Clinical Evaluation of a Loop-mediated Isothermal Amplification Test for Treponema Pallidum Pertenue: A Diagnostic Tool to Support Yaws Eradication
Conditions: Yaws
Sponsor: London School of Hygiene and Tropical Medicine

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Loop-mediated isothermal amplification test 
development, implementation and evaluation for 
yaws eradication 
 
 
LAMP 4 YAWS 
 
 
 
 
 
 
Version and date: 1.1 / 18/08/2020 
 
 
 
 
 
 
 
 
 

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STUDY SPONSOR: London School of Hygiene & Tropical Medicine  
SPONSOR ADDRESS: Keppel St, London, WC1E 7HT, United Kingdom 
CHIEF INVESTIGATOR: Dr Michael Marks 
INTERNATIONAL STUDY COORDINATOR: Rebecca Handley  
FUNDERS: European and Developing Countries Clinical Trials Partnership (EDCTP) 
STUDY COORDINATION CENTRE: London School of Hygiene & Tropical Medicine   
 
MAIN CONTACTS: 
Chief Investigator: Dr Michael Marks, Michael.marks@lshtm.ac.uk; Keppel St, London, WC1E 
7HT, UK, 0207 927 2457 
Study Coordination: Rebecca Handley, Rebecca.Handley1@lshtm.ac.uk, Keppel St, London, 
WC1E 7HT, UK, 0207 927 2866. For general queries, supply of trial documentation, and 
collection of data, please contact: Rebecca Handley. 
Sponsor: 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, UK; Tel: +44 207 927 2626,  
 
This protocol describes the LAMP4YAWs study and provides information about procedures 
for entering participants. Every care was taken in its drafting, but corrections or amendments 
may be necessary. These will be circulated to investigators in the study. Problems relating to 
this trial should be referred, in the first instance, to the study coordination centre.  This trial 
will adhere to the principles outlined in the International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) guidelines, protocol and all applicable local regulations.  
 

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Table of Contents 
1 STUDY OVERVIEW AND PROTOCOL SUMMARY 
6 
1.1 STUDY TITLE: 
6 
1.2 ACRONYM: 
6 
1.3 STUDY DESIGN: 
6 
1.4 STUDY SITES: 
7 
1.5 STUDY PARTICIPANTS AND PLANNED SAMPLE SIZE: 
7 
1.6 PLANNED STUDY PERIOD: 
7 
1.7 RESEARCH QUESTION/AIM: 
7 
1.8 PRIMARY OBJECTIVES: 
7 
1.9 SECONDARY OBJECTIVES: 
8 
1.10 COLLABORATING INSTITUTIONS: 
8 
1.11 FUNDING: 
9 
1.12 ROLE OF STUDY FUNDER: 
9 
2 BACKGROUND AND RATIONALE 
10 
2.1 BACKGROUND 
10 
2.1.1 YAWS 
10 
2.1.2 HISTORICAL AND CONTEMPORARY ERADICATION STRATEGIES 
10 
2.1.3 DIAGNOSTICS 
11 
2.1.4 SIGNIFICANCE AND SCIENTIFIC RATIONALE 
11 
3 RESEARCH AIMS AND OBJECTIVES 
13 
3.1 OVERALL STUDY OUTLINE AND RECRUITMENT 
13 
3.2 STUDY SITES 
14 
3.3 CONSENT AND INFORMATION FOR PARTICIPANTS 
14 
4 SUB-STUDY 1: MULTI-COUNTRY CASE SEARCHES AND EPIDEMIOLOGICAL ANALYSIS OF RISK 
FACTORS FOR YAWS 
16 
4.1 OBJECTIVES 
16 
4.2 INCLUSION AND EXCLUSION CRITERIA 
16 
4.3 SAMPLE SIZE CONSIDERATIONS 
16 
4.4 DATA COLLECTION 
17 

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4.5 SAMPLE COLLECTION AND STORAGE 
17 
4.6 LABORATORY TESTING 
18 
4.7 FOLLOW-UP 
18 
4.8 DATA ANALYSIS 
18 
5 SUB-STUDY 2: PERFORMANCE EVALUATION OF A TPHD LAMP ASSAY COMPARED TO QPCR FOR 
THE DIAGNOSIS OF YAWS-LIKE LESIONS 
19 
5.1 OBJECTIVES 
19 
5.2 INCLUSION AND EXCLUSION CRITERIA 
19 
5.3 SAMPLE SIZE 
19 
5.4 SAMPLE COLLECTION AND STORAGE 
19 
5.5 LABORATORY PROCEDURES 
20 
5.6 QUALITY ASSURANCE FOR LABORATORY PROCEDURES AT STUDY SITES 
21 
5.7 DATA ANALYSIS 
21 
6 SUB-STUDY 3: USE OF MOLECULAR TOOLS AND NEXT-GENERATION SEQUENCING TO 
UNDERSTAND THE AETIOLOGY OF YAWS-LIKE LESIONS, AND OPTIMISE SEQUENCING TOOLS FOR 
POINT OF CARE USE. 
22 
6.1 OBJECTIVES 
22 
6.2 INCLUSION AND EXCLUSION CRITERIA 
22 
6.3 SAMPLE SIZE 
22 
6.4 SAMPLE COLLECTION AND STORAGE 
22 
6.5 LABORATORY PROCEDURES 
22 
6.6 DATA ANALYSIS 
23 
7 SUB-STUDY 4: A HEALTH ECONOMIC AND SOCIAL SCIENCE EVALUATION OF HEALTHCARE 
WORKER AND COMMUNITY KNOWLEDGE-ATTITUDES AND PRACTICES IN RELATION TO YAWS AND 
RELATED DISEASES, AND THEIR DIAGNOSIS. 
24 
7.1 OBJECTIVES 
24 
7.2 INCLUSION AND EXCLUSION CRITERIA FOR OBJECTIVES 1 AND 2 
24 
7.3 DATA COLLECTION 
24 
7.4 DATA ANALYSIS 
25 
8 SAFETY CONSIDERATION AND REPORTING 
27 
8.1 POTENTIAL RISKS 
27 
8.1.1 MITIGATION FOR COVID-19 
27 
8.2 KNOWN POTENTIAL BENEFITS 
28 

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8.3 ADVERSE EVENTS 
28 
8.3.1 DEFINITIONS 
28 
8.3.2 REPORTING PROCEDURES 
28 
8.4 FOLLOW-UP 
29 
8.5 DATA QUALITY ASSURANCE AND MONITORING 
29 
8.6 RECORDS 
29 
8.7 DATA CAPTURE 
30 
8.8 IMMEDIATE AND LONG-TERM USE OF THE DATA 
30 
9 DISSEMINATION OF RESULTS AND PUBLICATION POLICY 
31 
10 DURATION OF THE STUDY 
32 
11 ANTICIPATED POTENTIAL PROBLEMS 
34 
12 ETHICAL CONSIDERATIONS AND PARTICIPANT CONFIDENTIALITY 
35 
13 SPONSOR 
36 
14 FUNDING 
37 
15 AUDITS AND INSPECTIONS 
38 
 
 
 
 

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1 
STUDY OVERVIEW AND PROTOCOL SUMMARY 
1.1 
 Study title:  
Loop-mediated isothermal amplification test development, implementation and evaluation for 
yaws eradication 
1.2 
Acronym:  
LAMP4Yaws 
1.3 
 Study design:  
This study will take place in three sub-Saharan African countries known to be endemic for yaws. 
We aim to screen approximately 60,000 participants, of all ages. Screened participants will 
undergo primary clinical examination for yaws-like lesions and will be classed as either suspected 
yaws or negative for active yaws. A proportion of the yaws free participants (≈10%) and all those 
with suspected yaws (≈6000 people) will be invited to answer a questionnaire relating to 
household demographics and possible risk factors for yaws. If resources allow they will also be 
asked to provide dried blood spots for future serological analysis of yaws and other co-endemic 
neglected tropical diseases. Rapid point of care (POC) serological tests will then be used to 
confirm the clinical yaws diagnosis in the field, to allow for recruitment into the diagnostic 
evaluation study. Those with suspected yaws lesions will be tested with the SD Bioline syphilis test 
and those who test positive will be confirmed with the Chembio Dual Path Platform Syphilis 
Screen and Confirm POC test (herein DPP).  As some cases of yaws occur in individuals who have 
not yet sero-converted, in order to improve the Treponema pallidum pertenue (TPE) prevalence 
estimate accuracy, we will collect lesion samples from all individuals with a dual seropositive DPP 
(treponemal and non-treponemal) and a subsample of the participants (1/10) negative for the SD 
Bioline or the DPP (either treponemal antibodies, non-treponemal antibiodies or both). Two 
swabs, collected simultaneously from the same lesion, will be used for the diagnostic evaluation 
of the T. pallidum and Haemophilus ducreyi Loop-mediated isothermal amplification (TPHD LAMP) 
assay. Up to three further swabs may be collected from the same or additional lesions of the 
participant and from individuals with non-seroreactive tests but yaws-like lesions. We will also 
collect swabs of asymptomatic skin and of fomites of the house from some participants with and 
without yaws. These additional swabs will allow us to investigate other pathogenic causes of 
lesions using specific nucleic acid amplification tests and next generation sequencing. We will also 
be able to perform supplementary laboratory analysis on swab collection and DNA extraction 
methodologies, investigate the role fomites may have in the transmission of TPE and H. ducreyi 
and further understand the role of H. ducreyi as a cause of skin ulcers. No more than five lesion 

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swabs will be collected from any one individual. Further to this, we will enrol people from yaws 
endemic communities, healthcare workers and key stakeholders into sub-studies examining 
knowledge, attitudes and practices in relation to yaws, skin disease, and their diagnosis. 
1.4 
 Study Sites:  
Field studies and laboratory testing will take place in three countries in West Africa: Cameroon, 
Cote d’Ivoire and Ghana. These countries have been selected as they have currently the highest 
reported incidence of yaws in Africa. Within each country, national reporting data will be used 
to select three districts that are highly endemic for yaws, to allow for more rapid attainment of 
the required number of participants with active yaws for the LAMP diagnostic evaluation.   
1.5 
 Study participants and planned sample size: 
Patients of all ages in endemic districts will be recruited into the study. In total, we plan to 
perform an initial clinical screen on approximately 60,000 participants of whom we expect 6,000 
will present with yaws-like lesions. Of these we expect around 600 individuals will have a dual 
positive DPP test and will therefore be eligible for the diagnostic evaluation. 
1.6 
Planned Study period:  
36 months 
1.7 
 Research question/Aim:  
LAMP4Yaws is a multidisciplinary study aiming to determine whether the T. pallidum and H. 
ducreyi Loop-mediated isothermal amplification (TPHD LAMP) test is suitable, feasible and cost-
effective for use in national yaws eradication programmes and/or routine yaws surveillance. The 
main aim is supported by additional, linked social science and laboratory analysis that we hope 
will allow us to learn more about risk factors associated with disease, local transmission dynamics, 
other skin conditions and build in country capacity to run efficient yaws eradication campaigns. 
1.8 
Primary objectives:  
1) Perform active case searching and collect household and community level data to identify 
risk factors associated with yaws transmission and disease.  
2) Assess the accuracy, acceptability and feasibility of the TPHD-LAMP assay for T. pallidum and 
H. ducreyi diagnosis under programmatic conditions in yaws endemic areas in Ghana, Cote 
d’Ivoire and Cameroon to determine its suitability as a diagnostic in yaws eradication 
programmes.   
3) Testing the Treponema pallidum macrolide resistance (TPMR)-LAMP assay in a field setting. 
 

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4) Establish other infectious causes of non-yaws ulcers using specific PCR tests and next 
generation sequencing techniques, including novel devices such as employing the Minion for 
nanopore sequencing. 
5) Collect data on community, patient and healthcare worker knowledge, attitudes and 
practices in relation to yaws.  
6) Determine the acceptability and feasibility and cost effectiveness of the TPHD-LAMP assay.  
1.9 
Secondary objectives:  
1) Build within-country capacity for future national yaws eradication campaigns.  
2) Refine and evaluate the current TPHD-LAMP assay to enable accurate detection of 
macrolide resistance in a field setting. 
3) If macrolide resistance is detected we will use whole genome sequencing to explore the 
evolution and transmission routes of resistant strains.  
4) Develop an external quality assurance scheme for molecular diagnosis of yaws across 
endemic countries. 
5) Collect an archive of swabs from people and fomites, which can be used to evaluate 
alternative methodologies for swab collection, DNA extraction, molecular testing, and 
investigations of bacterial transmission.  
6) Collect an archive of dried blood spots from participants for future serological analysis to 
detect other neglected tropical diseases and/or previous exposure to Treponemes. 
7) Establish which other skin conditions are commonly co-endemic in Ghana, Cote d’Ivoire and 
Cameroon. 
1.10 Collaborating institutions:  
 
Institute 
Principal 
investigator  
Contact details  
Primary Role 
Albert-Ludwigs-
Universität Freiburg, 
Germany 
Nadine Borst  
Nadine.Borst@Hahn-Schickard.de 
Refinement of LAMP 
assay  
Centre Pasteur du 
Cameroun, 
Cameroon  
Tania Crucitti  
 
crucitti@pasteur-yaounde.org 
Field evaluation and 
capacity building  
Fundación Privada de 
Lucha contra el sida 
(FLS), Spain  
Oriol Mitjà 
oriolmitja@hotmail.com 
Field study 
coordination  

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Georg-August-
Universität 
Goettingen Stiftung 
Oeffentlichen Rechts, 
Germany  
Sascha Knauf 
sascha.knauf@uni-goettingen.de 
External quality 
assurance scheme  
Institut Pasteur de 
Cote D’Ivoire, Cote 
d'Ivoire 
Solange Ngazoa  
 
 
ngazoa_solange@yahoo.fr 
Field Evaluation 
London School of 
Hygiene and Tropical 
Medicine  
Michael Marks 
Michael.marks@lshtm.ac.uk 
Lead institute: project 
management 
Field Evaluation 
Health economic and 
qualitative evaluation 
MAST Diagnostics  
Mohammed 
Bakheit 
bakheit@mast-diagnostica.de 
Provision and 
refinement of LAMP 
assay  
University of Ghana, 
Noguchi Memorial 
Institute for Medical 
Research (NMIMR), 
Ghana  
Kwasi Kennedy  
Addo  
 
kaddo@noguchi.ug.edu.gh 
Field Evaluation  
 
1.11 Funding:  
This study has been funded by an EDCTP grant (RIA2018D-2495)  
1.12 Role of study funder:  
The funder has not played any role in the study design.  
 
 
 

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2 
BACKGROUND AND RATIONALE 
2.1 
Background 
2.1.1 Yaws  
Yaws is a chronic infectious disease of the skin, bone and cartilage caused by the spirochete bacterium 
Treponema pallidum subsp. pertenue (TPE)1. TPE is closely related to the syphilis-causing bacterium 
Treponema pallidum subsp. pallidum 2 but unlike syphilis, yaws primarily affects children and is 
generally not sexually transmitted 3–5. It is one of the World Health Organization (WHO) neglected 
tropical diseases (NTDs) and under the WHO’s new roadmap is targeted for eradication by 2030 6. 
Yaws presents initially as a bacteria-rich papilloma, which if left untreated, can ulcerate. In some cases, 
the disease progresses to secondary yaws and it is at this stage that it can cause further skin 
manifestations as well as inflammation of bones and joints. In rare cases tertiary yaws develops; this is 
often characterised by destructive and disfiguring lesions 3. Untreated patients are susceptible to 
developing a latent infection, which can relapse, commonly within five years of the initial infection. 
During the latent stage of the disease there are no clinical manifestations 3.  It is thought that for every 
clinical case of yaws there are six latent cases 7, which has serious implications for eradication 
programs.  
2.1.2 Historical and contemporary eradication strategies  
Yaws was once reported to be endemic in more than 80 countries, but global prevalence was reduced 
by over by 95% as the result of a 1950’s to 60’s WHO/UNICEF eradication campaign involving mass 
drug administration (MDA) of single dose parenteral penicillin4,8 . Most likely, a lack of resources, 
reduced political will and premature discontinuation of efforts led to a resurgence of yaws and the 
disease 9,10. A single dose of oral azithromycin has since been shown to be highly effective at treating 
yaws11–14.  This resulted in a revised WHO yaws eradication strategy, which consists of azithromycin 
MDA at the district or sub-district level, followed by active-case finding and treatment of remaining 
cases 15. Human yaws is currently known to be endemic in 14 countries, with the majority of cases 
reported in West Africa, South East Asia and the Pacific 16,17.  Prevalence estimates are likely to be an 
underestimate due to a lack of systematic mapping data and under-reporting of the disease 1,17.  To 
meet eradication targets it is essential that all endemic areas are accurately mapped to allow for 
targeted interventions. However, this is dependent on the availability of an accurate diagnostic 
method, that can be deployed in all suspected yaws-endemic districts in a standardised manner. 
 

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2.1.3 Diagnostics   
Accurately diagnosing yaws will be key to the success of the global eradication campaign.  
Traditionally, serological tests have been the mainstay of yaws diagnosis, consisting of detection of 
treponemal and non-treponemal antibodies. Anti-treponemal antibodies are long-lived so are useful 
for indicating exposure but cannot distinguish between current and past infections, nor can they 
distinguish between infection with any of the T. pallidum subsp. pallidum (syphilis), pertenue (yaws), 
endemicum (bejel) or T. carateum (pinta) 1. Non-treponemal tests are used to predict a current 
infection, but lack specificity 1. When used in conjunction non-treponemal and treponemal tests are a 
better indicator of a current treponemal infection, but also fail to distinguish between different species 
of Treponemes, and have been shown to miss early cases of yaws1,  which could be important for 
identifying resurgence of the disease after MDA.   
 
Clinical diagnosis based on lesions alone is unreliable, often due to the presence of co-endemic ulcer-
causing diseases. Recent studies using molecular tools have demonstrated only one third of yaws-like 
ulcers are positive for T. pallidum18. Studies from Papua New Guinea 18  and Solomon Islands 19 have 
shown H. ducreyi, the causative agent of chancroid, to be responsible for a further third of these 
clinically-indistinguishable lesions. Often neither T. pallidum nor H. ducreyi are detected in these 
lesions 18,19 and it is possible another infectious agent is responsible. Therefore, we will conduct further 
investigations to determine if other pathogens may be routinely detected that could influence the 
design of yaws eradication programs.  
 
The urgent need for novel diagnostics is perhaps most essential in light of the emergence of 
azithromycin-resistant TPE. These strains have been shown to emerge independently, and have led to 
localized transmission 20. Highly sensitive and specific nucleic acid amplification tests (NAATs) which 
include tests for azithromycin resistance are available 18,21.  However, these tests are often inaccessible 
and have limited use in the field setting. In light of this emerging macrolide resistance it is clear that 
more robust laboratory-based surveillance is required. 
2.1.4  Significance and Scientific Rationale 
Improved understanding of yaws transmission and novel yaws diagnostics are urgently needed to 
support the WHO eradication campaign. Current WHO strategy is based on clinical case searches 
combined with serological point of care (POC) assays 15. An improved understanding of yaws 
epidemiology, informed by diagnostics, would help refine and improve case search approaches and 
contact tracing as part of eradication efforts. Given the limitations of existing diagnostics, improved 
diagnostic tests are needed. These tests should be designed to fulfil the WHO REASSURED criteria, i.e. 
allow for real-time connectivity, easy to collect specimens, be affordable, sensitive, specific, user-

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friendly, rapid, equipment-free and deliverable to end users 22. The TPHD LAMP test (Mast 
Diagnostica GmbH, Germany) is a POC test that is practical to use in resource-limited areas. LAMP 
does not require expensive equipment, such as thermocyclers required for traditional NAATs, and the 
reagents have been designed to be stored lyophilised for field conditions. The TPHD LAMP test can 
rapidly identify an infection with T. pallidum or H. ducreyi and has been shown to have high sensitivity 
and specificity in the laboratory 23.  It can also be adapted to include azithromycin resistance 
detection 24. This test may, therefore, provide a suitable solution to the problems faced by current 
yaws diagnostics and if accurate and feasible to use in the field, has the potential to be used by yaws 
eradication programs, allowing for systematic real-time detection of T. pallidum and H. ducreyi, as 
well as azithromycin-resistant strains.  
 
 
 

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3 
RESEARCH AIMS AND OBJECTIVES 
LAMP4Yaws is a multidisciplinary study aiming to determine whether the TPHD LAMP test is a 
suitable, feasible and cost-effective diagnostic tool for yaws eradication campaigns and 
surveillance. The main aim is supported by additional, linked social science and laboratory analysis 
that will allow us to learn more about risk factors associated with disease, local transmission 
dynamics, other endemic skin conditions and build in country capacity to run efficient yaws 
eradication campaigns. This project consists of four linked sub-studies: 
1. Multi-country case searches and epidemiological analysis of risk factors for yaws and other 
causes of yaws-like skin lesions (SECTION 4) 
2. Performance evaluation of a TPHD LAMP assay compared to qPCR for the diagnosis of 
yaws-like lesions and optimisation of sample collection and processing (SECTION 5) 
3. Use of molecular tools and next-generation sequencing to understand the aetiology of 
yaws-like lesions, and optimise sequencing tools for POC use (SECTION 6) 
4. A health economic and social science evaluation of healthcare worker and community 
knowledge-attitudes and practices in relation to yaws and related diseases, and their 
diagnosis (SECTION 7)  
 
By performing these four linked studies we hope to achieve a better understanding of the 
epidemiology of yaws in our chosen districts including identifying key risk factors and transmission 
dynamics, and determining the main aetiologies of lesions. The evaluation of the TPHD LAMP assay 
and the social science analyses will result in data that allow us to conclude if the current assay is a 
useful tool for routine diagnostics and use in yaws eradication programmes.  An outline of each 
specific sub-study is given below.  
 
3.1 
Overall Study Outline and recruitment 
 
 
 
  
 
 
 
 
 

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3.2 
Study Sites  
In each of the three countries, national reporting data will be used to select at least three districts that 
are highly endemic for yaws, facilitating rapid fulfilment of the sample size required number of yaws 
cases for the diagnostic accuracy study. The number of villages/communities selected will depend on 
prevalence of the disease. This exercise will be conducted with the yaws programme national 
coordinators in the three endemic countries.  
3.3 
 Consent and information for participants 
In each site we will conduct active case finding activities in collaboration with Ministry of Health 
departments and National Yaws Programmes. For sub-studies 1-3, all residents in selected 
communities will be eligible to participate in the study. Prior to any study procedures, written 
informed consent will be obtained from participants or, from parents, guardians, or legal 
Figure 1: Flow chart of participant recruitment across all three-study countries. Initially we will screen 60,000 people or 
whom we expect 6000 will present with yaws-like lesions. These 6000 participants, along with 6000 without clinical 
evidence of yaws will be invited to take part in sub-study 1, the epidemiological analysis. All participants with clinically 
suspected yaws will be tested with a serology POC test, with positive results being confirmed by second POC test. Those 
with clinical and serological evidence of yaws will be invited to participate in sub-studies 2 and 3. Additionally, some 
participants from studies 1-3 along with healthcare workers and key stakeholders will be invited to enrol in study 4.  
* A DPP test is considered positive only if sero-reactive for both treponemal and non-treponemal antibodies.  

Page 15 of 41 
representatives if the participant is under the age of consent. Following national recommendations 
informed consent will be conducted in English or French, with the assistance of a community 
representative translating if necessary. If a person is illiterate the study information and consent 
procedure will be clearly explained to them by a member of the study team or a local translator. If a 
translator is used they will be required to witness the informed consent procedure. Children and 
minors over ten years of age and under the age of consent, will also be required to give verbal assent 
for enrolment. If consent is not given for future research, it will be indicated under the appropriate 
section in the consent form. If any participants wishes to withdraw from the study at any point post-
enrolment this will be indicated to study coordinator and data managers for complete elimination of 
that participants information. Consent forms, approved by all relevant ethical review boards, will be 
available in English and French, as appropriate. Individuals at each site will be consented for 
participation into each sub-study based on the relevant inclusion/exclusion criteria (see sections: 4.2; 
5.2; 6.2; 7.2).  
 
 

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4 
SUB-STUDY 1: MULTI-COUNTRY CASE SEARCHES AND 
EPIDEMIOLOGICAL ANALYSIS OF RISK FACTORS FOR YAWS  
4.1 
Objectives  
1) Determine the prevalence of active and latent yaws in each setting using combined clinical, 
serological and molecular tools 
2) Identify risk factors associated with active and latent yaws 
3) Collect a representative dried blood spot sample library for future serological testing 
 
We will undertake integrated case searches for individuals with yaws-like lesions. We will collect 
baseline clinical, demographic and risk factor information from participants. Individuals with clinical 
evidence of yaws will be tested with point-of-care serological tests (SD Bioline and Chembio DPP) in 
line with standard WHO recommendations, and will have lesion samples collected which will form the 
sample set required for sub-studies 2 & 3. We may record the presence of other skin conditions seen.  
4.2 
Inclusion and exclusion criteria 
Inclusion Criteria 
• 
Resident in selected community 
• 
Willingness to provide informed consent to participate in the study 
 
Exclusion Criteria 
• 
Refusal of village chief (for village inclusion), or refusal of individual and/or guardian (for 
individual inclusion)  
• 
Not willing or able to give informed consent for the study 
• 
Permanent disability that prevents or impedes study participation and/or comprehension 
4.3 
Sample Size Considerations 
We aim to screen approximately 60,000 individuals, 20,000 in each country. Of these we estimate 
6000 individuals will present with yaws like lesions and 600 will have active yaws (i.e. present with 
yaw-like lesions and confirmed serologically). The remaining participants will be classed as either 
negative for yaws due to the absence of clinical signs or negative serology or suspected latent yaws if 
sero-reactive.  We will aim to enrol all 6000 participants with yaws like lesions and a further 6000 
without clinical yaws into this sub study. This will give us  >80% power to detect risk factors associated 
with yaws with an odds ratio of 3. If resources allow, we will collect dried blood spots of half of the 
people enrolled into sub-study 1 giving us roughly 6000 dried blood spots samples. This would allow us 
to detect 5% seroprevalence with 0.5% precision, and a seroprevalence of 50% with a precision of 
1.3%, which is deemed more than sufficient for a convenience sample 25.   

Page 17 of 41 
4.4 
Data Collection 
In conjunction with Ministry of Health and local collaborators we will undertake case searches for 
individuals with yaws and yaws-like lesions in selected communities. All study staff involved in the 
field surveys will be extensively trained in the clinical, diagnostic, and public health aspects of yaws. 
The initial screening will involve a quick examination to determine if people have yaws lesions 
present. All people with lesions and around 6000 people without any clinical evidence will then be 
invited into sub study 1. Those enrolled will provide demographic data and risk factor data (Appendix 
A and B). Those with yaws will undergo a more thorough skin examination to look for evidence of 
clinically active yaws and this will be recorded into a case report form (CRF) (see Appendix B) and may 
note the presence of other skin conditions. Yaws-like lesions will be assessed using WHO 
recommended procedure 26 (Appendix C), with a simplified system of WHO yaws pictorial guide27 
used to define each yaws-like lesion.  These will be marked on a diagram and lesions will be 
photographed for quality control purposes (Appendix B).  Each participant will be assigned a unique 
study identification number and all data will be recorded and analysed with this unique identification 
number. Study data will be electronically entered into a Good Clinical Practice (GCP)-compliant, 
offline, trial-specific mobile application [Research Electronic Data Capture (REDCap), Nashville, TN, 
USA] or Open Data Kit (ODK).   
 
4.5 
Sample collection and storage 
As per WHO recommendations, people with clinically suspected yaws lesions will be screened using a 
T. pallidum POC test, and those that test positive will be confirmed with the DPP Screen and Confirm 
POC test detecting both treponemal and non-treponemal antibodies. In individuals with a positive DPP 
assay, we will collect lesion swabs for molecular testing according to WHO procedure 26 (Appendix C).  
As some cases of yaws occur in individuals who have not yet sero-converted we will also collect swabs 
from 1/10 people with suspected yaws but non-reactive serological tests. Two swab samples will be 
simultaneously obtained from each suspected yaws lesion (i.e. rolling both swabs over the lesion at the 
same time); one to conduct the TPHD-LAMP assay at the district level hospital, the other for PCR at the 
national reference laboratory for the detection of T. pallidum and H. ducreyi and monitoring resistance 
to antibiotics. A maximum of three other swabs may be taken, from the same or different lesions from 
people with or without yaws, and further swabs may be collected from asymptomatic skin or from 
fomites in the house, for additional infection detection analysis.  Swab samples will be collected dry, or 
in suitable transport medium, and all tubes will be labelled with the participant’s unique identification 
numbers and collection date. Half of those enrolled into sub-study 1 will be asked to provide a dried 
blood spot sample. This will give us an archive of around 6000 dried blood spots with which we can 
perform serological testing in the future. For this we will collect capillary finger blood onto a filter 

Page 18 of 41 
paper designed specifically for blood collection following published procedures (see Appendix D). 
These will be air dried for at least 12 hours prior to being stored with desiccant sachets at ambient 
temperature.  
4.6 
Laboratory testing 
The dried blood spots will be used to conduct multiplex serological assays on the Luminex platform to 
look for antibodies to Treponemes and other infectious diseases. By measuring levels of anti-
Treponemal antibodies in children we will be able to estimate the number of latent yaws cases within 
the community.  We will choose a panel of infectious diseases that we know, or suspect, to be endemic 
in our populations and measure antibody responses specific to these pathogens in order to indicate 
exposure and seroprevalence at the individual, community and district level 28.  
 
4.7 
Follow-up  
All samples for the primary analysis are collected at the first visit and therefore we do not anticipate 
loss to follow-up or non-compliance to be an issue. All individuals with a clinically suspected yaws 
lesion or ulcer, regardless of screening-assay result, will be offered treatment with azithromycin or 
referred to the relevant services for treatment in line with national guidelines. If other skin conditions 
are seen, participants will also be referred to the relevant services. As per WHO guidance, we will 
review patients with suspected yaws lesions at week four, when unhealed lesions will be swabbed and 
treatment with benzathine benzylpenicillin offered. If azithromycin resistance is detected through the 
laboratory analysis at any point during the study, we will work with yaws eradication programmes to 
implement interventions for study participants and communities. 
 
4.8 
Data Analysis 
Quantitative data analysis will be performed with R, STATA and other software packages.  
Initially we will perform univariable logistic regression analyses on each potential risk factor. If any 
factors are found to be associated with yaws we will include these in multivariate logistic regression 
models with age and gender included a priori in order to calculate odds ratio for any factors associated 
with active yaws. We will also calculate the 95% confidence interval for each odds ratio.  The 
multivariate analysis will allow us to determine the strength of associations between active yaws and 
each factor, or combinations of factors.  All statistical analysis will be performed to the 95% 
significance level. For the Luminex assay, we will use appropriate statistical tools and published 
literature to determine cut-off values for each antibody and calculate seroprevalence with 95% 
confidence intervals.  

Page 19 of 41 
5 
SUB-STUDY 2: PERFORMANCE EVALUATION OF A TPHD 
LAMP ASSAY COMPARED TO QPCR FOR THE DIAGNOSIS OF 
YAWS-LIKE LESIONS 
5.1 
Objectives 
 
1) Evaluation of a TPHD LAMP assay performed in local hospitals compared to qPCR/PCR results 
obtained at the national reference laboratory in each country.  
2) Testing the Treponema pallidum macrolide resistance (TPMR)-LAMP assay in a field setting 
3) Development of an external quality assurance scheme for molecular diagnosis of yaws across 
endemic countries 
4) Evaluating alternative methodologies for swab collection, DNA extraction and molecular 
testing  
 
5.2 
Inclusion and exclusion criteria 
This study will consist exclusively of a subset of the individuals enrolled into sub-study 1 (case-finding). 
Participants will be consented for inclusion into sub-studies 2&3 at the same time as consent and 
enrolment into sub-study 1.  
 
5.3 
Sample size 
We will collect samples for the TPHD LAMP evaluation from the 600 individuals with clinical and 
serological evidence of yaws, and an additional 10% of participants with clinical yaws but negative 
serology (sub-study 1). Based on the assumption that 35% of seropositive cases will also be positive by 
qPCR, this will result in approximately 210 qPCR positive T. pallidum cases and a similar number of H. 
ducreyi cases 18,19,29. This sample size will allow us to confirm that the TPHD-LAMP has both a sensitivity 
and specificity of at least 85% compared to the reference qPCR assays for both targets. This will also 
provide us with about 200 samples with unknown lesion aetiology to evaluate using next-generation 
sequencing as part of sub-Study 3. 
 
5.4 
Sample collection and Storage 
Samples will consist of lesion samples collected in sub-study 1 to be tested using both the TPHD LAMP 
assay (performed at the district/regional level) and qPCR/PCR (performed at each country’s National 
Reference Laboratory). We will also test all Treponema pallidum positive samples for azithromycin 
resistance genes using the multiplex TPMR-LAMP assay and the gold standard resistance detection 
techniques performed at reference laboratories. Samples from the study will also be used to 

Page 20 of 41 
undertake objectives 3-5; including development of an external quality assurance (EQA) scheme for 
yaws molecular diagnostics and evaluating optimal sample collection and processing approaches. 
These samples will also be used for sub-study 3. 
 
5.5 
Laboratory Procedures        
For the diagnostic evaluation, samples will be transferred to both district and reference laboratories 
for testing under routine programmatic conditions. For reference PCR testing at the reference 
laboratory, DNA will be extracted from lesion samples. In the first instance, we will run PCRs targeting 
T. pallidum and H. ducreyi. A negative (no-template) control, positive controls for T. pallidum and H. 
ducreyi DNA and an extraction control will be included in each PCR run. In samples where T. pallidum is 
detected further PCRs targeting macrolide resistant point mutations A2058G and A2059G in the 23s 
RNA genes and to distinguish between different species of Treponemes will be run.  
 
TPHD-LAMP and TPMR-LAMP will be performed at district laboratory level. DNA will be extracted 
using a commercial extraction kit deemed suitable for use in the field, the same kit will be used in all 
district laboratories.  The TPHD-LAMP assay will be run in accordance with the manufacture-provided 
instructions. SOPs will be written by members of the LAMP4yaws consortium and provided to each of 
the laboratories. Incubation will be performed using the field compatible Tubescanner platform, 
which reads fluorescence signals for up to 4 different dyes. Final test results in terms of “positive”, 
“negative” or “invalid/retest” will be directly displayed on the screen using test methods developed 
by Mast (Mast Diagnostica GmbH, Germany). 
 
All results, including raw and cleaned data from both reference and local laboratories will be 
electronically uploaded and deposited in the secure, encrypted central study database at LSHTM. 
Staff at district laboratories performing the TPHD-LAMP will be blinded to results from the national 
reference laboratories, and staff performing the PCR/qPCR at the national reference laboratories will 
be blinded to the TPHD LAMP results. 
 
A subset of the additionally collected swabs taken from suspected yaws patients, the fomite swabs and 
some aliquots of DNA extracts will be shipped to the laboratories of the LAMP4YAWS consortium (see 
1.11) for use in sub-study 3 and to run additional investigations into optimal sample collection and 
processing methods. These will include testing fomite swabs for the presence of T. pallidum or H. 
ducreyi and preforming additional viability PCRs to determine if living bacteria can be detected on 
fomites in order to highlight if fomites are a key route of transmission. We will also use the TPHD-
LAMP assay and reference qPCR to investigate if diagnostic accuracy can be enhanced by taking swabs 

Page 21 of 41 
from multiple lesions of an infected patient. A selection of additional swabs and/or DNA extracts may 
be sent to various collaborating laboratories to enable the development of an external quality 
assurance (EQA) scheme.  
 
5.6 
Quality assurance for laboratory procedures at study sites 
The TPHD LAMP will be used at local hospital facilities close to the enrolled communities. Extensive 
training for this will take place prior to participant recruitment and we will conduct both internal and 
EQA activities throughout the study. Some aliquots of extracted DNA or additional lesion swabs will 
be exported to the European consortium laboratories and rechecked using both the TPHD-LAMP and 
gold standard (qPCR/PCR) for each pathogen. We will purposively select a higher proportion of 
samples for External Quality Assurance in the first six months of the laboratory testing to allow early 
detection of significant implementation problems. In case of significant differences between the 
reported results and the international reference laboratory results, an on-site evaluation will be 
initiated to identify the cause and solve the problem. All participating laboratories will work according 
to the GCLP guidelines, including internal quality assurance activities.  
 
5.7 
Data Analysis  
For the evaluation of the TPHD-LAMP assay compared to qPCR for the diagnosis of yaws, we will 
calculate the sensitivity and specificity of the T. pallidum and H. ducreyi targets individually compared 
to the respective reference qPCR assays. We may perform additional sub-analyses to assess if the 
performance of the TPHD-LAMP assay is related to bacterial load as measured by qPCR. The positive 
predictive value and negative predictive value will also be determined against the qPCR results.  
 
 
 

Page 22 of 41 
6 
SUB-STUDY 3: USE OF MOLECULAR TOOLS AND NEXT-
GENERATION SEQUENCING TO UNDERSTAND THE 
AETIOLOGY OF YAWS-LIKE LESIONS, AND OPTIMISE 
SEQUENCING TOOLS FOR POINT OF CARE USE.  
6.1 
Objectives 
 
1) Use nucleic acid amplification tests to investigate the presence of Mycobacterium ulcerans, 
Leishmania or Corynebacterium diphtheria DNA in TPE and H. ducreyi negative swabs to 
determine if these pathogens are a significant cause of yaws-like lesions 
2) Perform metataxonomic microbial analysis to determine other bacterial causes of yaws-like 
lesions when targeted pathogens are not detected through NAATs 
3) Explore the evolution and transmission routes of resistant strains using whole genome 
phylogenetic analyses if macrolide resistant TPE are isolated  
4) Optimise the Oxford Nanopore Minion sequencing for detection of TPE, H. ducreyi, and 
macrolide resistance strains of both pathogens, in the field 
5) Perform investigations into the role of H. ducreyi as a significant cause of yaws-like lesions by 
measuring the presence of H. ducreyi on the skin of asymptomatic patients of all ages, using 
culture and molecular methods and genotyping any H. ducreyi detected from symptomatic or 
asymptomatic cases.  
6) Detail the baseline resistance profiles of H. ducreyi using drug susceptibility assays 
6.2 
Inclusion and exclusion criteria 
As with sub-study 2, this study will consist exclusively of a subset of the individuals enrolled into sub-
study 1 (Case-finding). Participants will be consented for inclusion into sub-studies 2&3 at the same 
time as consent and enrolment into sub-study 1.  
6.3 
Sample size 
There is no formal sample size for this sub-study.  
6.4 
Sample collection and storage  
As with sub-study 2, this study will utilise the samples collected from participants in sub-study 1. These 
will be stored in country and a sub-set will be sent to the laboratories of the consortium in Europe.  
6.5 
Laboratory Procedures 
Tests will be performed in the laboratories having the assays, platform and equipment in 
place or under development for the purpose. For all NAATs and sequencing techniques, DNA will 
be extracted from lesion swabs using a suitable commercial kit specially designed or adapted for swab 

Page 23 of 41 
extraction. Targeted tests for Mycobacterium ulcerans, Leishmania or Corynebacterium diphtheria will 
be performed using qPCR. To detect H. ducreyi from fomite swabs or asymptomatic skin, swabs will be 
inoculated on enriched culture media before being tested using qPCR. Genotyping using whole 
genome sequencing (WGS) or multilocus sequence typing (MLST) will be performed on the H. ducreyi 
cultures obtained. Genotyping directly from swab DNA extracts, without the need for enrichment 
culturing, will also be tested. These activities will be led by the Centre Pasteur du Cameroun. 
 
For Illumina and Minion WGS sequencing, qPCR will be used to select samples with a higher bacterial 
presence.  DNA fragmentation and library preparation will be performed using a suitable commercial 
kit for the sequencing platform. We will then sequence using manufacture guidelines or methodology 
optimised within house.  A range of open source software will be used to highlight low quality reads, 
clean data and complete assembly of genomes using established reference strains.  
 
For metataxonomic sequencing, the variable region 4 (V4) of the 16S ribosomal RNA gene will be 
targeted. A blank control (microbial DNA-free water) and a mock control sample will be included into 
the analysis. Amplicon triplets will be pooled and purified using AMPure XP beads (Beckman Coulter). 
Sequencing will be done using Illumina technology (MiSeq 2x250 bp paired-end sequencing), following 
published guidelines 30.   
 
6.6 
Data Analysis  
Whole genome sequencing: We will compare any sequences T. pallidum or H. ducreyi to reference 
strains to highlight single nucleotide polymorphisms, or any other structural differences. We will create 
phylogenetic trees to compare relatedness. We will use the same technology to explore how antibiotic 
resistance may have spread around the participating communities and if it is caused by the two 
common resistance mutations or novel mutations. Data management will fall under the responsibility 
of the lead investigator of the laboratory conducting each test and will be shared with other members 
of the consortium if requested.  
 
 
 
 
 
 
 

Page 24 of 41 
7 
SUB-STUDY 4: A HEALTH ECONOMIC AND SOCIAL SCIENCE 
EVALUATION OF HEALTHCARE WORKER AND COMMUNITY 
KNOWLEDGE-ATTITUDES AND PRACTICES IN RELATION TO 
YAWS AND RELATED DISEASES, AND THEIR DIAGNOSIS. 
7.1 
Objectives 
 
1) Collect data on community, patient and healthcare worker knowledge, attitudes and 
practices in relation to yaws and identify specific needs among the community including 
challenges in accessing prevention, care, and treatment services  
2) Determine the acceptability and feasibility of TPHD-LAMP assay routine use 
3) Determine cost-effectiveness of the TPHD-LAMP assay 
 
7.2 
Inclusion and exclusion criteria for objectives 1 and 2   
Inclusion criteria:  
• 
Willingness to provide informed consent and assent to participation in the study 
• 
Male or female and over the age of consent  
• 
Male or female >12 and under the age of consent if accompanied by a parent or guardian 
• 
Community members should be permanent residents in the selected communities 
 
Exclusion  
• 
Not able to give informed consent  
• 
Under the age of consent with no parent/guardian available  to accompany 
• 
Not a permanent resident in the community  
 
7.3 
Data collection  
Attitudes and practices in relation to yaws and identifying community needs: In each community, we 
will invite a sample of key groups including community health workers, opinion leaders and parents 
and guardians of children to participate in focus group discussions and semi-structured interviews 
about yaws and related skin diseases. As this is a qualitative evaluation, we have not performed a 
formal sample size calculation but will aim to achieve data saturation. For the community, members 
will be selected with an equal portion of males and females and a range of ages to ensure a 
representative sample. All focus group discussion and interviews will be conducted in English or French 
with local translators available if another language is preferred. All interviews and focus groups will be 
recorded, transcribed and translated if necessary. After being transcribed, recordings will be deleted.  
Common themes identified from the focus group discussions will be used to develop a questionnaire 
relating to their experiences and perceptions of: the cause of the disease and its transmission; its 

Page 25 of 41 
diagnosis; healthcare seeking attitudes and factors (barriers/facilitators) influencing this.  This 
questionnaire will be circulated to other communities and key stakeholders enabling us to obtain 
quantitative data to validate the hypotheses generated from the focus group discussions.  
 
Acceptability and feasibility:  To assess the acceptability and feasibility of the TPHD-LAMP assay to 
healthcare workers and laboratory staff, we will conduct semi-structured interviews and/or use 
standardised questionnaires to gather their overall impressions of performing traditional molecular 
tests and the TPHD- LAMP assay.  Again focus group discussion and interviews will be conducted in 
English or French with local translators available if another language is preferred. All interviews and 
focus groups will be recorded, transcribed and translated if necessary. After being transcribed, 
recordings will be deleted. For the feasibility aspect of the study, we will collect a range of information 
such as: determining the amount of training required to perform the test, for both field workers and 
laboratory staff, practicality of running the LAMP test in terms of laboratory space, reagent storage, 
electricity, sample transport and storage and other factors such as the time taken between sample 
collection and result and how easy it is to get the result back to the patient.  
 
With patients and other key stakeholders, the test acceptability, will be explored via focus group 
discussions and in-depth interviews, with a focus on local knowledge and perceptions: of yaws and 
other skin conditions; diagnostics and available treatments, perceptions of healthcare workers’ 
decisions in relation to yaws; and barriers to acceptability of the new test.  
 
Cost-effectiveness: For the health-economic analysis we will use an “ingredients approach to costing”, 
gathering unit costs for each category of costing (training, personnel, capital costs, consumables, 
results storage and reporting, EQA, etc.). The costs of the LAMP assay will be compared with running 
the gold standard qPCR at national reference laboratories. The measure of effectiveness will be the 
number of additional yaws cases diagnosed and the number of additional yaws cases appropriately 
managed. If the macrolide resistance test can be successfully incorporated, then these costs will also 
be included, including the cost-savings of avoiding inappropriate antibiotic treatment.   
 
7.4 
Data Analysis  
 
1) This will involve a mixed methods analysis. We will perform thematic analyses to identify 
common themes arising from focus group discussions and in-depth interviews. The 
quantitative questionnaires will be analysed using standard statistical analyses, including Chi 

Page 26 of 41 
squared tests and logistic regression to analyse relationships between reported knowledge, 
attitudes and practices, and demographic variables such as gender, age and location.  
2) For the feasibility and acceptability analysis we will also perform thematic analysis to generate 
themes of acceptance of the TPHD-LAMP assay amongst healthcare workers, laboratory staff 
and patients.   
3) Cost-effectiveness: This analysis will involve using a decision tree model to weigh up the costs 
attributed to either test. The model will include the sensitivity and specificity of both tests, 
costs of person time required to perform the test and other variables that will affect if the 
TPHD-LAMP assay is suitable for routine diagnostics and surveillance.  
 
 
 
 
 
 

Page 27 of 41 
8 
SAFETY CONSIDERATION AND REPORTING 
8.1 
 Potential risks 
 
Risks associated with participation in this study are minimal. Swabs are non-invasive and are routinely 
used programmatically. Finger prick blood samples may cause slight discomfort and bruising; 
however, these too are routinely used programmatically. Loss of privacy is a potential risk associated 
with participation in any research project. In this study, it is necessary to confirm cure at four weeks 
post-treatment, as per WHO guidelines, so it is essential personal details are recorded at the time of 
recruitment. Potential for loss of confidentiality will be minimized by use of coded study numbers on 
data collection forms and laboratory samples, and all data will be stored in locked filing cabinets and 
on encrypted, password-protected computer files. As with the use of any antibiotic, there is a risk that 
resistance may evolve in either TPE, H. ducreyi or in a host’s commensal microbiota. There have also 
been reports of microbial gut community disturbances with azithromycin administration which could 
have long term implications 31,32. However, the data suggest that azithromycin antimicrobial 
resistance is not long-lived 33 .  
8.1.1 Mitigation for COVID-19 
COVID-19 transmission is occurring globally. As participation in this study entails interactions with staff 
from the Ministry of Health and the research team as well as interactions with other members of the 
community there might  be an increased risk of COVID-19 transmission to staff and/or study 
participants. In order to mitigate these risks we will at all times  follow guidance published by WHO on 
restarting of NTD related activities in the context of COVID-19 
(https://www.who.int/publications/i/item/WHO-2019-nCoV-neglected-tropical-diseases-2020).   
These considerations include but are not limited to:   
1) We will appoint a COVID-19 focal person in each study site to ensure appropriate SOPS are available 
and followed, that PPE is available, and liaise with other relevant stakeholders such as Ministries of 
Health 
2) We will provide dedicated training to all staff on COVID-19 transmission, infection control and use of 
PPE.  
3) Risk-benefit assessments will be carried out for each individual study activity using the framework 
laid out by WHO. Research activities will only proceed where the benefit is felt to outweigh the risk.  
4) Before each activity participants and staff will be screened symptoms suggestive of COVID-19 and 
study staff will use PPE and other appropriate infection prevention and control measures at all times. 
5) The majority of study activities including screening, enrolment, sample collection and treatment will 
be conducted outside (as is routine for community NTD screening activities) and will be conducted 

Page 28 of 41 
using physical distancing. In-depth interviews and focus groups will be conducted using appropriate 
physical distancing between study staff and participants.  
 
These steps will be reviewed in each country in line with national guidance and any further guidance 
provided by WHO. 
8.2 
 Known potential benefits 
All individuals with a skin ulcer will be referred to relevant health services for treatment with 
azithromycin, regardless of screening-assay result, as this is an effective treatment for both T. 
pallidum and H. ducreyi. Due to the activity of azithromycin against a broad range of bacterial 
pathogens there is likely to be collateral benefits to taking the prescribed dose of azithromycin for 
yaws, as has been shown previously 34. By conducting this study we hope to improve in-country 
capacity for running field and laboratory studies. If the LAMP tests are shown to be an effective tool 
each country will have the capacity to use the assay in future studies and eradication campaigns.  
 
8.3 
 Adverse events 
As this is an observational study the likelihood of encountering adverse events is low.  
8.3.1 Definitions 
I. Adverse event (AE): Any untoward medical occurrence in a patient or study participant, 
which occurs between enrolment and follow up (four weeks post enrolment). 
II. Serious adverse event (SAE): Any untoward medical occurrence that: 
• results in death; 
• is life-threatening; 
• requires inpatient hospitalization or prolongation of existing hospitalization; 
• results in persistent or significant disability/incapacity; 
• consists of a congenital anomaly or birth defect. 
Serious adverse events are only likely to occur in participants with a severe allergy to penicillin; anyone 
requiring penicillin treatment will have their history checked. SAEs are rarely associated with 
azithromycin 13,35. 
8.3.2  Reporting Procedures 
Any questions concerning adverse event reporting will be directed to the Chief Investigator in the first 
instance. An SAE form will be completed and submitted to the Study Coordination Centre with as much 
detail as available at that time. If awaiting further details, a follow up SAE report should be submitted 
promptly upon receipt of any outstanding information.   

Page 29 of 41 
8.4 
 Follow-up 
No formal study follow-up is planned for participants after the completion of study activities, 
however, as mentioned in section 5.6, participants will be re-visited four weeks after initial treatment 
and offered benzathine benzylpenicillin if treatment failure is thought to have occurred.  
 
8.5 
Data quality assurance and monitoring 
Study staff will be trained on the study protocol prior to the start of study activities at each site. For 
laboratory testing, this will include centralised and localised training of the TPHD-LAMP technique for 
laboratory staff working in the local laboratories. Staff at reference laboratories will be trained. Data 
collection forms will be reviewed by the Study Coordinators and Principal Investigators (PIs) for 
completeness and accuracy and tested prior to the pilot study. As well as being trained about how to 
diagnose and assess yaws, field staff and healthcare workers will also be taught about the 
epidemiology of yaws, treatment options and the importance of good hygiene for combating the 
disease. All field workers will be trained to perform and interpret serological POCTs, collect lesion 
swabs and blood spots and other relevant components of the protocol. Refresher training of field and 
laboratory staff followed by a 2-week pilot period will be conducted before beginning the study at 
each site, which will allow the investigators and study personnel to identify and resolve potential 
logistical and technological problems prior to beginning data collection. Standardized protocols and 
SOPs will be followed for quality control/quality assurance of clinical evaluations, biological sample 
procurement and preparation, and all laboratory procedures. As well as regular internal audits and 
monitoring of quality indicators, a central quality management team consisting of the international 
study coordinator and other team members involved in field coordination, will help to ensure that all 
laboratory procedures are performed according to SOPs at all sites and with high quality standards. 
PIs and study coordinators from the different countries will maintain regular contact to ensure 
consistency in protocol implementation at each site, and will meet regularly with study staff at their 
site/s to ensure consistency in the collection of data. PIs and study coordinators will participate in 
regular study group meetings to assess progress of the study, address any difficulties and provide 
feedback to members of the study group.  
8.6 
Records 
All participants will be identified throughout the study by a unique identifying number (UID) that will 
be assigned at recruitment using uniquely numbered and barcoded consent forms.  This number will 
only be traceable to identifiable data by the study coordinator. All data will be backed up regularly to 
an off-site secure facility.  Electronic data will be stored on password-protected and encrypted media.  
Access to the records will be limited to study staff. The investigators and study staff will allow all 
requested monitoring visits, audits or reviews by relevant ethical review boards. 

Page 30 of 41 
 
8.7 
Data capture 
Clinical data will be collected using REDCAP and/or ODK. Data connections with the encrypted server 
can be made over mobile connections and data are transferred automatically to the server whenever 
an internet access point is available. This allows continuous backup of data and quality monitoring in 
real time. 
 
8.8 
Immediate and long-term use of the data 
Data collected in this study will be compiled, analysed, and made available to collaborating partners 
and to ministries of health in participating countries in an anonymised way. Research data will be 
prepared for publication in scientific open-access peer-reviewed journals. Anonymised data may be 
made available to research students in participating countries and at participating institutions for 
educational use. The investigators will comply with international standards and guidelines regarding 
open access to research data. Upon completion of the study all study documents including CRFs and 
laboratory notes will be stored at study sites for at least ten years. Electronic data records will be 
stored indefinitely.  
  
 
 
 

Page 31 of 41 
9 
DISSEMINATION OF RESULTS AND PUBLICATION POLICY 
A high degree of public engagement is an integral part of the research plan for this study, and forms 
the basis of sub-study 4. Stakeholders include research participants, clinicians, policy-makers, and the 
general public. In the first year of the study meetings will be held with community leaders and with 
the public at each site to inform local communities about the aims of the study and the methods to be 
used. Seminars will be organised at participating health facilities to inform medical and nursing staff 
about the study before it begins.   
 
 

Page 32 of 41 
10 
DURATION OF THE STUDY 
This study has been funded for 36 months. The duration of participant recruitment is expected to take 
no more than 12 months. Laboratory testing will coincide with the fieldwork at each site. We 
anticipate data analysis and dissemination to take one year. The total study is expected to take 36 
months.  
ACTIVITY 
Year 1 
Year 2  
Year 3 
1 
2 
3 
4 
1 
2 
3 
4 
1 
2 
3 
4 
Final Version of Study 
Protocol 
  
 
 
 
 
 
 
 
 
 
 
 
Data and Material Transfer 
Agreements established 
  
  
  
  
Appoint of Key Project 
Staff 
 
  
  
  
 
 
 
 
 
 
 
 
Project Steering Group 
Meetings 
 
 
 
  
  
  
  
  
 
 
 
 
Training needs assessment 
 
 
 
 
 
  
  
  
  
 
 
 
Training 
 
 
 
 
 
 
 
 
 
 
 
 
Monitoring Visits 
 
 
 
 
 
 
 
 
  
  
  
  
Production of TPHD LAMP 
assay for field study 
 
 
 
 
  
  
  
  
 
 
 
 
LAMP SOP development  
  
 
 
 
 
 
 
 
  
  
  
  
Assessment of Reference 
Labs diagnostic capacity 
 
 
 
 
 
 
 
 
 
 
 
 
Laboratory procurement  
 
 
 
 
 
 
 
 
 
 
 
 
Sub study 1-3 enrolment 
 
 
 
 
 
 
 
 
 
 
 
 
Sample Testing at District 
and National Laboratories 
 
 
 
 
 
 
 
 
 
 
 
 
Laboratory monitoring 
 
 
 
 
 
 
 
 
 
 
 
 
Sub-study 4 - Patient and 
Stakeholder Interviews on 
Diagnostic Testing 
 
 
 
 
 
 
 
 
 
 
 
 
Sub-study 4 - Health-
Economic Data Collection 
and Analysis 
 
 
 
 
 
 
 
 
 
 
 
 
Lab analyses for sub-
studies 1-3  
 
 
 
 
 
 
 
 
 
 
 
 

Page 33 of 41 
 
 
 
 
 
  
 
 
 
Sub study 1-3 Data 
Analysis 
 
 
 
 
 
 
 
 
 
 
 
 
Community Dissemination 
Meetings 
 
 
 
 
 
 
 
 
 
 
 
 
Final Study Findings 
Publication 
 
 
 
 
 
 
 
 
 
 
 
 

Page 34 of 41 
11 
ANTICIPATED POTENTIAL PROBLEMS 
Table 5. Potential study problems and proposed solutions 
Potential problem 
Proposed solution 
Delayed start of study activities 
due to delays in ethics approvals, 
procurement, other logistic 
impediments or due to the Covid-
19 outbreak 
1) Work with relevant personnel/agencies, local and national 
authorities as needed to minimize impact on project timelines 
2) If necessary, discuss with EDCTP re: adjustment of deliverables 
timeline 
3) Streamline field collection by increasing number of 
fieldworkers  
Insufficient sample size (for cases 
and/or controls), or slower than 
expected enrolment 
1) Work with local community leaders, health facility staff and 
other relevant personnel to identify and address possible 
causes 
2) Increase number of clinical sites (within the same 
geographical/population area) 
Adverse events and/or protocol 
violations 
1) Report to appropriate ethical committee and EOC 
2) Prevention as appropriate through protocol amendment 
and/or personnel training 
Loss of or damage of specimens 
during storage or transport 
1) Minimize risk with use of good-quality equipment, SOPs and 
training at sites, and use of well-established international 
courier 
 
 

Page 35 of 41 
12 
ETHICAL CONSIDERATIONS AND PARTICIPANT 
CONFIDENTIALITY  
Ethics approval will be sought from the London School of Hygiene & Tropical Medicine ethics review 
committee and from national/institutional review committees in all participating countries (Ghana 
Health Services Ethics Committee, Cote d’Ivoire National Health Ethics Committee, Cameroon 
National Health Ethics Committee). In addition, we will obtain relevant data-transfer and material-
transfer agreements to cover the transfer of samples and study data between countries. All data 
will be coded through the use of unique study IDs and held on a secure server. We may provide 
participants with compensation or travel expenses according to each country’s ethical committee 
requirements.  

Page 36 of 41 
13 
SPONSOR 
London School of Hygiene & Tropical Medicine (LSHTM) will act as the main sponsor for this study. 
Delegated responsibilities will be assigned locally. 

Page 37 of 41 
14 
FUNDING  
This study has been funded by an EDCTP grant, number RIA2018D-2495 

Page 38 of 41 
15 
AUDITS AND INSPECTIONS 
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 Good Clinical Practice. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
REFERENCES: 

Page 39 of 41 
1.  
Mitjà O, Asiedu K, Mabey D, 2013. Yaws. Lancet. 381(9868):763-773. doi:10.1016/S0140-
6736(12)62130-8. 
2.  
Čejková D et al., 2012. Whole Genome Sequences of Three Treponema pallidum ssp. pertenue 
Strains: Yaws and Syphilis Treponemes Differ in Less than 0.2% of the Genome Sequence. 
Lukehart S, ed. PLoS Negl Trop Dis. 6(1):e1471. doi:10.1371/journal.pntd.0001471. 
3.  
Marks M, Lebari D, Solomon AW, Higgins SP, 2015. Yaws. Int J STD AIDS. 26(10):696-703. 
doi:10.1177/0956462414549036. 
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