDT can be used for effective ACD in Myanmar. The proposed study may shed light on the
development of national standard operating procedures (SOPs) for ACD as recommended by
the NMCP. The development of these SOPs are ongoing, and our study may provide timely
and relevant contribution for their development, particularly assessing the detection accuracy
and utility of the new hsRDTs when used as a screening tool in the RCD strategy. Additional
useful information can be obtained on the applicability, cost, and operational challenges faced
during the field implementation.
The proposed study also addresses PMI elimination or priority to “Evaluate the utility and
scalability of more sensitive diagnostic tools (e.g. highly-sensitive RDTs, PCR, etc.) in an
elimination context, including their utility for reactive or proactive case detection.” It will also
contribute to current PMI research activities comparing RCD with RDTs to focal Mass Drug
Administration (MDA) planned for Madagascar and Ethiopia. The Madagascar study will also
include the hsRDT in their RCD arm. If the test of this hsRDT in detecting additional malaria
infections around index cases shows good field performance, further trials can be planned in
Myanmar to evaluate its role in malaria elimination.

5DWLRQDOHIRUWKHVWXG\
Preliminary data in the development of a new hsRDT indicated that this hsRDT has a
significantly higher sensitivity than cRDT and will perform better in detecting low density
malaria infections (Personal communication; Jonathan Cox, Bill and Melinda Gates
Foundation), and we anticipate that it may play a critical role in reactive case detection strategy.
The current tools such as microscopy or cRDT are insufficient in identifying these infections
and missing them may pose a great drawback for the progress of preparation for malaria
elimination. In addition to its high sensitivity, hsRDT is a point of care test, therefore it is
convenient for large scale testing and sustainable in resource-limited settings. This tool is
urgently needed in the current surveillance activities in Myanmar during the time of preparation
for the pre-elimination and subsequent elimination phase. Findings from the proposed study
will also contribute useful data in the development of national standard operating procedures
for ACD with potential value of the new hsRDTs as a screening tool in the RCD strategy.

3RWHQWLDO5LVNVDQG%HQHILWV
3.3.1. Potential risks
(IIHFWVRIILQJHUVWLFNV. Very small risks of bruising, bleeding, infection and fainting
can accompany these procedures. Study-related blood collection for malaria diagnostic
testing (blood smear on microscope slides or rapid diagnostic test) and dried blood spot
sample can be accomplished by using finger-stick.
3RWHQWLDO ULVNV WR VWXG\ SHUVRQQHO. The main risks to study personnel are from
accidental exposure to blood and body fluid borne infections. SOPs for staff safety are

hsRDT reactive case detection
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used in clinical and laboratory areas, including sharps management, hazardous waste
management, etc. Universal precautions are used for handling all body fluids.
Because the described analyses pose minimal appreciable additional risks to the trial
participants but present potential benefits to their communities and others living in
malaria endemic areas, the risk-to-benefit ratio is very low.
3.3.2. Known potential benefits
Participants may not receive any direct benefit from their participation in the study.
However, if symptoms of other illnesses are noted, participants will be referred for
evaluation and treatment at the appropriate local clinic.
If malaria parasites were identified by cRDT or hsRDT, participants will receive
appropriate anti-malaria treatment promptly and potential transmission will be
interrupted, benefiting the community. The benefits to the community could include
improved tools for surveillance and diagnosis of malaria. Results from this study may
help malaria control programs improve understanding the degree and distribution of
“silent” reservoir that cannot be readily detected using current diagnostic tests.
 
6WXG\REMHFWLYHV

*HQHUDOREMHFWLYH To evaluate the performance of the highly-sensitive rapid diagnostic
test (hsRDT), developed by SD Bioline, versus conventional RDT (cRDT), compared
with PCR as the gold standard, in reactive case detection to identify additional malaria
infections in Rakhine State, Myanmar
6SHFLILFREMHFWLYHV:
1. To evaluate the prevalence of malaria infections identified by the new hsRDT in
comparison with that detected by cRDT and PCR
Outcomes: malaria test positivity rate by cRDT, hsRDT and PCR, respectively
2. To assess the diagnostic performance characteristics of hsRDT YHUVXV cRDT, using
PCR as gold standard, in the detection of 3IDOFLSDUXP infections
Outcomes: Sensitivity, specificity, positive and negative predictive values of hsRDT
and cRDT
3. To evaluate correlation of detection capability between cRDT and hsRDT
Outcomes: Correlation of test positivity rate by the three different study tests
4. To identify risk factors associated with malaria infection, including but not limited to,
socio-demographic factors and travel history related with malaria index cases
Outcome: Relative risk of malaria in association with different risk factors identified
 0HWKRGRORJ\




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
6WXG\'HVLJQ	2YHUYLHZ
This is a prospective community-based single-center reactive case detection (RCD) study to
assess the performance of hsRDT versus cRDT in identifying individuals with malaria
infection (“Secondary case”) in a population living and/or working in a close physical
proximity to an “index case.”
“Index case” is defined as a malaria infection identified by passive surveillance by local
health services (rural health center, sub-centers) or village malaria workers (VMWs), by
cRDT or microscopy.
“Secondary case” is defined as any malaria infection identified by any of the study diagnostic
test: cRDT, hsRDT or PCR, regardless of the presence of acute clinical illness. The
secondary cases will be identified actively and systematically around the residence, or work
place if relevant, of the index case.
The study will be initiated after obtaining administrative (Myanmar MoHS, NMCP, PMI)
and ethical approval (Myanmar DMR ERC, CDC IRB and UMB IRB) from all relevant
institutions. Consent, and assent and adult permission when appropriate, will be sought for all
index cases and secondary cases. Only those who are eligible and provide informed consent
will be enrolled in the study.
All index cases parasitologically confirmed by cRDT or microscopy will be appropriately
and promptly notified to the study team, using mobile phone technology where possible, to
ensure timely enrollment. The home of the index case, or work place when relevant, will be
visited within three days of the diagnosis, and s/he will be interviewed with a standardized
case investigation form. During the visit, all members of the index case household and all
members of the nearest 10 households, aged 5 years or above, will be invited to participate in
the study. All participants in the study will be interviewed with a structured questionnaire,
tested with cRDT and hsRDT, and a dried blood spot (DBS) will be collected for subsequent
PCR analysis. All cases parasitologically confirmed by cRDT or hsRDT will be appropriately
treated, following the national treatment guideline. DBS samples will be collected, processed
and stored until analysis following the World Wide Antimalarial Resistance (WWARN)
Molecular Testing for Malaria Standard Operating Procedures DBS Sample Collection and
Transportation (http://www.wwarn.org/learning/procedures/dbs-sample-collection-and-
transportation).
The prevalence of secondary malaria cases (number positive /number tested), by cRDT and
hsRDT will be compared, using PCR as gold standard. Using simple analysis of performance
characteristics, the sensitivity and specificity of hsRDT will be estimated in the study
population. Potential correlation between the detection rate by cRDT, hsRDT and PCR will be
assessed.

6WXG\3HULRG
The study will be conducted from June, 2017 to May, 2018, after obtaining administrative and
ethical approval from appropriate authorities. 

6WXG\$UHD
The proposed study will be conducted in the village tract of Sakhanmaw, in Ann township in
South Rakhine State. This village tract encompasses 63 villages located in a hilly forested area

hsRDT reactive case detection
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with small rivers and valleys, with a total population of about 23,000 residents. Sakhanmaw
village tract is covered by one rural health center (RHC) and seven sub-Centers. In addition to
basic health services, malaria volunteers conduct malaria case detection and management in 52
villages. The volunteers are trained by National Malaria Control Program, University of
Research Cooperation and Myanmar Medical Association.
Of the 63 villages, the research study will be carried out in about 35 villages with low malaria
transmission with an annual parasite incidence (API) less than 5 per 1,000 population at risk.
Based on the epidemiological data collected during 2016, the test positivity rate in the study
villages ranged from 0% to 13%, and more than 80% of the malaria infections were caused by
3IDOFLSDUXP. The primary malaria vectors are $QRSKHOHVPLQLPXV and $QGLUXV.

6WXG\SRSXODWLRQ
All adult and children at least 5 years old in the study villages under Sakhamaw RHC will be
recruited and enrolled in the study.
,QFOXVLRQFULWHULD
- Age at least 5 years old
- Resident of the villages, or temporary visitors, or co-workers or co-travelers of the
index case
- Willingness to participate in the study evident by informed consent
([FOXVLRQFULWHULD
- Presence of severe clinical illness including severe malaria
- Non-resident index cases
- Refusal to participate in the study
Initial meetings will be organized with all partners involved in the research (NMCP, VBDC
Rakhine, DMR, Defeat Malaria partners, local authorities and community leaders) to clarify
the scope of the study, and prompt their active collaboration.
Additionally, specific information and sensitization activities will be organized for the
communities involved in the study to explain the nature of the research, address possible
concerns, and encourage their motivated participation.

5HFUXLWPHQW 3URFHGXUHV ,GHQWLILFDWLRQ LQGH[ FDVHV DQG VHOHFWLRQ RI VHFRQGDU\
FDVHV
Suspected malaria cases attending local health services (rural health center and sub-centers) or
village malaria workers (VMW) will be tested with a cRDT as in routine practice. All cases
parasitologically confirmed by cRDT will be treated according to national guidelines, and
asked for informed consent to participate in the study. If the patient gives informed consent,
the VMW or basic health staffs will promptly notify the study team, using mobile phone.
Additionally, finger prick blood will be collected from the index case to test with hsRDT, and
on dry blood spot (DBS) for later PCR analyses.
The study team will conduct the investigation visit at the residence of the index case within 3
days, and the index cases will be interviewed using a standard case investigation form (Annex
1). All members of the index case’s household will be invited to participate in the study and

hsRDT reactive case detection
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interviewed using the participants’ questionnaires (Annex 2) to assess a history of malaria in
previous year, travel information, and utilization of malaria prevention measures. The nearest
ten households around the index case’s household will be identified by using household listing.
All members, including temporary visitors, of these ten households will be invited to
participate in the study and interviewed using the participants’ questionnaires (Annex 2), tested
with cRDT and hsRDT, and a DBS will be collected for subsequent analysis. If the index case
reports traveling to or working in the forest or other risk areas, co-travelers or co-workers will
be also recruited in the study, when feasible.
Study participants who tested positive on cRDT or hsRDT will be treated according to national
guidelines, in compliance with WHO recommendations and NMCP policies, which call for
treatment of any RDT-positive infection. Although the diagnostic performance of hsRDT is
under investigation, there is enough evidence that cRDT fails to detect low density infections,
therefore treatment of hsRDT-positive malaria is conservative and ethically justifiable.
PCR results will not be available immediately, and there are no recommendations for
retrospective malaria treatment based on ultrasensitive testing. Those who are ill but found to
be negative on RDTs will be provided with information to seek further care and referral to an
appropriate medical professional will be made.
Study team will be equipped with basic
medical supplies and provide as much care as possible.


6HHNLQJ,QIRUPHG&RQVHQW$VVHQWIURPSRWHQWLDOSDUWLFLSDQWV
Prior to the study data and sample collection, preliminary discussion meetings will be
organized with all partners involved in the research (NMCP, VBDC Rakhine, DMR, Defeat
Malaria partners, local authorities and community leaders) to explain the nature and scope of
the study, and prompt their active collaboration. Community outreach activities will be
conducted to ensure the community response and study participation. Study time line will be
disseminated to the targeted communities in a locally appropriate manner.
The principles of research ethics in the current edition of the Declaration of Helsinki will be
applied, and protocol-specified procedures will be initiated only after obtaining informed
consent. 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.
Information about the study will be given to prospective participants, in a simple lay language
both in Myanmar and Rakhine, in both oral and written forms whenever possible. Independent
witnesses will be used to attest that illiterate potential participants have understood the contents
of the informed consent document.
The UMB investigators and staff will closely work with the NMCP and other relevant local
research staff at the study sites, providing training, and re-training if necessary, in Protection
of human subjects, Good Clinical Practice, and responsible conducts of research to assure study
information and informed consent procedures meet requirements of the community, sponsor
and all relevant ethic review oversight committees. UMB investigators have published detailed
descriptions of the processes that have been used to obtain community “permission to enter”
and individual informed consent [42]. We consider informed consent to be a dynamic, ongoing

hsRDT reactive case detection
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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. Even though subject participation will be brief for this study, these same principles
will be followed.
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.
In Myanmar, individuals who are at least 18 years old are considered adults and will provide
consent for themselves; children 6 months to ≤7 years of age are too young to provide assent;
however, written consent will be provided by their guardian; children 8 to ≤12 years of age
will provide verbal assent, in addition to written consent provided by their guardian; children
13 to ≤17 years of age will provide written assent, in addition to written consent provided by
their guardian. In rural Myanmar 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.
5.7.
'DWDDQG%ORRG6DPSOH&ROOHFWLRQ
Participant will be given a unique study identification number which, instead of the
participant’s name or other personal identification, will be used for his/her case record forms,
other study related documents and for labelling the study samples. This will minimize a
potential loss of confidentiality for the participant.
A trained study personnel will clean with antiseptic, and prick the tip of the participant’s finger
(or the heel if and when appropriate for children 6–12 months old) using a lancet, and collect
5-7 drops of blood (a quarter of teaspoon). Two to three drops will be used to check for malaria
by cRDT and hsRDT, and 3-4 drops will be collected on to filter papers for malaria PCR
testing. The filter paper will be placed at the prick site on the finger to absorb the blood. For
infants 6 months to one years of age, a heel prick might be more appropriate.
The PCR analysis will be carried out at the DMR in Yangon, with technical assistance and
quality management from the University of Maryland. Additional molecular, antigen and
antibody-based testing for malaria may be conducted depending availability of funding. The
DBS, packed in individual plastic bags with desiccant, will be appropriately stored at Defeat
Malaria’s field office in Ann Township and sent to DMR in Yangon every 2-3 weeks.
 6DPSOH0DQDJHPHQWDQG/DERUDWRU\(YDOXDWLRQ

6SHFLPHQ&ROOHFWLRQ3UHSDUDWLRQ+DQGOLQJDQG6KLSSLQJ
DBS specimens will be collected using standard methods (see
http://www.wwarn.org/learning/procedures/dbs-sample-collection-and-transportation). DBS
samples will be labeled with participant unique ID. No names and other personal identifiers
will be recorded. After collection, DBS will be air dried and stored in zip-lock plastic bags
with desiccant at each site (see Appendix B: Molecular Testing for Malaria Standard
Operating Procedures DBS Sample Collection and Transportation) at +4 ºC, -20 ºC or room
temperature in a cool, dry, dark location.

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
3&5DVVD\IURPGULHGEORRGVSRW
DNA will be extracted dried blood spots using a validated new extraction method developed
by our group (Zainabadi et al. “A dried blood spot based ultrasensitive detection method for
asymptomatic malaria” Oral presentation at the 57the annual meeting of American Society of
Tropical medicine and Hygiene, November 2016). As much of the molecular analyses as
possible will be done at the Advanced Molecular Research Center of the Department of
Medical Research, under the Myanmar Ministry of Health. Extracted DNA will be subjected
to ultra-sensitive qPCR using well-established techniques that currently have a lower limit of
detection of 22 parasites/mL (9). PCR will be done in Myanmar with some duplicate samples
analyzed at the University of Maryland for quality control purposes.
Future studies of parasite DNA derived from samples may be conducted without restriction.
1RRWKHUVWXGLHVRQDQ\WRSLFVEH\RQGZKDWLVGHVFULEHGLQWKLVSURWRFRODQGFRQVHQWDVVHQW
IRUPVRURQKXPDQ'1$ZLOOEHGRQHXQOHVVVSHFLILFSHUPLVVLRQIRUDGGLWLRQDOVWXGLHVLV
REWDLQHGIURPWKHUHOHYDQW,5%V Study participants will have the right to withdraw their
permission for further use of their samples at any time during and after the study.
Samples will be batched and shipped periodically to the DMR laboratory and/or the
laboratories at the University of Maryland in Baltimore or CDC-Atlanta. DBS specimens will
be shipped at room temperature and desiccant will be replaced upon receipt. Specimen receipt
will be logged using a system designed at UMB using the Freezer works program. The
laboratory contact at DMR is Dr. Kay Thwe Han, E-mail: drkaythwehan@yahoo.com. The
laboratory
contact
at
UMB
is
the
lab
supervisor
Matthew
Adams
E-mail:
madams@som.umaryland.edu.
 6WDWLVWLFDOFRQVLGHUDWLRQDQG'DWD0DQDJHPHQW

3RZHUFDOFXODWLRQZLWKDYDLODEOHVDPSOHVL]H
The primary limitation is the size of population that needs to be screened since malaria burden
may be low in the study sites, and associated financial costs and logistical difficulties.
The primary endpoint of this proposed study is the prevalence of secondary malaria cases
identified by hsRDT in comparison with usPCR prevalence. Based on our current data on
prevalence of 3IDOFLSDXP in 2016, and logistical and financial considerations, the recruitment
of 50 index cases is feasible in a given study period. This will give us a total of 1980 individuals
for testing of secondary cases in ACD, assuming the average household member of 4.5, 11
households for each index case (counting the household of the index case) and a refusal rate of
20% (4.5 persons/HH x 11 HH x 50 index cases x 0.80=1980).
We conservatively estimate that hsRDT will detect 20% of usPCR+ 3IDOFLSDUXP infections
(i.e., sensitivity of hsRDT=0.2 using usPCR as gold standard). We expect very few recently
treated individuals to be enrolled in the study, so that hsRDT should only rarely give a
positive result when usPCR does not. Hence we assume that the specificity of hsRDT is
0.995. Table 1 shows estimated statistical power that can be achieved with the proposed
sample size of 1980 for comparing the prevalence by hsRDT and the prevalence by usPCR,
at different malaria prevalence by usPCR and different sensitivity of hsRDT, using a two-
sided McNemar test with a Type I error of 5%. (PASS version 11. NCSS, LLC. Kaysville,
Utah, USA)

hsRDT reactive case detection
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As shown in Table 1, if the sensitivity of hsRDT is 0.2 and the specificity of hsRDT is 0.995,
a sample size of 1980 subjects achieves at least 92% power to detect the difference in
prevalence between hsRDT and usPCR when the malaria prevalence is 2% or higher. If the
sensitivity of hsRDT is 0.5 (see the last four columns of Table 1), a sample size of 1980
subjects achieves at least 86% power when the malaria prevalence is 3% or higher. If the
prevalence of malaria is higher, the performance of hsRDT will be better with a higher
sensitivity and positive predictive value.


7DEOH6WDWLVWLFDOSRZHUH[SHFWHGZLWKSURSRVHGVDPSOHVL]HRIDWGLIIHUHQW
PDODULD
SUHYDOHQFHDQGGLIIHUHQWVHQVLWLYLW\RIKV5'7DVVXPHGVHQVLWLYLW\RIRUDQG
VSHFLILFLW\

Prevalence
by usPCR
Sensitivity
of hsRDT
Specificity
of hsRDT
Estimated
prevalence
by hsRDT
Power
Sensitivity
of hsRDT
Specificity
of hsRDT
Estimated
prevalence
by hsRDT
Power
0.02
0.2
0.995
0.009
0.92
0.5
0.995
0.015
0.45
0.03
0.2
0.995
0.011
>0.99
0.5
0.995
0.020
0.86
0.06
0.2
0.995
0.017
>0.99
0.5
0.995
0.035
>0.99
0.12
0.2
0.995
0.029
>0.99
0.5
0.995
0.065
>0.99

Table 2 below shows changes in the power of the study, if the specificity of hsRDT is 0.95
(increased false positive by hsRDT), while the sensitivity of hsRDT and baseline true
prevalence remain the same.
7DEOH6WDWLVWLFDOSRZHUH[SHFWHGZLWKSURSRVHGVDPSOHVL]HRIDWGLIIHUHQW
PDODULD
SUHYDOHQFHDQGGLIIHUHQWVHQVLWLYLW\RIKV5'7DVVXPHGVHQVLWLYLW\RIRUDQG
VSHFLILFLW\

Prevalence
by usPCR
Sensitivity
of hsRDT
Specificity
of hsRDT
Estimated
prevalence
by hsRDT
Power
Sensitivity
of hsRDT
Specificity
of hsRDT
Estimated
prevalence
by hsRDT
Power
0.02
0.2
0.95
0.009
0.053
0.5
0.95
0.059
>0.99
0.03
0.2
0.95
0.011
0.055
0.5
0.95
0.064
>0.99
0.06
0.2
0.95
0.017
0.059
0.5
0.95
0.077
0.79
0.12
0.2
0.95
0.029
0.068
0.5
0.95
0.104
0.61




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
$QDO\WLFDO3ODQ
All data will be double-entered into Epi-Info and any discrepancies will be reviewed against
the paper forms. All analyses will be conducted using Stata/SE (version 14), using data from
the participant interviews and the three parasitological tests.
The malaria test positivity rate will be calculated as a proportion of samples tested that are
positive using cRDT, hsRDT and PCR, respectively. Corresponding 95% confidence
intervals (CIs) will be computed using exact method. A 2x2 table method will be used to
calculate the sensitivity, specificity, positive and negative predictive values of hsRDT (and
cRDT) and their corresponding 95% CIs using PCR as gold standard. The malaria prevalence
by each test (e.g., the rate of malaria test positive) will be compared between each two tests
(hsRDT vs PCR; cRDT vs PCR; hsRDT vs cRDT) using McNemar test. All statistical tests
will be performed with a two-sided significance level of 0.05.
Descriptive analysis will be conducted to assess socio-demographics characteristics.
Univariate analysis will include t-test and non-parametric methods for continuous variables
and chi-square test for comparison of categorical variables. Multi-level regression models
will be developed to examine risk factors significantly associated with malaria infections,
accounting for clustering by index case.

'DWD0DQDJHPHQW
The principal investigator will ensure that the study protocol is strictly adhered to and that all
data are collected and recorded correctly on the data collection forms and logs. Any change
or correction to a form should be dated and explained and should not obscure the original
entry. All forms will be checked for completeness. The paper forms will be kept in a locked
file cabinet when not in use.

'DWD&ROOHFWLRQ0HWKRG
- Record review of malaria case registers of village malaria workers and health
facilities
- Face to face interview of index case, all members of index case household members
and all members of nearby 10 households
- Finger prick blood sample collection for cRDT, hsRDT, DBS collection of all
participants including short face to face interview to be recorded on the household
screening form

'DWD&ROOHFWLRQ7RRO
- The routine malaria surveillance data from the study villages will be collected from
malaria patient register used by volunteers or basic health staffs.
- Index case will be interviewed and recorded by respective VMWs/BHS using case
investigation form (Annex 1) and household listing and participant questionnaire form
(Annex 2).
- All the index household members and nearby household members will be interviewed
and recorded by respective VMWs/BHS using household listing and participant
questionnaire form (Annex II).
- Information of results of the conventional RDT and hsRDT screening as well as
personal risk factors such as travel history, forest related work, use of malaria
prevention tools, etc. will be collected from all recruited household members.

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- Original data collection forms or database will be handled only by staff members and
kept under locked storage until completely coded, checked and transported for data
entry. Once data entry and cleaning are completed the original forms will be stored
under lock and key at the Defeat Malaria Office until final analyses and reports have
been prepared. They will then be destroyed.

4XDOLW\&RQWURO

7UDLQLQJDQGVXSHUYLVLRQRIILHOGWHDPVDQGTXDOLW\PDQDJHPHQW
A field research coordinator (FRC) will be dedicated to ongoing supervision and
monitoring of study implementation. Clinical procedures and data collection will be
evaluated regularly by the FRC. Other quality control (QC) measures will include
daily review of patient records, observation of interviews and clinical procedures,
adherence to the approved protocol and Standard Operating Procedures (SOPs) and
ongoing evaluation of malaria laboratory procedures according to standardized
checklists. Refresher training will be provided as necessary. The PI will lead the
preparation of the study together with the field team, prior to implementation, and
supervise and support the field team throughout the study. Regular visits will be
scheduled every 3 months and make additional support visits as needed.
The UMB-DMR collaborative team will provide training, and re-training if necessary,
prior to the study initiation. Monitoring of the data and sample quality will be ongoing
throughout the study, while study procedures are carried out and when the samples
are delivered. Feedback, and re-training as needed, will be provided based on the
observed quality of the data and sample.

'LDJQRVWLFVFRQYHQWLRQDODQGKLJKO\VHQVLWLYHPDODULD5'7
Routine quality control procedures will include close monitoring and supervision of
RDT performance against standardized monitoring checklists on a monthly basis.

4XDOLW\PDQDJHPHQWRIGDWDDQGVDPSOHV
Established procedures for monitoring and documenting the quality of both clinical
and molecular data will be followed, using a protocol-specific Data Monitoring Plan.
This plan will include monitoring of source documents and a subset of case record
forms, systematic sliding scale evaluation of study documents, systematic assessment
of the quality of DBS specimens, and an external quality assurance exercise organized
by the laboratories at the University of Maryland in Baltimore to validate the results
of molecular assays done at in-country laboratories.

(WKLFDO&RQVLGHUDWLRQV
The study will be performed in accordance with ethical principles based on Declaration of
Helsinki and International Conference on Harmonisation (ICH)/Good Clinical Practice
(GCP) and all applicable regulatory requirements.

,QIRUPHG&RQVHQW$VVHQW
The principles of research ethics in the current edition of the Declaration of Helsinki will be
applied, and protocol-specified procedures will be initiated only after obtaining informed
consent. The written consent documents will embody the elements of informed consent as

hsRDT reactive case detection
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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.
Information about the study will be given to prospective participants, in a simple lay
language both in Myanmar and Rakhine, in both oral and written forms whenever possible.
Independent witnesses will be used to attest that illiterate potential participants have
understood the contents of the informed consent document.
All informed consent, assent and adult guardian permission will be seek in local language.
The written forms will be translated in to the respective local languages (Myanmar) and then
translated back to English by a second translator to verify accuracy of information or certified
by an official translator. These will be provided to participants and/or their caretakers in their
respective local language. If a participant or parent/guardian is illiterate, the consent form
will be read to them in their respective local language and a thumbprint will be accepted as a
legally effective signature. In such situation a witness signature will be sought. Consenting
participants and/or their caretakers will be advised that they are free to decline any question
or procedure and that they may terminate their participation at any time.
All adult participants (18 years of age and above) will be asked to provide informed consent.
Children 13 to ≤17 years of age will be asked to provide written assent, in addition to written
consent provided by their guardian.
Children 8 to ≤12 years of age will be asked to provide verbal assent, in addition to written
consent provided by their guardian.
For children aged 5 to ≤7 years of age, adult guardian permission will be obtained.
The study investigators and staff will closely work with the NMCP and other relevant local
research staff at the study sites, providing training, and re-training if necessary, in Protection
of human subjects, Good Clinical Practice, and responsible conducts of research to assure
study information and informed consent procedures meet requirements of the community,
sponsor and all relevant ethic review oversight committees. UMB investigators have
published detailed descriptions of the processes that have been used to obtain community
“permission to enter” and individual informed consent [42]. During the consent process,
emphasis will be made to ensure that the participant understand that the study participation is
entirely voluntary, refusal to participate will not have any negative consequences or
repercussion and free to withdraw from the study at any time. 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. Even though subject participation will be
brief for this study, these same principles will be followed.

&RQILGHQWLDOLW\
Participant’s 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.

hsRDT reactive case detection
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Study records including case record forms and consent forms will be stored in locked
cabinets in secure facilities only accessible to authorized investigators.
The data generated from each study sample will be linked to the coded, unique identifier
provided by study investigators on the original sample container, as well as any relevant
information such as parasite counts. Study data will be stored on password protected
computers. Data from this study may be submitted to publicly available databases if and
when appropriate, but will not be linked to any identifying information from study
participants. Participants will not be identified in any publications resulting from the study.

9XOQHUDEOH3RSXODWLRQV
Children and other (those living in poverty, limited education or poor literacy), considered
vulnerable, will be enrolled in the study. This is justified since all are at risk for malaria, all
must be included in malaria surveillance, and children may have a different parasitological
profile than adults. The study team will undergo proper training in human subject protection
and good clinical practice to understand ethical issues related to conducting research in
vulnerable populations.
No justification for waiver or alteration of informed consent is being sought.

3RWHQWLDO5LVNVDQG%HQHILWVDQG3URFHGXUHVWR0LQLPL]H5LVNV
5LVNVThere are risks of bruising, bleeding, infection and fainting from blood collection, of
potential loss of confidential and private information. There are also risks to study personnel
such as accidental exposure to blood and body fluid borne infections. We will take proper
procedures to minimize all the risk. We will provide protocol-specific didactic and hands-on
training including good clinical practice and human subject protection prior to the study
initiation, careful evaluation during the early stage of the study, and re-training based on our
evaluation as needed. Standard Operating Procedures (SOPs) for staff safety are used in
clinical and laboratory areas, including sharps management, hazardous waste management,
universal precautions are used for handling all body fluids. Since no sensitive information
will be collected, social or economic risks are considered low.
%HQHILWVParticipants may not directly benefit from their participation in the study. However,
if symptoms of malaria or other illnesses are noted participants, they will be referred for
evaluation and for prompt treatment at the appropriate local clinic. There will be benefits to
the community could include improved tools for surveillance and diagnosis of antimalarial
drug resistant malaria. The finding of the study will be an important input for the national
malaria control program in Myanmar as they develop the strategies to conduct RCD.
We believe that the study poses minimal appreciable additional risks to the study participants,
but may provide potential benefits to their communities and others living in malaria endemic
areas, and the risk-to-benefit ratio is acceptable and justifiable to conduct this study.

&RQIOLFWRILQWHUHVW
The study team members have been trained on the conflict of interest, and none of the study
team members has declared any financial or other conflicts.


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
&RRSHUDWLRQZLWKQDWLRQDODQGORFDOSDUWQHUV
We have been working with and will continue to seek guidance from the Myanmar national,
regional and township administrative and technical leadership including, but not limited to,
the regional and township administration, Ministry of Health and Sports, Vector-borne
Disease Control, National Malaria Control Program, Township Medical Services and related
networks. We will seek appropriate administrative and ethical approval from all relevant
review committees in Myanmar and in the USA before the initiation of the study. We will
hold initial meetings led by the NMCP and organized with all partners involved in the
research (NMCP, VBDC Rakhine, DMR, Defeat Malaria partners, local authorities and
community leaders) to explain the nature and scope of the study, and prompt their active
collaboration, and maintain sufficient communication and information flow throughout the
study.

5HVXOW'LVVHPLQDWLRQ'DWD3XEOLFDWLRQDQG$XWKRUVKLS
After the completion of all data analysis, preliminary data will be presented and discussed
with the relevant Myanmar leadership, VBDC, NMCP, Defeat Malaria partners and the
development partner. A final dissemination workshop will be organized to present and
discuss the results to a large audience involving all national and international organizations
involved in malaria control and elimination in Myanmar.
Findings and data will be used to indicate the possible role of this hsRDT in the RCD strategy
during the elimination phase, and in the development of national SOPs on active case
detection. Publication of the results in a peer-reviewed journal will be considered.
Data generated from this study are highly valuable for the whole public health community,
particularly for those deeply involved in malaria elimination, and timely and prompt
dissemination of data (before peer-review publication which may take a long time) is critical.
After proper review, discussion and getting approval from the country’s leadership and the
development partner, we will share the data in several venues (USAID/PMI databases, web-
based media) with known good accessibility by a large public, as well as with international
agencies such as WHO and USAID/CDC. Together with NMCP, URC and DMR, the UMB
team will provide a lead role in data analysis, interpretation and preparation and publication
of manuscript, and presentation of data in international public health arena such as the annual
meeting of American Society of Tropical Medicine and Hygiene. Authorship will be
discussed and decided collectively based on the multiple means of contribution to the success
of the study.



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7LPHOLQH
$FWLYLW\


May
June
July
Aug
Sept
Oct
Nov
Dec
Jan
Feb
Mar
Apr
Proposal development
Ethical submission
Training/advocacy/Pilot
testing
Data collection
Data entry/ analysis
Report writing
Dissemination of results



5()(5(1&(6
1. 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:e1001942.
2. malERA Consultative Group on Drugs: A research agenda for malaria eradication: drugs.
PLoS Med 2011, 8:e1000402.
3. Stephens JWW, Christophers MB: The Malarial Infection of Native Children. In Reports
to the Malaria Committee of The Royal Society. pp. 2-14: The Royal Society; 1900:2-14.
4. 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, 14:520.
5. Cheng Z, Wang D, Tian X, Sun Y, Sun X, Xiao N, Zheng Z: Capture and Ligation Probe-
PCR (CLIP-PCR) for Molecular Screening, with Application to Active Malaria
Surveillance for Elimination. Clin Chem 2015, 61:821-828.
6. Okell LC, Ghani AC, Lyons E, Drakeley CJ: Submicroscopic infection in Plasmodium
falciparum-endemic populations: a systematic review and meta-analysis. J Infect Dis
2009, 200:1509-1517.
7. 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, 52:3303-3309.
8. 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, 14:520.

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