Culture Free Diagnosis and Follow-up of Multidrug Resistant Tuberculosis Patients
Conditions: Tuberculosis, Multidrug-Resistant
Sponsor: Dissou AFFOLABI

 
 
 
 
Culture free diagnosis and follow-up  
of multidrug resistant tuberculosis patients  
(DIAMA) 
 
Version 1.2, 21-Dec-2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
Sponsor/Commissioner: 
Centre National Hospitalier de Pneumo-Phtisiologie, 
Laboratoire de Reference des Mycobactéries (LRM) 
01, BP 817 Cotonou, Bénin 
(Coordinating) Investigator: 
Dissou Affolabi 
 
 

Laboratoire de Reference des Mycobactéries (LRM), Cotonou  
 
  
 
               DIAMA 
 
 
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Protocol Number: 
DIAMA 
Title: 
Culture free diagnosis and follow-up  
of multidrug resistant tuberculosis patients  
Version: 
Version 1.2  dd 21-12-2016  
Partnering institutions: 
 
- 
Centre 
National 
Hospitalier 
de 
Pneumo-Phtisiologie, 
Laboratoire de Reference des Mycobactéries (LRM), Dissou 
Affolabi, Benin, Cotonou 
- Rwanda Biomedical Center (RBC), Jean Claude Semuto 
NGABONZIZA, Rwanda, Kigali 
- The Tuberculosis Reference Laboratory Bamenda, Melissa 
Sander, Cameroon, Bamenda 
- Institut National de Recherche Biomédicale (INRB), Michel 
Kaswa, DRC, Kinshasa 
- Jimma University, Gemeda Abebe, Ethiopia 
- Service de Pneumophtisiologie, Oumou Sow, Guinea, Conakry 
- Université des Sciences, des Techniques et des Technologies de 
Bamako, SEREFO, Bassirou Diarra, Mali, Bamako 
- Damian Foundation, Osman Eltayeb, Ibadan, Nigeria 
- Université Cheikh Anta Diop, Aissatou Gaye-Diallo, Senegal, 
Dakar 
- Institute of Tropical Medicine (ITM), Bouke de Jong, Belgium, 
Antwerp 
- Special Program for Research and Training in Tropical Diseases, 
World 
Health 
Organization 
(WHO/TDR), 
Corinne 
Merle, 
Switzerland, Geneva 
- London School of Hygiène and Tropical Medicine (LSHTM), Sian 
Floyd, United Kingdom, London 
- Genoscreen, Philip Supply, France, Lille 
 
 
 
Funder: 
EDCTP2 
Sponsor: 
Laboratoire National des Mycobactéries, Benin 
Coordinating 
Investigator: 
Dissou Affolabi 
Department: 
 
Address: 
01 BP817 Cotonou Benin 
Telephone: 
+229 66 61 48 62 
Fax: 
+229 21 33 70 57 
Email: 
affolabi_dissou@yahoo.fr 
 

Laboratoire de Reference des Mycobactéries (LRM), Cotonou  
 
  
 
               DIAMA 
 
 
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TABLE OF CONTENTS 
 
STATEMENT OF COMPLIANCE & CONFIDENTIALITY ......................................................... 4 
SYNOPSIS ............................................................................................................................................. 5 
INTRODUCTION ................................................................................................................................. 6 
1. 
Background .................................................................................................................. 6 
2. 
Rationale ...................................................................................................................... 9 
STUDY OBJECTIVES ....................................................................................................................... 10 
1. 
Overall Aim:............................................................................................................... 10 
2. 
Primary objectives:..................................................................................................... 10 
3. 
Secondary objectives:................................................................................................. 10 
METHODS .......................................................................................................................................... 10 
1. 
Study design: .............................................................................................................. 10 
2. 
Study settings ............................................................................................................. 11 
3. 
Study population ........................................................................................................ 11 
4. 
Sample size................................................................................................................. 11 
5. 
Study outcomes .......................................................................................................... 12 
6. 
Data management & Data Collection ........................................................................ 12 
7. 
Patient management ................................................................................................... 13 
8. 
Laboratory procedures ............................................................................................... 14 
9. 
Data Analysis ............................................................................................................. 15 
ETHICAL CONSIDERATIONS ....................................................................................................... 16 
1. 
Ethical (and Regulatory) Review ............................................................................... 16 
2. 
Informed Consent ....................................................................................................... 17 
3. 
Confidentiality ........................................................................................................... 17 
4. 
Insurance .................................................................................................................... 17 
STUDY COMMITTEE AND STUDY MANAGEMENT ............................................................... 17 
DISSEMINATION OF RESULTS .................................................................................................... 18 
REFERENCES ...............................................................................ERREUR ! SIGNET NON DÉFINI. 
LIST OF ABBREVIATIONS ............................................................................................................. 20 
 
 
 
 

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               DIAMA 
 
 
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Statement of Compliance & Confidentiality 
 
The information contained in this study protocol is privileged and confidential. As such, it may 
not be disclosed unless specific permission is given in writing by the principal investigator or 
when such disclosure is required by federal or other laws or regulations. These restrictions on 
disclosure will apply equally to all future information supplied which is privileged or 
confidential. 
 
Once the final protocol has been issued and signed by the Investigator(s) and the authorized 
signatories, it cannot be informally altered. Protocol amendments have the same legal status 
and must pass through the mandatory steps of review and approval before being 
implemented. 
 
By signing this document, the Investigator commits to carry out the study in compliance with 
the protocol, the applicable ethical guidelines like the Declaration of Helsinki and consistent 
with international scientific standards as well as all applicable regulatory requirements. The 
Investigator will also make every reasonable effort to complete the study within the timelines 
designated. 
 
 
 
PROJECT COORDINATOR: 
 
 
Title, Name: Prof Dissou Affolabi 
Date: 
24 Nov 2016 
 
Signed: 
 
 
 
 
 
 
 
 
 
 
 
PRINCIPAL INVESTIGATOR OF THE PARTNER INSTITUTION: 
 
 
Title, Name: Prof Bouke de Jong 
Date: 
24 Nov 2016 
 
Signed: 
 
 
 
 
 
 
 
 
 

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               DIAMA 
 
 
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Synopsis 
The proposed DIAMA (DIAgnostics for Multidrug resistant tuberculosis in Africa) study aims 
to address current gaps in the diagnosis and management of patients with Multi-Drug- 
Resistant (MDR) tuberculosis (TB). Building on existing networks and research collaborations, 
the project aims to evaluate and implement rapid and accurate molecular tests for 
resistance to anti-TB drugs, to replace the current dependency on phenotypic drug 
resistance testing (DST), which takes up to 4 months and is technically so demanding that 
few laboratories can perform these tests correctly. 
 
The project builds on the World Health Organization (WHO) recommended continuous 
surveillance of patients for rifampicin resistance. Two African partners with advanced 
molecular laboratories will establish reference laboratories for the ‘Deeplex’ assay, a novel 
multiplex deep sequencing-based drug resistance diagnostic platform that simultaneously 
provides sequence information of genes that confer resistance to key TB drugs. Partners will 
recruit all patients with RR TB, and a subset of those with rifampicin sensitive TB. In a first 
phase, sputum will be shipped for the Deeplex assay, for comparison against phenotypic DST 
to 1st and 2nd line drugs. In a second phase, InSilixa and Cepheid 2nd line Xpert, two ‘lower 
tech’ tests at the last stages of laboratory validations, will be implemented in all countries 
that have established recruitment of retreatment patients. The InSilixa lab-on-chip assay, 
powered by a smartphone, is able to query 117 drug resistance conferring mutations at 
around $20 per test. The Cepheid Xpert 2nd line cartridge can be implemented in existing 
Xpert machines used for the Xpert MTB/Rif assays. These two tests will be compared versus 
the Deeplex assay. 
 
Using the latest advances in e-Health software ‘DataToCare’, molecular results would be 
communicated in real time to the National TB Programs, so that MDR patients can swiftly 
start appropriate treatment.  
 
Lastly, once patients have initiated MDR treatment, they will be monitored for treatment 
success by faster alternative approaches to the WHO recommended monthly cultures: serial 
sputum samples will have Fluorescein DiAcetate (FDA) vital stain microscopy and 
measurement of the bacterial load using the Xpert MTB/Rif.  
 
Together, these advances are expected to dramatically improve the currently dismal 
prognosis of MDR-TB in health systems in resource-poor settings. 

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Introduction 
1. 
Background 
Background on MDR-TB diagnosis 
The majority of MDR-TB patients worldwide today are neither detected nor accessing 
appropriate treatment (1). Recent advances in molecular diagnostics, especially the Xpert 
MTB/Rif test (2), have reduced the time to diagnose MDR-TB, and improved treatment 
regimens are rolled-out concurrently (3-5). Despite these advances, many questions remain. 
Only rifampicin resistance is diagnosed by the Xpert MTB/Rif test, leading to presumptive 
diagnosis of resistance to isoniazid and maybe other drugs. Resistance to other anti-TB 
drugs, linked to a range of mutations in other genes, affects the patients’ prognosis and 
optimal treatment composition (6). Unpublished results from Institute of Tropical Medicine 
(ITM, Antwerp, Belgium) suggest that certain specific mutations in the gyrase gene confer 
high-level resistance to fluoroquinolones, and are associated with poor MDR TB treatment 
outcome. Patients with RR TB therefore need treatment adjusted towards other potent 
drugs. However, assessing resistance to other drugs currently takes weeks to months (7). In 
the present study, we aim to swiftly and accurately diagnose resistance to rifampicin and 
other drugs at country and regional levels by: 
- 
Validating a higher complexity assay (Deeplex) that would be available in two 
regional reference laboratories for the benefit of the whole regions  
- 
Validating lower complexity assays that, albeit less informative, could be available 
country-wide at district level (Xpert 2nd line) and/or as point of care tests (InSilixa) 
- 
Identifying the thresholds of proportions of drug resistant mutants directly in sputum 
that predict clinical resistance and poor outcome. 
- 
Informing the potential addition of phenotypically and/or clinically validated 
mutations identified by Deeplex to the InSilixa point-of-care assay 
 
Technical background – Deeplex assay 
The Deeplex test, developed by Genoscreen (8), consists of a targeted Next Generation 
Sequencing (NGS) assay, coupling multiplex PCR to deep sequencing. This approach is 
designed to be directly applicable on clinical samples, providing species- and M. tuberculosis 
lineage identification, and detection of drug resistance. The Deeplex test can simultaneously 
detect mutations in numerous genes that confer resistance to multiple anti-TB drug classes:  
The amplicons are deep sequenced on a MiniSeq platform, This molecular assay is coupled 
to a dedicated secured, cloud-based bioinformatics application, including analysis of deep 
sequencing data and final reporting of identification of resistance associated mutations. The 
MiniSeq based Deeplex test however requires specific infrastructure, equipment and 
technical skills and will only be deployed in well-established reference laboratories such as 
Laboratoire de Référence des Mycobactéries (LRM, Cotonou), Rwanda Biomedical Center 
(RBC, Kigali), and ITM in Antwerp. A sample referral system will be set up between the 
different countries to ensure shipment to the two African laboratories; ITM will serve as a 
back-up and external referent. Once concurrent phenotypic assays will not be needed any 
longer in the context of this project, sputum referral systems can include inactivation, such 
as in ethanol, to facilitate sample shipment (non-infectious, and no cold chain required).. 
The Deeplex assay will in a second phase also serve for validation of the point of care tests, 

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as it will detect all mutations in the respective genes. Moreover, it may identify novel 
mutations, including those unique to strains circulating in Sub-Saharan Africa.  
 
Table 1. Antibiotics, and their gene targets, to which resistance is detected in Deeplex 
Gene 
target 
Antibiotic 
Deeplex 
aphC 
Isoniazid  
+ 
eis 
kanamycin 
+ 
embB 
ethambutol 
+ 
ethA 
ethionamide 
+ 
fabG1 
isoniazid 
+ 
gidB 
streptomycin 
+ 
gyrA 
Fluoroquinolones* 
+ 
gyrB 
Fluoroquinolones* 
+ 
inhA 
isoniazid 
+ 
katG 
isoniazid 
+ 
pncA 
pyrazinamide 
+ 
rplC 
linezolid 
+ 
rpoB 
rifampicin 
+ 
rpsL 
streptomycin 
+ 
rrl 
linezolid 
+ 
rrs 
Injectable agents 
+ 
tlyA 
capreomycin 
+ 
*including ofloxacin, levofloxacin, moxifloxacin and gatifloxacin 
 
 
Technical background – InSilixa ‘HYDRA-1K’ chip  
The InSilixa ‘HYDRA-1K’ chip (9) can detect 117 resistance conferring single nucleotide 
polymorphisms (SNPs), in addition to the wild-type sequence, all in triplicate, in a disposable 
chip and a hand held reader that can be operated on a smart phone (10). This test, rapid and 
cheap, including integrated sample processing, is at the last stage of laboratory validation 
and is expected to be ready for field testing in the second phase of the project. The platform 
is flexible, like Deeplex, and can have novel SNPs added. 
 
Preliminary results – InSilixa ‘HYDRA-1K’ chip  
InSilixa’s chip has been validated on the WHO Tropical Disease Research (TDR) TB strain 
collection of 229 strains and showed good results.  
Sputum processing will be done in an integrated platform and has been completed 
successfully on artificial sputum spiked with different concentrations of Mycobacterium 
tuberculosis complex bacteria. 
 
Technical background- Xpert 2nd line  
The Xpert MTB/Rif (developed by Cepheid) has already revolutionized the rapid detection of 
resistance to rifampicin (2). As this platform is rapidly expanded, and is based on a closed 
system that limits contamination, a cartridge for 2nd line drugs will be easily implementable 
and provide rapid indications on resistance to 2nd line drugs. Based on communications by 
Cepheid representatives, the Cepheid 2nd line cartridge is expected to be released in early 

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2017. It will be implemented for patients who test RR in the Cepheid Xpert MTB/RIF and will 
be compared against the Deeplex and InSilixa tests.  
 
Background on continuous surveillance 
WHO recommends to perform continuous surveillance for rifampicin resistance among 
retreatment patients, in whom such resistance occurs in around 15-20% in most Sub 
Saharan African settings (1). This tends to be done using the Xpert MTB/Rif, which in most 
settings is highly sensitive for rifampicin resistance, yet does not detect additional 
resistance to other antibiotics. The Xpert moreover misses the rpoB mutations outside of 
the test region, of which mainly the rpoB572 mutation is clinically relevant and is included 
in the novel assays tested in this proposal.  
 
Background on MDR notification rates at partner institutes 
The partners in the proposal expect to recruit at least 80% of the eligible retreatment 
patients (those with rifampicin resistance and the next rifampicin sensitive patient) 
presenting for diagnosis and care. The expected numbers of rifampicin-resistant TB are 
as follows:  
 
Table 2. Annual new Rifamipicin-resistant (RR) TB diagnoses among patients at the African 
partner institutions 
  
Estimated 
RR-TB 
patients 
diagnosed /year 
Benin, LRM 
30 
Cameroon, TBRL (*) 
80 
DRC, INRB  
150 
Ethiopia, JU 
55 
Guinea, GNRL 
100 
Mali, SEREFO 
20 
Nigeria, DF 
55 
Rwanda, RBC 
90 
Senegal, CHUD 
10 
Total  
590 
 
Background on MDR treatment monitoring  
Currently, culture on monthly sputum samples is recommended by WHO for follow-up of 
MDR-TB patients under treatment (11), who are monitored through the end of their 
treatment. Unfortunately, culture is often not locally available and samples need to be 
transported from field to culture laboratories. The associated transport delays lead to high 
rates of contamination and false negative culture, thus leading to missing and unreliable 
data, particularly in laboratories in low resource settings. Even when locally available, 
negative cultures require 42 days of incubation, with the associated delay in knowing 
whether a patient is responding to therapy (5). 
 
 
 

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Technical background – FDA 
Fluorescein DiAcetate (FDA) microscopy is a low cost diagnostic based on the FDA ester that 
needs to be cleaved by enzymes in live bacteria before it fluoresces, and slides can be read in 
the same fluorescent microscopes used for auramine microscopy (6). Preliminary results 
suggest that FDA vital stain microscopy correlates strongly with culture in MDR patients on 
follow-up.  
 
Technical background – Xpert MTB/Rif 
Similarly, we hypothesize that the now widely available Xpert MTB/Rif assay can be used to 
follow the decline in bacterial load, akin to the measurement of viral loads for the 
monitoring of HIV patients on therapy (12). We expect that the slope of the decline will be 
predictive of successful outcome. 
 
Technical background on the connectivity solutions for GeneXpert 
The Xpert has revolutionized the rapid diagnosis of RIF resistance in endemic settings, yet 
the logistics of data reporting can be challenging. 
The GeneXchangeTM system, now improved as DataToCare is an information technology 
solution for capturing and analyzing data linked to Xpert, developed for low resource 
countries. This software permits to capture data directly from the laptop of the Xpert 
machine, such as the test results, including precise information such as cycle threshold (Ct) 
and individual probe results, basic epidemiological information and basic clinical data. This 
information is then sent by SMS or internet to a central server, which allows consultation, 
analysis and exportation of data using a web interface. The connectivity solutions will target 
the Xpert results and Specific systems will be developed to allow data capture and 
transmission of the novel tests.  
 
 
2. 
Rationale 
We anticipate that the impact of the DIAMA project, if the results are as promising as 
expected, will be potentially paradigm-shifting. We foresee that MDR-TB will move from an 
often terminal illness with grave prognosis to a readily recognizable and treatable disease, in 
which treatment success can be predicted early, so treatment can be modified in ‘real’ time. 
The novel tests that identify specific resistance to particular drugs used for the treatment of 
resistant TB allow for a way of ‘personalized medicine’, and by monitoring the impact of 
specific mutations on the outcome of MDR treatment, we anticipate learning which 
mutations should lead to treatment modifications in order to enhance the chance of 
treatment success. In addition, we will learn whether heteroresistance, which is the 
simultaneous occurrence of bacterial populations with and without resistance, is of clinical 
relevance. Lastly, the fact that the Deeplex assay gives full sequence information on a large 
number of genes allows us to detect, for the first time, whether different mutations are 
common in circulating drug resistant strains in the participating countries in Sub-Saharan 
Africa. Such results will thus not only inform the treatment algorithms used in clinical trials 
for MDR-TB, yet also provide fundamental insights into the pathogenesis underlying the 
development of TB resistance. 
Once the validation of the novel diagnostic tests for TB resistance mutations will be 
completed in the DIAMA proposal, the companies that have developed these tests will 

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include these results in their respective applications for approval by regulatory authorities, 
and for endorsement by WHO. The monitoring of MDR patients on treatment with FDA 
microscopy is readily implementable in most peripheral microscopy centers. 
 
Study Objectives 
1. 
Overall Aim: 
To improve diagnosis and management of MDR-TB patients with culture-free approaches 
 
2. 
Primary objectives: 
1. To diagnose TB resistance to 1st and 2nd line drugs through novel molecular multiplex 
assays (Study 1) 
a. To validate the Deeplex assay and establish a network for shipment of sputum 
samples in ethanol to regional reference laboratories (Study 1 – phase 1) 
b. To validate the InSilixa chip-based detection of resistance as a point of care test (Study 
1 – phase 2) 
c. To validate the Cepheid Xpert 2nd line cartridge at the district level  
2. To setup alternative culture-free approaches for the monitoring of patients’ response to 
MDR treatment (Study 2), with: 
a. FDA microscopy 
b. Measurement of bacterial load by following Cycle threshold (Ct) values in Xpert 
MTB/Rif 
 
3. 
Secondary objectives: 
1. To estimate the proportion of additional resistance in patients resistant to 
RIF (isoniazid, and other drugs) in the countries involved using the novel 
validated tests and identify associations of programmatic outcome in MDR 
patients. 
2. To measure the association of specific mutations against INH and PZA with 
programmatic treatment outcome  
3. To pilot whether implementation of DataToCare for real-time monitoring of 
molecular test results can reduce delay between diagnosis and treatment of RR-TB 
patients  
Methods 
 
The study is expected to start in the first quarter of 2017, and will end May 31st, 2021. 
1. Study design:  
Two multi center observational studies will be conducted:  
 
 Study 1: Cross sectional study for TB cases aged ≥ 15 (all RIF resistant (RR) ones (new 
cases or retreatment patients) and equal number of RIF sensitive retreatment 

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patients from the same country) with comparison of TB resistance diagnostic test 
performance at baseline. There are 2 phases in this project; phase 1 is the 
comparison of Deeplex results against the phenotypic results (Gold standard), phase 
2 is the comparison of Insilixa and Xpert 2nd generation results against Deeplex (gold 
standard). 
 
 Study 2: Cohort study of RR-TB patients (recruited in Study 1) under treatment with 
comparison of the performance of FDA or Xpert compared to solid culture 
 
The study flow chart is placed in annex 1 of this protocol 
2. Study settings  
The study takes place in nine African countries (Benin, Cameroon, DRC, Ethiopia, Guinea, 
Mali, Nigeria, Rwanda and Senegal) with patients recruited at a national or regional level 
(depending on country size), and the samples tested in the respective reference laboratory 
(see partners). All partners involved in this study have or will implement(ed) a routine 
system of screening all TB patients at risk for resistant TB aged ≥ 15 with Xpert/MTbRif.  
 
3. Study population 
In order to be eligible for study 1 and 2, study participants must meet the following criteria: 
- 
Being ≥ 15 year old 
- 
Having a positive test on GeneXpert (M. tuberculosis) with or without resistance 
detected to rifampicin 
- 
Willing and able to provide written informed consent, or for minors: assent from 
these patients and consent from a legal representative 
 
In addition, for the study 2 (cohort study), only patients for whom RIF resistance was 
detected with GeneXpert at baseline and who started MDR-TB treatment will be eligible.  
 
4. Sample size 
The partners in the proposal expect to recruit at least 80% of the eligible retreatment 
patients (those with RIF resistance -considered as MDR- and equal numbers of RIF sensitive 
patients) presenting for diagnosis and care. Based on previous years data, over 4700 
patients at risk of RR TB are on average collectively being screened annually out of which 
590 (15%) are diagnosed as being RR-TB (Table 2, in Background. 
For the validation of the novel tests (Study 1), we will recruit all RR patients and equal 
numbers of RIF susceptible patients, for a total of 1420 RR- and 1420 RIF susceptible 
patients. In the table below, we summarize the power we expect to have to detect 
resistance to the key drugs: 
 
Table 3. Sample size considerations against phenotypic gold standard (Phase 1), and the 
genotypic gold standard (Phase 2, see study flow chart in Annex 1), with each phase 
including 1420 patients, half of whom will have RR TB, for a total of 2840 patients. 
 
 

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Drug 
Expected 
prevalence of 
resistance in 
RIF R 
(n=710 each 
phase) 
Expected 
total 
prevalence of 
resistance 
(n=1420 each 
phase) 
Expected 
sensitivity 
and precision 
against 
phenotypic 
test 
Expected 
specificity 
and precision 
against 
phenotypic 
test 
Expected 
sensitivity 
and precision 
against 
genotypic 
test 
Expected 
specificity and 
precision 
against 
genotypic test 
RIF 
710 (100%) 
710 (50%) 
98% +/- 1% 
98% +/-1% 
98% +/- 1% 
98% +/-1% 
INH 
675 (95%) 
923 (65%) 
90% +/- 1% 
98% +/-1% 
98 +/- 1% 
98% +/-1% 
FQ 
43 (6%) 
43 (3%) 
85% +/- 8% 
98% +/-1% 
98%, 
range 
88%-100% 
98% +/-1% 
RIF= rifampicin, INH=isoniazid, FQ=fluoroquinolones 
The estimated prevalence of resistance is based on the WHO report (1) and personal 
communications. This sample size will be ample to validate these tests for resistance to RIF, 
isoniazid, and fluoroquinolones, according to established guidelines (9).  
 
5. Study outcomes 
 
 For the study 1 (validation of deeplex test, Insilixa and Xpert 2nd line):  
 
Drug resistance to INH, RIF, ofloxacin, kanamycin as measured by phenotypic methods and 
Deeplex method. 
Secondary outcome: for both RR- and RS patients programmatic treatment outcome will be 
recorded. 
 
 For the study 2 (cohort study, on RR patients only):  
Three types of unfavorable treatment outcomes will be considered:  
 Early treatment failure defined as a culture positive at month 6 of the TB treatment 
(+/- 2 weeks) not followed by negative consecutive cultures and with molecular 
confirmation of the similarity of the strain with the baseline one 
 End of treatment failure defined as a positive culture at the end of the TB treatment 
(+/- 2 weeks) not followed by negative consecutive cultures and with molecular 
confirmation of the similarity of the strain with the baseline one 
 Relapse defined as a positive culture after the end of the TB treatment not followed by 
negative consecutive cultures and with molecular confirmation of the similarity of the 
relapse strain with the baseline one.  
 
6. Data management & Data Collection 
 
Data collected:  
For each patient enrolled in study 1 & 2, an expanded laboratory request form will be 
completed.  
 
For all patients the following data will be collected:  

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 Baseline characteristics (mostly those collected from routine monitoring, including 
demographic- and contact information, detailed treatment history including details 
such as failure vs relapse prior MDR treatment, return after loss to follow-up), risk 
factors for (resistant) TB 
 Phenotypic drug sensitivity tests (phase I of Study 1 only) 
 Deeplex results (phase I and II of the Study 1) 
 Programmatic treatment outcomes 
In addition:  
 for the phase 2 of the study 1 (validation of InSilixa and 2nd line Xpert test) the results 
of these 2 tests (InSilixa and 2nd line Xpert) will also be recorded  
 
 for all patients enrolled as RR, the following additional information will be recorded:  
o monthly FDA results,  
o monthly GeneXpert and culture results,  
o treatment outcomes  
 
Database:  
A Master database will be developed by Savics company together with  the central study 
Data Manager (DM) of Benin. A unified data entry system will be used, with a unique 
identifying number for each patient who is recruited in the study.  
 
 
Data entry and control:  
Each partner will be responsible for regular data entry with built-in validation checks, 
internal quality control and regular back-up. All data collected will be kept in a secure place 
with access restricted to study staff.  
 
The Study Data Manager of Benin will remotely follow data entry. Quality control checks will 
be performed. After review of the files, necessary queries, if arising, will be immediately 
addressed to the country-site data manager for clarification using patient query forms. After 
resolution of the queries, corrected data will be entered in the database.  All databases will 
be secured with password-protected access systems.  
 
 
7. Patient management 
An informed consent form will be required to be signed by all patients enrolled in the 
studies. 
 
For minors, an assent will be sought for participating in the studies and a legal 
representative will provide consent.  
 
If patients or legal representative are illiterate a literate witness will check that the consent 
process is done as per scheduled and will sign the consent form.  
 

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Patients found to have RR-TB will be referred to initiate MDR-TB treatment and will be 
eligible to participate in both Study 1 and Study 2, while the RIF sensitive patients will 
participate in Study 1 only.   
 
RR TB patients will be followed during the whole course of the MDR-TB treatment, as per 
routine recommendations (study flow-chart in annex 1).  
 
8. Laboratory procedures 
 
Sampling schedule 
The DIAMA project is based on routine diagnosis and patient management.  
While country TB programs recommend collecting sputum samples, a maximum of two 
additional samples will be asked at the same visit. 
 
All patient interventions and associate laboratory tests are summarized in Table 4 below. 
 
Table 4. Schedule of assessment 
 
 Screening 1 
Enrollment 2 
Follow up3 
Medical/demographic history 
X 
 
 
Informed Consent 
 
X 
 
Sputum collection 
X 
X 
X 
Programmatic outcome follow-up1 
 
 
X 
1 Includes routine programmatic interventions 
² May be combined with screening 
3 RR patients only 
 
As no additional visit is required, and intervention is minimal, no compensation fee will be 
given to the participants. 
 
Specific tests 
In all settings, smear microscopy with Ziehl-Neelsen (ZN) or auramine/rhodamine (A/R), 
culture, 1st line drug susceptibility testing (DST) and Xpert MTB/Rif are routinely used. Where 
2nd line DST on solid culture is not yet available, the project will help implementing the 
recommended tests on a routine basis. If implementation is not possible in a reasonable 
framework, respective samples will be tested in one the study reference labs (LRM Cotonou, 
RBC Kigali or ITM Antwerp), together with the Deeplex.  
  
Samples will undergo direct auramine microscopy and direct FDA microscopy. All samples 
(including AFB negative) will undergo Xpert MTB/Rif testing and culture. 
 
As all partners already conduct phenotypic DST, the culture and first-line phenotypic DST will 
be conducted on-site, following training for second line DST by the Lowenstein Jensen 
proportion method as necessary. Participating laboratories will receive periodic External 
Quality Assessment (EQA) panels from the ITM in Antwerp. Surplus sputum will be stored at 

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-20C and serves for testing additional bacterial diagnostic tests, such as the InSilixa and 
Cepheid 2nd line tests available in the second phase of study 1. 
 
Partners are moreover encouraged to follow WHO guidelines regarding the use of Line 
Probe Assays (LPA) for FQ and injectable resistance for RR patients, which is not part of the 
DIAMA study protocol. Given the intrinsic delays of shipments in phase I, and the fact that 
the Deeplex is not yet validated in phase I, the use of such LPAs will support the appropriate 
choice of treatment regimen for RR patients. The partner laboratories will however be able 
to request results from Deeplex testing to weigh these ‘unvalidated’ results in the choice of a 
salvage regimen in case patients fail their RR treatment. 
 
Shipment samples  
The shipment of samples to the reference laboratories in Cotonou and Kigali will be 
organized, based on parallel sputum aliquots in validated sputum preservative, to avoid 
contamination, for phenotypic testing if not completed on-site; as IATA Category A 
shipment), and for Deeplex testing. Quality control and troubleshooting will be provided by 
shipping samples to ITM, where the same Deeplex tests will be set-up, as well as to 
Genoscreen, which will offer support to all three laboratories using the test.  
Sample shipping has been reinforced through organizing a 2-day IATA shipping course in 
Cotonou, followed by an examination for IATA certification for all partners. 
 
DataToCare 
To help increase the GeneXpert connectivity, we will assess the landscape and their eventual 
existing connectivity solutions in all countries at the start of the project. This will allow us to 
develop a plan for implementation and upgrade of connectivity solutions in selected sites 
adapted to the local context. In the selected sites, we will provide ongoing support after 
installation, and monitor the Xpert results, diagnostic delay, and users’ satisfaction. 
 
9.  Data Analysis 
For the phase I and II of the study 1 (validation of Deeplex against phenotypic DST & 
validation of InSilixa and Xpert 2nd generation against Deeplex results), we will validate the 
molecular assays for their performance (Se and Sp) on the diagnosis of RIF resistance, 
against phenotypic DST on solid medium as gold standard, with resolution of discordances 
by classic target sequencing of the rpoB gene. Similar analyses will be conducted to 
determine the performance for the diagnosis of isoniazid and fluoroquinolone resistance. 
 
An interim analysis will be performed after 350 RR-TB patients have been recruited in order 
to test our assumptions regarding resistance proportions and consider adjustment of 
sample size or recruitment strategy.  
 
For the study 2, the results will be analyzed with time dependent Cox regression to identify 
the optimal time point to identify patients who are at risk of failing treatment.  
 
 

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Ethical considerations 
 
This study is mainly based on routine procedures, already in place or to be implemented as 
capacity building. All patients will receive the diagnostic testing as per recommendations. 
The only intervention concerns the request of two additional sputum samples after 
inclusion in the study. Minors from 15 years old will be included, as they produce sputum 
samples like adults. During follow-up, MDR patients will have to give two samples, i.e. an 
additional one to the one requested for routine management. The production of an 
additional sputum sample is very unlikely to cause any major harm to the patient. Personal 
data will be protected, with a unique study identifier number used in the password 
protected database. 
1. 
Potential risks 
The risk to the participants from the standard collection of additional sputum 
specimens is minimal. There is a small risk of disclosure of personal information 
obtained from the clinical interviews.  
2. 
 Protection against potential risks 
Confidentiality among participants will be protected by assigning each patient a 
unique identifying number, and no patient identifying information will be included in 
study database. Care will be taken to ensure that patients understand that study 
documents will be kept in strict confidence by the investigator, though absolute 
confidentiality cannot be guaranteed.  
3. 
Risks in relation to benefits 
Patients will benefit from intensive screening for resistance to the standard TB/MDR-
TB drug regimens; drug resistance is a significant cause of mortality for patients with 
TB. These benefits significantly outweigh the minimal risks involved from specimen 
collection and the low risk of disclosure of patient information. 
 
4. 
Ethical (and Regulatory) Review  
This study will be submitted for formal review and approval to the Benin ethics committee 
(i.e. the central ethics committee), and the Ethics Committee(s) and/or Competent 
Authorities of the nine countries where the study is carried out and of selected 
collaborating partners.  
The study will be carried out according to the principles stated in the Declaration of Helsinki, 
all applicable regulations and according to established international scientific standards. 
 
Each partner will submit the protocol of the studies, including the information sheet and 
consent form as well as a Material Transfer Agreement on the shipment of samples to the 
competent body of their country for independent examination of the scientific merits and 
ethical acceptability. No participants will be enrolled or subject related activities performed 
in a site, before written approval from the local- as well as the central ethics committees is 

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obtained.  The studies will moreover not start at each site until a copy of a written and dated 
approval has been received by the project coordinator.  
 
The choice of treatment for the patient will not depend on the results of the test under 
validation but on locally conducted tests. In case study results are suggestive of discordant 
results with the local site laboratory, the interpretation of all available results will be 
discussed with experts in the field to determine whether the patient’s standardized MDR 
regimen would best be adjusted. 
 
5. 
Informed Consent 
An informed consent form will be required to be signed by all patients enrolled in the studies 
1 through 3. For participants aged 15-17 years of age, assent will be sought, as well as 
consent from their parent or legal representative. Appropriate information as to the purpose 
and the nature of the studies will be available in French, English and other local languages 
when appropriate. When patients are unable to read, an independent witness will be 
present to ensure that the patients receive complete information. Patients may be enrolled 
in other studies and still participate in this low risk diagnostic study. 
 
6. 
Confidentiality 
All study procedures will be conducted in private, and every effort will be made to protect 
participant privacy and confidentiality.  A standard operating procedure for confidentiality 
protection that reflects the study implementation plan will be established. In addition, the 
procedures described below will be implemented:  
 All study-related information will be stored securely at the study site.   
 All participant information will be stored in locked filing cabinets in areas with access 
limited to study staff.  
 Data collection, administrative forms, laboratory and other reports, will be 
anonymised to maintain participant confidentiality.   
 All records that contain names or other personal identifiers, such as locator forms 
and informed consent forms, will be stored separately from study records identified 
by the randomisation number.  
 All databases will be secured with password-protected access systems.  
 
7. 
Insurance  
Given the observational nature of the study with minimal risks no insurance will be obtained 
by the sponsor. 
 
 
Study Committee and Study management  
The project will be coordinated by LRM, which has close collaborative links with all research 
teams. Overall coordination will be the responsibility of the Project Coordinator (Prof Dissou 
Affolabi), who will be assisted in this task by an LRM based Study manager, an Administrator 
and the Project Steering Committee (PSC). The PSC will include the Principal investigator 

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from each of the collaborating partners, a representative of Savics company, and the 
financial administrator of the project (LRM). 
The project manager under the supervision of the project coordinator will monitor the 
project on a daily basis. PSC meetings will occur at regular intervals (at the start of the 
project, and every 6 months). All salient issues will be discussed within the PSC in order to 
take full advantage of the expertise of the whole team, and to find the most appropriate 
solution to deal with the issue. 
 
Dr Corinne Merle (WHO/TDR) and Bouke de Jong (ITM) will assist the project Coordinator 
(Prof Dissou Affolabi) for the overall coordination of the project. The coordination 
mechanism between the partners is shown through the organogram in Annex 2. 
 
Monitoring visits will be conducted at the sites by the study manager, assisted by other 
partners as needed, to review the recruitment procedures and numbers and the laboratory 
activities and sample shipments. Depending on the findings and recommendations made at 
the monitoring visit, the schedule of follow-up visits will be determined for each site. 
 
Dissemination of Results 
Data and results will be made available to the consortium partners as early as the specific 
study requirements will allow. The rules/guidelines regarding authorship will be discussed 
and decided jointly by the members of the PSC prior to the start of the project. 
 
Both the design and the results of the studies will also be discussed with representatives of 
the national TB programs of the respective countries in order to favor change in practices (in 
case of positive findings).  
 
The involvement of WHO TDR in the design and execution of the project will facilitate links 
with WHO Global TB and the ultimate translation of the findings into policy. 
 
At the start of the project, an agreement will be prepared and signed between all partners, 
which will balance the access to scientific results and the protection of Intellectual Property 
for the Deeplex- and InSilixa diagnostics, based on the premise that drug resistance 
mutations, and their prevalence, as identified in this project, were the results of public 
funding and should be exploited for scientific advancement as widely as possible. Any such 
data sharing will be done without patient identifiers and will be included in the informed 
consent procedure and proposals for ethics approvals. 
 
 
 
 
 

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References 
1. 
WHO. 
Global tuberculosis 
report 
2014. World 
Health 
Organization 
Document. 
2014;WHO/HTM/TB/2014.08:1-171. 
2. 
Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al. Rapid molecular 
detection of tuberculosis and rifampin resistance. N Engl J Med. 2010;363:1005-15. 
3. 
Van Deun A, Kya Jai Maug A, Halim MA, Kumar Das P, Ranjan Sarker M, Daru P, et al. Short, 
highly effective, and inexpensive standardized treatment of multidrug-resistant tuberculosis. Am J 
Respir Crit Care Med. 2010;182:684-92. 
4. 
Piubello A, Harouna SH, Souleymane MB, Boukary I, Morou S, Daouda M, et al. High cure rate 
with standardised short-course multidrug-resistant tuberculosis treatment in Niger: no relapses. The 
international journal of tuberculosis and lung disease : the official journal of the International Union 
against Tuberculosis and Lung Disease. 2014;18(10):1188-94. 
5. 
Aung KJ, Van Deun A, Declercq E, Sarker MR, Das PK, Hossain MA, et al. Successful '9-month 
Bangladesh regimen' for multidrug-resistant tuberculosis among over 500 consecutive patients. The 
international journal of tuberculosis and lung disease : the official journal of the International Union 
against Tuberculosis and Lung Disease. 2014;18(10):1180-7. 
6. 
Bastos ML, Hussain H, Weyer K, Garcia-Garcia L, Leimane V, Leung CC, et al. Treatment 
outcomes of patients with multidrug-resistant and extensively drug-resistant tuberculosis according 
to drug susceptibility testing to first- and second-line drugs: an individual patient data meta-analysis. 
Clin Infect Dis. 2014;59(10):1364-74. 
7. 
Canetti G, Fox W, Khomenko A, Mitchison DA, Rist N, Smelev NA. Advances in techniques of 
testing mycobacterial drug sensitivity, and the use of sensitivity tests in tuberculosis control 
programmes. Bull World Health Organ. 1969;41:21-43. 
8. 
Genoscreen. 
2015. 
Available 
from: 
http://www.genoscreen.fr/en/dedicated-
solutions/microbiology/genome-analysis/resistance-prediction. 
9. 
InSilixa. 2015. Available from: http://www.insilixa.com/Home_Page.html. 
10. 
Hassibi 
A. 
2014. 
Available 
from: 
http://www.hotchips.org/wp-
content/uploads/hc_archives/hc26/HC26-11-day1-epub/HC26.11-3-Technology-epub/HC26.11.335-
BioChip-Hassibi-InSilixa_08112014_FINAL.pdf. 
11. 
Companion handbook to the WHO guidelines for the programmatic management of drug-
resistant tuberculosis(2014). 
12. 
Tucker JD, Bien CH, Easterbrook PJ, Doherty MC, Penazzato M, Vitoria M, et al. Optimal 
strategies for monitoring response to antiretroviral therapy in HIV-infected adults, adolescents, 
children and pregnant women: a systematic review. AIDS. 2014;28 Suppl 2:S151-60. 
 
 
 
 

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List of Abbreviations 
 
AFB 
 
Acid Fast Bacillus 
CNHPP  
CENTRE NATIONAL HOSPITALIER DE PNEUMO-PHTISIOLOGIE 
DST 
 
Drug Susceptibility Testing 
EDCTP  
European & Developing Countries Clinical Trials Partnership 
FDA 
 
Fluorescein Di-Acetate 
FM 
 
Auramine Fluorescent Microscopy 
HNID  
Hôpital National Ignace Deen, Conakry, Guinea 
INRB  
Institut National de Recherche Biomédicale du Zaire 
ITM 
 
Institute of Tropical Medicine 
LED 
 
Light emitting Diode 
LBV 
 
Laboratoire de Bacteriologie Virologie, Dantec, Dakar  
LRM   
Laboratoire de Référence de Mycobactéries 
MDR  
Multi Drug Resistant 
MTB 
 
Mycobacterium tuberculosis  
NTP 
 
National TB Programme  
RBC 
 
Rwanda Biomedical Center 
REG 
 
Register 
RIF 
 
Rifampicin 
SEREFO 
Centre de Recherche et de Formation, Mali 
SOP 
 
Standard Operating Procedure 
TB 
 
Tuberculosis 
TBD 
 
To be determined 
TBRL  
Tuberculosis Reference Laboratory Bamenda 
USTTB  
Université des Sciences, des Techniques et des Technologies de Bamako 
  
 
12. ANNEXES 
 
1. Study Flow chart 
2. Organogram 
3. Patient information sheet and ICF 
 

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ANNEX 1:  Study flow chart  
 
 
 
 
Data2care 
evaluation 
Recruit all Rif R 
(15%)  
+ as many Rif S 
M6-M18 : Phase 1 
 
DEEPLEX 
 
Comparison with phenotypic 1st 
and 2nd DST 
 
710 Rif resistant 
710 Rif sensible 
M18 – M30: phase 2 
 
DEEPLEX 
 
Comparison with InSilixa 
and Xpert 2nd generation 
710 Rif resistant 
710 Rif sensible 
MDR (15%) 
1420 patients 
Rif sensible (15%) 
1420 patients 
Identification of 
the assay and time 
point most 
predictive of 
treatment failure 
 
Failure (10%) 
FDA (1/month) 
Xpert (1/month) 
Culture (1/month) 
Study 1 
Study  2 
Sputum microscopy + culture 
Xpert (4800 / year) 
TB patients at risks of RR-
TB 
Study 1  

Annex 2> DIAMA Organigram 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TBRL: Tuberculosis Reference Laboratory Bamenda, Cameroon 
CHUALD: Centre Hospitalier et Universitaire Aristide Le Dantec, Dakar, 
Senegal 
HNID: Hôpital National Ignace Deen, Conakry, Guinea 
DF : Damien Fondation, Ibadan, Nigeria 
Genoscreen, Lille, France 
INRB : Institut National Biomédicale, Kinshasa, Democratic Republic of 
Congo 
ITM: Institute of Tropical Medicine, Antwerp, Belgium 
 
 
 
JU : Jimma University, Jimma, Ethiopia 
LRM :Laboratoire de Reference des Mycobatéries, Cotonou, Benin 
LSHTM: London School of Hygiene & Tropical Medicine, London, UK 
RBC : Rwanda Biomedical Center, Kigali, Rwanda 
SAVICs company, Belgium 
SEREFO: Bamako, Mali 
WHO/TDR : Special programme of Tropical Disease Research at WHO, 
Geneve
Project Steering Committee 
Project Coordinator, CNHPP 
(Prof Dissou Affolabi) 
WP1 
(LRM-Benin)/ 
G
WP2 
(SEREFO-Mali) 
WP5 
(ITM, Belgium) 
 
BTRL, CHUALD, HNID, DF, Genoscreen, INRB, ITM, JU, LRM, LSHTM, RBC, SAVICs, SEREFO, WHO/TDR 
WP4 
(LRM-Benin) 
WP3 
 (LRM-Benin/ LSHTM, UK) 
Financial project management 
(LRM, Benin) 
WHO/TDR & ITM Belgium 
Support for Project coordination 
(C. Merle / B. de Jong) 

     
 
DIAMA - Patient Information Sheet - Informed Consent Form [Country X] 
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1. RESEARCH PARTICIPANT INFORMATION SHEET 
 
Diagnostics for Multidrug resistant tuberculosis in Africa 
 
Investigator:  
<name of the Principal Investigator> 
Organization: 
<name local hospital, organization,…> 
 
Sponsor: 
 
Laboratoire National des Mycobactéries, Benin 
 
 
You are invited to participate in a research study on drug resistant tuberculosis, which is difficult to 
diagnose. Before you decide to be part of this study, it is important that you understand the information 
in this form, because it explains your rights, as well as our responsibilities. In this information and 
consent form, the purpose, examinations, possible advantages, risks and inconveniences related to this 
study are explained to you. The right to withdraw your consent at any time to participate is also 
described below. You have the right to ask questions at any time, for example about the possible 
benefits and possible risks related to this study. Your participation is completely voluntary. You may talk 
to anyone you feel comfortable with about the research and you can take your time to think about 
whether you want to participate or not. If you consent to participate in the study, we suggest you keep 
this background information with you throughout the entire study period. 
 
PURPOSE AND DESCRIPTION OF THE STUDY 
This research study is being done to learn more about tuberculosis (TB), and especially about the 
detection of TB germs that do not respond to the drugs normally used to treat them, which is called 
“drug-resistant TB”. We want to evaluate new tests, which will give more information about why some 
TB germs may not respond to treatment. Approximately 2800 people will participate in this study in nine 
different countries.  
 
HOW THE STUDY IS DONE 
If you accept to participate in this study and if you meet the conditions to participate, you will be asked 
to provide extra sputum samples. Also, we will record how well your treatment is working. If you have 
drug-resistant tuberculosis, you will also be asked to give one extra sputum sample at each of your 
follow-up visits. This sample will be used for extra testing to see how well your treatment is working.  
 
RISKS AND INCONVENIENCES 
The production of sputum is not dangerous to you. You can produce the extra sputum in the same way 
as you did for the first sample(s) you gave. It may be a minor inconvenience for you to spend extra time 
to produce more specimens. 
 
BENEFITS 
You will receive the results of the lab tests currently done in your health center as usual, and some 
additional lab tests will also be done. For a small number of people, the results of the additional tests 
may help to improve your treatment. You may also contribute to improving health care for others if the 
results of the tests under evaluation in this study help to improve treatment management for future 
tuberculosis patients. 
 
COMPENSATION  
You will not have to pay any fee for any test done for this study. You will also not be given any fee for 
participating in this study. 
 

     
 
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PROTECTION OF YOUR CONFIDENTIALITY 
We will do everything we can to protect your confidentiality. The information collected about you will 
be stored in an electronic database. This information will not be shared with anybody except the study 
researchers and your doctor, and it may be written down in your medical record. This information may 
also be examined by the organizer of this study to check that the information is correct. Your identity 
will remain secret since your personal information will be given a unique code. Your name will not 
appear in any reports or publication resulting from this study. 
 
ETHICS COMMITTEE 
Before the start, this study was reviewed and approved by the regional/national Ethics Committee(s) in 
all the participating countries.  
 
VOLUNTARY PARTICIPATION 
Your participation in this study is entirely voluntary. It is your choice whether you want to take part in 
the study. Whether you choose to participate or not, all the services you receive at this clinic will remain 
the same.  
 
You also have the right to stop your participation in the study at any time, even after you have signed 
this Informed Consent Form. You do not have to give a reason for wanting to stop being part of the 
study. 
 
The study investigator can stop your participation in this study at any moment as well, even without 
having to request your permission, if he or she judges this in your best interest or if you do not follow 
the instructions for participation in the study. 
 
CONTACT PERSON IN CASE OF QUESTIONS  
If you have any questions concerning your participation in this study, your rights or think you have been 
harmed as a result of the study, you can contact, now, during, or after the study: 
 
Principal investigator: Pr Dissou Affolabi, at Laboratoire de Référence des Mycobactéries, Cotonou, 
Benin. Phone : +229 21331533. Fax : +229 21337057. Email: affolabi_dissou@yahoo.fr. 
 
Local investigator: [adapt for each country] 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

     
 
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2. INFORMED CONSENT FORM 
 
Diagnostics for Multidrug resistant tuberculosis in Africa 
 
Part which is destined only to the participant (and his/her legal representative if applicable) 
 
I confirm that I have been informed about the study and that I have received a copy of the research 
participant information sheet and the Informed Consent Form. I have understood the information. I 
have been given sufficient information concerning the conditions, the duration of the study and any 
possible inconvenience to me. In addition, I have received sufficient time to consider the information 
and to ask questions, to which I have received satisfying answers. 
I freely consent to participate in this study and consent to cooperate in the examinations/activities 
requested. I am willing to give information concerning my medical history, use of medication and 
participation in other studies if any. 
I agree that my doctor and other healthcare professionals involved in my treatment are informed about 
my participation in this study.  
I agree that my samples may be used for additional scientific studies on TB or other germs. 
I understand that I will give extra sputum samples.  
 
 I agree that my samples may be stored after completion of this study. 
 
Date:  ……………………………                    Name:  ……………………………………………………………………………………. 
 
Signature (or thumbprint) of participant:  
__________________________________________________________________________________ 
 
Name of the witness: ………………………………………… 
 
Signature:  
__________________________________________________________________________________ 
 
Name of legal representative (for minors aged 15-17): ………………………………………… 
 
Signature (or thumbprint) of participant: 
 
 
 
Part only destined to the investigator’s team 
 
I, the undersigned, ……………………………………… confirm that I have informed the participant (and/or the legal representative) about all the 
relevant aspects of this study. I confirm that he/she has consented voluntarily to participate in the study. 
 
Date: …..……………………………                                          Signature: