Clinical Investigation Study to Evaluate the Consistency and Reproducibility of Two Consecutive Mosquito Feeding Assays in Adults With Varying Plasmodium Falciparum Gametocyte Densities
Conditions: Malaria
Sponsor: PATH

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Clinical investigation study to evaluate the consistency and 
reproducibility of two consecutive mosquito feeding assays in adults 
with varying Plasmodium falciparum gametocyte densities 
 
Protocol Identifying Number: CVIA 085 
 
Principal Investigator: Dr. Ben Andagalu 
 
Sponsor: PATH 
 
Funded by: PATH with support from the Bill & Melinda Gates Foundation 
 
Version Number: 1.3 
 
WRAIR Number:  2739 
 
KEMRI Number: 4082 
 
Trial registration: ClinicalTrials.gov: NCT04666350 
 
Date: 07MAY2021 
 
 
Confidentiality Statement 
This document is confidential and is to be distributed for review only to investigators, potential investigators, consultants, 
study staff, and applicable independent ethics committees or institutional review boards.  The contents of this document 
shall not be disclosed to others without written authorization from PATH (or others, as applicable), unless it is necessary to 
obtain informed consent from potential study participants. 
 
 
 

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TABLE OF CONTENTS 
 
TABLE OF CONTENTS ..................................................................................................................................... 2 
LIST OF ABBREVIATIONS ............................................................................................................................... 6 
STATEMENT OF COMPLIANCE ...................................................................................................................... 8 
PROTOCOL SUMMARY .................................................................................................................................. 9 
2. 
KEY ROLES ....................................................................................................................................... 12 
3. 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE .............................. 14 
3.1 
Background information ......................................................................................................... 14 
3.1.1 
Biological basis for transmission-reducing interventions ............................................. 14 
3.1.2 
Regulatory approaches for testing interventions that interrupt transmission ............ 14 
3.1.3 
Accelerated approval using biomarker assay(s) ........................................................... 15 
3.1.3.1 
Standard Membrane Feeding Assay (SMFA) ................................................................ 15 
3.1.3.2 
Direct Skin Feeding Assay (DSFA) ................................................................................. 16 
3.1.3.3 
Direct Membrane Feeding Assay (DMFA) .................................................................... 17 
3.1.3.4 
Biomarker validation for future clinical trial design ..................................................... 18 
3.2 
Rationale for approach and hypothesis .................................................................................. 19 
3.3 
Potential Risks and Benefits .................................................................................................... 21 
3.3.1 
Known Potential Risks and Risk Management ............................................................. 21 
3.3.1.1 
Risks of Accidental Disclosure of Private Information .................................................. 21 
3.3.1.2 
Risks of Phlebotomy ..................................................................................................... 21 
3.3.1.3 
Risks of Direct Skin Feeding Assay ................................................................................ 21 
3.3.1.4 
Risks of receipt of Primaquine ...................................................................................... 21 
3.3.1.5 
Risks of Receipt of Artemether/Lumefantrine (Coartem®) .......................................... 22 
3.3.1.6 
Risk of infectious mosquito bite to lab personnel ........................................................ 23 
3.3.1.7 
Other Health Risks of Participation in the Study .......................................................... 23 
3.3.2 
Known Potential Benefits ............................................................................................. 24 
4. 
OBJECTIVES AND PURPOSE ............................................................................................................. 24 
5. 
STUDY DESIGN AND ENDPOINTS .................................................................................................... 25 
5.1 
Description of the Study Design .............................................................................................. 25 
5.2 
Study Endpoints ...................................................................................................................... 26 

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5.2.1 
Primary Endpoint .......................................................................................................... 27 
5.2.2 
Secondary Endpoints .................................................................................................... 27 
5.2.3 
Exploratory Endpoints .................................................................................................. 27 
6. 
STUDY ENROLLMENT AND WITHDRAWAL ...................................................................................... 27 
6.1 
Participant Inclusion Criteria ................................................................................................... 27 
6.2 
Participant Exclusion Criteria .................................................................................................. 28 
6.3 
Strategies for Recruitment and Retention .............................................................................. 28 
6.3.1 
Community information ............................................................................................... 28 
6.3.2 
Recruitment .................................................................................................................. 28 
6.3.3 
Reasons for Withdrawal or Termination ...................................................................... 29 
6.3.4 
Handling of Participant withdrawals or termination .................................................... 29 
6.4 
Premature Termination or Suspensions of Study ................................................................... 29 
7. 
STUDY TREATMENT ......................................................................................................................... 29 
7.1.1 
Acquisition .................................................................................................................... 30 
7.1.2 
Product Storage and Handling ...................................................................................... 30 
7.1.3 
Dosing and Administration ........................................................................................... 30 
7.1.4 
Justification for treatment of asymptomatic parasite carriers .................................... 30 
8. 
STUDY PROCEDURES ....................................................................................................................... 31 
8.1 
Study procedures and evaluations .......................................................................................... 31 
8.1.1 
Study site ...................................................................................................................... 31 
8.1.2 
Recruitment, informed consent and enrolment .......................................................... 32 
8.1.3 
Blood sample collection ............................................................................................... 32 
8.1.4 
Enrolment of gametocytemic participants ................................................................... 33 
8.1.5 
Mosquito feeding assays (DSFA & DMFA) .................................................................... 33 
8.1.6 
Management of study participants .............................................................................. 35 
8.1.7 
Standard of care study procedures .............................................................................. 36 
8.1.8 
Clinical laboratory procedures ..................................................................................... 36 
8.1.9 
Other assays or procedures .......................................................................................... 36 
8.1.9.1 
Malaria blood film microscopy ..................................................................................... 36 
8.1.9.2 
Plasmodium detection molecular assays ..................................................................... 36 
8.1.9.3 
Oocyst and sporozoite detection and quantification ................................................... 37 
8.1.10 
Specimen Preparation, Handling, and Storage ............................................................. 37 
9. 
ASSESSMENT OF SAFETY ................................................................................................................. 37 

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9.1 
Adverse Events (AE) ................................................................................................................ 37 
9.2 
Serious Adverse Event ............................................................................................................. 38 
9.3 
Classification of An Adverse Event .......................................................................................... 38 
9.4 
Time Period and Frequency of Event Assessment and Follow-up .......................................... 38 
9.5 
Reporting Procedures ............................................................................................................. 38 
9.5.1 
Adverse event and Serious Adverse Event reporting ................................................... 38 
9.5.2 
Unanticipated problems and UPIRTSOs ....................................................................... 39 
9.5.3 
Pregnancies .................................................................................................................. 39 
9.5.4 
Continuing Review/Progress Reports ........................................................................... 39 
9.5.5 
Inspections and audits .................................................................................................. 39 
9.5.6 
Protocol deviations ....................................................................................................... 39 
10. 
STATISTICAL CONSIDERATIONS ................................................................................................... 40 
10.1 
Overview and General Design ................................................................................................. 40 
10.2 
Statistical Hypothesis .............................................................................................................. 40 
10.3 
Sample Size ............................................................................................................................. 41 
10.4 
Definitions of Populations to be Analyzed .............................................................................. 44 
10.4.1 
Enrolled population ...................................................................................................... 44 
10.4.2 
Full analysis population ................................................................................................ 44 
10.4.3 
Per Protocol population ............................................................................................... 44 
10.5 
Analysis Methodology ............................................................................................................. 45 
10.5.1 
Analysis of Safety Data ................................................................................................. 45 
10.5.2 
Analysis of Primary Objectives ..................................................................................... 45 
10.5.3 
Analysis of Secondary Objectives ................................................................................. 45 
10.6 
Handling of Dropouts and Missing Data ................................................................................. 46 
10.7 
Timing of Analyses .................................................................................................................. 47 
11. 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS ....................................... 47 
12. 
QUALITY ASSURANCE AND QUALITY CONTROL .......................................................................... 47 
13. 
ETHICS/PROTECTION OF HUMAN SUBJECTS .............................................................................. 47 
13.1 
Ethical Standard ...................................................................................................................... 47 
13.2 
Institutional Review Board ...................................................................................................... 47 
13.2.1 
Ethics Approval ............................................................................................................. 47 
13.2.2 
Protocol Amendments .................................................................................................. 47 
13.3 
Informed Consent Process ...................................................................................................... 48 

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13.3.1 
Consent and Other Informational Documents Provided to Participants ..................... 48 
13.3.2 
Consent Procedures and Documentation .................................................................... 48 
13.4 
Participant and Data Confidentiality ....................................................................................... 48 
13.4.1 
Future Use of Stored Specimens .................................................................................. 49 
13.5 
Compensation ......................................................................................................................... 49 
13.5.1 
Compensation for injury ............................................................................................... 49 
13.5.2 
Compensation for subject participation ....................................................................... 49 
14. 
DATA HANDLING AND RECORD KEEPING ................................................................................... 50 
14.1 
Data Collection and Management Responsibilities ................................................................ 50 
14.1.1 
Source Data .................................................................................................................. 50 
14.1.2 
Source documents ........................................................................................................ 50 
14.1.3 
Data Capture Methods ................................................................................................. 50 
14.1.4 
Data Storage ................................................................................................................. 50 
14.2 
Study Records Retention and Disposal ................................................................................... 51 
14.3 
Publication and Data Sharing Policy........................................................................................ 51 
15. 
KEY PERSONNEL ROLES ............................................................................................................... 51 
16. 
MILITARY RELEVANCE ................................................................................................................. 52 
17. 
BUDGET ....................................................................................................................................... 52 
18. 
LITERATURE REFERENCES ........................................................................................................... 53 
 
  
 
 

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LIST OF ABBREVIATIONS 
 
 
ACL-2
Arthropod Containment Level 2
AE
Adverse event
CDMS
Clinical Data Management System
cDNA
Complementary DNA
CFR
Code of Federal Regulations
Coartem®
Artemether/Lumefantrine
CRC
Clinical Research Center
CRF
Case Report Form
DBS
Dried blood spot
DMFA
Direct Membrane Feeding Assay
DMP
Data Management Plan
DNA
Deoxyribonucleic acid
DSFA
Direct Skin Feeding Assay
ECCT
Expert Committee on Clinical Trials (ECCT)
EDTA
Ethylenediaminetetraacetic
ERC
Ethical Review Committee
GCP
Good Clinical Practice
GPS
Global Positioning System
HDSS
Health and Demographic Surveillance System
HIV
Human immunodeficiency virus
HRPO
Human Research Protections Office
HRPP
Human Research Protection Program
HSPB
Human Subject Protection Branch
IB
Investigator's Brochure
ICH
International Council for Harmonisation 
ICMJE
International Committee of Medical Journal Editors
IEC
Institutional Ethics Committee
IRB
Institutional Review Board
KEMRI
Kenya Medical Research Institute
MBF
Malaria Blood Film
MDC
Malaria Diagnostic Center
MDR
Malaria Drug Resistance
MoH
Ministry of Health
NIH
National Institutes of Health
ORP
Office of Research Protections
PBS
Phosphate Buffered Saline
P. falciparum
Plasmodium falciparum

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Pf
P. falciparum
PATH REC
PATH’s Research Ethics Committee
PCR
Polymerase chain reaction
PHI
Protected Health Information
PI
Principal Investigator
PII
Personal identifying information
QC
Quality Control
qPCR
Quantitative PCR
REC
Research Ethics Committee
RNA
Ribonucleic Acid
rRNA
Ribosomal ribonucleic acid
RT-PCR
Reverse Transcriptase PCR
SAE
Serious Adverse Event
SERU
Scientific and Ethics Review Unit
SMFA
Standard Membrane Feeding Assay
SOP
Standard Operating Procedure
SSA
Sub-Saharan Africa
SSC
Scientific Steering Committee
TRIs
Transmission Reducing Interventions
UPIRTSO
Unanticipated Problem Involving Risk to Subjects or Others
US FDA
United States Food and Drug Administration
USAMRD-A
United States Army Medical Research Directorate - Africa
WHO
World Health Organization
WRAIR
Walter Reed Army Institute of Research
 
 

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STATEMENT OF COMPLIANCE 
 
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 ICH-GCP (E6) guidelines. 
 
Site Investigator: Ben Andagalu 
Title: Co-Director, Malaria Drug Resistance Laboratory 
United States Army Medical Research Directorate/KEMRI 
Signed:  
 
Date:  
 
 
 
 
 

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PROTOCOL SUMMARY  
 
Title
Clinical investigation study to evaluate the consistency and reproducibility of two 
consecutive mosquito feeding assays in adults with varying Plasmodium falciparum 
gametocyte densities 
Design
The proposed trial design has been developed to assess the consistency and reproducibility 
of two consecutive direct skin feeding assays (DSFA) at 24-hour interval. The results will 
determine the type of pivotal trial design for a follow-on Phase 2b trial whose objective is 
to bridge the standard membrane feeding assay (SMFA) to the direct skin feeding assay 
(DSFA) and direct membrane feeding assay (DMFA) using a monoclonal antibody 
intervention, TB31F mAb,  which interrupts transmission from human to mosquito. The 
results from this experimental medicine study will inform whether the preferred “Before-
After” trial design in which each human volunteer serves as their own internal control can 
be utilized for a follow-on Phase 2b trial.  
The rationale for this experimental medicine trial is described in the background section of 
the protocol.  
 
Figure 1. Experimental medicine clinical study design 
 
 
The sample size to be included in this study is shown below in Figure 2. 
 
 
 
 
 
 
 

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Figure 2. Estimated sample size screening and enrollment for experimental medicine trial. 
 
 
Objectives
Primary Objective: We would like to better understand the relationship between parasite 
transmission from humans to mosquitoes both within a person and across persons by 
assessing the variation in the proportion of infected mosquitoes with at least one oocyst 
(oocyst prevalence) in DSFA and DMFA performed at two consecutive time points in the 
same human subject with P. falciparum gametocytemia using microscopy 
 
Secondary Objectives: 
1. To assess variation in oocyst density in DSFA and DMFA at two consecutive time points 
in same subject using microscopy. 
2. To assess variation in the proportion of infected mosquitoes with at least one 
sporozoite (sporozoite prevalence) in DSFA and DMFA performed at two consecutive 
time points in the same human subject using microscopy 
3. To assess the variation in sporozoite density in DSFA and DMFA at two consecutive 
time points in the same subject using microscopy. 
 
Exploratory Objective: 
1. To assess oocyst and sporozoite density in DSFA and DMFA at two consecutive time  
points in the same subject using molecular quantitative PCR (qPCR) 
2. To summarize the relationships between DSFA and DMFA results for oocyst and 
sporozoite density 
 
 
 
Endpoints
Primary Endpoint:  

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Oocyst prevalence in DSFA and DMFA in mid-guts of mosquitoes performed at two 
consecutive time points in the same human using microscopy 
Secondary Endpoints: 
1. Oocyst density in DSFA and DMFA at two consecutive time points in same subject using 
microscopy. 
2. Sporozoite density and prevalence in DSFA and DMFA performed at two consecutive 
time points in the same human subject using microscopy 
Exploratory Endpoint: 
Oocyst and sporozoite density in DSFA and DMFA at two consecutive time points using 
qPCR 
Population
Adults 18 to 55 years old
Number of 
sites 
enrolling 
participants
One site; Kombewa Clinical Research Center, Kenya
Description 
of study 
agent 
No investigational agent in this study
Study 
duration 
Estimated 9 months from when the study opens to enrollment until completion of last 
subject last visit and study data analysis 
Participant 
Duration  
 
Estimated 4 days it will take for each individual participant to complete all participant visits
 
 
 
 

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2. KEY ROLES  
PATH Clinical Lead 
Protocol Co-author 
Christian F. Ockenhouse, MD, PhD 
PATH
Center for Vaccine Innovation and Access 
455 Massachusetts Avenue NW, Suite 1000, Washington, DC 
20001, United States 
Phone: +1 (202) 716-5849 
Email: cockenhouse@path.org 
Principal investigator(s) 
Dr. Ben Andagalu 
Co-Director, Drug Resistance Laboratory  
KEMRI
P.O. Box 54-40100, Kisumu, Kenya 
Phone: +254 716004851 
Email: Ben.Andagalu@usamru-k.org 
Clinical Investigators 
Dr. Lucas Otieno Tina,
Deputy Director Clinical Operations,
Kombewa Clinical Research Center,
KEMRI,
P.O. Box 54-40100,
Kisumu, Kenya
Phone: +254 720 597654/0705 251 819
Email: Lucas.Tina@usamru-k.org 
 
Nathanial Copeland, MD, MTM&H, MAJ, MC  
Director, Kombewa Clinical Research Center 
US Army Medical Research Directorate-Africa (USAMRD-A) 
P.O. Box 54-40100, Kisumu, Kenya 
Phone: +254 705 251782 
Email: nathanial.copeland@usamru-k.org  
 
Dr Bernhards Ogutu 
KEMRI 
P.O. Box 54 – 40100, Kisumu, Kenya 
Phone: +254733812613 
Email: ogutu6@gmail.com  
MDR/MDC Investigators 
Benjamin Opot
KEMRI 
P.O. Box 54 – 40100, Kisumu, Kenya 
Phone: +254735412812  
benjamin.opot@usamru-k.org 
 
Dennis Juma 

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KEMRI
P.O. Box 54 – 40100, Kisumu, Kenya 
Phone: +254 720 326 111 
dennis.juma@usamru-k.org 
 
Hoseah Akala 
KEMRI 
P.O. Box 54 – 40100, Kisumu, Kenya 
Phone: +254 722 329 845 
hosea.akala@usamru-k.org 
 
Amanda Roth 
USAMRD-A 
P.O. Box 54 – 40100, Kisumu, Kenya 
Phone: +254 705 251 693 
amanda.roth@usamru-k.org 
Entomology Investigators 
James Mutunga
KEMRI 
P.O. Box 54 – 40100, Kisumu, Kenya 
Phone: +254 725 238 395 
james.mutunga@usamru-k.org 
 
Jaree Johnson 
USAMRD-A 
P.O. Box 54 – 40100, Kisumu, Kenya 
Phone: +254 705 251 622 
jaree.johnson@usamru-k.org 
WRAIR IRB 
Walter Reed Army Institute of Research
Office of Research Management
503 Robert Grant Avenue
Silver Spring, MD 20910-7500
USA
Tel: +001.301.319.9940
Fax: +001.301.319.9961
email: usarmy.detrick.medcom-wrair.mbx.hspb@mail.mil  
KEMRI 
Scientific 
and 
Ethical Review Unit 
Secretary 
KEMRI SERU, c/o Kenya Medical Research Institute 
P.O. Box 54840, Nairobi, Kenya. 
Phone: +254.20.272.25.41 
Email : ERCAdmin@kemri.org; seru@kemri.org 
Funder 
PATH with support from the Bill and Melinda Gates 
Foundation 
 
Dr. Ben Andagalu, Principal Investigator will be responsible for the overall conduct of the trial at this 
study site, administrative actions between USAMRD-A/K, Institutional Review Boards, and the sponsor, 

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oversight of field activities and patient care. He contributed to the protocol development and will 
contribute to the statistical analysis and writing of the scientific report(s). 
  
All Clinical Investigators listed above will be responsible for the clinical conduct of the trial, the care 
provided to patients, oversight of field activities, and will contribute to the interpretation of data and 
analysis and writing of scientific report(s). Dr. Copeland also contributed to the protocol development. 
 
3. INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE  
3.1 Background information 
3.1.1 Biological basis for transmission-reducing interventions 
New tools are needed to accelerate the path toward eventual elimination of Plasmodium falciparum 
malaria. Interventions, such as vaccines, drugs, or biologics which can break the cycle of malaria parasite 
transmission between humans and mosquitoes, are viewed as being of particular importance in this 
regard and are endorsed by the WHO 
(https://www.who.int/immunization/sage/meetings/2013/april/7_Malaria_Vaccine_TRM_Final.pdf?ua=
1). Such interventions harbor the potential to prevent mosquitoes becoming infected with parasites, 
even after feeding on infectious individuals, thereby breaking the cycle of transmission.  
One of the more novel strategies being pursued is the interruption of transmission of malaria parasites 
through the mosquito vector, thereby reducing the number of secondary infections (4).  The 
transmission of malaria requires the survival and development of Plasmodium parasites within the 
vertebrate host and the mosquito vector. Female anopheline mosquitoes ingest the sexual stage 
gametocytes when taking a blood meal from infectious vertebrate hosts. Within the mosquito’s midgut, 
the parasite must overcome both the vector’s innate defense mechanisms and the vertebrate host’s 
immune mechanisms that include anti-parasite antibodies found within the blood meal following a 
mosquito bite (5). The conserved nature of the parasite’s proteins expressed in the sexual stages, not 
under natural selective pressure, renders the sexual stages of the parasite attractive targets for 
transmission reducing interventions (TRIs).  
 
TRIs interfere with this process of parasite transmission from the vertebrate host to the vector in various 
ways. Anti-malarial drugs could reduce gametocyte carriage or infectivity directly by killing circulating 
gametocytes in human blood, or human antibodies directed against gametocyte-specific surface 
proteins could prevent sporogonic development within the mosquito’s midgut, preventing sporozoite 
formation.  
3.1.2 Regulatory approaches for testing interventions that interrupt transmission  
The absence of direct and immediate benefit to the recipient, and the complexity of the Plasmodium 
falciparum life cycle make clinical development of this class of interventions challenging.  The regulatory 
pathway for interventions that interrupt malaria transmission is complex and challenging.  
Since 2010, PATH has convened a series of workshops with malaria experts, policy makers, and 
regulators. The discussions in those meetings indicated that: 

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 
There is no clear legal bar to approving a transmission-blocking intervention (such as a vaccine, 
drug, or biologic) that confers no direct benefit on recipients 
 
Regulations governing biological product licensing do not on their face require that a biological 
product confer direct clinical benefit on recipients. However, designing clinical studies for such 
vaccine may raise ethical issues which need to be appropriately addressed in clinical 
development of such product.  
 
Two Technical Consultation Groups (TCG) convened by PATH discussed the potential 
development pathways of a tool which interrupts malaria transmission. TCG-1 focused on a 
more conventional approach considering the pros and cons of a large single Phase 3 cluster 
randomized trial approval pathway, while TCG-2 focused on an Accelerated Approval pathway 
based on an analytically and biologically validated surrogate marker of clinical efficacy.  
 
A Type C meeting with the US FDA indicated that an accelerated approval pathway in which a 
biomarker endpoint of efficacy substitutes for a clinical endpoint of population-based efficacy 
may be acceptable. The US FDA may grant licensure on the basis of adequate and well-
controlled clinical trials establishing that the biological product has an effect on a surrogate 
endpoint that is reasonably likely to predict clinical benefit. The advantages of such an approach 
includes feasibility, reduced trial costs, and shortened timelines to study outcomes. 
 
3.1.3 Accelerated approval using biomarker assay(s) 
The development of an accelerated approval pathway (to be evaluated under future clinical trials) 
depends upon a biomarker assay that would reasonably likely predict clinical benefit. Assays that can 
accurately and quantitatively determine malaria transmissibility are therefore crucial in the evaluation 
of the efficacy of TRIs.  
 
3.1.3.1 Standard Membrane Feeding Assay (SMFA) 
The SMFA, which has been used by groups worldwide measures whether a drug or antibody added 
exogenously to an in vitro assay can completely block the development of P. falciparum malaria 
parasites in the Anopheles mosquito when quantified early as oocyst stage parasites (days 7 to 9), or 
later as sporozoites within the mosquito salivary glands (days 14-21).  The SMFA is shown in the 
schematic below. 

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Figure 3: Standard Membrane Feeding Assay 
 
The Standard Membrane Feeding Assay (SMFA) was developed from an experimental laboratory 
protocol that measures in vitro the inhibition of transmission of malaria parasites to their vectors (6,7) 
when a test drug, biologic, or antibody-containing sera are introduced.  Although the SMFA assay is 
considered to be a surrogate assay that would recapitulate the activity as it would occur in vivo in a 
human host given the intervention,  the assay does not directly involve the testing within a human 
volunteer provided a drug, biologic, or vaccine, and therefore direct linkage between the SFMA and an 
effect in vivo would need to be evaluated in future trials comparing and bridging the in vitro SMFA to 
assays that measure the same intervention in vivo (DSFA) described below.  The present study does not 
include SMFA testing.  
 
Briefly, laboratory-reared anopheline mosquitoes (typically A. stephensi) are permitted to feed on 
cultured P. falciparum gametocytes (NF54 isolate/3D7 strain) through a membrane. The fed mosquitoes 
are maintained in cages to permit any ingested parasite gametocytes to develop into oocysts. After 
approximately 7-9 days mosquitoes are dissected and the number of oocysts is used to report the 
success of infection and the percentage of parasite-infected mosquitoes are quantified. A portion of 
infected mosquitoes are also dissected between days 14 and 21 to assess salivary gland sporozoites 
which are the infective stages of the parasite following a mosquito bite which injects sporozoites into 
the skin thereby initiating the parasite’s life cycle. 
 
SMFA has been utilized widely to assess the transmission-blocking potential of test antibodies and anti-
malarial drugs in preclinical and clinical vaccine development by mixing the cultured P. falciparum 
gametocytes with test antibodies/drugs and allowing Anopheles mosquitoes to feed. The transmission 
reducing activity of the test intervention is then calculated based on comparison of infection prevalence 
and oocyst density with that obtained in mosquitoes fed gametocytes mixed with control serum (8,9).  
 
 
3.1.3.2 Direct Skin Feeding Assay (DSFA) 

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The DSFA has been developed to directly measure the transmissibility of parasites directly from human-
to-mosquito without an in vitro membrane step as illustrated below. Although DSFA is biologically more 
relevant with regard to the functional activity that a drug/antibody has on malaria transmission, the 
assay is complex requiring human volunteers and the inherent variability in human hosts, parasite 
strains, and anopheline vector competencies rendering regulatory approval for such an approach 
unsustainable. 
Figure 4: Direct Skin Feeding Assay 
 
The DSFA is desirable since it closely mimics what would happen in nature, but also has important 
drawbacks: (i) the number of mosquitoes that can be fed on a human at any one time is restricted (~<30 
per skin feed) by volunteer acceptability. (ii) it is difficult to quantify the number of gametocytes 
circulating in the human blood and in blood meal from a mosquito after they have fed on the human 
host.  (iii) the DSFA may not be ethically used in certain age groups (i.e. young children) depending upon 
country-specific and community acceptability;  (iv) the DSFA can be affected by inter-individual variation 
in innate attractiveness to mosquitoes; the DSFA is limited to geographical locations with a source of 
gametocyte-positive P. falciparum-infected persons and advanced laboratory and insectary support.  
 
3.1.3.3 Direct Membrane Feeding Assay (DMFA) 
A third type of assay named the Direct Membrane Feeding Assay (DMFA) is an assay that is positioned 
between the SMFA and DSFA. In the DMFA, instead of using cultured gametocytes of a single strain of P. 
falciparum as in the SMFA, venous blood samples from gametocytemic individuals previously 
administered with anti-malarial drugs or antibodies are placed into a membrane feeder and fed to 
mosquitoes. Such an assay illustrated below is performed in malaria endemic regions with direct access 
to naturally-infected gametocyte carriers and advanced laboratory support and insectary facilities. In the 
DMFA, oocysts in the mosquito midgut and sporozoites in the salivary glands are quantified. 

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Figure 5: Direct Membrane Feeding Assay 
 
 
3.1.3.4 Biomarker validation for future clinical trial design 
The search for a biomarker assay, such as the SMFA, will be the objective of a follow-on clinical trial. This 
experimental medicine protocol is focused on testing the reproducibility of two sequential direct skin 
feeding assays to inform trial design for future trials that assess biomarker feasibility. The section below 
describes how future trials will assess the feasibility of a biomarker SMFA to substitute for conventional 
trials that measure the interruption of malaria transmission. 
 
The SMFA, DMFA, DSFA have advantages and limitations. As stated previously, the SMFA is the gold 
standard used by multiple laboratories around the world. The assay is reproducible and robust. 
However, the SMFA is limited in terms of the number of malaria parasite strains and anopheline vector 
compatibility. Nevertheless, if the SMFA could recapitulate the transmission dynamics and functionality 
of the DSFA, the validation of the SMFA may meet the standards set by regulatory agencies for 
accelerated approval of new tools that interrupt malaria transmission as it does not include the 
requirement of using natural acquired P. falciparum parasites not naturally caught Anopheles species 
that transmit malaria.   
 
DSFAs are quantitatively more sensitive than membrane feeding assays in detecting positive oocyst-
infected Anopheles mosquitoes after having fed on low-density numbers of gametocytes in a feeding 
assay. The reasons for this are unknown and do not appear to be a function of sequestration of 
gametocytes in the skin of an infected person (unpublished data).  
 
Our ultimate objective for evaluation of TRIs would be to substitute an in vivo DSFA for a surrogate in 
vitro SMFA assay.  The suitability of the SMFA over the DSFA would need to be validated by bridging the 
two assays against each other. In order to evaluate these two assays against each other in a streamlined 
cost-effective manner, we have proposed a bridging study to be performed at a later date (not the 
subject of this experimental medicine trial), that capitalizes upon a single person serving as their own 

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internal control in a “before-after” study design. The quantitation of baseline variability in the current 
proposed trial would be used to inform trial design of a follow-on study. 
Figure 6. Rationale of experimental medicine trial of baseline variability in transmission of parasites from 
human-to-mosquito 
 
It is unknown whether the numbers of infected mosquitoes or the density of midgut oocysts are similar 
(i.e. variability) after having been fed on the same gametocyte-positive person at two consecutive short 
time intervals. This unknown is critical in our understanding and design of an interventional trial in 
which each human subject serves as their own internal control in a classic “before-after” trial 
intervention. 
 
The “before-after” trial design depends upon the assumption that there is minimal variability in parasite-
to-mosquito transmission dynamics in the same person when measured via DSFA at two time points 
separated by a 24-hour interval when no other intervention is applied (no TRI) and forms the hypothesis 
for carrying out the current experimental medicine trial. 
 
3.2 Rationale for approach and hypothesis 
It is crucial that variation in DSFA and DMFA be measured by quantifying the prevalence of infected 
mosquitoes and/or oocyst density in the mosquitoes at two sequential time points. The data obtained 
from this study will inform the methodology for future field studies of TRIs. 
The illustration below depicts the strategy to determine variability in same person undergoing DSFA at 
two consecutive 24-hour intervals. 

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Figure 7: Schematic of study 
 
 
The use of internal controls is a strategy that has been widely used in clinical trials. The advantages of 
this approach include the need for a smaller sample size to detect differences in efficacy of 
interventions. This experimental medicine trial proposes therefore to measure the baseline variation in 
the infectivity of mosquitoes in both the DSFA and DMFA when performed in the same individual at two 
consecutive time points. 
 
The United States Army Medical Research Directorate – Africa (USAMRD-A) in Western Kenya has 
successfully piloted a platform within the Kombewa Clinical Research Center’s (CRC) Health and 
Demographic Surveillance System (HDSS) study area that has been designed to study malaria 
transmission dynamics in a holoendemic setting. Study activities were executed under IRB-approved 
protocol between July 2015 and June 2016. The general objective was to monitor trends in Plasmodium 
spp. (asexual and sexual stage) carriage and transmissibility in a population exposed to high transmission 
through the seasonal variation of one calendar year. Among the specific study objectives was the need 
to approximate the infectiousness of the human population within the study area to mosquitoes by 
quantifying oocysts and sporozoites in lab-reared Anopheles gambiae mosquito midguts and salivary 
glands respectively after mosquito feeding assays. Over 1000 mosquito feeding assays (DFMA and/or 
DSFA) were performed during this study with high proportion of mosquitoes demonstrating positive 
midgut dissections and positive mosquito salivary gland dissections. The proposed experimental 
medicine trial will leverage the expertise that exists at USAMRD-A/K to ensure successful completion of 
the study objectives. 
 
 

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3.3 Potential Risks and Benefits 
 
3.3.1 Known Potential Risks and Risk Management 
3.3.1.1 Risks of Accidental Disclosure of Private Information  
The study will be collecting private information and there is risk this could be exposed and reveal the 
status of the subject in the study or some other personally identifying information (PII) of protected 
health information (PHI). In order to ensure that all information collected on study volunteers is kept 
confidential, the following safeguards will be applied: Access to study files and personal information will 
be limited to study personnel, ethics committees, regulatory authorities, and sponsor. Study information 
will be kept in locked rooms when not in use. All information or samples that leave USAMRD-A will be 
labeled with a unique study identification number and have no PII. Any link between individual study 
identification number and an individual’s PII (e.g., and individual’s study file and associated documents) 
will be maintained at the USAMRD-A facilities in accordance with site standard operating procedures 
(SOPs) to maintain each individual’s confidentiality. 
 
3.3.1.2 Risks of Phlebotomy  
Venipuncture is a routine clinical procedure the medical community commonly uses to obtain blood 
samples. Immediate complications may be slight pain during the entry of the needle into the skin, very 
rarely possible dizziness and syncope. Additionally, a hematoma may result from the venipuncture, but 
this has minimal risk. Late complications might include thrombosis of the vein due to trauma or 
infection. These complications are extremely rare. Participant monitoring, aseptic technique, including 
sterile disposable blood collection apparatus and adherence to standard medical precautions reduce any 
risk to a minimum. A credentialed phlebotomist or member of the clinical team experienced in 
venipuncture techniques will perform all venipunctures. The amount of blood to be taken for sampling 
will not be harmful to the subject’s health. 
 
3.3.1.3 Risks of Direct Skin Feeding Assay  
DSFA exposes an individual to mosquito bites. In addition to mild expected discomfort from a bite itself 
and pruritus following the bite(s), there is also a risk of a local site reaction (e.g., allergic reaction; 
including pain, swelling, and erythema), secondary infection, or systemic allergic reaction (e.g., 
anaphylaxis). Mild local site reactions are common and can be treated with symptomatic medication as 
needed and subjects will be monitored for 30 minutes following DSFA for evidence of a severe systemic 
reaction and treated for any complications by the study team or with medical referral as needed.  
There is the additional risk of mosquitoes transmitting other infectious agents.  This risk will be at the 
very minimum for this project since cow’s blood is used to maintain the colonies and only new progenies 
of female mosquitoes will be used for skin feeding assays. Additionally, 10% of each new generation of 
female mosquitoes will be pooled and tested for presence of arboviruses by PCR at the USAMRD-
A/K/KEMRI laboratories. In USAMRD-A/K’s experience, there have been no known cases of the 
laboratory-reared mosquitoes transmitting other infectious agents. 
 
3.3.1.4 Risks of receipt of Primaquine  

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Subjects who receive primaquine generally may experience the following adverse events:  
Cardio-vascular: Cardiac arrhythmia and QT prolongation, mainly with high dose.  
Gastrointestinal: Nausea, vomiting, epigastric distress, and abdominal pain.  
Hematologic: Leukopenia, hemolytic anemia especially in G-6-PD deficient individuals and 
methemoglobinemia especially in NADH methemoglobin reductase deficient individuals.  
Nervous system disorders: Dizziness.  
Skin and subcutaneous tissue disorders: Rash maculopapular, pruritus. 
Standard dose primaquine is contraindicated in subjects with severe glucose-6 phosphate 
dehydrogenase (G6PD) deficiency due to the risk of hemolytic anemia. However, a single low 
dose (15 mg primaquine base) is safe in individuals with G6PD deficiency and recommended by 
the WHO for the clearance of sexual stage gametocytes of P. falciparum without specific testing 
for G6PD adults (except in pregnant or breastfeeding women) (10). The side effects that may be 
experienced are mostly transient and usually do not require any specific treatment. The 
management of any persistent side effects is usually symptomatic. 
 
3.3.1.5 Risks of Receipt of Artemether/Lumefantrine (Coartem®)  
Adult subjects who receive Coartem® may experience the following adverse events: 
Metabolism and nutrition disorders 
Very common: Decreased appetite 
Psychiatric disorders 
Very common: Sleep disorder 
Nervous system disorders 
Very common: Headache, dizziness 
Common: Clonus 
Uncommon: Somnolence, hypoesthesia, ataxia 
Cardiac disorders 
Very common: Palpitations 
Respiratory, thoracic and mediastinal disorders 
Common: Cough 
Gastrointestinal disorders 
Very common: Vomiting, abdominal pain, nausea 
Common: Diarrhea 
Skin and subcutaneous tissue disorders 
Common: Rash, pruritus 
Uncommon: Urticaria 
Musculoskeletal and connective tissue disorders 
Very common: Arthralgia, myalgia 
General disorders and administration site conditions 
Very common: Asthenia, fatigue 
Uncommon: Gait disturbance 
Investigations 
Uncommon: Electrocardiogram QT prolonged, liver function tests increased 

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Symptoms are generally mild and self-limited without any treatment required. To ensure compliance, 
subjects will receive the first dose of Coartem® under the supervision of a medical provider at the CRC 
and sent home with the remaining doses and instructions on how to complete the course. The 
management of any persistent side effects is usually symptomatic. 
3.3.1.6 Risk of infectious mosquito bite to lab personnel 
There is a small but potential risk of occupational exposure to the bite of an infectious A. gambiae 
mosquito that escapes from carton in insectary or during dissection procedures for determination of 
sporozoite prevalence. Laboratory personnel will be instructed to inform the PI upon a mosquito bite 
and every attempt will be made to capture and dissect or evaluate the mosquito for the presence of 
salivary gland sporozoites. Laboratory personnel will be counselled and followed up periodically for the 
emergence of blood stage parasitemia and treated for malaria according to the Kenya Ministry of Health 
National Guidelines for the Diagnosis, Treatment and Prevention of Malaria. 
3.3.1.7 Other Health Risks of Participation in the Study 
There is the risk of exposure to respiratory infections such as COVID-19 when potential participants visit 
health facilities. The Kenya Ministry of Health has put in place guidelines that help reduce the risk of 
exposure, such as screening all individuals presenting at health facilities, implementing physical 
distancing measures and ensuring healthcare workers have the appropriate personal protective 
equipment (PPE). The study team will follow local guidelines in order to minimize risk of exposure of 
both the participants and the study staff.  
Briefly, staff will do self-screening for illness at home before they report on duty as per KEMRI 
guidelines, and will also be screened when they arrive at the duty station. Staff who are unwell will not 
be allowed to be on duty. Staff will use full PPE in accordance with WHO guidelines (mask, eye 
protection, disposable gown, gloves) when handling study participants. Staff will wash or sanitize hands 
before encounter, after any contact with the participant, and at the end of the encounter. Hand 
sanitizers and handwashing stations will be made available by the study. The staff will routinely disinfect 
the surfaces of the research center as well as the sample shipment boxes prior to their dispatch to the 
central lab. Central lab staff in full PPE will disinfect the surfaces of sample shipment boxes that are 
received from the study site prior to opening them. Central lab staff will work in full PPE whenever they 
are working with the samples. 
Potential participants will be screened for symptoms of COVID-19 during the recruitment process. Those 
suspected to have COVID-19 will not be allowed to participate in the study. Participants who meet the 
case definitions of COVID-19 while in the study will not continue with study activities and will be advised 
to seek medical care as per Kenya Ministry of Health guidance. Study participants will sanitize hands 
upon entry and at exit from the research center and use face masks while at the center. Hand sanitizers 
and handwashing stations will be made available by the study. The study will also provide the 
participants with face masks.  
The waiting areas for study participants as well as work stations for staff will be re-arranged to ensure 
physical distancing of 1.5 – 2 meters. It will be difficult to maintain physical distancing between 
participants and study staff during the execution of study procedures such as physical examination, 
measuring of vital signs and drawing of blood. For this reason, study staff will don full PPE, including face 
shields, while study participants will be provided with face masks.  

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Participants and staff suspected to have COVID-19 (meeting the case definitions of COVID-19 as per 
Kenya Ministry of Health guidelines) while at the study site will be kept in separate isolation rooms as 
they await to be processed as per Kenya Ministry of Health protocols on COVID-19. After they leave the 
site, study staff donned in full PPE, in accordance with Ministry of Health guidelines, will disinfect the 
study site, including all rooms they had visited.  
While no other health risks are expected as part of participation in this study, if a participant is hurt as a 
direct result of participating in this study, the medical care will be provided by the study team at the 
respective research centers and the study will pay for the expenses. If the injury requires hospital 
admission, the participants will be admitted at the Kombewa Sub-County Hospital or the Jaramogi 
Oginga Odinga Teaching and Referral Hospital (JOOTRH), or another local area facility, as appropriate. If 
specialized treatment is required, they will be referred to relevant hospitals for management and the 
study will pay for medical expenses. 
 
  
3.3.2 Known Potential Benefits 
 
All volunteers for this study will receive the following benefits for their participation: 
 
 
All volunteers will undergo a medical examination at screening free of charge. All volunteers, 
whether accepted for enrolment into the trial or not, will benefit from this free health check-up. 
The results of all tests will be communicated to all volunteers. Where illnesses are newly 
diagnosed, a referral to an appropriate health provider will be made for the volunteer. 
 
 
For the short duration of their participation, subjects will receive free health care for acute 
conditions. In case of chronic conditions, subjects will be referred to appropriate health 
providers.  
 
• 
All gametocytemic individuals will receive Coartem® and low dose primaquine therapy 
(excluding pregnant or breast-feeding women for the latter) for the clearance of their malaria 
infection as the conclusion of their participation in the study. This does not constitute standard 
of care since asymptomatic individuals do not typically receive malaria treatment. However, 
clearance of asymptomatic parasitemia is associated with significant health benefits (such as 
reduction of incidence of chronic anemia and reduction of ongoing malaria transmission). 
 
4. OBJECTIVES AND PURPOSE  
Primary Objective: 
To assess variation in the proportion of infected mosquitoes with at least one oocyst (oocyst prevalence) 
in DSFA and DMFA performed at two consecutive time points in the same human subject with P. 
falciparum gametocytemia using microscopy 
 
Secondary Objectives: 

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1. To assess variation in oocyst density in DSFA and DMFA at two consecutive time points in same 
subject using microscopy. 
2. To assess variation in the proportion of infected mosquitoes with at least one sporozoite (sporozoite 
prevalence) in DSFA and DMFA performed at two consecutive time points in the same human 
subject using microscopy. 
Exploratory Objectives: 
1. To assess oocyst and sporozoite density in DSFA and DMFA at two consecutive time points in the 
same subject using molecular quantitative PCR (qPCR). 
2. To summarize the relationships between DSFA and DMFA results for oocyst and sporozoite 
density 
5. STUDY DESIGN AND ENDPOINTS  
5.1 Description of the Study Design 
This is a clinical study with an entomological component. Study participants will be recruited to 
participate in mosquito feeding assays.   
The proposed trial design has been developed to assess the consistency and reproducibility of two 
consecutive direct skin feeding assays (DSFA) at 24-hour interval. The results will determine the type of 
pivotal trial design for a follow-on Phase 2b trial whose objective is to bridge the standard membrane 
feeding assay (SMFA) to the direct skin feeding assay (DSFA) and direct membrane feeding assay (DMFA) 
using a monoclonal antibody intervention, TB31F mAb,  which interrupts transmission from human to 
mosquito. The results from this experimental medicine study will inform whether the preferred “Before-
After” trial design in which each human volunteer serves as their own internal control can be utilized for 
a follow-on Phase 2b trial.  
The rationale for this experimental medicine trial is described in the background section of the protocol.  
 
Figure 1. Experimental medicine clinical study design 

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The sample size to be included in this study is shown below in Figure 2. 
 
Figure 2. Estimated sample size screening and enrollment for experimental medicine trial. 
 
 
 
 
 
 
 
 
5.2 Study Endpoints 

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5.2.1 Primary Endpoint 
The primary study endpoint will be oocyst prevalence in DSFA and DMFA in mid-guts of mosquitoes 
performed at two consecutive time points in the same human using microscopy.  Oocyst prevalence is 
defined as the proportion of mosquitoes in a cup with at least one oocyst detected in the mid-gut 
among the mosquitoes (in the same cup) that underwent the feeding assays. The within and between 
subject variability will be described and the differences within and between DSFA and DMFA prevalence 
will be quantified.  
5.2.2 Secondary Endpoints 
 
1) Oocyst density in DSFA and DMFA at two consecutive time points in same subject using microscopy. 
Oocyst density is defined as the mean number of oocysts detected in infected mosquitoes that 
underwent feeding assays on the same subject. The variability and change in oocyst prevalence and 
density between the two consecutive feeds as measured by DSFA and DMFA will be determined.   
2) Sporozoite prevalence and intensity 
a) Sporozoite prevalence in DSFA and DMFA performed at two consecutive time points in the same 
human subject using microscopy. Sporozoite prevalence is defined as the proportion of 
mosquitoes in a cup with at least one sporozoite detected in the salivary glands among the 
mosquitoes (in the same cup) that underwent feeding assays.  
b) Sporozoite density in DFSA and DFMA performed at two consecutive time points in the same 
human subject using microscopy. Sporozoite density is defined as the mean number of 
sporozoites detected in infected mosquitoes that underwent feeding assays 
The change in sporozoite prevalence and density between the 2 consecutive feeds will be 
determined. 
5.2.3 Exploratory Endpoints 
The exploratory endpoint will be oocyst and sporozoite density using qPCR and their comparison to data 
obtained through microscopy.  
 
6. STUDY ENROLLMENT AND WITHDRAWAL  
6.1 Participant Inclusion Criteria 
In order to be eligible to participate in this study, an individual must meet all of the following criteria:  
 
 
Provision of signed or thumb printed and dated informed consent form  
 
Stated willingness to comply with all study procedures and availability for the duration of the 
study  
 
Male or female aged between 18 years and 55 years inclusive. 
 
Resident within the study area  
 
In good general health as evidenced by medical history and clinical examination before entering 
the study 

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 
Ability to take oral Coartem and low-dose primaquine anti-malarials upon conclusion of day 2 
(2nd direct skin feed) and be willing to adhere to the medication regimen  
 
For females, she must be of non-childbearing potential or use appropriate measures to prevent 
pregnancy for 30 days after receiving Coartem and primaquine. Non-childbearing potential 
means she is surgically sterilized or at least one year post-menopausal.  Appropriate measures 
to prevent pregnancy include abstinence or adequate contraceptive precautions (i.e. 
intrauterine contraceptive device; oral contraceptives; diaphragm or condom in combination 
with contraceptive jelly, cream or foam; Norplant or Depo-Provera).  
 
For males, he must be willing to ensure that he does not get his partner(s) pregnant for at least 
3 months after treatment with primaquine. Appropriate measures to prevent pregnancy include 
abstinence or adequate contraceptive precautions in either the participant or the partner. 
 
Positive for P. falciparum gametocytes as measured by PCR with cT value < 31. 
6.2 Participant Exclusion Criteria 
An individual who meets any of the following criteria will be excluded from participation in this study:  
 
 
Presence of any signs or symptoms of malaria 
 
Presence of contraindications to administration of Coartem and primaquine as indicated in the 
respective drug package inserts 
 
History of severe allergic reactions to mosquito bites (other than pruritus and local swelling)  
 
Pregnant (i.e. a positive pregnancy test)  
 
Current or recent (within the preceding 2 weeks) use of antimalarial treatment 
 
Current participation in a malaria vaccine study 
 
Any other findings that the investigator feels would increase the risk of having an adverse 
outcome from participation in the trial. 
6.3 Strategies for Recruitment and Retention 
 
6.3.1 Community information 
 
Community sensitization through public meetings (“barazas”) convened by project staff, whenever 
possible, will precede the actual human subject recruitment process. The community in which the study 
will take place will be informed about the nature and design of the study. Community leaders (chiefs, 
local village elders, and opinion leaders) and local health authorities will be formally briefed in their own 
language on the nature and purpose of the study. They will have the opportunity to ask questions of the 
PI or his designees. The community leaders can then disseminate the information within the community 
using their usual channels. 
6.3.2 Recruitment 
The target sample size for subjects with gametocytemia is approximately 45 individuals. Approximately 
400 participants will be screened for gametocyte carriage by PCR to achieve the target sample size. 
Screening of subjects will continue until the desired sample size of gametocytemic individuals who 
undergo all study procedures is attained. Adults aged 18 – 55 years will be recruited from the villages 
in the Kombewa Health and Demographics Surveillance System (HDSS) consisting of half of Kisumu 

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West and all of Seme Sub-Counties of Kisumu County, Kenya. Informed consent will be obtained from 
all subjects. Language and illiteracy will not be impediments in the recruitment process, all briefings 
and explanations will be in the understandable language of the participant. 
6.3.3 Reasons for Withdrawal or Termination 
Subjects can leave the study at any time for any reason if they wish to do so without any penalty or loss 
of benefits to which they are otherwise entitled. Subjects can also be withdrawn from the study 
procedures at the discretion of the clinical investigator. The following reasons may lead to withdrawal of 
individual subjects: 
 
Withdrawal of informed consent by volunteer;  
 
Any serious adverse event;  
 
Any adverse event that, according to clinical judgment of the investigator, is considered as a 
definite contraindication to proceeding with the study procedures such as severe allergic 
reaction to mosquito bites from DSFA on day 1 of trial activities that would preclude DSFA on 
day 2.   
 
Volunteer non-compliance with study procedures  
 
6.3.4 Handling of Participant withdrawals or termination 
Screening of potential subjects will continue until the desired sample size of gametocytemic individuals 
who undergo all study procedures is attained. Subjects who are withdrawn or terminated will be 
replaced by recruiting new ones. If the reason for withdrawal or termination is a medical event, then the 
subjects will be referred for management at an appropriate Kenya Ministry of Health (MoH) treatment 
facility. 
6.4 Premature Termination or Suspensions of Study 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause as determined by the PATH, USAMRD-A, KEMRI or WRAIR. Written notification, documenting the 
reason for study suspension or termination, will be provided to the investigator, WRAIR IRB, and to 
Kenyan regulatory authorities. If the study is prematurely terminated or suspended, the PI will promptly 
inform the IRB and will provide the reason(s) for the termination or suspension. Clinical evaluation and 
management of emergent clinical malaria that results in early termination of and individual study 
participation will be done according to the Kenya MoH guidelines. In the case of consent withdrawal of 
participants who are known to harbor gametocytes, malaria treatment will be offered to clear any 
parasites that could be circulating within the participant. There will be no further evaluation of 
gametocyte negative participants or participants in whom gametocyte testing has not been carried out. 
 
7. STUDY TREATMENT 
There will be no investigational agents in this study. The only medications that will be used are 
antimalarial medications, Coartem® and primaquine provided upon completion of day 2 DSFA in order 
to clear any asexual and sexual stage parasites. Package inserts for Coartem® and primaquine will be 
provided to the ethical review committees.  

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7.1.1 Acquisition 
Coartem® and primaquine will be procured by USAMRD-A from reputable vendors. 
 
7.1.2 Product Storage and Handling 
All anti-malarial medications should be stored below 30oC in the pharmacy at KCRC. Drugs will be 
provided to each subject upon completion of day 2 procedures. 
 
7.1.3 Dosing and Administration 
Refer to dosing and administration of Coartem® and primaquine in Table 1. The dosage for Coartem® 
and primaquine are as per the manufacturer’s package insert. Coartem® is part of the usual treatment 
prescribed for malaria as per the Kenya standards of treatment for malaria. The addition of primaquine 
is not part of standard of care, however the participant will benefit from the reduction of gametocyte 
carriage as a result of primaquine administration. The administration of Primaquine and the first dose of 
Coartem will be done under direct observation by clinic staff. According to standard of care in Kenya for 
malaria treatment, doses of Coartem on days 2 and 3 will be taken by the subject as instructed by study 
staff. The KCRC Pharmacy will maintain accountability logs for dispensed drugs. 
7.1.4 Justification for treatment of asymptomatic parasite carriers 
Plasmodium spp. asymptomatic carriers, defined as individuals with detectable asexual or sexual 
parasites in their blood and an absence of any acute clinical symptoms of malaria, asymptomatic carriers 
are not yet targeted by national malaria intervention strategies in many malaria-endemic countries, 
including Kenya. Local malaria treatment guidelines are currently focused on the management of 
symptomatic malaria: the starting point for treatment algorithms is symptoms such as fever, with an 
emphasis on testing for malaria prior to treatment. Asymptomatic carriers do not seek treatment for 
their infection. They and, therefore, constitute a reservoir of parasites for maintaining the parasite life 
cycle and transmission by the anopheline vector and thus a real public-health risk. Studies have 
suggested that a proportion of individuals with asymptomatic blood-stage malaria infection may 
eventually become symptomatic, although reinfections or other causes of fever cannot always be ruled 
out. These asymptomatic parasites have also been linked with chronic anemia and co-infections with 
invasive bacteria. As such, the treatment of asymptomatic malaria carriers who will be identified in this 
study will have both individual and public health benefits. At the individual level, parasite clearance may 
reduce the risk of getting symptomatic malaria later on and may also reduce the risk of adverse health 
outcomes associated with parasite carriage, such as chronic anemia and co-infections with invasive 
bacteria. From a public health point of view, clearance of parasitemia may reduce the risk of malaria 
transmission to others, especially to other members of the household, some of whom may be 
susceptible to severe malaria. Good clinical judgment strongly encourages that persons who are 
asymptomatic and infected with malaria parasites when found to be positive by molecular diagnostic 
techniques be provided antimalarial medications to clear both the asexual parasites and sexual stage 
gametocytes.  
 
Table 1. Anti-malarial Medications 
 

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Study Agents 
Dosage forms and strength 
Body 
weight 
Day 
1**** 
(AM) 
Day 1 
(+8hr) 
Day 2 
(AM) 
Day 2 
(PM) 
Day 3 
(AM) 
Day 3 
(PM) 
COARTEM * 
artemether (20 mg) and 
lumefantrine (120 mg)** 
> 35 kg 
4 tabs 
4 tabs 
@ 8 
hrs. 
4 tabs 
4 tabs 
4 tabs 
4 tabs 
  
  
  
  
  
  
  
  
  
PRIMAQUINE*** 
primaquine phosphate tablets 
26.3 mg primaquine phosphate 
(equivalent to 15 mg primaquine 
base) 
> 35 kg 
1 tab 
- 
- 
- 
- 
- 
  * Coartem should be taken with food 
** Coartem is also available as artemether (80mg) and lumefantrine (480mg) tablet for adults. The dosage of this strength is 1 
tablet at each dosing time point.  
***Primaquine may be taken with food 
**** Refers to first day of drug taken after 2nd DSFA 
8. STUDY PROCEDURES 
8.1 Study procedures and evaluations 
Table 2. Time and event 
 
Procedures 
Visit Day -1 to 0 
(Screening) 
Visit Day 1 
(Baseline/Enrollment) 
Visit Day 2 (Final) 
Window (hours)
–
–
+/-3 
Obtain informed consent of potential participant 
● 
 
 
Obtain demographic information 
● 
 
 
Obtain medical history 
● 
 
 
Obtain medications history 
● 
 
 
Perform medical examinations 
● 
● 
● 
Collect blood for gametocyte detection tests (2 mL) by PCR
● 
 
 
In females, urine pregnancy test 
● 
 
● 
Verify inclusion/exclusion criteria
● 
● 
● 
Verify gametocyte detection (RT-PCR) test results
 
● 
 
Record vital signs 
● 
● 
● 
Blood sample for serology – 5 mL*
 
● 
 
Blood sample for DMFA and microscopy (1ml)
 
● 
● 
DSFA
 
● 
● 
Admit the study participant for observation overnight
 
● 
 
Record local and unsolicited AEs as reported by participant or observed by 
investigator before DSFA and 30 minutes after DSFA 
 
● 
● 
Administer low dose primaquine* and first dose of Coartem® and give 
remaining Coartem® dose with instructions 
 
 
● 
 
Sera collected will be assayed in membrane feeding assay (risk mitigation) only if gametocyte-positive subject fails to 
transmit any parasites to mosquitoes in either the DSFA or DMFA performed either on day 1 or day 2. Sera will be 
destroyed before study close-out. 
  
8.1.1 Study site 

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The study will be conducted within the Kisumu West and Seme sub-counties of Kisumu County, western 
Kenya. Study activities will be coordinated from the USAMRD-A/K Kombewa CRC located in Kombam 
village, Kombewa. The study site is located about 40 km west of Kisumu city, the administrative capital 
of Kisumu County. The study sub-counties cover an area of about 369 km2 stretching on the North 
Eastern shores of Lake Victoria. The study area has been cartographically mapped by the USAMRD-A/K 
KCRC HDSS using Global Positioning System (GPS) technology. The HDSS is a longitudinal population 
registration system designed to track the evolving demographic and health status of the study 
populations over time. The HDSS population is monitored by field staff visiting each household bi-
annually to capture health and demographic information (birth rates, death rates, causes of death, 
pregnancies, immunization status, in-and out-migrations, etc.). Various studies nested on this platform 
take advantage of the sampling frame inherent in the HDSS, whether at individual, 
household/compound or regional levels. 
8.1.2 Recruitment, informed consent and enrolment 
 
Field workers (who will include nurses and/or clinical officers) will approach potential participants within 
their homesteads and give them a brief overview of what this protocol intends to undertake and request 
them to undergo a formal consenting session using a standard recruitment script. A typical field team 
will have one nurse or clinical officer and one or two field assistants. Those potential subjects who 
accept will undergo a formal and private consenting process that will be conducted by the nurse or 
clinical officer. Only those who accept to participate (as evidenced by the signing or thumb printing of 
the informed consent form) will be considered for enrolment in the study. Privacy will be achieved by 
conducting the consenting sessions in a secluded area within the compound of a given homestead. In 
the event that the potential participant is illiterate, an individual not associated with the study will be 
identified from within the participant’s village by the participant to act as an independent witness to the 
consenting process.  
After informed consent is obtained, the field team will assign the participant a study identification 
number (study ID). The list of study IDs (format CV85###) available will be prepared by the study 
coordinator and availed to the field teams. As the field teams recruit participants, they would 
communicate with the coordinator so that double assignment of IDs does not occur in the event more 
than one field team is doing recruitment. The field team would then collect demographic data, a brief 
malaria history and assess eligibility.  In addition to a general medical history, the clinician in the field 
team will specifically ask questions that would elucidate any contraindications to the administration of 
Primaquine and Coartem. The clinician will err on the side of caution whenever there is doubt regarding 
the presence or absence of a specific contraindication. 
Women of child-bearing potential will have a pregnancy test done within the homestead. The 
nurse/clinical officer will instruct the participant on how to obtain the urine specimen and then will 
proceed to conduct the pregnancy test using a commercially available human chorionic gonadotropin 
(hCG) based test kit.  
Individuals who meet eligibility criteria at this stage will have a blood sample collected for detection of 
gametocytemia.   
 
8.1.3 Blood sample collection 
The nurse or clinical officer in the field team will obtain 2ml of whole blood from participants. This will 
happen while the study team is still in the participant’s homestead. Blood will be collected in a tube 
containing Ethylenediaminetetraacetic acid (EDTA) using the aseptic technique, placed in a sample 

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transportation box at +4 to +80C for shipment to the central Malaria Drug Resistance Laboratory (MDR) 
for the gametocyte detection assays by PCR. Blood sample collection will conclude the field visit. All 
study documents that will be filled out while in the field will be transported back to the KCRC. A signed 
copy of the ICF will be given to the study participant. The blood samples will be batched and transported 
to the MDR, to arrive within 6 hours of collection. In the event that the field team anticipates that the 6 
hour window to ship the blood samples to MDR is closing in, the team will contact the transport 
dispatcher at the KCRC to pick up the sample transportation box from the field.  
 
8.1.4 Enrolment of gametocytemic participants 
Participants who are found to harbor gametocytes as per the PCR test (with a cT value of less than 31) 
will be enrolled to participate in the mosquito feeding assays. The MDR expects to turn around the 
results of the PCR tests within approximately 48 hours of sample collection. The results of the tests will 
be communicated to the study team in Kombewa, who will then contact the participants. For 
participants who do not have gametocytes detected, they will be informed of the results and the study 
team will explain to them that their participation has ended. Participants who are gametocytemic will be 
invited to participate in the mosquito feeding assays. Participants who are found to have very low 
density gametocytemia (PCR cT value of 31 and above) will not be invited to participate in mosquito 
feeding assays. They will, however, be offered treatment to clear their parasitemia as described in 
section 7 of this protocol. The team in Kombewa will facilitate the movement of the participants from 
their homesteads to the KEMRI/USAMRD-A facilities where the feeding assays will take place as 
described in section 8.1.5. 
 
8.1.5 Mosquito feeding assays (DSFA & DMFA) 
Mosquito feeding assays involve feeding unfed, sterile (pathogen-free), insectary-reared Anopheles 
gambiae mosquitoes on malaria infected humans for the purposes of obtaining oocyst and sporozoite 
counts in mosquito’s midgut and salivary glands, respectively, thereby allowing an accurate evaluation 
of malaria transmission. The KEMRI/USAMRD-A/K entomology laboratory maintains a colony of An. 
gambiae sensu stricto (KISUMU strain) that were originally collected at Lwanda village, Siaya County in 
western Kenya. Mosquitoes are provided a 10% water infused sucrose meal daily, maintained at 30o C 
(70-80% relative humidity), and reared under very stringent conditions. Cow’s blood is used to maintain 
the colony, and only the progeny (3-5 day old female mosquitoes) are used for the feeding assays. 
Additionally, 10% of each new generation of mosquitoes are pooled and tested for arboviruses. 
This study will conduct two types of mosquito feeding assays using study participants concurrently: 
Direct Skin Feeding Assay (DSFA) and Direct Membrane Feeding Assay (DMFA).  
 
DMFA will be performed using the general procedures of Bousema et al. and DA et al. (13,14) for 
membrane feeding, with the notable exception that serum in collected subject blood samples will not be 
replaced with non-immune serum. Briefly, 0.12 mL of whole blood collected from study participants just 
prior to the feeding assays will be transferred to 2ml-capacity glass receptacles that comprise the 
artificial feeders warmed at 36-37 0C using a circulating water bath and provided to 3-5-day old starved 
female An. gambiae sensu stricto mosquitoes (KISUMU strain; n= 30 per cup; 2 cups per subject). 
 
DSFA will be performed using the protocol described by Bonnet et al. and Bousema et al. (11,12) 
(studies successfully completed in Sub-Saharan Africa). Sixty sterile 3-5-day old An. gambiae sensu 

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stricto female mosquitoes (KISUMU strain) will be starved for five hours and transferred to a cardboard 
cup covered with netting prior to use. The mosquito containing cup will be placed on the calf or arm of 
each volunteer to feed for 15 minutes, after which antihistamine ointment will be applied to the 
volunteer’s skin. (KISUMU strain; n=60 per cup; 1 cup per subject). 
 
 
Day 1 procedures: Consenting human subjects identified as gametocytemic by RT-PCR will be 
transported to the KEMRI/USAMRD-A/K laboratory in Kisian within 48 hours of being identified in order 
to participate in the mosquito feeding assay. A blood sample (~ 1 mL) for DMFA will be collected from 
each subject (Table 2) prior to the DSFA.  A blood sample (~ 5 mL) for serum collection will be collected 
and a membrane feeding assay may be performed (risk mitigation) only if gametocyte-positive subject 
fails to transmit any parasites to any mosquito in either the DSFA or DMFA on days 1 or day 2. Sera will 
be destroyed before study close-out. Upon completion of the first DSFA, the subjects will stay overnight 
under the supervision of KCRC staff at KCRC-affiliated district hospital. Subjects will not return to their 
home at the end of day 1 procedures. 
 
Day 2 procedures: Subjects will be transported to the KEMRI/USAMRD-A/K laboratory in Kisian for 
the second DMFA and DSFA assay within +/- 3 hours of the time when the first DSFA was performed. 
The two sequential mosquito feeding DSFA and DMFA assays on day 1 and day 2 ) will be performed in 
all study participants within 24 hours (+/- 3 hours) to mimic a potential follow-on study design proposed 
to evaluate infectiousness of individuals before and after the administration of transmission-blocking 
intervention (drug and/or monoclonal antibody). A different skin site will be selected for DFSA on Day 2. 
The remainder of the blood sample collected for DMFA will be used to prepare malaria blood films 
(MBFs). Dried blood spots (DBS) for RT-PCR for Plasmodium detection will be collected and stored for a 
maximum of 30 days as a back-up diagnostic assay and then destroyed. There will be no future use 
archiving of biological samples.   
Upon completion of day 2 of DSFA, subject will take day 1 of both Primaquine and Coartem anti-
malarials. Coartem tablets for days 2 and 3 will be provided to subjects per standard operating 
procedures. Upon release from the clinic, the participation of each subject will be ended.  
 
Mosquito and insectary procedures: All fed (and potentially infectious) mosquitoes will be held in 
locked environmental chambers located at the ACL-2 insectary at the USAMRD-A/K entomology facilities 
for security and optimum developmental conditions. Any unfed mosquitoes will be transferred to a 
second container and killed by placing in a -20oC freezer for 48 hours prior to disposal. 
 
Out of the 60 mosquitoes in each cup, it is estimated that approximately 5% will not feed. Therefore 29 
will be held up to Day 9 or Day 10 post feed for oocyst count determination and the remaining 28 will be 
held up to Day 14 or Day 15 post feed for sporozoite count determination. The maximum anticipated 
mortality for mosquitoes held to D9/D10 is approximately 15%, while for those held to D14/D15 is 
approximately 50%. Thus, at least 25 mosquitoes will be available for oocyst counts and at least 15 for 
sporozoite counts as described in section 8.1.9.3 below. This is summarized in the figure below. 
 

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Figure 8: Disposition of mosquitoes after the feeding assays 
 
 
 
8.1.6 Management of study participants 
Subjects found to harbor gametocytes will be admitted for a period of approximately 24 hours to an 
observation ward at the Kombewa CRC or the Kombewa sub-county hospital located across the street 
from the CRC while awaiting their second feed as described above. While in the ward, they will be 
encouraged to dress in clothing that covers most of the skin so as to minimize contact with mosquitoes. 
In order to allow for the successful execution of the mosquito feeding assays, all gametocytemic 
participants will be offered antimalarial treatment only after completion of all feeding assays. A full 
course of artemether/lumefantrine 480/80mg (Coartem®) along with a single low dose of primaquine 
(15 mg primaquine base) will be administered as per the package insert – primaquine and the first dose 
of Coartem® will be witnessed by study staff. Primaquine will not be given to pregnant or breastfeeding 
women. Given that PCR testing tends to detect parasite levels much lower than the detection limit of 
standard clinical tests for malaria (RDT and microscopy), standard clinical testing will not be performed 
prior to administration of antimalarial treatment to clear parasitemia. Administration of primaquine to 
malaria patients is not part of standard care in Kenya. Primaquine, however, is specifically effective 
against gametocytes and therefore serves the purpose of ensuring clearance of circulating gametocytes 
in the participant. The administration of antimalarials to asymptomatic individuals is currently not part 
of standard of care in Kenya. 
Gametocytemic participants who show signs of malaria while under observation will be managed in 
accordance to the Kenya MoH National Guidelines for the Diagnosis, Treatment and Prevention of 
Malaria. Such participants will not participate in further feeding assays and will be replaced until the 
desired sample size is achieved. 
Study participants who withdraw consent while under observation and are still asymptomatic will be 
offered antimalarial treatment to clear parasitemia as described above prior to leaving the observation 

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ward. If they are symptomatic, they will be offered the choice to be managed by the study clinical team 
in accordance to the Kenya MoH National Guidelines for the Diagnosis, Treatment and Prevention of 
Malaria, or be referred to the Kenya Ministry of Health clinician for further management. 
 
8.1.7 Standard of care study procedures 
According to the Kenya MoH National Guidelines for the Diagnosis, Treatment and Prevention of 
Malaria, patients presenting with signs and symptoms of malaria will get tested for malaria (either by 
rapid diagnostic test or microscopy) and treatment offered immediately upon receiving the test results. 
Artemisinin based combination therapy (ACT) (for example artemether lumefantrine) would be typically 
prescribed for uncomplicated malaria, and intravenous artesunate would be prescribed for severe 
malaria. In certain cases, presumptive treatment of malaria is allowed. Additionally, drugs for alleviating 
symptoms, such as antipyretics for fever, are also typically prescribed. 
For this study, asymptomatic participants will be recruited and tested for presence of gametocytes. They 
will receive malaria treatment as described in section 8.1.5. 
 
8.1.8 Clinical laboratory procedures 
A urine pregnancy test using a commercially available human chorionic gonadotropin (hCG) based test 
will be done in all females of child-bearing potential at screening and repeated no more than 24 hours 
prior to administration of primaquine, with all pregnant women being excluded. Clinical laboratory tests 
that are needed to guide subject treatment for emergent clinical malaria will be requested by the 
attending clinician in accordance to the Kenya MoH guidelines. 
 
8.1.9 Other assays or procedures 
 
8.1.9.1 Malaria blood film microscopy 
Two glass microscope slides will be prepared by the project laboratory technician from the whole blood 
sample obtained from each volunteer at the screening visit, and prior to mosquito feeding assays in 
accordance to site SOPs, each with one thick and one thin blood film. The slides will be batched and read 
at a later date and thus will not influence the decision of whether to perform feeding assays or not. The 
primary purpose of collecting the slides is to classify gametocyte carriage as microscopic or sub-
microscopic during data analysis. The slides will also be used as a quality control tool for the molecular 
assays described in section 8.1.8.2. An additional slide may be collected in an acutely ill subject to guide 
management – this slide will be read immediately, and the results shared with the study clinician. 
A minimum of 200 microscopic fields will be examined to determine presence or absence of sexual 
(gametocytes) and asexual malaria parasites on the thick smear. If asexual parasites are found on the 
thick smear, the thin smear will be evaluated for Plasmodium speciation. If gametocytes are present, the 
male to female ratio will be determined. The findings of two expert microscopists will be considered. A 
third expert microscopist will be used to resolve any discrepant results. 
 
8.1.9.2 Plasmodium detection molecular assays 
Gametocyte RT-PCR will be performed overnight or on the day following blood collection on all eligible 
subjects that consent for the study and results will be used to guide mosquito feeding assays to be 
performed on gametocytemic individuals. Total nucleic acids (DNA/RNA) will be isolated from blood 

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samples and then PCR amplification of the Plasmodium 18s small subunit ribosomal RNA (rRNA) 
metabolic gene will be performed to detect P. falciparum, P. ovale and P. malariae parasites. For 
gametocyte RT-PCR, RNA will be reverse-transcribed and the resulting cDNA will be used to amplify 
gametocyte-specific gene targets. Among the markers that shall be used are pfs16, pfs25 and pfGMET. 
For quantification, positive control standards with known densities will be included and used to 
generate a standard curve. Parasite density of unknown samples will then be extrapolated from the 
standard curve. These assays will specifically help quantify both parasitemia and gametocytemia below 
the current level of detection of microscopy. Participants who are found to have gametocytes by the RT-
PCR will be invited to participate in mosquito feeding assays. 
8.1.9.3 Oocyst and sporozoite detection and quantification 
 
Approximately half of fed mosquitoes will be maintained for 9-10 days post feeding for detection and 
quantification of oocysts in the midgut. Briefly, midguts will be dissected from mosquitoes, stained with 
1% mercurochrome and examined by microscopy. Oocyst numbers in each midgut will be recorded. 
The remainder of fed mosquitoes will be maintained for 14-15 days post-feeding for detection and 
quantification of sporozoites. Briefly, salivary glands will be dissected from live mosquitoes submerged 
in phosphate-buffered saline (PBS) in order to visualize motile sporozoites by microscopy. Sporozoite 
prevalence will be recorded. 
The dissected mosquitoes will be processed for detection and quantification of oocysts and sporozoites 
by molecular methods. After microscopy, slides containing midguts will be flooded with PBS to allow 
easy removal of coverslips, and excess mercurochrome will be removed by dragging the midgut gently 
through a clean PBS droplet. Mosquito carcasses will then be homogenized in glass grinding tubes with 
180 μl of PBS and stored in 180 µL of DNA Tissue Lysis buffer at -800C in preparation for PCR analysis.  
Nucleic acid extraction will be performed using commercial extraction kits following the manufacturer’s 
instructions. P. falciparum parasite detection and quantification will be undertaken using the 18S rRNA 
gene target. To quantify the number of genomes detected in the mosquito midguts and salivary glands 
using the 18S qPCR assay, a standard curve generated using synthetic plasmid DNA will be used. 
8.1.10 Specimen Preparation, Handling, and Storage 
Whole blood collected in EDTA tubes from the field will be sent to the MDR central laboratory located 
on the KEMRI campus for processing (see section 8.1.3 for details).  Specimens will be stored at 
KEMRI/USAMRD-A facilities. Filter paper and whole blood samples will be stored at -800C.  DNA and RNA 
extracts will be stored at +4OC if intended for immediate use or -20OC if intended for near future use. 
The remaining DNA and RNA will be stored appropriately at -800C and retrieved whenever they need to 
be analyzed. Serum will be stored at -800C until required for analyses. At the end of the study, after all 
analyses have been completed, any remaining samples will be destroyed.  
 
9. ASSESSMENT OF SAFETY 
9.1 Adverse Events (AE) 

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Local solicited AEs, as defined in this protocol, include pruritus and erythema at the site of the mosquito 
bite, in a study participant following DSFA on days 1 and 2. Unsolicited AEs will be captured in study 
participants after informed consent is obtained and until the subject completes all study procedures.  
 
9.2 Serious Adverse Event 
An AE or suspected adverse reaction is considered "serious" if, in the view of either the investigator or 
sponsor, it results in any of the following outcomes: death, a life-threatening adverse event, inpatient 
hospitalization, a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or a congenital anomaly/birth defect. Important medical events that may not 
result in death, be life-threatening, or require hospitalization may be considered serious when, based 
upon appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition.  
 
9.3 Classification of An Adverse Event 
For AEs, the following guidelines will be used to describe severity.  
 
 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning.  
 
Severe – Events prevents a participant’s usual daily activity. 
 
9.4 Time Period and Frequency of Event Assessment and Follow-up 
The investigator will record all reportable events with start dates occurring any time after informed 
consent is obtained until the last day of study participation. All AEs not meeting the criteria for severe 
adverse events (SAEs) will be captured on the appropriate case report form.  
 
9.5 Reporting Procedures 
9.5.1 Adverse event and Serious Adverse Event reporting 
All serious adverse events (SAEs) will be promptly (within 48 hours) reported to the KEMRI SERU and to 
the WRAIR IRB. Prompt (within 48 hours) reports will be submitted to the KEMRI SERU by email 
(ERCAdmin@kemri.org; seru@kemri.org) and to the WRAIR IRB by phone (301) 319-9940, or by email 
(usarmy.detrick.medcom-wrair.mbx.hspb@mail.mil), or by facsimile (301) 319-9961. The Principal 
Investigator will then submit written reports within 10 working days to the WRAIR IRB at the following 
address: Walter Reed Army Institute of Research, ATTN: Human Subject Protection Branch (HSPB), 503 
Robert Grant Ave, Silver Spring, MD 20910.  
 
Any SAEs or other events that meet PATH Research Ethics Committee’s (REC) expedited reporting 
requirements will be promptly (within 24 hours) reported to the PATH Study Representative by email. 
The PATH Study Representative will then promptly (within 48 hours) submit the report to PATH REC 
using their online submission and reporting website (www.irbnet.org). 

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Follow up reports will be submitted as additional information becomes available. A summary of the non-
serious adverse events and the SAEs (both related and unrelated) that occurred during the reporting 
period should also be included in the continuing review report to the KEMRI SERU, and the WRAIR IRB. 
The WRAIR HSPB will report SAEs to the USAMRMC ORP HRPO as per WRAIR SOP UWZ-C-636. 
9.5.2 Unanticipated problems and UPIRTSOs 
All non-serious unanticipated problems (events not involving risk to participants or others) will be 
reported in the continuing review/progress report to the WRAIR IRB and KEMRI SERU. 
All serious unanticipated problems involving risk to participants or others should promptly (within 48 
hours) reported to the WRAIR IRB and KEMRI SERU. The Principal Investigator will then submit a written 
report within 10 working days to the WRAIR IRB and KEMRI SERU. 
Follow up reports should be submitted as soon as additional information becomes available. A summary 
of the serious unanticipated problems will also be included in the continuing review/progress report 
submitted to the WRAIR IRB and KEMRI SERU. 
9.5.3 Pregnancies 
Each pregnancy must be reported promptly (within 48 hours of identification) by email to the WRAIR 
IRB and KEMRI SERU. 
9.5.4 Continuing Review/Progress Reports 
The PI or designee will be responsible for submitting the required continuing review/progress reports 
and supporting documentation, as appropriate, to the WRAIR IRB and KEMRI SERU, allowing sufficient 
time for review and continuation determination prior the established continuing review/progress report 
date.  A closeout report will be submitted at the end of the study upon completion of all the study 
activities or 5 years, whichever comes first. 
9.5.5 Inspections and audits 
Knowledge of any pending compliance inspections/visits by Office for Human Research Protections 
(OHRP) or other government agency concerning clinical investigation or research, the issuance of 
Inspection Reports, warning letters or actions taken by any Regulatory Agencies including legal or 
medical actions and any instances of serious or continuing noncompliance with the regulations or 
requirements will be reported immediately to the KEMRI SERU (ERCAdmin@kemri.org; seru@kemri.org) 
and WRAIR IRB by phone (301) 319-9940, or by email (Usarmy.detrick.medcom-
wrair.mbx.hspb@mail.mil), or by facsimile (301) 319-9961.  The WRAIR HSPB will report to USAMRDC 
ORP HRPO as per SOP UWS-HP-636. 
9.5.6 Protocol deviations 
Major protocol deviations are a subset of protocol deviations that may significantly impact the 
completeness, accuracy, and/or reliability of the study data or that may significantly affect a subject's 
rights, safety, or well-being. The principal investigator will not implement any deviation from or changes 
to the protocol without the agreement of PATH and prior review from all IRBs expect where necessary 
to eliminate an immediate hazard to trial subjects or when the change involves only logistical or 

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administrative aspects of the trial. The PI will ensure that deviations from the protocol are documented 
and reported to PATH. 
Major/significant deviations must be reported to the WRAIR IRB and KEMRI SERU within 48 hours of 
becoming aware of the event.  A written report must be submitted within 10 working days of knowledge 
of the event to the WRAIR IRB and KEMRI SERU.  
 All deviations, including minor/non-significant deviations, will be reported to the WRAIR HSPB/IRB and 
KEMRI SERU in a cumulative summary report with each continuing review/progress report and with the 
study closeout report. These reports will include descriptions of the deviation, any actions taken in 
response to the deviation and an assessment of the impact of the deviation. 
All subject-specific deviations from the protocol (e.g., failure to return for follow-up visits or blood 
collection within the time indicated in the protocol) are to be documented. The PI or designee will be 
responsible for identifying and reporting all deviations, which are defined as isolated occurrences 
involving a procedure that did not follow the study protocol or SSP. Action taken in response to the 
deviation, and the impact of the deviation will be assessed by the PI or sub investigator and recorded as 
Major or Minor as per the WRAIR HSPB definitions. Deviations will be reported in the continuing 
review/progress report to the WRAIR IRB and the closeout report. 
Any protocol deviation that adversely affects the safety or rights of a subject or scientific integrity of the 
study, the deviation will be reported within 48 hours of recognizing the deviation, to the WRAIR IRB and 
KEMRI SERU with a written report submitted within 10 working days. 
 
10. STATISTICAL CONSIDERATIONS  
10.1 Overview and General Design 
This is a single center and single group study designed to better understand the relationship between 
parasite transmission from humans to mosquitoes, within a person, across persons, and by both DSFA 
and DMFA performed at two consecutive time points.  
10.2 Statistical Hypothesis 
 
We are interested in assessing the equivalence of oocyst prevalence measured in the same subject 
undergoing DSFA at two consecutive 24-hour time periods.  If the difference in oocyst prevalence 
measured on two consecutive days is represented as Δ, the null (H0) and alternative (H1) hypotheses of 
the primary efficacy endpoint analysis are as follows: 
H0:  
Δ> ΔE or Δ<- ΔE 
H1:  
- ΔE< Δ< ΔE 
Where Δ¬E represents a value such that range that would support the use of a “before-after” trial 
design in a future mAb study. 
 

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10.3 Sample Size 
This is a clinical investigation study where there is no prior knowledge related to the oocyst prevalence 
or density on a repeated DSFA or DMFA in a human subject. As such, the sample size chosen for this 
study was primarily based on logistical and budgetary considerations that would estimate the variability 
in DSFA. The results of this experimental medicine trial would be to ascertain the feasibility of using a 
paired “before-after” study design in same individual in a future trial when a TRI is introduced.   
There are two unknowns and a major constraint in arriving at a meaningful sample size for the current 
trial challenging. First, there is no prior data in metrics of DSFA in same person at 2 consecutive time 
points. Secondly, there is a wide range of initial gametocyte densities in persons at baseline screening 
which may markedly influence the transmission and development of parasites from man-to-mosquito 
and detection of oocysts in the mosquito midgut 9 days after a blood meal. 
Prior data obtained from the same site and same investigators in Kenya indicate that: 
Approximately 25% of persons at any one time will have positive P. falciparum gametocytes by PCR 
Of those that are positive for P. falciparum gametocytes and who have undergone DSFA, there will be a 
distribution of oocyst-positive mosquitoes (defined as positive event) detected 9 days after a mosquito 
feed as shown below. 
 
Figure 9: Prevalence of infected mosquitos. Unpublished data from previous study 
 
 
It is important to note that in approximately 16% of experiments with PCR-positive gametocytes, there 
will be no (zero) infected mosquitoes detected. 
Greater than 50% of DSF assays have < than 5 infected mosquitoes out of 25 that were found to have 
oocysts detected in the midgut 9 days after a blood meal. 

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As the budget for the trial is constrained, we estimate a sample size below that will have 45 consented 
individuals out of 400 screened with positive P. falciparum gametocytes of whom will have an estimated 
transmissibility on day 1 indicated in the figure below. It is unknown whether the same level of 
transmissibility occurs on day 2 in the same person which is the subject of the proposed trial. The 
sample size proposed below provides for a reasonable and logistically feasible number of subjects 
(n=36) that will have two consecutive DSFA performed across a range of positive events.  
 
Figure 10: Study sample size 
 
 
With these limitations in mind a simulation approach was used to understand the power to reject 
various equivalence null hypotheses (-ΔE, ΔE) assuming either high correlation within subjects for 
consecutive DSFA or independence between measurements.   
 
Briefly, we assume that the number of positive mosquitos for any DSFA is binomially distributed 
xit~Bin(25,pit ) where pit~Beta (1.5,6). The parameters for the Beta distribution were selected to generate 
a distribution of positive mosquitos which achieves, approximately, the characteristics listed in points 3 
and 4 above (16% with 0 positive mosquitos and 50% less than 5).  For a single draw of xt=x1t,…,x36t, the 
distribution of mosquitos looks similar to the data observed above.   
 
We considered two scenarios in our simulations. First, we assume that observations within person on 
consecutive days are independent. That is, xi1~Bin(25,pi1 )  and xi2~Bin(25,pi2 ) where pi1 and pi2 are 
independent draws from the Beta distribution.  Second, we assume the underlying transmission 
probability within a person is constant for the two consecutive feeds and xi1~Bin(25,pi )  and xi2~Bin(25,pi 
) where  pi=pi1=pi2.  A simulated example for these two scenarios is shown in the figure below. 

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Figure 11. Simulating scenarios with independent daily transmission probabilities within subjects (left) or 
constant transmission probabilities over 24 hours within subjects (right). 
 
We simulated this experiment based on both of the scenarios and then used a paired t-test to test for 
equivalence in oocyst prevalence at various equivalence intervals (-ΔE, ΔE). We find that with the current 
sample size of 36 subjects and 25 mosquitos per feeding assay, we will have 90% power to reject the 
null hypothesis that the difference of oocyst prevalence between consecutive feeding assays is greater 
than +/-7% for correlated data (scenario 2).  However, the same equivalence interval (ΔE=0.07) has only 
4% power assuming independent data (scenario 1). Power for the equivalence test over a range of 
equivalence intervals and both scenarios are shown in the figure below. 
 
Figure 12. Power calculation for equivalence test 
 
 

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The data collected in this study will help inform the design of follow on studies by informing parameter 
values of interest, such as the correlation between subsequent feeds, to assess the power to measure 
monoclonal antibody or drug efficacy with before-after or two-group designs under various samples 
sizes and efficacy assumptions. For example, in the figures below independent data has an average 
correlation of 0.0 and is compared with a t-test and correlated data has an average correlation of 0.5 
and is tested with a paired t-test. Using the parameters of the simulated data above we can investigate 
the impact of sample size and design on the power to detect varying levels of efficacy.  The real data 
from this study will help inform this parameter space. 
 
Figure 13. Estimated power calculation for follow-on Phase 2b trial design 
 
10.4 Definitions of Populations to be Analyzed 
10.4.1 Enrolled population 
All screened participants who provide informed consent. 
10.4.2 Full analysis population 
All participants in the enrolled population who participated in at least one direct skin feed. 
10.4.3 Per Protocol population 
All participants in the full analysis population who have no major protocol violations that are 
determined to potentially interfere with DSFA or DMFA results. This population will serve as the primary 
analysis population for the DSFA and DMFA assessments. 

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10.5 Analysis Methodology 
All statistical methods shall be detailed in a statistical analysis plan (SAP) that will be finalized prior to 
database lock. 
The number of subjects enrolled and having undergone DSFA will be summarized. Demographic data will 
be summarized by descriptive statistics. Except where otherwise indicated in this document or the SAP, 
summary statistics will be composed of the mean, standard deviation, 1st, 2nd, and 3rd quartile, and the 
minimum and maximum for continuous variables.  For categorical variables, the count and proportion 
(using one digit beyond the decimal point) will be presented.  Correlation coefficients will be used to 
summarize associations between measurements.  Scatter plots and Bland-Altman plots will be used to 
graphically explore relationships between measurements.  All study data will be presented in listings.   
 
A full description of analyses will be described in the SAP, but brief descriptions have been provided in 
the subsequent sub-sections.  
10.5.1 Analysis of Safety Data 
All safety assessments will take place in the full analysis population.  All subject-level percentages will be 
supplemented with two-sided 95% CIs computed via the Clopper-Pearson method.  Summaries will 
include all events occurring on or after the date of first skin feed.  Individual summaries (denominators 
for percentages) will be limited to the number of subjects within the appropriate analysis population 
with data available. 
All unsolicited AEs, including serious and/or severe AEs will be coded according to the Medical 
Dictionary for Regulatory Activities (MedDRA) version 20.1 or later. A summary table will be prepared 
for unsolicited AEs presenting incidence of any AE, any related AE, any serious AE, any severe AE, any AE 
of grade ≥2, any related AE of grade ≥2, and any AE leading to study withdrawal where a subject only 
contributes once. 
10.5.2 Analysis of Primary Objectives 
Oocyst prevalence 
The variation in the proportion of infected mosquitoes with at least one oocyst (oocyst prevalence) in 
DSFA and DMFA performed at two consecutive time points in the same human subject will be assessed 
with the per protocol population.  Summary statistics will be provided for each assay and by time point 
(first and second).  Additionally, Pearson correlation coefficients and estimates of total variation 
decomposed by between and within person variation will be summarized for both DSFA and DMFA.  A 
paired t-tests will be used to evaluate the equivalence hypothesis for DSFA and DMFA.  Mean 
differences and the corresponding 95% confidence intervals (CIs) will be provided. 
10.5.3 Analysis of Secondary Objectives 
Oocyst density 
The arithmetic mean oocyst density will be calculated for each subject by time point and assay. 
Distributions of subject-level means will be summarized by time point and assay.  Medians will also be 
considered as a summary measure by subject.  A generalized linear mixed model framework will be used 

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to incorporate the data from all mosquitos for each subject and summarize within and between person 
variability as well as summarize differences between time points.  Variability and difference estimates 
(with 95% CIs) will be summarized for DSFA and DMFA.  These analyses will be conducted on the per 
protocol population.  A sensitivity analysis will be conducted using the full analysis population. 
 
Sporozoite prevalence 
The variation in the proportion of infected mosquitoes with at least one sporozoite in DSFA and DMFA 
performed at two consecutive time points in the same human subject will be assessed with the per 
protocol population.  Summary statistics will be provided for each assay and by time point (first and 
second).  Additionally, Pearson correlation coefficients and estimates of total variation decomposed by 
between and within person variation will be summarized for both DSFA and DMFA.  A paired t-tests will 
be used to evaluate the equivalence for DSFA and DMFA.  Mean differences and the corresponding 95% 
confidence intervals (CIs) will be provided. 
 
Analysis of Exploratory Objectives 
Oocyst and sporozoite density measured via qPCR 
The arithmetic mean sporozoite and oocyst density as measured by qPCR will be calculated for each 
subject by time point and assay. Distributions of subject-level means will be summarized by time point 
and assay.  Medians will also be considered as a summary measure by subject.  A generalized linear 
mixed model framework will be used to incorporate the data from all mosquitos for each subject and 
summarize within and between person variability as well as summarize differences between time 
points.  Variability and difference estimates (with 95% CIs) will be summarized for DSFA and DMFA.  
These analyses will be conducted on the per protocol population.  A sensitivity analysis will be 
conducted using the full analysis population. 
 
Assessing the relationship between oocyst and sporozoite density measured by DSFA and DMFA via 
microscopy and qPCR 
Pearson correlation coefficients and Bland-Altman plots will be used to explore relationship between 
density measures as measured by DSFA and DMFA via microscopy and qPCR.  The regression framework 
described above will be expanded to explore mean differences in measures.  Estimated mean 
differences and corresponding 95% CIs will be reported.  These analyses will be conducted on the per 
protocol population.  A sensitivity analysis will be conducted using the full analysis population. 
 
10.6 Handling of Dropouts and Missing Data 
In this experimental medicine study, missing data will be assumed to be missing completely at random, 
and only observed data collected from participants and available in the appropriate study population 
will be used for analysis. 

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10.7 Timing of Analyses 
Upon completion of the second DSFA and DMFA for the final participant, a topline analysis will be 
initiated to include the results of the primary and secondary oocyte prevalence and density results. 
Following collection of the remainder of the data and database lock, all results will be described in a 
peer reviewed publication. 
11. SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS  
The KCRC site will maintain appropriate medical and research records for this trial. Source data are all 
information, original records of clinical findings, observations, or other activities in a clinical trial 
necessary for the reconstruction and evaluation of the trial.  
12. QUALITY ASSURANCE AND QUALITY CONTROL 
The KCRC site has SOPs for quality management that include staff training methods, product 
accountability records, specimen tracking logs, questionnaires, audio or video recordings.  
 
13. ETHICS/PROTECTION OF HUMAN SUBJECTS  
13.1 Ethical Standard 
This study will be conducted in accordance with the latest revision of the Declaration of Helsinki (2013), 
the Medical research Involving Human Subjects act (WMO), the ICH Good Clinical Practice, Department 
of Defense (DoD) requirements as outlined in 32 CFR 219, 45 CFR 46 and local rules of KEMRI SERU. The 
investigators are responsible for obtaining all relevant ethical approvals of the protocol and any 
subsequent amendments in compliance with local law before the start of the study.  
13.2 Institutional Review Board 
13.2.1 Ethics Approval 
Submission of the protocol and supporting documents will occur in accordance with local regulatory 
requirements. KEMRI SERU is the IRB of record for the Kenyan Site, Kombewa in Kisumu. This protocol 
will be submitted to KEMRI Center Scientific Committee for review and approval. It will also be 
submitted to the KEMRI SERU where ethical aspects of the protocol will be reviewed before the protocol 
can be approved. 
The Walter Reed Army Institute of Research IRB will also be involved in the approval process for this 
protocol. WRAIR IRB is the IRB of record for US Army Medical Research Directorate-Africa (USAMRD-A). 
For this reason, approval from the WRAIR IRB will be sought alongside the KEMRI SERU approval prior to 
implementation of the protocol or protocol amendments for this study. 
PATH will delegate ethical review and oversight to KEMRI SERU. 
13.2.2 Protocol Amendments 
All amendments and modifications will be submitted to the WRAIR IRB and KEMRI SERU for review and 
approval.  No changes in protocol conduct will be implemented until approval is obtained from the 

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WRAIR IRB and KEMRI SERU, as applicable, unless required to eliminate apparent immediate hazards to 
the study subjects. Amendments and modifications cannot be implemented until the WRAIR 
Commander’s Memorandum has been issued. The WRAIR HSPB will submit amendments to USAMRMC 
ORP HRPO as per WRAIR SOP UWZ-C-636.  
Non-significant amendments from the sponsor that are defined as amendments that do not affect the 
safety and wellbeing of the subjects at the site, and that are more administrative will also require both 
WRAIR IRB and KEMRI SERU approval prior to implementation. 
13.3 Informed Consent Process 
 
13.3.1 Consent and Other Informational Documents Provided to Participants 
Consent forms describing in detail the study procedures, and risks and benefits are given to the 
participant and written documentation of informed consent is required prior to starting the 
intervention.  
 
13.3.2 Consent Procedures and Documentation 
Discussion of risks and possible benefits of participation will be provided to the participants and their 
families. Consent forms will be IRB-approved and the participant (or the consent witness) will be asked 
to read and review the document. The investigator or designee will explain the research study to the 
participant and answer any questions that may arise. All participants will receive a verbal explanation in 
terms suited to their comprehension of the purposes, procedures, and potential risks of the study and of 
their rights as research participants. Participants will have the opportunity to carefully review the 
written consent form and ask questions prior to signing. Thereafter, signature of the written informed 
consent will be obtained from each participant who wants to participate in the study. The participant 
and the study staff conducting informed consent explanation will sign and date the consent form. The 
participant signature confirms that (s)he has understood the information. For illiterate individuals, the 
informed consent process will be conducted in the presence of a literate impartial witness selected by 
the participant but not a member of the CRC staff. The participant will thumb print the consent form 
and the witness will write the participant’s name and sign and date the consent form. In case of any 
amendments to the protocol resulting in changes in study procedures, the participant will be informed, 
and additional informed consent will be obtained if necessary. In accordance with Good Clinical Practice, 
the volunteer may terminate participation in the study at any time for any reason without penalty. 
Additionally, if the participant is unable or unwilling to adhere to the protocol design, the investigator 
may terminate volunteer’s participation. The study team will use the participant’s preferred language 
usually English, Kiswahili or Luo, for informed consent. The participant will sign the informed consent 
document prior to any procedures being done specifically for the study. A copy of the informed consent 
document will be given to the participants for their records.  
13.4 Participant and Data Confidentiality 
Participant confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor(s). This confidentiality is extended to cover testing of biological samples in addition to the 
clinical information relating to participants. Therefore, the study protocol, documentation, data, and all 
other information generated will be held in strict confidence to the extent possible. No information 

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concerning the study, or the data will be released to any unauthorized third party without prior written 
approval of the sponsor. When the results of the research are published or discussed in conferences, no 
information or names will be used that would reveal identity. Authorized representatives of the 
Sponsor, KEMRI/Walter Reed Project (WRP), or representatives of the USAMRDC, as part of their 
responsibility to oversee the research and protect human subjects, are eligible to review records of 
individual subjects. As a result, they may see one or more names, but they will be bound by rules of 
confidentiality not to reveal anyone’s identity to others. No names will be used in any report resulting 
from this study. 
 
All study documentation containing personal information relating to study subjects like consent forms 
and other documents that might link subject ID with other subject personal details will be kept in a 
secure locked area with limited access at USAMRD-A/Kenya facilities. Such documentation will only be 
made available to authorized personnel. These study documents will be made available to the 
investigators, clinical personnel who require this information to treat the subject and the above-
mentioned personnel for inspection or auditing reasons. In addition, access to subjects’ medical records 
will be granted to representatives of the Sponsor, regulatory agencies and ethics committees for the 
purpose of validating data. All electronic data kept at the investigator’s site will be kept secure. 
Computer access will only be made available to authorized personnel  
The study participant’s contact information will be securely stored at the study site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location for as 
long a period as dictated by local IRB and Institutional regulations.  
 
13.4.1 Future Use of Stored Specimens 
There will be no future use of stored specimens. Dried blood spots and sera collected from subjects will 
be destroyed at the end of the study. 
13.5 Compensation 
13.5.1 Compensation for injury 
Participants will be insured against injury caused by the study according to legal requirements and 
compensation for research related injury, should it occur.  If a participant suffers injury directly 
attributable to participation in this study, the participant is asked to contact the PI using the emergency 
contacts that will be provided on the consent forms. They will also be provided the contacts of KEMRI 
SERU secretariat in the event that they would like to speak to someone independent of the trial. 
Appropriate treatment during the trial will be provided by the site personnel.  
13.5.2 Compensation for subject participation 
Study participants will receive KSh. 1,000 for each day they participate in the feeding assays as 
compensation for time lost. A participant who participates on the first day only will receive KSh 1000, 
while a participant who participates for two days will receive KSh. 2000. Transport reimbursement will 
be provided separately if required (when unit transportation is not available) taking into consideration 
the average cost of travel to the clinic: they will receive between KSh. 500 and 1,000 for scheduled visits, 
and 300 and 500 Kenya Shillings for unscheduled visits as determined by the study team on a case by 
case basis.  

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14. DATA HANDLING AND RECORD KEEPING  
Data collection is the responsibility of the clinical study staff at the site under the supervision of the site 
Principal Investigator (PI). The PI is responsible for assuring that the data collected is complete, legible, 
attributable, accurate, and recorded in a timely manner. Data recorded in the CRF should be consistent 
with the data recorded on the source documents. All source documents and laboratory reports must be 
reviewed by the site team, who will ensure that they are accurate and complete. 
14.1 Data Collection and Management Responsibilities 
14.1.1 Source Data 
All information in original records and certified copies of original records or clinical findings, 
observations, or other activities in a clinical trial necessary for the reconstruction and evaluation of the 
trial. Source data are contained in source documents (original records or certified copies). [ICH 
E6 section 1.51]. 
 
14.1.2 Source documents 
Original documents, data and records (e.g., hospital records, clinical and office charts, laboratory notes, 
memoranda, subjects' diaries of evaluation checklists, pharmacy dispensing records, recorded data from 
automated instruments, copies or transcriptions certified after verification as being accurate and 
complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject files, and 
records kept at the pharmacy, at the laboratories, and at medico-technical departments involved in the 
clinical trial are considered Source Documents. [ICH E6 section 1.52] 
 
14.1.3 Data Capture Methods  
Clinical data is recorded in source documents, should be completed in a neat, legible manner to ensure 
accurate interpretation of data. Source documentation supporting the CRF data should document the 
dates and details of study procedures and subject status. The PI will ensure that all information in the 
CRFs and all source documents that support the data collected from each subject are maintained in a 
secure area and treated as confidential material. 
The CRFs will be developed by local staff at KCRC and data entered into the database by KCRC staff.  
14.1.4 Data Storage 
All study documentation containing personal information relating to study subjects like consent forms 
and other documents that might link subject ID with other subject personal details will be kept in a 
secure locked area with limited access at the KCRC. Such documentation will only be made available to 
authorized personnel. These study documents will be made available to the investigators, clinical 
personnel who require this information to treat the subjects and the above-mentioned personnel for 
inspection or auditing reasons. In addition, access to subjects’ medical records will be granted to 
representatives of the Sponsor, regulatory agencies, ethics committees, and the USAMRDC for the 
purpose of validating data. 

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14.2 Study Records Retention and Disposal 
Study documents should be retained for the duration required by PATH, applicable local regulations, and 
in according to U.S. DoD requirements, in a safe and secure facility. No records will be destroyed 
without the written consent of PATH. Upon study completion, all records will be stored safely at one of 
USAMRD-A’s archiving facilities or at a secure contracted storage facility for at least 5 years. If storage at 
an alternative facility is considered, this will be documented and the documentation will be filed in the 
study’s regulatory file. Should longer storage be recommended by the sponsor or relevant authorities, 
the site will contact the reviewing IRBs for consideration and approval. Study files can be stored either 
as paper or digitally. Digitization, if done, will consist of the scanning of all paper records according to 
site SOPs to ensure accurate and complete representation of the same. This may be done by the site 
staff of a contracted third-party. All digitized records will be maintained on a secure, password 
protected computer system with access limited to those with access to study as previously listed in this 
protocol as well as USAMRD-A’s archivist(s) and IT personnel who will maintain the database. Upon 
completion of digitization of study records, and receipt of a sponsor’s approval, paper copies may be 
destroyed by incineration according to site SOPs. 
Paper or digitized records may be disposed of after storage duration has been met (5 years or as 
otherwise specified as above) and with the sponsor’s approval. Any remaining paper records will be 
incinerated and/or digitized records will be securely deleted. The final disposal of any remaining paper 
or digitized records will be witnessed by a representative of USAMRD-A’s regulatory affairs department 
and will be documented. The documentation of records disposal will be provided to the sponsor and a 
copy will be stored at the Regulatory Affairs department. 
14.3 Publication and Data Sharing Policy 
Public availability of data at the end of this trial will be posted on a recognized clinical trial registry such 
as www.clinicaltrials.gov.  
 
This study will comply with PATH policy, which ensures that the public has open access to the published 
results of Gates Foundation funded research, as well as US DoD publication and data sharing policies. 
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted a 
clinical trials registration policy as a condition for publication.  
The first publication or disclosure of study results shall be a complete publication or disclosure 
coordinated by PATH.  
 
15. KEY PERSONNEL ROLES 
Dr. Ben Andagalu, Principal Investigator: will be responsible for the overall conduct of the trial at this 
study site, administrative actions between USAMRD-A/K, Institutional Review Boards, and the sponsor, 
oversight of field activities and patient care. He contributed to the protocol development and will 
contribute to the statistical analysis and writing of the scientific report(s). 
Kombewa CRC Sub Investigators: will be responsible for the clinical conduct of the trial, the care 
provided to patients, oversight of field activities. They contributed to the protocol development and will 
contribute to the statistical analysis and writing of scientific report(s). 

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MDR/MDC/Entomology laboratory Sub Investigators:  will be responsible for the execution of the 
various research assays of the trial. They contributed to the development of the protocol and will 
contribute to the statistical analysis and writing of the scientific report(s). 
Study pharmacist: will be responsible for the accountability of medications used in the study – including 
dispensing and maintenance of accountability logs 
Study clinical team (clinical investigators, nurses and clinical officers): will be responsible for recruitment 
and administering consent procedures 
 
16. MILITARY RELEVANCE 
US Service Members conduct operations in regions of the world where they are at risk of malaria 
acquisition. Acquiring malaria is significantly detrimental to individual Service Members and their units 
resulting in lost duty hours, increased utilization of medical resources in the field (often in austere 
settings), potential requirement for medical evacuation, risk of death to individual Service Members, 
and risk to a unit's mission if a significant proportion or key individuals become ill. As such medical 
countermeasures to malaria are essential in malaria endemic regions and include effective treatment, 
effective prophylaxis, and immunoprophylaxis (such as a vaccine). Additionally, efforts to decrease or 
eliminate malaria transmission in endemic regions (such as transmission blocking interventions) would 
also support the Warfighter through decreasing or eliminating the risk of malaria in a given region. 
Efforts such as this which are aimed at facilitating the development of transmission blocking 
interventions contribute to the medical readiness of US Service Members indirectly. While it is unlikely 
that an agent that would be tested with assays such as the DMFA would be directly used in a Service 
Member, the overall effort is directed at interventions that do contribute to the health and readiness of 
our Service Members, thus giving it relevance to the US Military. Additionally, this supports the efforts 
of the US Africa Command as their major lines of effort include building sustainable partnerships, 
improving the health infrastructure of the countries in their Area of Responsibility (AOR), and “setting 
the theatre” (in which, decreased malaria risk is directly relevant to Service Members deploying to the 
AOR).  
17. BUDGET 
Budget USD 
Budget KSh 
  
Reimbursable budget: 
  
 
 Personnel
 $   668,053.34 
66,805,334
 
 Transportation
 $         29,085 
2,908,500
 
 Other project costs
 $           4,200 
420,000
 
 Indirect - reimbursable costs
 $                  - 
 
  Subtotal - reimbursable costs
 $       701,338 
70,133,800
 

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Milestone  budget: 
 
 Clinical supplies
 $         35,960 
3,596,000
  
 
 Laboratory supplies
 $       224,162 
22,416,200
 
  Subtotal - milestone budget
 $       260,122 
26,012,200
  
 
Total Direct Project Costs
 $       961,460 
96,146,000
  
 
Indirect Costs (WRAIR & KEMRI O/H) 24%
 $       230,750 
23,075,000
  
 
TOTAL PROJECT COSTS
 $    1,192,211 
119,221,100
 
18. LITERATURE REFERENCES  
1. WORLD MALARIA REPORT 2018 ISBN 978 92 4 156565 3 [Internet]. [cited 2019 Oct 11]. Available 
from: www.who.int/malaria 
 
2. Olotu A, Fegan G, Wambua J, Nyangweso G, Leach A, Lievens M, et al. Seven-year efficacy of RTS, 
S/AS01 malaria vaccine among young african children. N Engl J Med. 2016 Jun 30;374(26):2519–29.  
 
3. Lusingu J, Olotu A, Leach A, Lievens M, Vekemans J, Olivier A, et al. Safety of the malaria vaccine 
candidate, RTS,S/AS01E in 5 to 17 month old Kenyan and Tanzanian Children. PLoS One [Internet]. 
2010;5(11):e14090. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21124768 
 
4. Smith TA, Chitnis N, Briët OJT, Tanner M. Uses of mosquito-stage transmission-blocking vaccines 
against Plasmodium falciparum. Vol. 27, Trends in Parasitology. 2011. p. 190–6.  
 
5. Bousema T, Sutherland CJ, Churcher TS, Mulder B, Gouagna LC, Riley EM, et al. Human immune 
responses that reduce the transmission of Plasmodium falciparum in African populations. Int J 
Parasitol. 2011;41(3):293–300.  
 
6. Boyd MF, Stratman-thomas WK. Studies on benign tertian malaria. The clinical characteristics of the 
disease in relation to the dosage of sporozoites. Am J Epidemiol. 1933;17(3):666–85.  
 
7. Ponnudurai T, van Gemert GJ, Bensink T, Lensen AHW, Meuwissen JHET. Transmission blockade of 
Plasmodium falciparum: its variability with gametocyte numbers and concentration of antibody. 
Trans R Soc Trop Med Hyg. 1987;81(3):491–3.  
 
8. Li T, Eappen AG, Richman AM, Billingsley PF, Abebe Y, Li M, et al. Robust, reproducible, 
industrialized, standard membrane feeding assay for assessing the transmission blocking activity of 

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vaccines and drugs against Plasmodium falciparum. Malar J [Internet]. 2015 Dec 9 [cited 2019 Oct 
30];14(1):150. Available from: http://www.malariajournal.com/content/14/1/150 
 
9. Swihart BJ, Fay MP, Miura K. Statistical Methods for Standard Membrane-Feeding Assays to 
Measure Transmission Blocking or Reducing Activity in Malaria. J Am Stat Assoc. 2018 Apr 
3;113(522):534–45.  
 
10. World Health Organization. Policy brief on single-dose primaquine as a gametocytocide in 
Plasmodium falciparum malaria [Internet]. [cited 2019 Nov 1]. Available from: 
https://www.who.int/malaria/publications/atoz/who_htm_gmp_2015.1.pdf?ua=1 
 
11. Bonnet S, Gouagna C, Safeukui I, Meunier JY, Boudin C. Comparison of artificial membrane feeding 
with direct skin feeding to estimate infectiousness of Plasmodium falciparum gametocyte carriers to 
mosquitoes. Trans R Soc Trop Med Hyg. 2000;94(1):103–6.  
 
12. Bousema T, Dinglasan RR, Morlais I, Gouagna LC, van Warmerdam T, Awono-Ambene PH, et al. 
Mosquito feeding assays to determine the infectiousness of naturally infected Plasmodium 
falciparum gametocyte carriers. PLoS One. 2012 Aug 22;7(8).  
 
13. Bousema T, Dinglasan RR, Morlais I, Gouagna LC, van Warmerdam T, Awono-Ambene PH, et al. 
Mosquito feeding assays to determine the infectiousness of naturally infected Plasmodium 
falciparum gametocyte carriers. PLoS One [Internet]. 2012;7(8):e42821. Available from: 
http://www.ncbi.nlm.nih.gov/pubmed/22936993 
 
14. Da DF, Dixit S, Sattabonkot J, Mu J, Abate L, Ramineni B, et al. Anti-Pfs25 human plasma reduces 
transmission of plasmodium falciparum isolates that have diverse genetic backgrounds. Infect