A Within-subject Laboratory and Field Trial to Test the Use of Commercially Available Insect Repellents Against Contact From Musca Sorbens, the Putative Vector of Trachoma
Phase: PHASE2
Conditions: Trachoma
Sponsor: London School of Hygiene and Tropical Medicine

Stronger-SAFE – Phase II – Clinical Trial Protocol – Repellent testing  
 
                           
 
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Clinical Trial Protocol
STRONGER SAFE: PHASE II
A within-subject laboratory and field trial to test the use of 
commercially available insect repellents against contact from 
Musca sorbens, the putative vector of trachoma
Version 2.3, 12th December 2018
Wellcome Trust
London School of Hygiene & Tropical Medicine is the main research 
sponsor for this study.  For further information regarding the 
sponsorship conditions, please contact the Research Governance 
and Integrity Office:
London School of Hygiene & Tropical Medicine
Keppel Street
London WC1E 7HT
Tel: +44 207 927 2626
Email: RGIO@lshtm.ac.uk
1

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1
Table of Contents
2
ADMINISTRATIVE INFORMATION
 
                                                                                                                         
 
 
...............................................................................................................
 
 6  
3
DATE AND VERSION IDENTIFIER
 
                                                                                                                           
 
 
.................................................................................................................
 
 7  
4
LIST OF ABBREVIATIONS
 
                                                                                                                                          
 
 
...............................................................................................................................
 
 8  
5
SUMMARY
 
                                                                                                                                                                         
 
 
...........................................................................................................................................................
 
 9  
6
CONTRIBUTORSHIP
 
                                                                                                                                                  
 
 
......................................................................................................................................
 
 11
  
7
SPONSOR AND FUNDER
 
                                                                                                                                           
 
 
................................................................................................................................
 
 11
  
8
ORGANISATIONAL STRUCTURE AND RESPONSIBILITY
 
                                                                            
 
 
.....................................................................
 
 12
  
9
INTRODUCTION
 
                                                                                                                                                           
 
 
..............................................................................................................................................
 
 13
  
9.1
TRACHOMA....................................................................................................................................................... 13
9.2
TRACHOMA TREATMENT, PREVENTION AND CONTROL...................................................................................13
9.3
TRACHOMA IN ETHIOPIA................................................................................................................................. 14
9.4
FLIES AND TRACHOMA..................................................................................................................................... 16
9.5
RATIONALE FOR THE USE OF REPELLENTS AGAINST MUSCA SORBENS............................................................17
10
RESEARCH HYPOTHESIS
 
                                                                                                                                       
 
 
............................................................................................................................
 
 18
  
11
CHOICE OF COMPARATORS
 
                                                                                                                                 
 
 
......................................................................................................................
 
 18
  
12
STUDY OBJECTIVES
 
                                                                                                                                                 
 
 
.....................................................................................................................................
 
 19
  
12.1
PRIMARY OBJECTIVE..................................................................................................................................... 19
12.2
SECONDARY OBJECTIVES............................................................................................................................... 19
13
TRIAL DESIGN
 
                                                                                                                                                            
 
 
...............................................................................................................................................
 
 19
  
13.1
LABORATORY TRIAL...................................................................................................................................... 19
13.2
FIELD TRIAL................................................................................................................................................... 20
14
STUDY SETTING AND POPULATIONS
 
                                                                                                              
 
 
.....................................................................................................
 
 21
  
14.1
AGE OF PARTICIPANTS................................................................................................................................... 21
14.2
LABORATORY TRIAL (LONDON SCHOOL OF HYGIENE & TROPICAL MEDICINE)..........................................21
14.3
FIELD TRIAL (OROMIA, ETHIOPIA)............................................................................................................... 21
15
TRIAL ELIGIBILITY AND WITHDRAWAL CRITERIA
 
                                                                                  
 
 
...........................................................................
 
 21
  
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15.1
LABORATORY TRIAL ELIGIBILITY CRITERIA..................................................................................................21
15.2
FIELD TRIAL ELIGIBILITY CRITERIA............................................................................................................... 22
15.3
PARTICIPANT WITHDRAWAL......................................................................................................................... 23
15.4
PARTICIPANT RETENTION.............................................................................................................................. 23
16
INTERVENTIONS
 
                                                                                                                                                       
 
 
..........................................................................................................................................
 
 23
  
16.1
INVESTIGATIONAL PRODUCTS........................................................................................................................ 23
16.1.1
TOPICAL INSECT REPELLENT PRODUCTS.....................................................................................................................23
16.1.2
WEARABLE REPELLENT DEVICES.................................................................................................................................23
16.2
APPLICATION OF TOPICAL PRODUCTS........................................................................................................... 24
16.2.1
LABORATORY TRIALS APPLICATION RATE..................................................................................................................24
16.2.2
FIELD TRIALS APPLICATION RATE................................................................................................................................24
17
STUDY OUTCOMES
 
                                                                                                                                                   
 
 
......................................................................................................................................
 
 24
  
17.1
PRIMARY OUTCOME MEASURES..................................................................................................................... 24
17.2
SECONDARY OUTCOME MEASURES................................................................................................................. 24
18
PARTICIPANT TIMELINE
 
                                                                                                                                       
 
 
...........................................................................................................................
 
 25
  
19
SAMPLE SIZE
 
                                                                                                                                                              
 
 
.................................................................................................................................................
 
 25
  
19.1
LABORATORY TRIALS SAMPLE SIZE............................................................................................................... 25
19.2
FIELD TRIALS SAMPLE SIZE............................................................................................................................ 26
20
ENROLMENT, RANDOMISATION AND ALLOCATION
 
                                                                                 
 
 
.........................................................................
 
 26
  
20.1
LABORATORY TRIALS..................................................................................................................................... 26
20.2
FIELD TRIALS................................................................................................................................................. 27
21
STUDY PROCEDURES
 
                                                                                                                                              
 
 
..................................................................................................................................
 
 27
  
21.1
LABORATORY TRIALS..................................................................................................................................... 27
21.1.1
TEST INSECTS.................................................................................................................................................................. 27
21.1.2
TESTING ROOM............................................................................................................................................................... 27
21.1.3
TOPICAL REPELLENTS.................................................................................................................................................... 28
21.1.4
WEARABLE REPELLENT DEVICES.................................................................................................................................29
21.2
FIELD TRIALS................................................................................................................................................. 30
21.2.1
TEST INSECTS AND TESTING SITE................................................................................................................................30
21.2.2
PILOT PHASE.................................................................................................................................................................. 30
21.2.3
TOPICAL REPELLENTS.................................................................................................................................................... 30
21.2.4
WEARABLE REPELLENT DEVICES.................................................................................................................................31
21.2.5
ACCEPTABILITY MUSCA SORBENS REPELLENTS..........................................................................................................32
22
RISKS, BENEFITS AND BURDEN
 
                                                                                                                         
 
 
...............................................................................................................
 
 32
  
22.1
RISK FROM EYE-SEEKING FLIES, MUSCA SORBENS.........................................................................................32
22.1.1
LABORATORY TRIALS..................................................................................................................................................... 32
22.1.2
FIELD TRIALS.................................................................................................................................................................. 33
22.2
RISK FROM INSECT REPELLENT PRODUCTS...................................................................................................33
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22.2.1
DEET..................................................................................................................................................................................33
22.2.2
IR3535...............................................................................................................................................................................34
22.2.3
PICARIDIN........................................................................................................................................................................ 35
22.2.4
PMD....................................................................................................................................................................................35
22.2.5
PERMETHRIN.................................................................................................................................................................. 36
22.2.6
VANILLIN......................................................................................................................................................................... 36
22.2.7
SPECIFIC RISK FROM APPLICATION OF TOPICAL REPELLENTS TO THE FACE (FIELD TRIAL ONLY).....................37
22.3
RISK FROM WEARABLE REPELLENT TECHNOLOGIES......................................................................................37
22.4
BURDEN ASSOCIATED WITH PARTICIPATION................................................................................................37
22.4.1
LABORATORY TRIALS..................................................................................................................................................... 37
22.4.2
FIELD TRIALS......................................................................................................................................................................37
22.5
BENEFITS ASSOCIATED WITH PARTICIPATION...............................................................................................38
23
MODIFICATIONS
 
                                                                                                                                                       
 
 
...........................................................................................................................................
 
 38
  
24
ADHERENCE
 
                                                                                                                                                                
 
 
..................................................................................................................................................
 
 38
  
24.1
TRIAL ADHERENCE......................................................................................................................................... 38
24.2
TRIAL ADHERENCE ASSESSMENT.................................................................................................................... 39
25
COMPARABILITY OF STUDY GROUPS (CONCOMITANT INSECT-REPELLING 
ACTIVITIES/FACTORS)
 
                                                                                                                                                    
 
 
.......................................................................................................................................
 
 39
  
26
DATA
 
                                                                                                                                                                              
 
 
...............................................................................................................................................................
 
 40
  
26.1
DATA COLLECTION......................................................................................................................................... 40
26.1.1
LABORATORY TRIALS..................................................................................................................................................... 40
26.1.2
FIELD TRIALS.................................................................................................................................................................. 40
26.2
DATA MANAGEMENT AND CONFIDENTIALITY................................................................................................40
26.2.1
LABORATORY TRIALS..................................................................................................................................................... 41
26.2.2
FIELD TRIALS.................................................................................................................................................................. 41
26.3
DATA ANALYSIS.............................................................................................................................................. 41
26.3.1
PROTECTIVE EFFICACY, P.............................................................................................................................................. 41
26.3.2
MEDIAN COMPLETE PROTECTION TIME (MCPT).....................................................................................................42
26.3.3
MEDIAN EFFECTIVE DOSE (ED50) AND MEDIAN EFFECTIVE TIME (ET50)...........................................................42
27
MONITORING
 
                                                                                                                                                             
 
 
................................................................................................................................................
 
 43
  
27.1
DATA MONITORING COMMITTEE.................................................................................................................. 43
28
SAFETY REPORTING
 
                                                                                                                                               
 
 
...................................................................................................................................
 
 43
  
28.1
ADVERSE EVENTS........................................................................................................................................... 43
28.2
DEFINITIONS.................................................................................................................................................. 43
28.3
REPORTING PROCEDURES............................................................................................................................. 44
28.3.1
NON SERIOUS AES......................................................................................................................................................... 44
28.3.2
SERIOUS AES.................................................................................................................................................................. 45
29
ETHICS AND DISSEMINATION
 
                                                                                                                            
 
 
..................................................................................................................
 
 46
  
29.1
RESEARCH ETHICS APPROVAL....................................................................................................................... 46
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29.2
PROTOCOL AMENDMENTS.............................................................................................................................. 46
29.3
CONSENT AND ASSENT................................................................................................................................... 46
29.3.1
LABORATORY TRIALS CONSENT....................................................................................................................................47
29.3.2
FIELD TRIALS CONSENT/ASSENT................................................................................................................................. 47
29.4
COMPENSATION............................................................................................................................................. 47
29.5
ACCESS TO DATA............................................................................................................................................ 47
29.6
DISSEMINATION POLICY................................................................................................................................. 47
30
TIMELINE
 
                                                                                                                                                                     
 
 
.......................................................................................................................................................
 
 48
  
31
ANTICIPATED OUTPUTS
 
                                                                                                                                       
 
 
............................................................................................................................
 
 49
  
32
REFERENCES
 
                                                                                                                                                               
 
 
.................................................................................................................................................
 
 50
  
5

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2
Administrative information
Primary  registry  and  trial 
identifying number
Not yet registered
Date  of  registration  in  primary 
registry
Not yet registered
Source of monetary support
Wellcome Trust (grant ITCRZL51)
Primary sponsor
London School of Hygiene & Tropical Medicine
Keppel Street, 
London, WC1E 7HT
Contact  for  public  or  scientific 
queries
Ailie Robinson
London School of Hygiene & Tropical Medicine, UK.
Tel: 0207 927 2718
Email: Ailie.robinson@lshtm.ac.uk
Public title
Testing insect repellents against Musca sorbens, the vector of trachoma
Scientific title
A  within-subject  laboratory  and  field  trial  to  test  the  use  of  commercially 
available insect repellents against contact from  Musca sorbens, the putative 
vector of trachoma
Countries of recruitment
Ethiopia, United Kingdom
Health problem to be studied
Trachoma transmission by fly contact to the eyes
Authors
Ailie Robinson 
James Logan
Anna Last
Matthew Burton
Adam Biran
Alexandra Czerniewska
Study site (UK)
London School of Hygiene & Tropical Medicine
Keppel Street
London, WC1E 7HT
Study site (Ethiopia)
Fred Hollows Foundation Office, 
Shashemene,
Nur 81 Building, 2nd floor, Office 209
Ethiopia
Chief Investigator
Prof. Matthew Burton
Co-Principle Investigator (Lab)
Laboratory Project Manager
Dr Ailie Robinson, 
London School of Hygiene & Tropical Medicine, UK.
Tel: 00 447 88 567 9592
Email: ailie.robinson@lshtm.ac.uk
Co-Principal Investigator (Field)
Field Project Manager 
Mr Oumer Shafi Abdurahman 
The Fred Hollows Foundation, Ethiopia
Tel: 00 251 91 204 8181
Email: oshafi@hollows.org 
LSHTM collaborators
Prof. James Logan, Professor of Entomology
Email:  james.logan@lshtm.ac.uk
 
  
Dr Anna Last, Associate Professor/Infectious Disease Physician  
Email: anna.last@lshtm.ac.uk
Prof. Matthew Burton, Professor of International Eye Health
London School of Hygiene & Tropical Medicine, UK.
Email: matthew.burton@lshtm.ac.uk
Dr Adam Biran, Assistant Professor in Environmental Health 
Email: adam.biran@lshtm.ac.uk 
Dr Katie Greenland, Assistant Professor in Environmental Health 
Email: katie.greenland@lshtm.ac.uk
Dr. Aalbertus Versteeg, Post-doctoral Research Fellow in Molecular Biology
Email:  aalbertus.versteeg@lshtm.ac.uk 
Ms. Alex Czerniewska, Research Assistant in Environmental Health 
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Email: Alexandra.czerniewska@lshtm.ac.uk 
Prof. Sandy Cairncross, Professor of Environmental Health 
Email: sandy.cairncross@lshtm.ac.uk 
Prof. Martin Holland, Professor of Microbial Immunity 
Email: martin.holland@lshtm.ac.uk
Dr. David Macleod, Post-doctoral Research Fellow in Statistics
Email: david.macleod@lshtm.ac.ul
Co-investigators and Partners
Federal Ministry of Health, Ethiopia
Mr Biruk Kebede Negash, Neglected Tropical Disease Team Leader
Email: biruck1@gmail.com
Oromia Regional Health Bureau, Ethiopia
Mr Hirpa Miecha, Neglected Tropical Diseases and Malaria Team Leader
Email: mirayeshma@gmail.com, 
The Fred Hollows Foundation, Ethiopia
Dr Yeneneh Mulugeta, Country Director
Email: ymulugeta@hollows.org
The Fred Hollows Foundation, Ethiopia 
Dr Zelalem Jemal, Deputy Country Director
Email: zjemal@hollows.org
The Fred Hollows Foundation, UK
Ms Virginia Sarah, Global Partnership Executive
Email:  vsarah@hollows.org
The Fred Hollows Foundation, Australia
Dr Sarity Dodson, Global Lead – Development Effectiveness 
Email: sdodson@hollows.org
Wellcome Trust Sanger Institute, UK
Prof. Nicholas Thomson, Professor of Bacterial Genomics and Evolution,  
Email: nrt@sanger.ac.uk
World Health Organization, Geneva
Dr Anthony Solomon, Medical Officer, Neglected Tropical Diseases 
Email: solomona@who.int
3
Date and version identifier
Chronology
Version
Revision
17 May 2018
1.7
Original
21st May 2018
1.8
Continuing edits for sponsorship submission
1.9
Continuing edits for ethics submission
26th May 2018
1.10
Edits following comments from LSHTM sponsorship panel
30th May 2018
1.11
Edits following comments from LSHTM sponsorship panel
24th July 2018
2.0
Edits following LEO feedback
3rd September 2018
2.1
Added “Field PI” to Oumer Shafi on p. 6 following his feedback
27th November 2018
2.2

Removed paragraph about Stronger-SAFE from p. 9

Edited Tables 1 and 2 to include links to supplier, added 
new supplier for PMD and permethrin

Added image credit for p. 18

Change  terminology  from  ‘patient’  to  ‘participant’ 
throughout
12th December 2018
2.3

Ensured OSA and AR were appropriately referred to as 
7

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“co-PI” throughout the document. 
8

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4
List of Abbreviations
Ct
Chlamydia trachomatis
FHF
The Fred Hollows Foundation
FMOST
Federal Ministry of Science and Technology
GTMP
Global Trachoma Mapping Project
LSHTM
London School of Hygiene & Tropical Medicine
NTD
Neglected Tropical Diseases
TF
Follicular Trachoma
WHO
World Health Organization
DEET
N,N-diethyl-3-methylbenzamide
PMD
para-Menthane-3,8-diol
IR3535
Insect Repellent 3535
CPT
Complete Protection Time
mCPT
Median Complete Protection Time
MED
Median Effective Dose
MET
Median Effective Time
MSDS
Material Safety Data Sheet
BSA
Body Surface Area
PSF
Participant Screening Form
GDPR
General Data Protection Regulation
ODK
Open Data Kit
PI
Principle Investigator
CI
Chief Investigator
SC
Steering Committee
DMC
Data Monitoring Committee
GLP
Good Laboratory Practice
GCP
Good Clinical Practice
ORHB
Oromia Regional Health Bureaux
FMHACA
Food,  Medicine  and  Health  Care  Administration  and  Control  Authority 
(Ethiopia)
RGIO
Research Governance and Integrity Office (LSHTM)
GHS 
Globally Harmonized System of Classification and Labelling of Chemicals
OPP
Office of Pesticide Programs
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5
Summary
Background: Trachoma is the commonest infectious cause of blindness worldwide, which leads to 
considerable ocular morbidity in children and adults. It is caused by ocular infection with the 
bacterium Chlamydia trachomatis (Ct). Trachoma is endemic in many areas of Ethiopia, which has 
the highest burden of this disease globally. Trachoma control requires implementation of the 
WHO-endorsed  SAFE  strategy:  Surgery  for  trichiasis;  Antibiotics  to  treat  infection;  Facial 
cleanliness and Environmental hygiene to reduce transmission. Although SAFE has been successful 
in reducing disease burden in many areas of the world, there is growing evidence that for 
hyperendemic regions, particularly in Ethiopia, implementation of SAFE (even under research 
study conditions) does not have the anticipated effect in reducing and eliminating disease. 
Clinical Trial rationale:  Musca sorbens, a fly that feeds from ocular and nasal discharge on 
humans, is thought to be the vector of trachoma. As part of Stronger-SAFE Phase II we are 
developing methods of fly control that specifically target this species, in the hope of interrupting 
Ct transmission. To our knowledge, the use of commercially available insect repellents has never 
been tested for prevention of Musca sorbens fly-eye contact (i.e. nuisance and landing in the peri-
ocular area). Given the likely necessity for prolonged and/or high frequency fly-eye contact for Ct 
transmission, the reduction of these contacts through the use of fly repellents presents an exciting 
opportunity for disease control. 
Clinical trial objective: To measure the protective efficacy (personal protection) of repellent 
products, by comparison of the inhibition of Musca sorbens contacts on participants before and 
after their application.
Study type: This is a within-subject, non-masked, trial of the use of commercially available insect 
repellents against Musca sorbens, with two consecutive participant groups in the laboratory and in 
the field, and a primary endpoint of measuring the protective efficacy of each repellent product. 
Study design: 
1. Laboratory trials
a. Target sample size: 17 participants (all participants test all product iterations)
b. Stage 1. Protective Efficacy. Determining the protection of repellent products. Only 
those products/concentrations that protecting against at least 30 % of fly contact 
will be carried on to stage 2.
c.
Stage 2. Persistence. The persistence of effect will be measured over a six-hour 
period.  For  slow-release  wearable  repellent  technologies,  this  period  will  be 
extended for follow-up at 1, 2, 3 and 4 weeks. Estimations of persistence will allow 
final selection of repellent products/concentrations to be tested in the field trials
2. Field trials
a. Target sample size: 29 participants per study arm (each participant tests only one 
product iteration), 10 participants in the Pilot Phase
b. Up to four groups (study arms) will test the effectiveness of only one topical 
repellent or slow-release wearable repellent device, at one concentration, against a 
control group who will receive no intervention. 
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Intervention: Repellent products will be chosen from: DEET (N,N-diethyl-3-methylbenzamide), 
IR3535  (3-[N-butyl-N-acetyl]-aminopropionic  acid  ethyl  ester),  Picaridin  (2-(2-hydroxyethyl)-1-
piperidinecarboxylic acid 1-methylpropyl ester); PMD (para-Menthane-3,8-diol) or permethrin (m-
Phenoxybenzyl)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate).  Products 
tested will be either (1) topical repellents, or (2) in long-lasting, plastic formulations of repellents 
that can be worn on the body (wearable repellent technologies). The insect repellent synergist 
Vanillin  (4-Hydroxy-3-methoxybenzaldehyde)  may  be  added  to  the  long-lasting  plastic 
formulations, to improve the duration of protection.
Main study outcomes/endpoints: Protective Efficacy, Complete Protection Time, Median Effective 
Time and Median Effective Dose
Key inclusion criteria (Laboratory trials, LSHTM): 
Ages eligible for study:  18 years and  65 years 
Sexes eligible for study: both
Health of volunteers: full health only, no known adverse reactions, or evidence at screening of 
adverse reactions, to the commercially available repellents DEET, PMD, IR3535, Picaridin or 
Permethrin, or to Vanilla
Inclusion criteria: willing to allow 100 laboratory-reared Musca sorbens flies to land and crawl on 
their arm, during the modified arm-in-cage assay, for periods of up to ten minutes at a time, as 
much as possible without disturbing fly behaviour.
Key inclusion criteria (Field trials, Ethiopia): 
Ages eligible for study:  3 years and  12 years
Sexes eligible for study: both
Health of volunteers: full health only, no known adverse reactions, or evidence at screening of 
adverse reactions, to the commercially available repellents DEET, PMD, IR3535, Picaridin or 
Permethrin, or to Vanilla
Inclusion criteria: willing to sit still on a chair outside their house, for sequential periods of up to 
ten minutes, allowing wild fly contact and landing on the body and face, as much as possible 
without disturbing fly behaviour. 
Nature and extent of the burden and risks associated with participation:
Benefits: Participants in the laboratory trial will receive no benefits from participation in the trial. 
Participants in the field trial will have the opportunity to have their vision and eyes checked by the 
Stronger-SAFE project team, and will receive appropriate referral for identified problems. There 
are no further benefits expected for any participants.
Burden: In the laboratory trials, participants will be required to make repeat visits to the LSHTM 
testing facility, to test each product and product formulation. During visits, they will be required to 
sit still for ten-minute observation periods, allowing flies to crawl freely over their forearm and 
hand. In the field trials, the participant’s face will be observed and filmed for ten-minute periods. 
During this time, the participant will be required to sit motionless and allow flies to crawl freely 
over their face and around their eyes, nose and mouth. In this study setting this burden would be 
considered ‘the norm’, with individuals rarely bothering to brush away flies due to their extreme 
persistence and prevalence. 
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Risks: There is a small risk of skin irritation or reaction following application of the repellent 
product. In the field trials, topical repellents must be applied to the face due to the nature of the 
target species (eye-seeking flies). The topical repellent active ingredients that will be tested 
topically have been chosen because they do not carry a specific risk of eye irritation beyond what 
would reasonably be expected through application of a chemical product. Topical repellents will 
be applied at a set distance away from the eyes, nose and mouth. Wearable repellent technologies 
will be formulated to contain insect repellent at doses within published limit of safe application. 
Because the repellent product will be formulated into plastic, most likely low density polyethylene 
(LDPE) or high density polyethylene (HDPE), the amount of product that is released onto the skin 
will be considerably lower than that which is experienced via topical application of a cream. Safety 
information regarding the repellent active ingredients used in the trial have been assessed, 
material safety data sheets (MSDS) and labels have been read to be sure they are safe for human 
use. Participants will be exposed to contacts by Musca sorbens. In laboratory trials, Musca sorbens 
will have been reared in captivity for over six generations and carry no risk of Ct transmission. 
Participants will only be exposed to fly contact on their arms, and after completion of testing, will 
immediately be instructed to wash their arm. Therefore, the modified arm-in-cage assay presents 
only negligible risk. In field trials, testing will occur outside the participant’s houses, therefore 
participants will not be exposed to any greater risk from fly contact than that which they 
experience day-to-day. 
6
Contributorship
AR, JL, MB, AL, AB and AC conceived of the study. AR and JL initiated the study design and MB, AL, 
OS, AC and AB assisted with implementation. DM provided statistical expertise in clinical trial 
design and AR is conducting the primary statistical analysis. All authors contributed to refinement 
of the study protocol and approved the final manuscript.
7
Sponsor and Funder
The Wellcome Trust (funder) had no role in the design of this study and will not have any role 
during its execution, analyses, interpretation of the data, or decision to submit results. The 
sponsor (LSHTM), principal investigators and collaborators accept full responsibility for all aspects 
of the study. 
SPONSOR
London School of Hygiene & Tropical Medicine will act as the main sponsor for this study. 
Delegated responsibilities will be assigned locally.  
INDEMNITY
London School of Hygiene & Tropical Medicine holds Public Liability ("negligent harm") and Clinical 
Trial ("non-negligent harm") insurance policies which apply to this trial
AUDITS AND INSPECTIONS 
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The study may be subject audit by the London School of Hygiene & Tropical Medicine under their 
remit as sponsor, the Study Coordination Centre and other regulatory bodies to ensure adherence 
to GCP. 
8
Organisational structure and responsibility
Chief investigator: Prof. Matthew Burton 
Co-Principal investigator (lab) and Laboratory Project Manager: Dr Ailie Robinson
Co-Principal Investigator (field) and Field Project Manager: Mr Oumer Shafi Abdulrahman 
Preparation of protocols and revisions: Dr Ailie Robinson, Prof. James Logan, Prof. Matthew 
Burton, Dr Anna Last, Dr Adam Biran, Ms. Alex Czerniewska
Organising steering committee meetings: Dr Ailie Robinson
Publication of study reports: Dr Ailie Robinson, Prof. James Logan, Prof. Matthew Burton, Dr Anna 
Last
Steering Committee (SC): Dr Ailie Robinson, Prof. James Logan, Prof. Matthew Burton, Dr Anna 
Last,  Mr Oumer Shafi Abdulrahman,  Dr Adam Biran, Dr Katie Greenland, Dr Esmael Ali, Dr 
Aalbertus Versteeg, Ms. Alex Czerniewska. Responsible for study planning, for reviewing the 
progress of the study, agreeing any changes to the protocol if and when required, and ensuring 
the smooth running of the study. Will report any SAEs [Serious adverse events] to the LSHTM 
ethics committee.
Data Monitoring Committee: Dr Vanessa Chen-Hussey, to be confirmed.
Agreement of final protocol: SC
Recruitment of participants and liaising with laboratory co-PI: Dr Ailie Robinson
Data manger: Dr Ailie Robinson
Lead investigator: Dr Ailie Robinson
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9
Introduction
9.1
Trachoma
Trachoma, a Neglected Tropical Disease (NTD), is the commonest infectious cause of blindness 
globally, affecting some of the world’s poorest communities(1). Trachoma is caused by repeated 
ocular  infection  with  the  bacterium  Chlamydia  trachomatis  (Ct).  Active  trachoma  begins  in 
childhood with recurrent episodes of follicular conjunctivitis (TF). Chronic inflammation results in 
immunologically  mediated  conjunctival  scarring  and  in-turned  eyelashes  scratching  the  eye: 
trichiasis. Eventually sight is lost from irreversible corneal opacification. 
Trachoma is currently endemic in 42 countries. The latest estimates from the Global Trachoma 
Mapping Programme (GTMP) suggest that 180 million people live in trachoma endemic areas and 
3.2  million  people  have  trachomatous  trichiasis  (2).  Around  2.2  million  people  are  visually 
impaired, of whom 1.2 million are blind (3). More than 80% of the burden of active trachoma is 
concentrated in 14 countries, mainly in the Sahel of West Africa and savannahs of East and Central 
Africa, where water supplies are often scarce(2). 
9.2
Trachoma treatment, prevention and control
Trachoma control requires community-wide measures. The  World Health Organization (WHO) 
Alliance  for  the  Global  Elimination  of  Trachoma  by 2020  (GET2020)  recommends the  SAFE 
Strategy:  Surgery  for  trichiasis,  Antibiotic  to  treat  Ct  infection,  Facial  cleanliness  and 
Environmental  improvements  to  suppress  transmission(1).  Many  endemic  countries  are 
implementing SAFE, and there has been a major effort to scale up activities, aiming to eliminate 
trachoma by 2020(2). 
Currently,  the  antibiotic  component  involves  mass  drug  administration  (MDA)  with  oral 
azithromycin to all community members older than six months. This is given as a single, annual 
dose, initially for 1-5 years, before reassessing the district-level TF prevalence in 1-9 year olds and 
deciding whether MDA can be discontinued(4). The F&E components are much more variable in 
content and application. If F&E are implemented at all, it usually involves improving water access, 
sanitation and hygiene (WASH) and fly-control(1). 
Unfortunately, there is now growing evidence, particularly from hyperendemic regions (>20% TF), 
that current approaches are not having the anticipated impact on infection and disease(5–8). This 
is a significant threat to the timely elimination of trachoma. Over 44 million live in districts with 
>30% TF (GTMP data). In hyperendemic areas, current antibiotic schedules appear insufficient to 
reliably achieve long-term control after treatment completion. For example, in Ethiopia, which has 
the greatest trachoma burden, despite seven years of annual or biannual high-coverage MDA, the 
prevalence of TF remains well above threshold for continuing MDA(5). Data on Ct after repeated 
MDA rounds in hyperendemic settings indicates that reliable long-term control is not consistently 
achieved, with re-emergence of infection being typical(6, 8). 
It is unknown which, if any, F&E measures, as applied programmatically, suppress Ct transmission. 
The trachoma literature is replete with studies (including several conducted by the applicants) 
which report associations between active trachoma and/or  Ct  infection and WASH indicators 
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(water and latrine access), fly-eye contact and clean faces. Based on these associations a recent 
meta-analysis concluded there is “strong evidence to support F&E components of SAFE”(9). 
However, we disagree with this conclusion. What has been demonstrated are associations, rather 
than causal relationships. There are few randomized-controlled trials in this area, which have 
demonstrated limited or no effect(10–15). Recent Cochrane Reviews of F&E intervention trials 
concluded there  is currently little or no evidence  that  the  tested interventions significantly 
impacted on trachoma(16, 17). 
Moreover, our understanding of how  Ct is transmitted within endemic communities is largely 
based on supposition. We believe that endemic trachoma is sustained by ongoing person-to-
person  Ct  transmission,  probably  through  a  combination  of  direct  contact  and  indirect 
transmission  on  fomites  and  flies  (Musca  sorbens).  However,  detailed  studies  investigating 
potential  transmission  routes  and  their  relative  importance  have  never  been  conducted. 
Therefore, we do not currently have a clear, evidence-based understanding of transmission 
biology or its socio-behavioural determinants, on which to base rational decisions about public 
health F&E interventions to eliminate trachoma.
There are at least three critical issues: 
1. Routes of  Ct transmission  and their relative importance  are poorly defined, as detailed 
studies have never been conducted, making it hard to focus F&E interventions.
2. The F&E intervention evidence base is very limited: there are few published randomized-
controlled trials, which have demonstrated limited or no effect, to guide programmes. 
3. Particularly in hyperendemic areas, current azithromycin schedules, with or without F&E, 
appear insufficient to control infection and disease.
To address these issues, we propose a sequence of interrelated studies in Ethiopia, conducted 
through a multi-disciplinary collaboration in three Phases, which will develop and test enhanced A, 
F & E strategies for trachoma elimination: Stronger-SAFE. In this protocol, we outline aspects of 
Phase II of this programme.
9.3
Trachoma in Ethiopia
Ethiopia remains the country with the greatest trachoma burden(2). It is estimated that 30% of 
Africa’s trachoma burden is in Ethiopia. More than 80% of its population of 90 million live in rural 
areas and 37% live on less than a dollar a day(18). Half the population travel significant distances 
to access safe drinking water, with 12 percent of the population still relying on untreated surface 
water(19). A national survey conducted in Ethiopia in 2010 showed that access to water supply 
and sanitation was 52% and 63% respectively(20). These environmental and living conditions are 
believed to create the ideal situation for trachoma to flourish. 
Recently collected Global Trachoma Mapping Project (GTMP) data from Ethiopia show that more 
than 76 million people are at risk of trachoma and the prevalence of TF in 1-9 year olds (TF1-9) 
ranges  from  0.2%  to 73.4%  (Figure  1).  In  Oromia, both  active  trachoma  and  trichiasis are 
significant public health problems. The most recent GTMP data published for this region shows an 
estimated overall prevalence of TF1-9 of 23.4% across 252 districts(21). In 46% of surveyed 
districts, TF1-9 prevalence was >30% (Figure 2) in 126 of 252 districts(22). Disabling sight loss and 
pain from trichiasis predominantly affects women. It has been estimated that trachoma causes up 
to  US$  8  billion/year  productivity  loss,  a  burden  that  falls  on  some  of  the  poorest 
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communities(23). Our recent work from Ethiopia found households of individuals with trichiasis 
are significantly poorer than their unaffected neighbours(24). Moreover, trichiasis has a profound 
impact on quality of life(25). 
Ethiopia is working towards eliminating trachoma by 2020 and began implementing the SAFE 
strategy as part of national policy in 2003. This has focused on the provision of improved trichiasis 
surgery, MDA and the distribution of public health messages by radio, video, and printed material. 
From 2001-2015 more than one million trichiasis surgeries were performed, over 170 million 
doses of azithromycin were given through MDA and more than 24 million latrines were built. 
Despite these encouraging efforts, trachoma remains a public health problem in many regions of 
the country, and the burden of disease is far above the elimination targets set by WHO. In many of 
these communities, despite seven years of annual or biannual high-coverage MDA, the prevalence 
of TF remains well above threshold for continuing MDA. Data on  Ct  prevalence after repeated 
rounds of MDA in hyperendemic settings such as Ethiopia, indicate that reliable long-term control 
is not consistently achieved, with gradual re-emergence of infection being typical(6). 
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Figure 2. Prevalence of TF in 1-9 year olds by evaluation unit from 2012-2014 (GTMP)(21)
9.4
Flies and Trachoma
Flies are likely to contribute to Ct transmission. Ct can be cultured from guts and limbs of Musca 
domestica fed on Ct-infected egg yolk(26). Using a tightly controlled guinea pig trachoma model, 
Chlamydia psittaci was transmitted by flies from infected to uninfected eyes(26). Infection was 
established consistently if the time between flies feeding on infected guinea pig ocular secretions 
and being exposed to uninfected guinea pigs was under one hour. Although there is some 
evidence that flies are involved in transmission, it is poorly understood.
In The Gambia,  Musca sorbens, which breeds most successfully in human faeces, accounts for 
>90% of fly-eye contacts(27, 28). In the “Flies and Eyes” study, intensive control through long-term 
insecticide spraying was associated with a significant reduction in active trachoma (infection not 
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tested)(12). In the same study, installing pit latrines was associated with a significant reduction in 
fly-eye  contacts  and  a  non-significant  reduction  in  active  trachoma(12).  However,  when 
azithromycin MDA was combined with intensive insecticide spraying in Tanzania, there was no 
additional benefit(11). Two studies tested M. sorbens caught leaving faces of Ethiopian children 
for  Ct by PCR; 15-23% of flies were positive(29, 30). These data suggest  M. sorbens may be a 
trachoma vector, however, its relative importance probably varies by setting. Interventions to 
control M. sorbens tried to date have met with variable success; intensive insecticide spraying is 
probably not a sustainable control measure. 
Our recent Gambian studies found M. sorbens is strongly attracted to odours produced by human 
faeces. Compared to other sources of excrement (dog, cow, horse), human excrement was up to 
sixty times more attractive. Volatile odours were collected from faeces and analysed by gas 
chromatography, mass-spectrometry and electrophysiology to identify chemicals detected by M. 
sorbens. Several putative attractants were identified in the odour of human faeces. The next step 
is to test these odours in behavioural experiments, with the aim of developing traps, which will 
locally suppress M. sorbens populations around human habitations. We have developed methods 
to establish M. sorbens laboratory colonies for behavioural studies. We have pilot-tested video 
observation of fly-eye contact for behaviour and transmission studies. 
Attractant and repellent technologies could be combined to create a “push-pull” strategy to 
reduce vector-host contact and attract flies to lethal oviposition traps to supress populations. 
More research is warranted in this area, particularly as our formative research indicates that 
control of flies would be a particularly popular public health measure. The Logan entomology 
research group at LSHTM group works in partnership with industrial collaborators, to investigate 
long-lasting wearable arthropod repellent technologies including clothing, wrist bands, necklaces 
and footwear.
To date there has been little investigation of the potential contribution of flies in the transmission 
of trachoma in Ethiopia, this warrants further study as this may be an important component.
9.5
Rationale for the use of repellents against Musca sorbens
The use of insect repellents is widespread world-wide to prevent nuisance biting by non-vector 
species, and to prevent disease transmission by vectors in disease-endemic regions. Although the 
use of plants with repellent qualities, either by burning leaves or presenting fresh foliage (31, 32), 
is prevalent in many regions, commercially available topical repellents are rarely used by people in 
low-income  and  disease-endemic  countries.  This  is  because  of  cost,  availability,  and  the 
practicalities of a product that requires repeat application. As such, there are limited studies on 
the  effectiveness  of  repellent  personal  protection  against  prevention  of  disease,  however, 
repellents have been used successfully to control arthropods of public health significance but 
which do not control disease, including lice (33) and the chigoe flea (34, 35). However, there is 
growing interest in the use of repellents as personal protection from disease transmission, 
particularly around the use of insecticide-treated clothing (36–39), which can repel biting insects. 
Insecticide  treated  clothing  has  been  shown  to  provide  protection  from  both  malaria  and 
leishmaniasis (40). Another study looked at the use of insecticide-treated headscarves for Afghan 
women in a Pakistani refugee camp, and found a reduction in the incidence of malaria in people 
under 20 years old (41). 
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As well as transmitting Chlamydia trachomatis, Musca sorbens flies can cause severe distress due 
to their eye-seeking behaviour (Figure 3). Therefore, reducing the number of  M. sorbens face 
contacts would not only contribute towards breaking the transmission cycle of Ct, but would also 
alleviate distress in regions where  M. sorbens are found. For these reasons, it is possible that 
personal protection against  M. sorbens by insect repellents could be highly successful, as the 
immediate  benefit  of  reduced  face  contact  would  encourage  continued  uptake  of  this 
intervention. 
Figure 3. Eye-seeking behaviour of Musca sorbens. Photo taken by A. Robinson, Faji Gole, Ethiopia, January 2018, reproduced with 
permission. 
Relative to other vectors of disease, very little is known about the biology and ecology of  M. 
sorbens, although limited studies are available (27, 28, 42–45). Particularly, the only M. sorbens 
control measures that have been robustly studied are that of insecticide, and breeding site/larval 
source management  (46). However, other closely related species are better understood, and 
repellents have been used with mixed success against the bush fly Musca vetustissima (47, 48), 
the  face  fly  Musca  autumnalis, and  the  housefly  Musca  domestica (48–50).  Insecticide-
impregnated ear-tags have also been developed for use in cattle, against horn- and face-flies (51). 
10 Research hypothesis
Commercially available insect repellent products can be used to decrease contact to the face, 
particularly the eyes, nose and mouth, by the eye-seeking fly  Musca sorbens.  The protection 
afforded by insect repellents will prevent transmission of Chlamydia trachomatis by infected flies, 
as well as reducing the nuisance caused by this species. 
11 Choice of comparators
It is well-established that individuals vary in their attractiveness to biting insects. A number of 
factors are thought to contribute to this variation in attractiveness, including body weight and/or 
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surface area (52), hormones (53), genetic factors (54) or disease (55, 56). Although Musca sorbens 
flies do not imbibe a blood meal, they are attracted to the face, and this attraction is presumably 
mediated via cues including odour and vision, that are highly person-specific. Further, there is 
evidence to suggest that flies are more attracted to individuals with ocular or nasal discharge, 
which is in turn influenced by the presence of trachoma. It is therefore reasonable to speculate 
that for a multitude of reasons some individuals are more attractive to  M. sorbens flies than 
others, and therefore a within-subject trial, which controls for such variation, is the optimal study 
design.
12 Study Objectives
12.1 Primary Objective
To measure the protective efficacy (personal protection) of repellent products, by comparison of 
the inhibition of Musca sorbens contacts on participants before and after their application.
The  products  will  be  some  or  all  of  the  following  insect  repellents:  DEET  (N,N-diethyl-3-
methylbenzamide), IR3535 (3-[N-butyl-N-acetyl]-aminopropionic acid ethyl ester), Picaridin (2-(2-
hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl ester); PMD (para-Menthane-3,8-diol) or 
permethrin
 
(m-Phenoxybenzyl)-cis,trans-3-(2,2-dichlorovinyl)-2,2-
dimethylcyclopropanecarboxylate). Repellents will be applied 1) topically on the skin, or 2) in long-
lasting, plastic formulations of that can be worn on the body (wearable repellent technologies). 
The insect repellent synergist Vanillin (4-Hydroxy-3-methoxybenzaldehyde) may be added to the 
long-lasting plastic formulations, to improve the duration of protection.
12.2 Secondary Objectives
1. To compare the duration of protection offered by different repellent products using the 
median Complete Protection Time (mCPT)
2. To compare the effectiveness of protection offered by different repellent products using 
the median effective dose and median effective time
3. To  assess  the  acceptability  of  the  repellent  products  tested  in  the  field  trials using 
qualitative data from participants. 
13 Trial Design
This is a within-subject, non-masked, trial of the use of commercially available insect repellents 
against Musca sorbens, with two consecutive participant groups in the laboratory and in the field, 
and a primary endpoint of measuring the protective efficacy of each repellent product. 
The trial is within-subject to allow comparison of M. sorbens contacts on the same participants 
both before (control) and after (test) application of the repellent or repellent device. This is to 
mitigate any possible inter-individual attractiveness effects. Control sampling will be conducted 
before test sampling, to preclude contamination of the control sampling by the test sampling. For 
this reason, the trial is not masked. 
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13.1 Laboratory trial
In preliminary laboratory clinical trials in London, 17 participants will test all products that have 
been found to exhibit repellency to Musca sorbens in benchmarking laboratory studies. These will 
be  chosen  from:  DEET  (N,N-diethyl-3-methylbenzamide),  IR3535  (3-[N-butyl-N-acetyl]-
aminopropionic  acid  ethyl  ester),  Picaridin  (2-(2-hydroxyethyl)-1-piperidinecarboxylic  acid  1-
methylpropyl ester); PMD (para-Menthane-3,8-diol) or permethrin (m-Phenoxybenzyl)-cis,trans-3-
(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate). Products tested will be either (1) topical 
repellents, or (2) in long-lasting, plastic formulations of repellents that can be worn on the body 
(wearable  repellent  technologies).  The  insect  repellent  synergist  Vanillin  (4-Hydroxy-3-
methoxybenzaldehyde) may be added to the long-lasting plastic formulations, to improve the 
duration of protection.
As all participants will test all products and all concentrations, this study is non-randomised. The 
combination of product and product format to be tested will be determined before the study 
commences. Participants will be asked to place their arm in a cage with 100 Musca sorbens flies, 
and the behaviour of the flies on the surface of the arm and hand will be filmed for ten minutes. 
This modified arm-in-cage assay has been developed specifically for use with Musca sorbens.
There will be a two-stage selection process to determine which repellent products, at which 
concentration (dose), should be carried forward to the field trial. In the first stage, a single 
measure of Protective Efficacy (PE) will be used to determine protection, by measuring the 
duration of fly-arm contact after application of a repellent product (test measurement) relative to 
that  before  application  of  the  product  (control  measurement).  By  only  selecting  repellent 
products/concentrations that protect against at least 30 % of fly contact immediately following 
application (at time zero), those with little or no effect will be disregarded.
In the second stage and using the same participants, those repellent products/concentrations that 
demonstrated at least 30 % PE will be measured for the persistence of effect, over a six-hour 
period. For the wearable repellent devices, tests will further be repeated at one, two, three and 
four weeks later. The duration of fly contact in a modified arm-in-cage assay will again be used. 
This stage will allow estimations of persistence including the Median Effective Dose (ED50), the 
Median Effective Time (ET50) and the Complete Protection Time. Estimations of persistence will 
allow final selection of repellent products/concentrations to be tested in the field trials.
13.2 Field trial
In the field clinical trial, eligible participants will be randomised between groups (study arms), each 
of which will test only one topical repellent or slow-release wearable repellent device, at one 
concentration. A maximum of four groups will be tested in addition to a control group, and each 
group will contain 29 children between the ages of three and 12 years. The additional control 
group, receiving no product, will allow for temporal comparison of fly contact across a day of 
testing. 
The combination of product and product format to be tested will be determined before the study 
commences. The PE will be determined by measuring the frequency of fly-eye, fly-nose, fly-mouth 
and fly-face contacts by Musca sorbens after application of the product, in a ‘field’ environment 
where these flies are naturally present at high density. Control measurements will be taken of fly 
contact on each participant prior to this. Test measures will be repeated over a period of six hours 
to determine the ET50, and for wearable repellent devices, participants will continue to wear the 
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product and tests will be repeated at one, two, three and four weeks later. Topical repellents will 
be applied by the Stronger-SAFE field team nurses, who will also demonstrate how to wear the 
repellent devices. A qualitative assessment of acceptability and barriers to use will be carried out 
at the end of the trial. 
14 Study setting and populations
14.1 Age of participants
This clinical trial has two consecutive participant groups, firstly adults in the laboratory trials and 
then children of age three to 12 years in the field trials. The first study will test proof-of-concept 
for the use of insect repellents against Musca sorbens, will narrow down repellent candidates, and 
will not replicate a naturalistic setting. The second study will take place in a trachoma-endemic 
setting, where  Musca sorbens are prevalent and likely contributing to disease transmission. 
Because fly harassment around the eyes, nose and mouth is not a problem that adults experience, 
it would neither be possible nor naturalistic to conduct the field study using adults. 
14.2 Laboratory trial (London School of Hygiene & Tropical Medicine)
Laboratory studies will be conducted in specialised, Good Laboratory Practice (GLP)-accredited 
insect testing facilities at LSHTM. A colony of field-collected, but laboratory-reared, Musca sorbens 
are maintained by the lead investigator in insectary facilities at LSHTM, these will be used for all 
laboratory trials. We will enrol adults (>18 years) of both sexes, among staff and students of 
LSHTM, to this component of the trial. 
14.3 Field trial (Oromia, Ethiopia) 
Field studies will be carried out in one woreda (district) in the West Arsi Zone of Oromia, Ethiopia, 
in  the  same  approximate  locality  that  the  other  Stronger-SAFE  Phase  II  studies  are  being 
conducted, but in villages (kebeles) that have not previously been enrolled to any other Stronger-
SAFE study component. Kebeles will be chosen where TF prevalence is believed to be low (TF1-
9<40%). We will perform trachoma screening in the selected kebeles to confirm TF prevalence. We 
will select low prevalence areas as these studies do not incorporate clinical or  Ct prevalence 
outcomes, requiring only fly populations, therefore we will aim to set the study site where there is 
minimal  disease  transmission  but  a  high  abundance  of  Musca  sorbens.  We  will  conduct  a 
preliminary assessment of the suitability of study sites by visiting and observing the extent of local 
fly-eye nuisance among children. We will enrol households with children aged three to 12 years, as 
they are at increased risk of TF relative to adults, and also tend to experience higher levels of face 
fly nuisance. This field study location will provide an excellent context for informing on the wider 
applicability of the study results, due to both the very high TF prevalence rates  (21) and fly 
population densities  (29) experienced in this area. Small-scale  field repellency trials will be 
conducted at the participant’s houses.
15 Trial eligibility and withdrawal criteria
Participants will be healthy individuals, and will be included in the study if they meet all of the 
following criteria:
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15.1 Laboratory trial eligibility criteria
1. Participant is aged  18 years and  65 years and in good health
2. Participant has a good understanding of the procedures of the study and agrees to abide to 
these procedures
3. Participant is able to communicate well with the investigator, and attend the laboratory for 
all aspects of the laboratory studies
4. Participant has no known adverse reactions, or evidence at screening of adverse reactions, 
to the commercially available repellents DEET, PMD, IR3535, Picaridin or Permethrin, or to 
Vanilla
5. Participant has no known history of skin allergies or hypersensitivity to topical creams
6. Participant agrees to a pre-trial skin reactivity test for all the repellents that will be used in 
the trial
7. If in the event of the participant experiencing an adverse reaction to a repellent during the 
trial, the participant agrees to inform his/her general practitioner and seek appropriate 
treatment if necessary
8. Participant is willing to allow laboratory-reared Musca sorbens flies to land and crawl on 
their arm, during the modified arm-in-cage assay, for periods of up to ten minutes at a time
9. Participant agrees not to use any perfumed or scented product, including bathing products, 
for a 24-hour period before each laboratory session
10. Participant has signed informed consent
11. Participant is not a smoker, and will agree to refraining from smoking for the 12 hours 
before each laboratory trial
15.2 Field trial eligibility criteria
1. Participant lives in the designated study site 
2. Participating households must be within a one-hour drive of Feya General Hospital
3. Participant considers themselves to be in good health, as does the parent or guardian
4. Participant is aged  3 years and  12 years 
5. Participant has a good understanding of the procedures of the study and agrees to abide to 
these procedures 
6. The parent or guardian of the participant has a good understanding of the procedures of 
the study and agrees to abide to these procedures 
7. Participant  is  able  to communicate  well  with the  investigator  or  fieldworker  who is 
conducting the study
8. Participant has no known adverse reactions to the commercially available repellents DEET, 
PMD, IR3535, Picaridin or Permethrin, or to Vanilla
9. Participant has no known history of skin allergies or hypersensitivity to topical creams
10. Participant agrees to a pre-trial skin reactivity test for all the repellents that will be used in 
the trial
11. If in the event of the participant experiencing an adverse reaction to a repellent during the 
trial, the participant can request medical advice from the Stronger-SAFE field team nurses 
if they wish
12. Participant is willing to sit still on a chair outside their house, for sequential periods of up to 
ten minutes, allowing wild fly contact and landing on the body and face, as much as 
possible without disturbing fly behaviour
13. Participant agrees not to use any perfumed or scented product, including bathing products, 
for a 24-hour period before each laboratory session
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14. Able and willing to give fully informed assent
15. The parent or guardian has signed informed consent
16. The participant does not become unacceptably upset during the procedures
15.3 Participant withdrawal
Participants can stop at any time without giving a reason for withdrawing. Data collected to the 
point of withdrawal will be used in the analysis of the study, unless the participant requests that 
their data is not used, in which case it will be removed from the database. Participants may also be 
removed at the discretion of the Chief Investigator, where continued participation may affect the 
safety of the participant or where there is a development of any condition which might interfere 
with study participation.
15.4 Participant retention
Once participants are enrolled to either the laboratory or field clinical trial, both study sites will 
make every reasonable attempt to ensure that these participants are followed for the entire study 
period, when repeat observations are necessary over a duration of one month for the wearable 
repellent devices. 
For the laboratory clinical trial, the loss-to-follow-up over this month is expected to be low, and 5 
% loss-to-follow-up has been allowed for the laboratory trial sample size (+ one child). For the field 
clinical trial, loss-to-follow-up over that month is expected to be higher, as it may be harder to 
locate young children and ensure that they are at home on the required days. Therefore, a 25 % 
loss-to-follow-up has been allowed for the field trial sample size (+ six children). Fieldworkers at 
this study site will be responsible for developing and implementing local standard operating 
procedures to achieve this level of follow-up.
16 Interventions
16.1 Investigational products
16.1.1 Topical insect repellent products
One or more of three insect repellent products (Table 1), previously determined by laboratory 
experiments at LSHTM to exhibit potential repellency to Musca sorbens, will be tested. 
Table 1. Insect repellent products that will be applied topically will be selected from these three actives
Generic name
Repellent active ingredient
CAS 
number
Manufacturer
DEET
N,N-Diethyl-meta-toluamide
134-62-3
Merck(1)
IR3535
3-[N-butyl-N-acetyl]-aminopropionic acid ethyl ester
52304-36-6
Merck (2)
Picaridin
2-(2-hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl 
ester
119515-38-
7
Alfa 
chemistry(3)
(1) https://www.sigmaaldrich.com/catalog/substance/deet1912713462311?lang=en&region=GB
(2) https://origin-webqws.sial.com/catalog/product/sial/34524?lang=en&region=GB&cm_sp=Insite-_-recent_fixed-_-recent5-3
(3) https://www.alfa-chemistry.com/sec-butyl-2-2-hydroxyethyl-piperidine-1-carboxylate-cas-119515-38-7-item-289774.htm
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16.1.2 Wearable repellent devices
One or more of five insect repellent products, with the possible addition of vanillin (Table 2), 
previously determined by laboratory experiments at LSHTM to exhibit potential repellency to 
Musca  sorbens,  and  formulated  into  long-lasting  plastic  formulations  made  of  low-density 
polyethylene (LDPE) or high-density polyethylene (HDPE). In the case of permethrin, the wearable 
repellent technology will be permethrin-treated fabric, e.g. a permethrin treated neckband. PMD 
will be allowed in a wearable repellent device, but not as a topical product, because of safety 
advice against its use on children’s faces (57). Vanillin is an organic compound that it is thought 
may enhance the protection time of insect repellents.
Table 2. Insect repellent products that will be formulated into long-lasting plastic formulations will be selected from these five 
actives
Generic name
Repellent active ingredient*
CAS 
number
Manufacturer
DEET
N,N-Diethyl-meta-toluamide
134-62-3
Merck(1)
Oil  of  Lemon 
Eucalyptus, 
PMD
para-Menthane-3,8-diol OR Citriodiol® (64% PMD [a mixture 
of the cis and trans isomers of p-menthane-3,8-diol) together 
with a number of minor constituents found in essential oil 
which enhance the efficacy further])
42822-86-6
Merck (2)
Citrefine (3)
IR3535
3-[N-butyl-N-acetyl]-aminopropionic acid ethyl ester
52304-36-6
Merck (4)
Picaridin
2-(2-hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl 
ester
119515-38-
7
Alfa 
chemistry(5)
Permethrin
(m-Phenoxybenzyl)-cis,trans-3-(2,2-dichlorovinyl)-2,2-
dimethylcyclopropanecarboxylate
52645-53-1
Merck(6) OR
Fagron Hellas(7)
*Vanillin (4-Hydroxy-3-methoxybenzaldehyde, CAS number 121-33-5, available from Merck) may be used as a synergist with these 
repellent actives (https://origin-webqws.sial.com/catalog/product/sial/v1104?lang=en&region=GB)
(1) https://www.sigmaaldrich.com/catalog/substance/deet1912713462311?lang=en&region=GB
(2)  https://origin-webqws.sial.com/catalog/product/aldrich/r751898?lang=en&region=GB
(3) https://www.citrefine.com/citriodiol/#what-is-citriodiol 
(4) https://origin-webqws.sial.com/catalog/product/sial/34524?lang=en&region=GB&cm_sp=Insite-_-recent_fixed-_-recent5-3
(5) https://www.alfa-chemistry.com/sec-butyl-2-2-hydroxyethyl-piperidine-1-carboxylate-cas-119515-38-7-item-289774.htm
(6) https://origin-webqws.sial.com/catalog/product/sial/45614?lang=en&region=GB
(7) https://gr.fagron.com/en-gr
16.2 Application of topical products
All topical products will be applied at the standard laboratory application rate of 1 ml product/600 
cm2. 
16.2.1 Laboratory trials application rate
The arm is an estimated 9 % of the adult body surface area (BSA), therefore the forearm and hand 
can be considered to be 4.5 %. With the average adult BSA of 19,000 cm 2, the surface area of the 
forearm can be taken to be 855 cm2. As such, 1.4 ml of solution will be applied to the forearm at 
the appropriate concentration, never exceeding 20 %.
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16.2.2 Field trials application rate
Topical repellent will be applied to a circular area on the cheek. An average diameter of 6 cm 
would give a surface area of 28.3 cm2. As such, 0.05 ml (50 L) of product will be applied to each 
cheek at the appropriate concentration, never exceeding 20 %. Topical repellents will be applied at 
least 3 cm (three finger-breadths) below the eye and positioned away from the nose and mouth.
Due to the differences in surface area under observation in the laboratory and field trials, with 
greater skin surface areas in the former (application on the arm) leading to greater amounts of 
active ingredient being applied, there is a risk that efficacy in the laboratory will be greater than 
that experienced in the field. However, it should be emphasised that the aim of the laboratory 
trials is to inform which repellents can be taken into the field trial for testing, as this is the context 
in which such a product would be used. Therefore, while both studies are merited, the Protective 
Efficacy and other outcome measures as calculated in the field trial will be those that are used to 
inform inclusion of repellents as a fly-control intervention in the Stronger-SAFE Phase 3 RCT.
17 Study outcomes
17.1 Primary outcome measures
The primary endpoint is the protective efficacy of the repellent products. Protective efficacy will 
be presented as a proportion, by comparing fly contact on the participants following application of 
the repellent product (test), relative to before application (control) (see section 26.3). Protective 
efficacy will be determined for all repellent products in both the laboratory and field clinical trials. 
17.2 Secondary outcome measures
1. Median Complete Protection Time (mCPT) in laboratory trials only
2. Median Effective Dose (ED50), in laboratory trials only
3. Median Effective Time (ET50), in both laboratory and field trials
4. Acceptability of the repellent interventions among children and their caregivers, in field 
trials only
18 Participant timeline
A timeline of participant recruitment and enrolment, consent, and completion of the clinical trials 
is given in Figure 4.
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Recruitment procedures: posters 
and website/ London-based
Potential participant makes contact for 
further information: email or phone
Online application
Assessment of eligibility 
based on online application
Excluded: doesn’t meet 
eligibility criteria
Eligible participant comes to 
study site. Complete assessment 
and informed consent. Skin 
reactivity test for all repellents
Eligible participant receives 
complete study information and 
informed consent documentation
Confirm continued willingness 
to participate. First laboratory 
repellency trial
Loss to follow-up 
or discontinued
Completion of laboratory trials 
and post-trial follow-up 
Loss to follow-up 
or discontinued
Loss to follow-up 
or discontinued
Recruitment procedures:
•
Meeting with kebele leaders and HEW
•
visiting households in suitable locality
Potentially participating HH re-
visited after several days
Assessment of eligibility 
based on discussion with 
primary caregiver
Excluded: doesn’t meet 
eligibility criteria
Eligible participant re-visited at 
HH after at least one day. 
Complete assessment and 
informed consent. Skin reactivity 
test for all repellents
Eligible participant and primary 
caregiver receives complete study 
information and informed 
assent/consent documentation
Confirm continued willingness 
to participate. First field 
repellency trial
Loss to follow-up 
or discontinued
Completion of field trials and 
post-trial follow-up 
Loss to follow-up 
or discontinued
Loss to follow-up 
or discontinued
LABORATORY TRIALS
FIELD TRIALS
Figure 4. Participant timeline for clinical trials of insect repellents against fly contact by Musca sorbens
19 Sample size
19.1 Laboratory trials sample size
For laboratory trials, estimations of the sample size required are dependent on the variability of PE 
between individuals. As such, a range of sample sizes were calculated that took both this, and 
variability  in  the  confidence  intervals  around  the  estimate,  into  consideration  (Table  3).  A 
conservative estimate of the PE standard deviation of 30 % was chosen, which when allowing for a 
confidence interval of ±15 % around the estimate, gives a sample of 16 people. When allowing for 
5 % loss-to-follow-up, the total sample size will be 17 people. Equal numbers of male and female 
participants are preferred.
Table 3. Estimation of sample size for determining the Protective Efficacy of repellent products in laboratory trials.
Standard deviation of Protective Efficacy 
10 %
15 %
20 %
25 %
30 %
35 %
Margin  of 
error (CI)
5 % 
16
35
62
97
139
189
10 %
4
9
16
25
35
48
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15 %
2
4
7
11
16
21
20 %
1
3
4
7
9
12
For stage two laboratory trials (repellent product persistence), multiple follow-ups on the same 
individuals will generate more repeat data points. As such, the sample size requirement for PE will 
be sufficient. If we observe in stage one that variability between individuals was much greater 
than expected then we will consider increasing the sample size for stage two.
19.2 Field trials sample size
This trial will be powered to detect a protective effect in the intervention (treatment) groups 
relative to the control groups. To test for 30 % protection (PE) in the intervention arm versus the 
control arm, assuming a standard deviation of 30% and using 90 % power, 23 children are required 
in each study group. When allowing for 25 % loss-to-follow-up, the total sample size in each study 
arm will be 29 people. Equal numbers of male and female participants are preferred.
Not all approximately participants in the wearable repellent device arms of the field trial will be 
interviewed for intervention acceptability. Based on previous experience, we anticipate that a 
sample of 15 to 20 child-caregiver pairs, purposively sampled to represent the range of child ages 
will be sufficient, and further data collection unlikely to yield additional information. However, we 
will review our data regularly during the data collection process and will adjust the sample 
accordingly.
20 Enrolment, randomisation and allocation
20.1 Laboratory trials
Participants will be recruited through standard recruitment methods, including emails, posters, 
leaflets and other advertising routes to staff and students of LSHTM and other members of the 
public. Participants will be fully informed before the study and it will be made clear that they can 
withdraw from the study at any time. Participants will be given and asked to read the Participant 
Information Sheet (Appendix 1) and Product Information Sheet (Appendix 2) which describes the 
tests which they will take part in, and a consent form (Appendix 3) which must be signed before 
the test begins.  Because in the laboratory trials all participants will test all products and all 
concentrations, this study is non-randomised.
20.2 Field trials
Prior to approaching members of the communities in which we wish to work there will be initial 
dialogues with the community leaders and local health officials to introduce the purpose and 
nature of the research project. Following this, participants will be recruited by visiting households 
in the study site that are home to children in the correct age bracket. Information about the study 
will be shared with potential participants by members of the field research team, who have 
previous experience in the participant information and consenting processes. During the visit, 
participants will be provided with Information Sheets (Appendices 4 and 5), a Product Information 
Sheet (Appendix 6) and Informed Consent and Assent forms (Appendix 7). Assent will be sought 
from the participant, and consent from the primary caregiver. This will be in Afaan Oromo, the 
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regional language. This will be read to those who are unable read. After verbal explanation of the 
relevant sections of the Information Leaflet and having the opportunity to ask questions, informed 
consent will be gained and evidenced by a signature or thumbprint signature (deemed acceptable 
locally due to high rates of illiteracy), in the presence of the study team and independent witness. 
Eligible participants will be randomised equally between the topical repellents and wearable 
repellent devices found to be protective in laboratory clinical trials, and a control group receiving 
no intervention.
21 Study procedures 
This clinical trial encompasses a series of laboratory studies, designed to determine which of a 
number of commercially available repellents provide protection against laboratory-reared Musca 
sorbens fly contact, followed by a field trial testing the protection afforded by these repellents and 
wearable repellent devices, from fly contact by wild M. sorbens on children aged three to 12 years. 
In the field trials, the acceptability of these products to the end-users (both children and their 
caregivers) will be assessed. 
Prior to both trials, preliminary benchmarking laboratory studies will be conducted to determine 
which of five repellent actives will be studied in the clinical trials. 
21.1 Laboratory trials
21.1.1 Test insects 
On the day before each test, Musca sorbens flies of between one and 14 days post-emergence will 
be brought into testing room and allowed to acclimatise overnight and for at least 12 hours. Flies 
will be starved of their sugar and protein source (milk or milk powder) during this time period. 
21.1.2 Testing room
The temperature and humidity in the room will be monitored and recorded for the duration of the 
study. Room temperature will be maintained at 20-27C, and relative humidity (RH) at 20-50 %, 
however, it has been noted in insect rearing that Musca sorbens are minimally affected by changes 
in temperature and humidity. All tests will be conducted in the diel phase between 09:00 and 
17:00.
21.1.3 Topical repellents
21.1.3.1
Protective Efficacy 
Consenting participants will be asked to avoid the use of fragranced cosmetic or washing products 
for 12 hours prior to each laboratory trial. Immediately before testing, the participant’s arm will be 
washed with unscented soap, rinsed with water, rinsed with 70 % ethanol in water, and towel 
dried. An analytical standard of the repellent will be tested at five incrementally increasing doses 
up to a maximum of 20 %, each diluted in ethanol. 
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For the first test, the diluent alone (1.4 ml) will be tested as a control. This will be applied to the 
participant’s arm and allowed to dry for one minute. The participant will then insert his/her arm 
into a purpose-designed insect cage, with a hole in the top allowing a camera lens to film the 
upper surface of the hand and arm. The cage will contain 100 test insects, and insect behaviour on 
the arm will then be filmed for ten minutes. This video footage will retrospectively be analysed for 
the number of fly contact, and the total duration of fly contact. The participant will be instructed 
to refrain from moving his/her arm, which will disturb landing flies. 
For the test to proceed, there must be five or more fly contacts, with the diluent (control,) in the 
ten-minute observation period. After this, the participant will remove their arm from the cage, 
carefully brushing off any flies as they leave. The lowest dose of repellent in ethanol (1.4 ml) will 
then be applied to the arm and allowed to dry for one minute. The participant will then re-insert 
his/her arm into the cage with the test insects, and insect behaviour on the arm will again be 
filmed for ten minutes.
This procedure will be repeated for each incremental dose of the repellent, up to a maximum of 
20 % active ingredient. Each dose will be tested serially and without delay. To determine the 
repellent dose, the doses applied to reach that which was effectively repellent will be summed. If 
at any point the fly-arm contact rate drops below five in 10 minutes, the test will be stopped. After 
all  repellent doses  have  been tested,  1.4  ml  of  the  diluent control  will  be  applied to  the 
participant’s other arm, and tested again as per the first ten-minute test, in order to verify 
continued fly contact/landings. If at this point there are less than five fly contacts, the results of 
the experiment will be discarded.
For a repellent product to be carried forward to the next phase of testing, a protective efficacy of 
30 % is required. While 50 % protection is often used as a benchmark for repellent testing, the 
total proportion of  Musca sorbens flies in endemic areas and carrying Ct has previously been 
estimated as 15 % (29). Therefore, it is plausible that reductions in fly-eye contacts of less than 50 
% could still have a significant effect on the transmission capability of this vector. 
21.1.3.2
Persistence 
Having established which repellent products are effective at which doses, the persistence of all 
repellent products at doses achieving at least 30 % protection (at time zero) will be determined. 
Persistence will be determined using an extended version of the protocol described in 21.1.3.1. 
Preparation, and control testing, will be conducted in the same manner, then the repellent 
products at the appropriate dose will be applied and tested for ten minutes as previously, and the 
observation will be repeated every hour for six hours (Figure 5). After testing, the participant will 
be given access to washing facilities to wash off the topical repellent. This data will allow 
calculation of the Median Protection Time/Effective Dose (ET50 and ED50). To determine the 
Median Complete Protection time (mCPT), the same protocol will be used however the first test 
sampling period will be extended until the first fly-arm contact. 
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Figure 5. Timescale for repellency complete protection time (CPT), and median protection time, testing in the laboratory.
21.1.4 Wearable repellent devices
The protection afforded by wearable repellent devices against M. sorbens contacts, formulated at 
the  effective  dose  determined  in  21.1.3.1,  will  be  measured  (protective  efficacy)  and  the 
persistence of this effect determined. Wearable repellent devices will be tested in an extended 
version of the protocol described in 21.1.3.2. Preparation and control testing will be conducted in 
the same manner but without the application of any diluent prior to the control test. The 
participant will then be given the wearable repellent device and asked to put it on (i.e. wear the 
necklace or neckband around their wrist), and a ten-minute test period will be conducted as 
previously described (21.1.3.1). After this, the observation will be repeated every hour for six 
hours. These procedures (control then tests over six hours) will be repeated every week for four 
weeks, and each timepoint participants will wear the repellent devices that they wore in the initial 
test at the first timepoint (Figure 6). 
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Figure 6. Timescale for wearable repellent device testing in the laboratory. 
21.2 Field trials
21.2.1 Test insects and testing site
Participants will be exposed to fly-face (including eye, nose and mouth) contacts outside their 
house. Previous  studies indicate  that  the  majority of such contacts will be  Musca  sorbens, 
although Musca domestica may be present (46); data thus far from our field site corroborates this 
prediction. Ambient temperature and humidity will be recorded at every fly-face observation 
period. Testing will be started between 09:00 and 10:00, as preliminary data from the same 
locality indicate that M. sorbens are less active earlier in the day, allowing for the six-hour testing 
duration. Should the fly density at the time of year of testing be very high, the test durations may 
be shortened (e.g. to five minutes), as a robust measure of protection will be obtained in a shorter 
interval. 
21.2.2 Pilot Phase
We will conduct an initial pilot study to refine and validate the proposed methodologies for Phase 
2 Repellent Testing. The pilot study will focus on 10 households each with one child aged three to 
12 years, on whom fly-eye observations will be conducted both before and after the application of 
diluent only.  Semi-structured pilot acceptability interviews will be conducted, to help refine and 
revise the topic guides. The Pilot will further enable us to determine whether fly density in the 
area is appropriate to the study. 
21.2.3 Topical repellents
Eligible participants will be randomly allocated in equal proportions to the study groups, as per 
section  13.  The repellent products and product formulations to be tested will be determined 
before the study commences. Each study participant will test only one product type, to minimise 
participant discomfort. Participants will be advised not to apply any cosmetics associated with a 
strong scent, such as perfume, hand cream, body wash, or other scented products. Additionally, 
volunteers will be asked not to consume spicy foods, i.e. curries, chillies and garlic for the 12 hours 
prior to the tests. This will be verified with the participants prior to the commencement of any 
tests.
The participants face will first be washed with unscented soap then rinsed with water and towel 
dried. The participant will then be seated comfortably on a chair outside their house, facing the 
investigators  (entomological field worker and nurse), and will be instructed to refrain from 
touching the face or brushing away landing flies. For the first test, the diluent alone (50 L) will be 
tested as a control. This will be applied to the participant’s face (cheeks) and allowed to dry for 
one minute. The participant’s face will then be videoed for ten minutes by the investigator or field 
laboratory assistant. During this time period, fly behaviour will be manually recorded and scored. 
The video footage will retrospectively be analysed for (i) fly-eye (ii) fly-nose (iii) fly-mouth and (iv) 
fly-face contact. After this ten-minute period of control measurements, the pre-allocated topical 
repellent will be applied to a circular area on the participant’s cheek by the Stronger-SAFE field 
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team nurses. Topical repellents will be applied in a 6 cm diameter circle on the cheek, at least 3 cm 
below the eye and positioned away from the nose and mouth. The repellent will be allowed to dry 
for one minute, then the videoing and observation will be repeated for another ten-minute period. 
Test measurements will then be repeated hourly for five further time points (Figure 7). After the 
final time point, the participant will be provided with soap and water to clean off the topical 
repellent. 
Figure  7. Study design for estimation of personal protection by topical repellents from fly-eye and fly-face contacts by  Musca 
sorbens. This process will be repeated for each of 29 participants.
21.2.4 Wearable repellent devices
Control measurements will be taken for a ten-minute period, exactly as with the topical repellent 
testing, but omitting the face washing step. After this, the pre-allocated wearable repellent device 
will immediately be administered. Field nurses will demonstrate how to wear the repellent device, 
which will then be given to the participant to wear, or his/her primary caregiver to put on to the 
participant. The participant will then be seated again, and the videoing and observation repeated 
for another  ten-minute period, as with the topical  repellent testing.  Again, the  ten-minute 
observation period will then be repeated hourly for five further time points. After the six-hour test 
period, the participant will be asked to continue to wear the repellent device. The investigators 
will return one week later to repeat test sampling, at which point they will measure for ten 
minutes with the wearable repellent device still in place, then remove the device and measure for 
a further (control) ten minutes (Figure 8).
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Child continues to wear device
for repeat measure in 7 days
Figure 8. Study design for estimation of personal protection by wearable repellent devices from fly-eye contacts by Musca sorbens. 
This process will be repeated for each of 29 participants.
21.2.5 Acceptability Musca sorbens repellents
Semi-structured interviews will be conducted with all participants over 5 years of age and their 
primary caregivers on completion of the trial. Interviews will be short (estimated 3 -15 minutes) 
and, for children, will use age-appropriate questions. The purpose of the interviews is to identify 
major barriers to adherence to the study protocol and/or to future use of the repellents within or 
outside of a trial setting. The findings may also point to issues for further exploration in a more 
naturalistic  setting.  Indicative  interview  guides  are  given  in  Appendix  8  (Semi-structured 
interview_Field), and these will be refined and revised during the pilot. 
For participants assigned to use the wearable devices, at the start of each data collection visit we 
will ask whether there have been any difficulties that prevented consistent use and whether any 
difficulties are foreseen. This will be done in addition to the final interview. Adherence to protocol 
(continued wearing of the device) will be encouraged according to section 24.
22 Risks, benefits and burden
22.1 Risk from eye-seeking flies, Musca sorbens
22.1.1 Laboratory trials
Participants will be exposed to laboratory-reared populations of Musca sorbens. These flies have 
been reared in captivity for over six generations and carry no risk of Ct transmission. Participants 
will only be exposed to fly contact on their arms, and after completion of testing, will immediately 
be  instructed  to  wash  their  arm.  Therefore,  the  modified  arm-in-cage  assay  presents  only 
negligible risk.
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22.1.2 Field trials
Participants will be exposed to natural populations of eye-seeking flies, primarily Musca sorbens. 
Because we will study the protective efficacy of these insect repellents outside the participant’s 
houses, they will not be exposed to any greater risk than that which they experience day-to-day. 
However, participants will be asked to remain seated and not brush away flies from their eyes 
during observation periods. Previous observations in the study setting indicate that not brushing 
away flies is normal behaviour. Further, field work will be conducted in part by the Stronger-SAFE 
team ophthalmic nurses. During recruitment, children will be screened for trachoma and their 
trachoma status recorded. After participation in the trial is complete, all children with trachoma 
will be treated with Azithromycin.
22.2 Risk from insect repellent products
Up to six active ingredients (repellent products and a repellent synergist) may be used in this 
study, the selection of which will be made following laboratory studies that will be conducted in an 
insect testing facility at LSHTM, London, using laboratory-reared Musca sorbens. These are:

DEET (N,N-Diethyl-meta-toluamide)

IR3535 (3-[N-butyl-N-acetyl]-aminopropionic acid ethyl ester)

Picaridin (2-(2-hydroxyethyl)-1-piperidinecarboxylic acid 1-methylpropyl ester)

PMD (para-Menthane-3,8-diol)

Permethrin
 
((m-Phenoxybenzyl)-cis,trans-3-(2,2-dichlorovinyl)-2,2-
dimethylcyclopropanecarboxylate)

Vanillin (4-Hydroxy-3-methoxybenzaldehyde)
A full assessment of the risk associated with the use of the above repellents is given in sections 
22.2.1 to  22.2.6 below. Here,  particular  attention  is given to adverse  incidents and  events 
associated with repellents in the eye or on the face. Safety information regarding the repellent 
active ingredients used in the trial have been assessed, material safety data sheets (MSDS) and 
labels have been read to be sure they are safe for human use. Participants will be given an 
information sheet explaining the details of the ingredients and what to do if they have a reaction 
to the product after completion of the test. Repellent products will not be applied to broken skin.
General risks to participants associated with involvement in this study will be addressed by 
adhering to ICH GCP (58), the Declaration of Helsinki (59), the Data Protection Act (60) and all 
applicable regulatory requirements. There will be no benefit to participants. The results of this 
study will be used to design the trachoma transmission-blocking intervention that will be rolled 
out in Stronger-SAFE Phase 3, a cluster-randomised controlled trial. 
22.2.1 DEET
N,N-Diethyl-meta-toluamide  (DEET)  is  a  colourless  liquid  that  is  the  most  common  active 
ingredient in insect repellents. It may cause eye and skin irritation and may be harmful if 
swallowed. 
Specifically with regards to the use of DEET on children, this repellent should not be applied to 
children under two years of age, further, this product should not be applied to the hands of 
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children under 12 years of age  (61). This is to reduce the risk of ingestion by hand to mouth 
behaviour. It is recommended that DEET should not be applied near the eyes and mouth, children 
should not be allowed to handle the product and when applying on children, it should first be 
applied to other hands and then put on the child (62). 
DEET has been found to cause serious eye effects in rabbits, where eye irritation and corneal 
opacity were observed but both cleared by day three and seven respectively  (62). The same 
document noted that LD50 values in this study were “quite high”, with four grams of test material 
being applied per kilogram of body weight. With reference to this trial, 18.85 mg of active 
ingredient would be applied to the cheeks at the highest dose, 20 %, which is 0.47 % of four 
grams. For a child of body weight 10 Kg this would equate to 4.59 % of the AEL (Appendix 9). The 
same guidelines stated “If used on the face, spray on hands first and then apply sparingly and 
avoid eyes. Do not spray directly onto face.”, indicating acceptability of use on the face. In 2010, 
the European commission conducted a risk assessment to human health for DEET. They found that 
an R statement of R36 (irritating to eyes) was not warranted, however, because of the scores for 
corneal opacity in rabbits, they gave a GHS  (Globally Harmonized System of Classification and 
Labelling of Chemicals) Category 2 (Figure 9), with an H statement of H319 (Causes serious eye 
irritation).
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Figure 9. A comparison of chemical hazard classification and labeling: Office of Pesticide Programs (OPP) and the Globally 
Harmonized System of Classification and Labelling of Chemicals (GHS) (63). DEET was found to be in GHS category 2 (level 
unspecified) (61).
DEET has caused adverse reproductive and fetal effects in animals, and may cause central nervous 
system effects. There are no known carcinogenic chemicals in this product. The safety of daily 
application of (DEET) in the second and third trimesters of pregnancy was assessed as part of a 
double-blind, randomized, therapeutic trial of insect repellents for the prevention of malaria in 
pregnancy. The results of the study suggest that the risk of DEET accumulating in the fetus is low 
and that DEET is safe to use in later pregnancy (64).
By having the ophthalmic nurse apply the DEET directly to the children’s face, we will avoid DEET 
application to the children’s hands. It is possible that the child may, over the course of the day’s 
testing, touch their face and then their mouth. However, DEET is commercially available for use on 
children above the age of two, despite the recommendation that it is not applied to their hands. 
From this, we can assume that the risk presented from skin (e.g. elsewhere on the child), to hand, 
to mouth is negligible, presumably because the amount of DEET that is transferred in this way is 
very small. Further to that, the amount that will be applied in this trial (a circle of 6 cm diameter) 
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will overall be much smaller than that which would be applied to the child’s whole body, for 
example to repel mosquitoes, which it is frequently used for. Finally, for topical repellent testing, 
the ophthalmic nurse and team will be present at the house and near the participant all day, and 
will try to dissuade the child from this type of behaviour.
Current European Union guidelines concerning the risk to consumers for the use of DEET sets the 
AELrepeated (acceptable exposure level for repeated use) at 8.2 mg/kg body weight (bw)/day (61). For 
an estimation of the percentage of this AEL that will be topically applied of active ingredient, for a 
hypothetical dose range of maximum 20 % and a range of hypothetical children’s body weights, 
see Appendix 9, for the material safety data sheet (MSDS) see Appendix 10. 
22.2.2 IR3535
Insect  repellent  IR3535  is  a  liquid  containing  98  %  active  ingredient  3-[N-butyl-N-acetyl]-
aminopropionic acid ethyl ester, and 2 % inert ingredients (65). This insect repellent is structurally 
similar to naturally occurring beta-alanine, and is itself a substituted beta amino acid. It has been 
used to repel mosquitoes, deer ticks, lice and biting flies (65). At initial assessment, IR3535 was 
found to have low acute toxicity, and with no reports of adverse health effects on humans. 
However, IR3535 has been found to cause conjunctival irritation at concentrations of 10, 15 and 
20 % in rabbits, further, some corneal opacities were observed which recovered in 8-21 days (66). 
At this time (2001) the LD50 given was >14,000 mg/kg orally and >10mg/kg dermally (66). In 2014 
the US EPA initiated review of IR3535. They found that all human health assessment data 
requirements had been addressed, and between 1991 and 2014 they found 211 reports of adverse 
incidents in humans, which included reports of running nose and eyes and eye irritation  (67). 
Following the review, subsequent fact sheets and technical documents state that there is reliable 
data regarding IR3535 to support the conclusion that this insect repellent is practically non-toxic to 
mammals, including infants and children (65). They found no threshold effect and therefore did 
not publish a margin of safe exposure. However, it was noted that eye irritation can occur if 
IR3535 enters the eyes (68).
Current European Union guidelines concerning the risk to consumers for the use of IR3535 sets the 
AOEL short term (Acceptable operator exposure level for short term use) at 6 mg/kg bw/day (69). For 
an estimation of the percentage of this AOEL that will be topically applied of active ingredient, for 
a hypothetical dose range of maximum 20 % and a range of hypothetical children’s body weights, 
see Appendix 9, for the material safety data sheet (MSDS) see Appendix 11. 
22.2.3 Picaridin
Picaridin, also known as icaridin, is a synthetic compound designed to resemble the natural 
compound piperine, which is found in the group of plants that are used to produce black pepper 
(70). It was first reviewed for toxicity by the WHO in 2001, and found to have a good safety profile 
with negligible dermal and limited ocular irritation capacity in rabbits (this was based on a 
summary of toxicity studies provided by Bayer AG, Germany) (66). The recommended target dose 
was 0.3 mg active ingredient/cm2 of skin. In 2004 the WHO published the results of OECD test 
number 405 (Acute eye irritation/corrosion) and found Icaridin to be a slight irritant. In 2014, the 
US EPA reviewed the use of Picaridin, to determine whether it met the federal insecticide, 
fungicide and rodenticide act (FIFRA). At that time, Picaridin was second only to DEET in use in the 
US (71). Previously reported oral and dermal toxicological effects were determined to be species-
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specific (conducted in rodents) and not relevant to humans. In a review of human incidents, 214 
minor incidents were reported between 2009 and 2014, which usually involved skin, eye or 
respiratory irritation. Importantly, the incidents were all of minor severity and resolved rapidly. In 
another database (Centers for Disease Control and Prevention/National Institute for Occupational 
Safety and Health (CDC/NIOSH)), 22 cases were reported between 1998 and 2010. Most cases (18 
of the 19 involving picaridin alone) were low in severity, largely involving dermal or eye irritation, 
however, in one case where picaridin was directly applied to the face of an infant, the case was 
moderate in severity. The US EPA identified no human health risks associated with the use of 
Picaridin. Most recently, Picaridin has been classified by the US EPA as being of low acute oral, 
dermal  and  inhalation  toxicity,  and  Toxicity  Category  III  for  primary  eye  and  skin irritation 
(Category III = slightly toxic. Toxicity category not specified but assumed to be OPP, Figure 9) (72). 
The toxicology database is considered complete and no additional studies are required. The US 
EPA stated that they believe that the normal use of Picaridin does not present a health concern to 
the general U.S. population (72). The acute dermal LD50 given was >2000 mg/kg (Limit test).
Current European Union guidelines concerning the risk to consumers for the use of Icaridin sets 
the AEL short term  (Acceptable exposure level for short term use) at 3.1 mg/kg bw/day (73). For an 
estimation of the percentage of this AEL that will be topically applied of active ingredient, for a 
hypothetical dose range of maximum 20 % and a range of hypothetical children’s body weights, 
see Appendix 9, for the material safety data sheet (MSDS) see Appendix 12. 
22.2.4 PMD
PMD, structurally similar to menthol, can be derived from the essential oil of the leaves of 
Corymbia citriodora, or from the synthetic citronellal. When C. citriodora oil is refined to increase 
the PMD content, it is known as Oil of Lemon Eucalyptus (OLE). PMD has been on the market as an 
insect repellent since 1998 (74), and is considered to be an eye irritant (Toxicity Category 1), and 
this is the only adverse effect that has been found in studies using lab animals (57). These adverse 
effects have led to precautions around the use of PMD, crucially, it is advised against the use of 
PMD on the face or hands of children (57). For this reason, PMD will not be assessed for use as a 
topical repellent in this clinical trial. However, the use of PMD in a wearable repellent device 
would be permitted, as (1) the device would not be worn on the face, and (2) exposure from a 
wearable repellent device is much lower as there is not direct dermal application. 
Other than eye irritation, PMD is not expected to pose any health risks to people, including 
children (57), and no AEL/AOEL are stated. Further, due to the avoidance of PMD as a topical 
repellent, such values would not be relevant here. For the material safety data sheet (MSDS) see 
Appendix 13. 
22.2.5 Permethrin
Permethrin, a member of the pyrethroid class of insecticides (75), is classed as a repellent when 
used to pre-treat clothing, and is the only insect repellent that is currently used for factory 
treatment of clothing (76). As with PMD, in this study Permethrin will only be considered for use in 
the wearable repellent devices.
In  2009,  the  US  EPA  evaluated  multiple  scenarios  for  permethrin  factory-treated  clothing, 
including toddlers wearing or mouthing the clothing, and found the product unlikely to pose any 
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significant immediate or long-term hazard to those wearing the clothing  (76). Multiple studies 
have demonstrated that maximum permethrin uptake from impregnated clothing is five times 
lower than the regulations established by the WHO and US EPA, and therefore have concluded 
that health impairments are unlikely (77–79). This study will focus on the use of polymer coated 
fabric, which has the lowest skin absorption rate when compared to other types of treated 
clothing (39). The majority of studies also show that impregnated clothing is usually comfortable, 
non-irritating and non-odorous (37).
When  used  at  appropriate  concentrations,  insecticide-treated  clothing  is  deemed  safe.  For 
example, one study showed that permethrin is transferred from clothing to skin at a rate of 0.49 % 
per day from fabric treated with permethrin at a rate of 0.125 mg/cm2. This equated to 0.00006 
mg/kg bw/day, with the permethrin AEL medium term (Acceptable exposure level for medium term use) 
0.05 mg/kg bw/day (80). For the material safety data sheet (MSDS) see Appendix 14. 
In summary, the amount of permethrin that is allowed in treated fabric is low, and from the fabric 
it is thought to be poorly absorbed through the skin.
The following international agencies recommend insecticide treated apparel:

The U.S. Centre for Disease Control and Prevention (CDC)

The World Health Organization (WHO) 

National Institute for Occupational Health and Safety 

The American Academy of Family Physicians 

The Public Health Agency of Canada 
22.2.6 Vanillin
Vanillin is  derived from  extract  of  the  vanilla bean.  Some  studies have  indicated that  this 
compound  can  increase  the  protection  time  of  insect  repellents  (81).  Because  vanillin  is 
considered to cause serious eye irritation, the use of vanillin in this trial would be restricted to use 
in the wearable repellent technologies, which would not be proximate to the eye. For the MSDS 
see Appendix 15. 
22.2.7 Specific risk from application of topical repellents to the face (field trial only)
Due to the nature of the eye-seeking vector in question, it is unavoidable that topical repellents 
must be applied to the face. As per section 16.1, the repellent active ingredients that will be tested 
topically (DEET, IR3535 and Picaridin) have been chosen because they do not carry a specific risk of 
eye irritation beyond what would reasonably be expected through application of a chemical 
product. 
It should be noted that commercially available repellent products do have instructions on the label 
that recommend application to the face. However, care must be taken when doing so. In the field 
trial, topical repellents will be applied in a 6 cm diameter circle on the cheek, at least 3 cm 
(measured by three finger-breadths) below the eye and positioned away from the nose and 
mouth. The repellents will be administered by Stronger-SAFE ophthalmic nurses, who will be 
present at all times during field testing, see section 28 for more information regarding participant 
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care during adverse events. The importance of preventing inadvertent transfer of topical repellent 
from the face to the eye will be fully communicated to participants and their caregivers. 
22.3 Risk from wearable repellent technologies
Wearable repellent technologies will be formulated to contain insect repellent at doses within 
published limit of safe application. Because the repellent product will be formulated into plastic, 
most likely low density polyethylene (LDPE) or high density polyethylene (HDPE), the amount of 
product that is released onto the skin will be considerably lower than that which is experienced via 
topical application of a cream.
22.4 Burden associated with participation
22.4.1 Laboratory trials
Participants will be required to make repeat visits to the LSHTM testing facility, to test each 
product and product formulation. During visits, they will be required to sit still for ten-minute 
observation periods, allowing flies to crawl freely over their forearm and hand. 
22.4.2 Field trials
The participant’s face will be observed and filmed for ten-minute periods. During this time, the 
participant will be required to sit motionless and allow flies to crawl freely over their face and 
around their eyes, nose and mouth. In this study setting this burden would be considered ‘the 
norm’, with children rarely bothering to brush away flies due to their extreme persistence and 
prevalence. 
At the first time-point of testing, the child will be asked to sit for ten minutes of control 
measurements, immediately followed by ten minutes of test measurements, with the application 
of insect repellent in-between. Ten-minute duration follow-up sessions will be conducted hourly 
for the rest of the day (six hours). As participants may be as young as three years old, sitting 
motionless for these periods may be difficult, additionally, the child may become distressed.
We will encourage the children to conform to protocol as far as possible but will make reasonable 
judgement around levels of distress. To be, and remain, eligible for the field trial, we state “15. 
The  participant  does  not  become  unacceptably  upset  during  the  procedures”.  The  same 
methodology ‘Fly-eye studies’ are currently underway as part of the Stronger-SAFE Phase 1 study 
of Ct transmission in the same study site. In Phase 1, the team are conducting ‘Fly-eye studies’ on 
two children in each of more than 200 households, and as such have gained a great amount of 
experience in this type of work. The Phase 1 ‘Fly-eye studies’ constitute two components, the ten-
minute observation/videoing period as described above in this protocol, followed by 15 minutes of 
catching flies from the child’s face. So far, we have not observed many instances in which the child 
becomes upset in the initial ten-minute period, with the latter fly-catching exercise generally 
proving more difficult. In instances where the child does become upset, the field team tries to 
calm the child with the help of the primary caregiver, or his/her friends and family, and encourage 
them to allow work to continue. If the child continues to be upset, sampling/participation is 
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discontinued for that child. In terms of managing distress, the same protocol will be followed for 
this study.
22.5 Benefits associated with participation
Participants  in  the  laboratory  trial  will  receive  no  benefits  from  participation  in  the  trial. 
Participants in the field trial will have the opportunity to have their vision and eyes checked by the 
Stronger-SAFE project team, and will receive appropriate referral for identified problems. There 
are no further benefits expected for any participants.
23 Modifications
Should it be necessary in the field trials, lower dose topical repellents can be substituted to 
improve tolerance to the facial application (e.g. in the event of discomfort), provided those lower 
doses were found to provide at least 30 % protective efficacy. 
24 Adherence
24.1 Trial adherence
The critical importance of both participant availability (for data collection), and sustained use of 
repellents, will be emphasised during recruitment, and participants and their parents/guardians be 
reminded of the importance of this when giving assent/consent.  It is expected that this will 
present a specific challenge in the field clinical trials, where participants who test the wearable 
repellent devices will be required for weekly repeat testing of the product over a four-week 
period. Therefore, in the field trial, the following measures will be implemented:

‘Adherence reminder sessions’ will be conducted at each testing time point (e.g. at each 
visit to the household). The importance of allowing topical repellent to remain on the face 
(i.e. without washing off or removing in any other way), and of wearing the repellent 
device,  for  the  full  testing  duration  (six  hours  or  seven  days  respectively)  will  be 
emphasised  (Appendix  16,  Adherence  reminder  sessions).  This  will  be  of  primary 
importance to the wearable repellent device interventions, as for the topical repellent 
trials investigators will be present throughout the six-hour duration, and can supervise.

In the six days between each testing session, for those households contactable by mobile 
phone,  text  messages  will  be  sent  to  remind  the  primary  caregiver  to  ensure  the 
participant continues to wear the repellent device

For the four, weekly follow-up testing sessions (wearable repellent devices only), on the 
day  prior  to testing one  of  the  fieldworkers will  visit the  household to remind the 
participant’s primary caregiver that the child should remain at home the following day for 
testing. During these ‘priming’ visits, the fieldworker will also discretely note whether the 
child is wearing the device or not, but will not comment on this or draw attention to their 
assessment.

On  days  in-between  those  four  weekly  follow-up  sessions,  a  limited  number  of 
unannounced  visits  by  a  member  of  the  project  team  may  take  place  to  allow  an 
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opportunity  for  problems  relating  to  repellent  use  to  be  identified  and  addressed. 
Participants will be informed about the possibility of those visits at recruitment and during 
consenting. 
24.2 Trial adherence assessment
Adherence to the protocol in the laboratory clinical trial will be simply recorded as participant 
presence/absence for follow-up testing.  
Adherence to the protocol in the field clinical trial will comprise either: (1) wearing the topical 
repellent on their face for the full testing duration of six hours, or (2) wearing the repellent device 
for four consecutive weeks. 
Adherence to protocol for topical repellents can be monitored and recorded by the field team, 
who will be present for the full six hours of testing. Adherence to the protocol for wearable 
repellent device use after the initial testing day (when the field team will be present) will be 
assessed by recording whether or not the participant is wearing the device at the time of each 
data collection visit, and at the time of each ‘priming’ visit on the day prior to testing. At each visit, 
participants will also be asked to report on any difficulties faced in continued use of the device.  
25 Comparability of study groups (concomitant insect-repelling activities/factors)
Co-intervention bias is precluded in the laboratory trial by the trial design being within-subject. 
To mitigate external influences on the outcome variables (frequency of fly contacts), participants 
in all trials will be asked to refrain from the use of any perfumed or scented product, including 
bathing products, for a 24-hour period before each testing session.
In the laboratory trial, co-intervention bias will be mitigated by asking participants to refrain from 
using any insect repellent products for a 48-hour period prior to any testing session.
In the field trial, co-intervention bias will be mitigated by asking participants and their families to 
refrain from the use of any insect-repelling activities for the duration of the trial. Previous 
observational work in the region has identified only one such activity, which involves scattering the 
leaves of Schinus molle trees on the floor and hanging these by the door. The leaves of this tree 
are considered to have anti-fly properties, although this belief is unsubstantiated.
26 Data 
26.1 Data collection
26.1.1 Laboratory trials
Data to be collected are: Participant name and ID number, address, phone number and email 
address, confirmation of informed consent, date of birth, eligibility details, topical repellent skin 
test details (active ingredient, amount, 24 h assessment), visit details (date and time of visit to 
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testing  facility,  investigator  conducting  testing,  person  variables  [body  weight,  tympanic 
temperature], temperature and humidity of testing room, repellent product and concentration 
tested, testing variables [Number of  Musca sorbens flies, number of different sex, age of flies, 
video file ID] and adverse event monitoring).
All arm-in-cage observation and videoing will be conducted by the laboratory co-PI or GCP trained 
staff, as will application of the repellent product. All investigators involved in the trial will be 
trained in the study requirements and will follow standard operating procedures, to ensure each 
participant is studied in a uniform and reproducible manner.
26.1.2 Field trials
Data to be collected are: Participant, household and kebele name and ID number, phone number, 
woreda name, confirmation of informed consent, date of birth, eligibility details, topical repellent 
skin test details (active ingredient, amount, 24 h assessment), visit details (date and time of visit, 
fieldworkers  conducting  testing,  person  variables  [ocular  or  nasal  secretions,  body  weight, 
tympanic temperature], environmental conditions, repellent product and concentration tested, 
number of fly-eye, -nose, -mouth and -face contacts, video recording of face), adverse event 
monitoring and qualitative data from the end-of-trial product acceptability interviews.
All fly-eye observation and videoing will be conducted by trained entomological fieldworkers with 
experience  in  fly-eye  studies.  Application  of  the  repellents  will  be  conducted  by  qualified 
ophthalmic nurses. All fieldworkers involved in the trial will be trained in the study requirements 
and will follow standard operating procedures, to ensure each participant is studied in a uniform 
and reproducible manner.
26.2 Data management and confidentiality
All data will be protected and stored in compliance with General Data Protection Regulation 
(GDPR). Specifics per site are given below. 
During screening, participants will view the participant screening form (PSF, see Appendix 17) as it 
is being completed. During screening and testing, all data collected (during both laboratory and 
field trials) will be recorded at the time of collection via electronic data capture using the Open 
Data Kit (ODK) secure data capture system provided by LSHTM http://opendatakit.lshtm.ac.uk/. 
The PSF and data collection forms will be created and managed in ODK, and study participants will 
not view the data collection forms. Automatic checks for invalid values, internal consistency and 
implausible responses will be programmed into ODK, and additional data validation checks will be 
run after data collection. ODK has an inbuilt audit trail. Encrypted data will be uploaded to a 
secure server at LSHTM for secure storage and analysis. Daily back-up of study data on central 
computers and servers, remote computers and hand-held devices will be conducted. Back-up data 
will be stored separately from the primary electronic storage, and video files (showing the 
participant’s arm or face for laboratory or field trials respectively) will be stored on encrypted 
external hard drives, or encrypted and uploaded to LSHTM secure server.
After study completion, all the relevant study documentation will be retained in accordance with 
the local legislation, for a minimum period of 10 years after completion of the study. The final 
dataset will be archived and maintained by the UK PI. Anonymised data sets will be made publicly 
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available after publication, to ensure the data are available for other investigators to explore. 
Specific permission for this is requested in the consent form.
26.2.1 Laboratory trials
Data from the study will be managed by the LSHTM PI. Paper records (Informed consent/assent, 
PSF, adverse event monitoring questionnaire, adverse event record) will be stored in locked 
cabinets in the locked Arctec office in LSHTM. Scanned electronic back-ups of these will be 
encrypted and uploaded to LSHTM secure server.
26.2.2 Field trials
Data from the study will be jointly managed by the LSHTM and Stronger-SAFE team in Ethiopia, 
coordinated by the UK and Ethiopian PIs. Paper records (Informed consent/assent, PSI, adverse 
event monitoring questionnaire, adverse event record) will be stored in locked cabinets in the 
secure/locked Stronger-SAFE project office. Scanned electronic back-ups of these will be stored in 
encrypted external hard drives, kept separately in the same office.
The UK and Ethiopian PIs will be responsible for ensuring a secure and appropriate location for 
storage of study related documentation present at the field study site, as well as for ensuring that 
only members of site staff who are authorised have access to the files. The site Investigator File 
will be held at the project office in Shashemene. The Investigator File will at all times remain 
available  for  internal  audits  and/or  inspections  of  regulatory  authorities,  including  after 
completion of the project.
26.3 Data analysis
26.3.1 Protective Efficacy, p
The protection (protective efficacy,  p) afforded by a repellent product will be presented as a 
percentage.  p  will  be estimated by comparing fly-arm contact duration and fly-eye contact 
frequency, in laboratory and field trials respectively, after application (or wearing) of the repellent 
product to that during the control period. 
Equation 1. Protective Efficacy, p
p = 100  ((C – T)/C) 
Where (laboratory trials):

C  is  the  total  duration  of  fly-arm  contact  before  application  of  repellent  (‘control’ 
measure), and 

T  is  the  total  duration  of  fly-arm  contact  after  application  of  repellent  (‘treatment’ 
measure)
Where (field trials):

C is the frequency of fly-eye contacts before application of repellent (‘control’ measure), 
and 
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
T is the frequency of fly-eye contacts after application of repellent (‘treatment’ measure)
26.3.2 Median Complete Protection Time (mCPT)
Median  CPT  will  be  estimated  in stage  two  (‘persistence’)  laboratory  trials only,  for  those 
repellents that demonstrated more than 30 % PE. The complete protection time for a specific dose 
will be estimated as the time elapsed until the first fly landing on the arm in each replicate, and 
based on repeat estimates of CPT, the mCPT will be estimated using a Kaplan–Meier function.
26.3.3 Median Effective Dose (ED50) and Median Effective Time (ET50)
ED50 and ET50 will be calculated in stage two (‘persistence’) laboratory trials, however, as only one 
dosage level will be used in the field only ET50 will be estimated there.
The relationship between Protective Efficacy and repellent dose and time since treatment can be 
estimated using a probit-plane regression model (Equation 2). The coefficient  b1 provides an 
estimate of the effect of repellent dose on p, and b2 provides the effect of the time since treatment 
on p.  
Once these coefficients have been estimated, then we can estimate the ED50, concentration (dose) 
of repellent product that affords 50 % protection from fly contacts at time zero (the time of 
application). This is done by setting  p=0.5 and  t1=0 and then solving equation 2. We can also 
estimate ET50, estimating the persistence of the protective effect of a repellent product for a given 
dosage, using the same method. 
Equation 2. Probit-plane regression model for ED50 and ET50
ln [p / (1 - p)] = a + b1(D0) + b2t1
Where:

p is as above, estimated from
o
Total duration of fly-arm contact (laboratory trials)
o
Total frequency of fly-eye contacts (field trials)

D0 is the dose calculated as the natural logarithm of the dose applied (ln[dose])

t is the time post-treatment in hours 

a, b1 and b2 are coefficients estimated using the probit–plane regression model
In the laboratory trials, p will be estimated by the total duration of fly-arm contact, while in the 
field trials p will be estimated using the total frequency of fly-eye contacts, as defined in section 
26.3.1.
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27 Monitoring
27.1 Data Monitoring Committee
A Data Monitoring Committee (DMC) of individuals that are independent of the study has been 
established. The  DMC will  be  supplied interim analyses (notably after  stage  1 and stage  2 
laboratory clinical trials), which will be used to monitor progress of the trial, the safety data, and 
critical efficacy end points (Protective Efficacy). The DMC will be able to advise the sponsor and the 
trial steering committee if the trial should be modified, or in the worst-case scenario, prematurely 
terminated. 
28 Safety reporting
28.1 Adverse events
Participants will be monitored throughout testing sessions by investigational staff (fieldworkers in 
the field clinical trials) for any adverse events. If any adverse events related to the repellent 
product are apparent at any time during the trial, testing will stop immediately. In laboratory 
trials, details of how to access treatment will be offered, and in field trials the participant will be 
assessed by the ophthalmic nurse in the field team. 
Volunteers can only participate in a test a minimum of 72 hours after the screening for skin 
sensitivity to the repellent product, and participants with known allergies to any of the product 
ingredients will not be eligible to take part. Within 72 hours after testing, the participant will be 
contacted and asked to report any adverse events that might have occurred since the end of 
testing. Adverse events that occur >72 hours after the end of participation in the trial will be 
passively monitored.
An adverse event which is ongoing at the time of participant withdrawal or completion will be 
followed up until it resolves or until 30 days after the participant terminates from the study, 
whichever comes first.
28.2 Definitions
A trained clinician will evaluate the severity of an AE (Table 4). Important AEs that are not 
immediately life-threatening or do not result in death or hospitalisation but may jeopardise the 
participant or may require intervention to prevent one of the other outcomes listed in the 
definition above, should also be considered serious.
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Table 4. Terms used to define adverse events
Term
Definition
Adverse Event (AE)/Adverse Reaction (AR)
Any untoward medical occurrence in a participant or 
study participant but which does not necessarily have 
a causal relationship with this treatment
Serious Adverse Event (SAE)
A serious event is any untoward medical occurrence 
that:

Results in death

Is life-threatening

Requires hospitalisation 

Results  in  persistent  or  significant 
disability/incapacity

Consists  of  a  congenital  anomaly  or 
birth defect
Other  ‘important  medical  events’  may  also  be 
considered serious if they jeopardise the participant 
or require  an  intervention  to prevent  one  of  the 
above  consequences,  or  is  an  important  medical 
event in connection with a clinical trial
Suspected  Unexpected  Serious  Adverse  Reaction 
(SUSAR)
Any adverse reaction that is classed as serious and is 
suspected to be caused by the product being tested, 
and is NOT consistent with the information about the 
product in the material safety data sheet (MSDS)
28.3 Reporting Procedures
All adverse events and serious adverse events will be reported to the appropriate regulatory 
authorities. Depending on the nature of the event the reporting procedures detailed below should 
be followed. Any questions concerning adverse event reporting should be directed to the CI in the 
first instance. For adverse event reporting, all data will be recorded via electronic data capture 
using ODK, then managed and stored as per section 26. 
 For all participants in both laboratory and field trials, the correct course of action for reporting 
adverse events is given in Appendix 18. Safety Reporting Flowchart_All. Any AEs occurring in 
participants  in  the  Field  Trial  be  reported  in  accordance  with  the  expectations  of  specific 
regulatory bodies in Ethiopia, for which the correct course of action for reporting is given in 
Appendix 19. Safety Reporting Flowchart_Ethiopia.
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28.3.1 Non serious AEs
All non-serious adverse events will be recorded in Appendix 20. Adverse Event Documentation 
Log_All.
All non-serious adverse events occurring in the Field Trial will additionally be recorded in Appendix 
21.Adverse Event Documentation Log_Ethiopia, which will constitute a tabulated summary of all 
non-serious adverse events occurring during the field trial. This summary will be issued as a report 
to the relevant regulatory authorities (ORHB, FMOST and Ethiopia’s Food, Medicine and Health 
Care  Administration  and  Control  Authority  [FMHACA])  in  accordance  with  their  reporting 
requirements, every three months, or at the end of the clinical trial if sooner. 
28.3.1.1
Non-serious AEs in laboratory trials
In the event of minor adverse reactions such as localised skin redness and swelling, volunteers will 
be directed to contact the nearby GP surgery at 20 Gower Street, London, WC1E 6DP (Tel. 020 
7637 7628) or their own GP. They will also be supplied with the mobile number of the PI, via 
whom they can contact the Stronger-SAFE clinical team.
28.3.1.2
Non-serious AEs in field trials
In the event of minor adverse reactions such as localised skin redness and swelling or minor eye 
irritation, volunteers will be advised by the Stronger-SAFE team field ophthalmic nurses, amongst 
whom  Mr  Muluadam  Abraham  will  be  given  specific  extended  training  in  adverse  event 
management.  The  eye(s) will be rinsed cautiously with saline  for several minutes, and the 
repellent washed off the face. For this eye irrigation, intravenous normal saline bags (1L) will be 
carried by all  field teams  at  all  times. Additionally, specific  medication for  treating  allergic 
reactions in the field will be available in the field medical kit to be administered by the nurses if 
clinically indicated. Should further care be required, ophthalmic nurses will immediately escort the 
participant to Feya hospital (the best hospital in Shashmene), and the event will be considered a 
SAE. In addition, contact with the clinicians Dr Esmael Ali (based in Ethiopia), or the clinical team in 
London (Prof. Matthew Burton or Dr Anna Last) will be available by telephone while fieldwork is 
being conducted.
28.3.2 Serious AEs
Regardless of the relation of the adverse event to study participation, the event must be reported 
as a serious adverse event if it meets any of the definitions in section 28.2. 
All SAEs will be recorded in Appendix 22.SAE Report_All. SAE reports will be submitted to Prof. 
Matthew Burton (CI) within 24 hours. Fatal or life-threatening SAEs that are assessed by the CI as 
being both related and unexpected (SUSAR) must be reported to RGIO and the LSHTM Ethics 
Committee within seven days. SUSARs that are not fatal or life-threatening should be reported to 
RGIO and the LSHTM Ethics Committee within 15 days of the CI becoming aware of the event.
All SAEs, SARs and SUSARs occurring in the Field Trial will additionally be recorded in Appendix 
23.SAE Report_Ethiopia. This report will be sent to the relevant regulatory authorities (ORHB, 
FMOST and FMHACA) within 48 hours. 
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28.3.2.1
SAEs in laboratory trials
In the case of a severe reaction such as anaphylaxis or a severe skin reaction, it will be treated as 
an emergency and an ambulance will be called immediately by dialling 999 directly from a mobile, 
or 555 from an internal phone (this is the emergency line at reception who will then dial 999). In 
addition, one of two of the clinicians on the Stronger-SAFE team, Prof. Matthew Burton or Dr Anna 
Last will be called. Alternatively, a trained First Aider within the Keppel Street building will be 
called. Designated First Aiders are Vanessa Chen-Hussey (ext. 2015), James Logan (ext. 2008) and 
Cheryl Whitehorn (ext. 2344), but if they are unavailable First Aiders are contactable through 
internal phones by typing in ‘first aid’ to the internal phone book which will bring up a list of 
registered First Aiders. 
28.3.2.2
SAEs in field trials
In the case of a severe reaction such as anaphylaxis or a severe skin or eye reaction, it will be 
treated as an emergency (SAE). For severe eye or skin reactions, the same rinsing and washing 
procedures will be followed as for 28.3.1.2 Non-serious AEs in field trials, then the participant will 
immediately be transported to Feya General Hospital (+251916301989 /+251911407518) by the 
field team car. All participating households will be within a 1-hour drive of this well-equipped 
private hospital in Shashemene (Field trial eligibility criteria, p.23). The field team will always 
include one ophthalmic nurse who can provide interim care until the car reaches the hospital and 
can access the advice of a clinician via the field co-PI (a direct mobile line to the clinicians Dr 
Esmael Ali (based in Ethiopia), Prof. Matthew Burton, or Dr Anna Last, who will be able to provide 
assistance remotely). 
29 Ethics and dissemination
29.1 Research Ethics Approval
Approval for the laboratory clinical trials will be sought from  the London School of Hygiene & 
Tropical Medicine Ethics Committee. Approval for the field clinical trials will be sought from the 
Federal Ministry of Science and Technology (FMOST), the Oromia Regional Health Bureau (ORHB) 
Ethics Committee and the London School of Hygiene & Tropical Medicine Ethics Committee. The 
Fred Hollows Foundation research review group will also review and endorse the protocol. All 
participants will provide written informed consent to take part in the study.
29.2 Protocol amendments
A  formal  amendment  to  the  protocol  will  be  required  for  any  protocol  amendments  or 
modifications that may impact either on the conduct of the study or may affect participant safety 
(including but not restricted to changes in: study objectives, study design, participant population, 
sample sizes, study procedures, or significant administrative aspects). Substantive1 amendments 
must be reviewed and agreed by the LSHTM ethics committee prior to implementation, and will be 
described  in  trial  reports.  Amendments  will  be  communicated  to  all  relevant  parties  via 
documented  (and  version  controlled)  amendments  to  protocols  and  standard  operating 
1 ‘Substantive’ is here defined as a protocol amendment that can affect the safety of trial participants or the scientific validity, 
scope, or ethical rigour of the trial 
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procedures. Minor amendments that have no effect on the way that the study will be conducted 
will be agreed by the TSC and appropriately documented.
29.3 Consent and Assent
Consent to enter the study must be sought from each participant only after a full explanation has 
been given, an information leaflet offered, and time allowed for consideration. Consent and 
Assent will be obtained under the jurisdiction of GDPR, that is, must be specific, freely given, 
granular (for a distinct purpose), clear, informed and unambiguous, properly documented and 
easily withdrawn.
29.3.1 Laboratory trials consent
Information about the study will be shared with potential participants by the laboratory co-PI or 
other GCP trained study staff. The same staff will be responsible for ensuring that all potential 
study participants fully understand the Participant and Product Information Sheet, the PSF and the 
consent forms, prior to formally agreeing to participate in the study.  
29.3.2 Field trials consent/assent
Prior to approaching members of the communities in which we wish to work there will be initial 
dialogues with the community leaders and local health officials to introduce the purpose and 
nature of the research project. Following this, participants will be recruited by visiting households 
in the study site that are home to children in the correct age bracket. Information about the study 
will be shared with potential participants by members of the field research team, who have 
previous experience in the participant information and consenting processes. During the visit, 
participants will be provided with Information Sheets (Appendices 4 and 5), Product Information 
Sheet (Appendix 6) and Informed Consent and Assent forms (Appendix 7). Assent will be sought 
from the participant, and consent from the primary caregiver. This will be in Afaan Oromo, the 
regional language. This will be read to those who are unable read. After verbal explanation of the 
relevant sections of the Information Leaflet and having the opportunity to ask questions, informed 
consent will be gained and evidenced by a signature or thumbprint signature (deemed acceptable 
locally due to high rates of illiteracy), in the presence of the study team and independent witness. 
Parents and guardians will be asked to provide Consent. Participants, all aged between three and 
12 years, will be asked for Assent (Appendix 7).
29.4 Compensation
We will not pay individuals to participate in research studies. 
29.5 Access to data
The Steering Committee will oversee data sharing between the two sites, with input from the Data 
Management Committee. The SC and DMC will both have access to project datasets, which will be 
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housed either in an Access database on the PI’s “H” drive (laboratory trials only) or in a secure 
server at LSHTM.
29.6 Dissemination policy
Results from this study will be disseminated at local, national  and international  levels. The 
Stronger-SAFE investigator group is well placed to do this as it involves leaders within Ethiopia at 
the national and regional level, WHO and a leading implementing NGO. Many of the investigators 
are involved in the WHO GET2020 Alliance for the elimination of Trachoma.
At the end of the study, we will inform the Ethiopian regional and Federal health authority and the 
community about the findings of the study via a written report and direct verbal communication. 
The findings will be shared directly with the communities that participated in the research through 
public meetings.
Formal reports will be written for the Ethiopian Federal and Regional health authority and the 
Federal Ministry of Science and Technology (FMOST). Reports will also be prepared for the 
Wellcome Trust and The Fred Hollows Foundation (Ethiopia and UK). 
To ensure operational uptake of the findings of the studies, we intend to present these data at the 
annual National Trachoma Task Force and NTD Research Symposium (Ethiopia). Additionally, we 
will present this research at the annual Trachoma Scientific Informal Workshop prior to the WHO 
GET2020 Alliance meeting. 
Scientific results will be published in Open Access in peer-reviewed journals and presented at 
relevant international conferences.  
The Sensitisation/Community Liaison Team will disseminate the results of the study to the study 
community in community dialogues and radio broadcasts in conjunction with The Fred Hollows 
Foundation Ethiopia Communications Team. 
Beyond this current phase of the work, the wider Stronger-SAFE programme will have a public 
engagement component, supported by the Wellcome Trust, to inform people about trachoma and 
share the outcomes of this work with the wider community in Ethiopia. Our concept for this is to 
involve community members to tell the story of trachoma in their community and how it can be 
controlled.
30 Timeline
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The work outlined in this protocol is anticipated to take place over an eight-month period (Table
5). 
Table 5. Proposed timeline for repellency trials, 2018
Month:
Jun
e
Jul
y
Augus
t
Septemb
er
Octobe
r
Novemb
er
Decembe
r
January
1
2
3
4
5
6
7
8
Ethical approval (LSHTM)
x
x
Ethical approval (Ethiopia)
x
x
x
Benchmarking  studies  (non-
clinical)
x
x
x
Recruitment and prep (Laboratory 
trials, LSHTM)
x
Laboratory trials (LSHTM)
x
x
Recruitment and prep (Field trials)
x
Field trials (Ethiopia)
x
x
x
31 Anticipated outputs
Results from this study will be disseminated at local, national  and international  levels. The 
Stronger-SAFE investigator group is well placed to do this as it involves leaders within Ethiopia at 
the national and regional level, WHO and a leading implementing NGO. Many of the investigators 
are involved in the WHO GET2020 Alliance for the elimination of Trachoma.
At the end of the study, we will inform the Ethiopian regional and Federal health authority and the 
community about the findings of the study via a written report and direct verbal communication. 
The findings will be shared directly with the communities that participated in the research through 
public meetings. Formal reports will be written for the Ethiopian Federal and Regional health 
authority and the Federal Ministry of Science and Technology (FMOST). Reports will also be 
prepared for the Wellcome Trust and The Fred Hollows Foundation (Ethiopia and UK). 
To ensure operational uptake of the findings of the studies, we intend to present these data at the 
annual National Trachoma Task Force and NTD Research Symposium (Ethiopia). Additionally, we 
will present this research at the annual Trachoma Scientific Informal Workshop prior to the WHO 
GET2020 Alliance meeting. Scientific results will be published in Open Access in peer-reviewed 
journals and presented at relevant international conferences.  
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