Prospective, Multicentre Trial to Assess the Diagnostic Accuracy of the Truenat Assays at Intended Settings of Use
Conditions: Tuberculosis, Pulmonary
Sponsor: Foundation for Innovative New Diagnostics, Switzerland

                                                                                                             CONFIDENTIAL 
 
Truenat Evaluation 
Protocol # 7212-3/2  
Version 3.3; Date 07 SEP 2018 
[Page 1 of 57]  
 
 
 
TRIAL PROTOCOL 
Prospective, Multicentre Trial to 
Assess the Diagnostic Accuracy of 
the Truenat Assays at Intended 
Settings of Use  
 
Protocol Number: 7212-03/2 
 
Amendment Number: NA 
 
Short title:  
Truenat Evaluation 
 
Version1.0 
Date: 07 SEP 2018 
Sponsor Name: FIND INDIA 
Funder: BMGF and ICMR 
Disease Programme: Tuberculosis 
Regulatory Agency Identifying Number(s):  
www.ClincalTrials.gov: NCT03712709 
 
 
FIND India 
Flat No. 6-14, 9th floor 
Vijaya Building 
17 Barakhamba Road 
New Delhi 110001 
India 
T: +91 (11) 4041 9517 
 
 
 
 

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Truenat Evaluation 
Protocol # 7212-3/2  
Version 3.3; Date 07 SEP 2018 
[Page 2 of 57]  
 
Confidentiality Statement:  
The information contained in this document, especially unpublished data, is the property 
of FIND (or under its control) and may not be reproduced, published or disclosed to 
others without prior written authorization from FIND.  
 

 
Truenat Evaluation 
Protocol # 7212-3/2  
Version 3.3; Date 07 SEP 2018 
[Page 3 of 57]  
 
Table of Contents 
Institutions/Partners Involved in the Trial* ........................................................................ 6 
Signature Page (Sponsor) ................................................................................................... 8 
Statement of Principal Investigator .................................................................................... 9 
Protocol History/Amendment Summary* ........................................................................ 10 
List of Abbreviations and Acronyms ................................................................................. 12 
Protocol Synopsis ............................................................................................................. 14 
Schedule of Activities ....................................................................................................... 16 
1 
Introduction ............................................................................................................. 17 
 
Trial Rationale .............................................................................................................. 17 
 
Background .................................................................................................................. 17 
 
Benefit/Risk Assessment .............................................................................................. 18 
2 
Trial Objectives and Endpoints ................................................................................. 19 
3. 
Trial Design .............................................................................................................. 20 
3.1. 
General Design ............................................................................................................. 20 
3.2. 
Scientific Rationale for Trial Design .............................................................................. 20 
3.3. 
End of Trial Definition .................................................................................................. 20 
4. 
Trial Population and Eligibility .................................................................................. 20 
4.1. 
Inclusion Criteria .......................................................................................................... 22 
4.2. 
Exclusion Criteria ......................................................................................................... 23 
4.3. 
Lifestyle Considerations ............................................................................................... 23 
4.4. 
Screen Failures ............................................................................................................. 23 
5. 
Trial Intervention ..................................................................................................... 24 
5.1. 
Medical Devices ........................................................................................................... 24 
5.2. 
Preparation/Handling/Storage/Accountability ............................................................. 24 
5.3. 
Minimization of Error and Bias ..................................................................................... 26 
5.3.1. 
Patient selection ...................................................................................................... 26 
5.3.2. 
Index test ................................................................................................................. 26 
5.3.3. 
Reference standard .................................................................................................. 26 
5.3.4. 
Flow and timing ....................................................................................................... 27 
5.3.5. 
Handling of indeterminate results ............................................................................ 27 
5.3.6. 
Blinding Procedure ................................................................................................... 27 
6. 
Discontinuation of Trial Intervention and Participant Discontinuation/Withdrawal .. 27 
6.1. 
Participant Discontinuation/Withdrawal from the Trial ............................................... 27 
6.2. 
Lost to Follow Up ......................................................................................................... 28 
7. 
Trial Procedures ....................................................................................................... 28 
7.1. 
Trial Workflow ............................................................................................................. 29 
7.2. 
Efficacy Assessments .................................................................................................... 29 
7.3. 
Safety Assessments ...................................................................................................... 29 
7.4. 
Health Economics ......................................................................................................... 29 
8. 
Specimen Collection, Handling, Transport and Storage ............................................. 29 
8.1. 
Reference Standard Test and Index Test Procedures .................................................... 30 
8.2. 
Trial supplies and incoming quality check ..................................................................... 32 

 
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8.3. 
Other Trial Procedures ................................................................................................. 32 
8.3.1. 
Assessment of patient important outcomes ............................................................. 32 
8.3.2. 
Participant follow-up ............................................................................................... 32 
8.3.3. 
Analysis of discordant results ................................................................................... 33 
8.4. 
Genetics ....................................................................................................................... 33 
8.5. 
Biomarkers .................................................................................................................. 34 
9. 
Safety and Incident Reporting .................................................................................. 34 
9.1. 
Time Period for Collecting SAE Information .................................................................. 34 
9.1.1. 
Reporting and Follow up of SAEs .............................................................................. 35 
9.1.2. 
Regulatory Reporting Requirements for SAEs .......................................................... 35 
9.2. 
Medical Device Incidents (including Malfunctions) ....................................................... 35 
9.2.1. 
Time Period for Detecting Medical Device Incidents ............................................... 36 
9.2.2. 
Follow-up of Medical Device Incidents ..................................................................... 36 
9.2.3. 
Reporting of Medical Device Incidents to Sponsor .................................................. 36 
9.2.4. 
Regulatory Reporting Requirements for Medical Device Incidents ......................... 37 
10. 
Statistical Considerations ...................................................................................... 37 
10.1. 
Statistical Hypotheses .................................................................................................. 37 
10.2. 
Sample Size Determination .......................................................................................... 38 
10.3. 
Populations for Analyses .............................................................................................. 39 
10.4. 
Statistical Analysis Plan ................................................................................................ 40 
10.5. 
Efficacy Analyses .......................................................................................................... 40 
10.5.1. 
General Approach ...................................................................................................... 40 
10.5.2. 
Analysis of Primary Outcomes .................................................................................. 41 
10.5.3. 
Analysis of Secondary Outcomes .............................................................................. 41 
10.5.4. 
Descriptive statistics .................................................................................................. 42 
10.5.5. 
Additional sub-group analyses .................................................................................. 42 
10.5.6. 
Planned Interim Analyses .......................................................................................... 43 
10.6. 
Safety Analyses ............................................................................................................ 43 
10.7. 
Independent Data Monitoring Committee (IDMC) ........................................................ 43 
11. 
Regulatory and Ethical Considerations .................................................................. 43 
11.1. 
Regulatory and Ethics Approvals .................................................................................. 43 
11.2. 
Financial Disclosure ...................................................................................................... 44 
11.3. 
Informed Consent Process ........................................................................................... 44 
11.4. 
Data Protection ............................................................................................................ 44 
11.5. 
Subject Confidentiality ................................................................................................. 45 
11.6. 
Other ethical considerations ........................................................................................ 45 
11.6.1. 
Use of stored specimens: .......................................................................................... 45 
11.6.2. 
Protocol deviations .................................................................................................... 46 
11.6.3. 
Termination of the Trial ............................................................................................ 46 
11.6.4. 
Site Performance and Data Submission .................................................................... 46 
12. 
Data Handling and Record Keeping ....................................................................... 46 
12.1. 
Source Data and Source Documents ............................................................................. 47 
12.2. 
Data to be captured ..................................................................................................... 47 
12.3. 
Data Management ....................................................................................................... 47 
12.4. 
Data Archiving ............................................................................................................. 48 
13. 
Quality Management ............................................................................................ 48 
13.1. 
Quality Control ............................................................................................................ 48 
13.2. 
Quality assurance ........................................................................................................ 48 
13.3. 
Training plan and piloting of laboratory and data collection procedures ...................... 49 

 
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13.4. 
Trial and Site Closure ................................................................................................... 50 
14. 
Work plan & timeline ........................................................................................... 50 
15. 
Publication Policy ................................................................................................. 51 
16. 
References ............................................................................................................ 51 
17. 
Appendices ........................................................................................................... 52 
Appendix 1:  Supply requirements for Truenat testing ..................................................... 53 
Appendix 2:  Safety Definitions and Reporting ................................................................. 54 
Appendix 3:  Incident Definition and Reporting ................................................................ 56 
Appendix 4: Protocol Amendment Summary ................................................................... 57 
 
 
 

 
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Version 3.3; Date 07 SEP 2018 
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Institutions/Partners Involved in the Trial* 
FIND India (Sponsor) 
Flat No. 6-14, 9th floor 
Vijaya Building 
17 Barakhamba Road 
New Delhi 110001 
India 
T: +91 (11) 4041 9517 
 
National Institute for Research in Tuberculosis, NIRT (Coordinating Site) 
No.1, Mayor Sathiyamoorthy Road 
Chetpet, Chennai 
Tamil Nadu 600031, India 
 
Hinduja National Hospital and Medical Research Centre (Investigational Site) 
Veer Sawarkar Marg, Asavari 
Shivaji Park, Mumbai 
Maharashtra 400016, India 
 
District TB Centre, Chennai (Investigational Site) 
Kanakaraya Thottam 
Pulianthope, Chennai 
Tamil Nadu 600012, India 
 
Commissionerate of Health Medical Services & Me (Hs), Gujarat (Investigational 
Site) 
Sector 10B, Sector 10 
Gandhinagar 
Gujarat 382010, India 
 
 
 

 
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Directorate of Health Services, Assam (Investigational Site) 
Hengerabari Health Complex 
Hengrabari Rd, Lichubagan, Hengrabari, Guwahati 
Assam 781036, India 
 
Revised National TB Control Programme 
State TB Control Department 
2nd Floor, Delhi Govt. Dispensary Building 
Gulabi Bagh, Delhi-110007 
 
Site 2 (Investigational Site) 
To be determined 
 
Site 3 (Investigational Site) 
To be determined 
 
Site 4 (Investigational Site) 
To be determined 
 
 
 
*Terms of references and nature of agreements are available from FIND on request. 

 
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Protocol # 7212-3/2  
Version 3.3; Date 07 SEP 2018 
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Signature Page (Sponsor) 
 
We, the undersigned, have reviewed and approved this Protocol, including 
Appendices. We will supervise and coordinate the clinical trial as described and 
ensure adherence to GCP/GCLP, the principles outlined in the Declaration of Helsinki 
and applicable regulatory requirements. 
 
HEAD OF TUBERCULOSIS PROGRAMME 
Name:  
Institution:  
 
Signature: 
 _____________________ 
 
Date:_______________ 
 
 
 
 
 
 
 
 
DD/MMM/YYYY 
HEAD OF CLINICAL & REGULATORY AFFAIRS 
Name:  
Institution:   
 
Signature: 
 _____________________ 
 
Date:_______________ 
 
 
 
 
 
 
 
 
DD/MMM/YYYY 
 
 

 
Truenat Evaluation 
Protocol # 7212-3/2  
Version 3.3; Date 07 SEP 2018 
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Statement of Principal Investigator 
All studies sponsored by FIND will be conducted in accordance with ICH-GCP Guidelines, to the extent 
possible in the research setting. 
 
In signing this page, I, the undersigned, agree to conduct the trial in compliance 
with all applicable regulations and guidelines as stated in the protocol and other 
information supplied to me. 
I will ensure that the requirements relating to obtaining Institutional Review Board 
(IRB)/ Independent Ethics Committee (IEC) review and approval are met. I will 
promptly report to the IRB/IEC any and all changes in the research activities covered 
by this protocol. 
I have sufficient time to properly conduct and complete the trial within the agreed trial 
period and I have adequate resources (staff and facilities) for the foreseen duration of 
the trial.   
I am responsible for supervising any individual or party to whom I delegate trial related 
duties and functions conducted at the trial site.  Further, I will ensure this individual or 
party is qualified to perform those trial-related duties and functions. 
I certify that Individuals involved with the conduct of this trial have completed GCP 
training within the past 3 years and, if applicable, Human Subjects Protection Training.  
I understand that all information obtained during the conduct of the trial with regard to 
the subjects’ state of health will be regarded as confidential.  No subject’s names or 
personal identifying information may be disclosed.  All subject data will be anonymized 
and identified by assigned numbers on all Case Report Forms, laboratory samples 
and source documents forwarded to FIND. Monitoring and auditing by FIND, and 
inspection by the appropriate regulatory authority(ies), will be permitted. 
I will maintain confidentiality of this protocol and all other related investigational 
materials. Information taken from the trial protocol may not be disseminated or 
discussed with a third party without the express consent of FIND.   
 
Name of Principal Investigator: ________________________ 
(Print) 
 
Signature: _________________________ 
        Date: ____________________ 
 
 
 
 
 
  
 
              DD/MMM/YYYY 
 
 
 

 
Truenat Evaluation 
Protocol # 7212-3/2  
Version 3.3; Date 07 SEP 2018 
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Protocol History/Amendment Summary* 
Protocol 
Version   
Date  
Description of changes 
Brief rationale 
Approve
d 1.0 
28 Dec-2018 
- 
Changed Draft 
versions to approved 
version 1.0 (Common 
protocol) 
- 
IRB approval at NIRT, Peru, 
EPHI and Burnet.  Pending 
PNG and Hinduja. 
Draft 3.3 
07 Sep-2018 
- 
Updated section 8.1, 
study flow, narrative 
and figure 
- 
Placement of a cap 
of 250 enrolled 
participants in the 
drug resistance 
group. 
- 
Sputum collection and test 
were altered to pool and 
homgenise S1 and S2 on Day 
1 (allow better distribution of 
bacilli) and separate S4 to 
prevent splitting sputum in the 
microscopy centre, thereby 
reducing risk of 
contamination. 
- 
Capped to not undermine 
enrolment of smear-negative 
culture positive participants 
for primary aim analysis. 
Draft 3.2 
27-Aug-2018 
- 
Updated section 8.1, 
study flow, narrative 
and figure 
- 
Sputum collection and test 
were altered to i) fit with 
national guidelines and ii) 
allow head-to-head 
comparison of Truenat assays 
to Xpert on unprocessed 
sputum. 
Draft 3.1 
05-Jul-2018 
- 
Removed use of 
frozen samples (RIF 
resistant). 
- 
Introduced 
differentiation of 
“Case Detection” 
group and “Drug 
Resistant TB” group. 
- 
Full RIF resistance will be 
performed in a separate 
laboratory study. 
- 
Increased enrolment of 
participants with presumed 
RIF resistance. 
Draft 3.0 
28-Jun-2018 
- 
Revised sample size 
- 
Added use of frozen 
samples (RIF-
resistant) to 
supplement testing 
- 
No differentiation of 
trial participants in 
two groups 
- 
Trial to be part of 
global trial 
- 
TB positivity increased based 
on pilot data Phase 1b 
- 
Mitigate the risk of not 
meeting targets to determine 
accuracy of RIF assay 
- 
A sub-analysis will be done 
based on prior history of TB 
independent of the patient 
group 

 
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- 
Updated timeline & 
list of investigators 
- 
Format changes 
- 
“Trial” replaces 
“study” 
- 
Modified exclusion 
criteria to having 
received TB 
treatment within the 
last 60 days prior to 
enrolment. 
- 
Results to be submitted to 
Indian health authorities as 
well as WHO, so need collect 
data outside India 
- 
Revised site selection 
- 
Changes in protocol template 
that was developed based on 
TransCelerate’s Common 
Protocol Template and 
modified for IVD clinical 
studies.  
- 
Updated FIND definition 
- 
Allows enhanced enrolment 
rate and inclusion of possible 
TB relapse cases.  
Draft 2.1 
05-Sep-2017 
- 
Truenat MTB & MTB 
Plus assays 
- 
Side by side assessment of 
MTB and MTB plus assays to 
complement India in-country 
trial 
Draft 2.0 
22-Aug-2017 
- 
Revised sample size 
- 
Removed global trial 
- 
Removed statistical 
analysis details 
- 
Added potential use 
of well-characterised 
samples in future 
- 
Updated timeline & 
list of investigators 
- 
Format changes 
- 
Preliminary data Phase 1a 
- 
Trial will be conducted only in 
India 
- 
Detailed information to be 
included in SAP 
- 
Better use of well-
characterised specimens and 
reduce need for ad-hoc 
collection 
- 
Update from IRB & site 
selection completed 
- 
Changes in protocol template 
Draft 
Version 
1.0 
(Original 
Protocol) 
27-Oct-2016 
NA 
NA 
 
*Refer to Appendix 3 for Protocol Amendment History  
 

 
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List of Abbreviations and Acronyms  
Abbreviation/acronym 
Meaning 
AE 
Adverse Event 
CRF 
Case Report Form 
DMC 
Designated Microscopy Centres 
DNA 
Deoxyribonucleic acid 
DR TB 
Drug Resistant Tuberculosis 
DST 
Drug Susceptibility Testing 
GCP 
Good Clinical Practice 
GCLP 
Good Clinical Laboratory Practice 
GDP 
Good Documentation Practice 
HIV 
Human Immunodeficiency Virus 
ICF 
Informed Consent Form 
IDMC 
Independent Data-Monitoring Committee 
ICH 
International Council on Harmonisation 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
IQC 
Income Quality Check 
ISF 
Investigator Site File 
ISO 
International Organization for Standardization 
MDR TB 
Multidrug Resistant Tuberculosis 
MGIT (SIRE) 
Mycobacterial Growth Indicator Tube (Streptomycin, 
Isoniazid, Rifampicin, Ethambutol) 
MTB 
Mycobacterium tuberculosis 
NAT 
Nucleic acid tests 
QA 
Quality Assurance 
QC 
Quality Control 
QMS 
Quality Management System 
RA 
Regulatory Authority 
RBM 
Risk Based Monitoring 
RIF 
Rifampicin 

 
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RM 
Risk Management 
SAP 
Statistical Analysis Plan 
SAE 
Serious Adverse Event 
SOA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
TB 
Tuberculosis 
TMF 
Trial Master File 
UPHC 
Urban Primary Health Centre 
 
 
 
 

 
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Protocol Synopsis 
Title 
Prospective, multicentre trial to assess the diagnostic accuracy of 
the Truenat assays at intended settings of use. 
Short title  
Truenat Evaluation 
Protocol 
version and 
date 
V3.1, 05-July-2018   
Background 
and rationale 
The Truenat MTB (including both MTB and MTB plus) and the 
MTB-RIF Dx reflex assays (Molbio Diagnostics; Bangalore, India) 
utilize chip-based real-time micro PCR for detection of tuberculosis 
(TB) and rifampicin (RIF) resistance from DNA extracted from 
sputum samples in about 25 minutes. 
A pilot trial conducted in India of the Truenat MTB assay found the 
assay to achieve high clinical performance. However, further 
evidence of the Truenat MTB as well as the Truenat MTB-RIF Dx 
assay, is needed prior to recommending the clinical use of the 
assays. 
Prior studies have been conducted as part of a three-phase/stage 
approach i) a validation of the Truenat MTB assays and MTB-RIF 
Dx assay diagnostic performance on frozen samples and ii) an 
operational assessment of the assays performance. The current 
trial corresponds to iii) the evaluation trial to confirm the diagnostic 
accuracy and to ensure that the performance characteristics will be 
consistent in the sites of intended use in a geographical diverse 
population (microscopy centre level). 
Further evidence on the diagnostic performance and patient 
important outcomes on the use of the Truenat MTB assays and 
MTB-RIF Dx assay at the microscopy centre level is needed to 
inform potential policy recommendations. 
Objectives 
1. Primary objectives: to determine the diagnostic accuracy of the 
Truenat MTB assays and MTB-RIF Dx assay using culture and 
phenotypic/genotypic drug susceptibility test (DST) as gold 
standard in the intended setting of use. 
2. Secondary objectives: to determine the diagnostic accuracy of 
the Truenat MTB assays and MTB-RIF Dx assay compared to 
Xpert MTB/RIF using culture and phenotypic/genotypic DST as 
gold standard and to assess patient important outcomes. 

 
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Trial design 
Prospective, multicentre assessment of Truenat MTB assays and 
MTB-RIF Dx assay diagnostic accuracy in the intended settings of 
use.  
The sample size has been calculated at 1,666 enrolled participants. 
Sites in India will enrol n= 1,110 participants.  Other three global 
sites will enrol n = 556 participants overall. 
Trial 
sites/setting 
India and three other global sites. In India the trial will be conducted 
at 9 designated microscopy centres (DMC) and 1 private 
laboratory. 
Trial population  Adults with pulmonary tuberculosis symptoms. 
Eligibility 
criteria  
Individuals who have symptoms consistent with pulmonary TB 
presenting to participating centres will be assessed and asked to 
participate. Participants will be categorised into 2 groups:   
Case Detection group - Only participants who have not received 
any form of TB treatment within the prior 60 days will be enrolled. 
Drug Resistant group – Non-converting TB cases presumed to be 
at risk of drug-resistance. 
Primary 
outcomes 
1. Primary outcomes 
1.1. Sensitivity of the Truenat MTB assays (including analysis 
by smear-status and by sample). 
1.2. Specificity of the Truenat MTB assays (including analysis 
by sample). 
1.3. Sensitivity of the Truenat MTB-RIF Dx assay. 
1.4. Specificity of the Truenat MTB-RIF Dx assay. 
Secondary 
outcomes 
2. Secondary outcomes 
2.1. Sensitivity of the Truenat MTB assays and MTB-RIF Dx 
assay compared to Xpert MTB/RIF. 
2.2. Specificity of the Truenat MTB assays and MTB-RIF Dx 
assay compared to Xpert MTB/RIF. 
2.3. Patient important outcomes (see details in section Patient 
important outcomes under Statistical analysis plan). 
Trial duration  
Approximately 12 months 
Time schedule 
Trial start expected Q4-2018 with enrolment completion by Q2-
2019. Follow-up after 2 months and testing of discordant cases 
completed Q4-2019. 
GCP statement 
All studies sponsored by FIND will be conducted in accordance 
with ICH-GCP Guidelines (ICH-GCP E6 R2), to the extent possible 
in the research setting as well as all national legal and regulatory 
requirements (as apply). 

 
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Schedule of Activities  
Procedure 
Enrolment 
Follow-up visit 1 
Notes 
Day 1 
Day 2 
Day 56 (±14) 
 
Informed consent 
X 
 
 
 
Inclusion and exclusion criteria 
X 
 
 
 
Demography 
X 
 
 
 
Medical history (includes prior 
history of TB) 
X 
 
 
 
Sputum sample collection 
X 
X* 
 
 
Blood sample collection (optional) 
X 
 
 
HIV testing will be offered to all participants as part of the trial procedures, unless 
a prior result is available (see Section 8) 
AE/SAE review 
ß======================à 
 
Assess interval medication history  
 
X 
• Culture-negative, Truenat MTB (and/or MTB plus) and Xpert discordant cases. 
• 20% of Case Detection Group participants with negative results for all test, 
including culture 
Spot sputum (microscopy and 
culture) 
 
X# 
• Culture-negative, Truenat MTB (and/or MTB plus) and Xpert discordant cases. 
• 20% of Case Detection Group participants with negative culture 
 
* Two sputum samples are collected on both Day 1 and Day 2.  On Day 1, two spot sputa are collected.  On Day 2, firstly, a morning sputum is self-collected in a pot taken 
home on Day 1.  Secondly, an additional sputum is collected when the participant returns to the clinic to deliver the sputum pot from home. 
# An additional spot sputum will be obtained at follow-up for smear microscopy and culture if i) not on TB treatment and ii) sputum can be produced spontaneously.

 
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Version 3.3; Date 07 SEP 2018 
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1 Introduction 
The Truenat MTB, MTB plus and MTB-RIF Dx assays are a set of new tests 
developed for the diagnosis of tuberculosis (TB) and detection of resistance to 
rifampicin (RIF), a commonly used drug for treatment of active TB. 
 
 Trial Rationale 
An estimated 4.1 million tuberculosis (TB) cases go undiagnosed globally each 
year1, leading to substantial morbidity and mortality. In most countries smear 
microscopy remains the only option for the rapid diagnosis of TB, though it 
detects only 45% of infections2-6. For these reasons, new molecular TB 
diagnostics that can be instituted at the microscopy level are a research and 
implementation priority. 
One such new molecular TB diagnostic is the Truenat including the MTB, MTB 
Plus and MTB-RIF Dx assays.  Further evidence on the diagnostic performance 
and patient important outcomes on the use of the Truenat MTB assays and the 
MTB-RIF Dx assay at the microscopy centre level is needed to inform potential 
policy recommendations. 
 
 Background 
The Truenat MTB and MTB plus assays and the MTB-RIF Dx (Truenat RIF or 
RIF assay) assay (Molbio Diagnostics; Bangalore, India) utilize chip-based real-
time micro PCR for detection of TB and rifampicin (RIF) resistance from DNA 
extracted from sputum samples in about 25 minutes7. Briefly, automated DNA 
extraction is performed using the Trueprep Auto sample prep device (Figure 1). 
The extracted DNA is then added onto the Truenat MTB (or MTB plus) chip 
which is pre-loaded with stabilized reagents. Automatic amplification and 
analysis is then done using a handheld battery-operated device, the Truelab 
UnoDx real time PCR analyser. If the Truenat MTB (or MTB plus) chip is 
positive- and dependent on the diagnostic algorithm being employed- the user 
can then take another aliquot of extracted DNA and add it onto a separate 
Truenat MTB-RIF Dx  chip to detect the presence of selected mutations 
associated with rifampicin resistance. A pilot trial conducted in India of the 
Truenat MTB assay found the assay to achieve 91% sensitivity and 100% 
specificity against a composite reference standard, including 99% sensitivity for 
smear-positive, culture-positive samples and 76% sensitivity for smear-
negative, culture-positive samples8. However, further evidence of the 
performance and operational characteristics of the Truenat MTB assay, as well 
as the Truenat MTB plus (alternative version including an additional gene target 

 
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for TB detection) and RIF Reflex assay, is needed prior to recommending the 
clinical use of the assays. 
 
Figure 1. Truenat MTB assays steps. A mixture of raw sputum and 
liquefaction buffer is directly loaded onto the Truepep Auto chip interface, which 
extracts MTB DNA in 25 minutes.  The extracted DNA is transferred to the 
Truenat MTB (or Truenat MTB Plus) chip and then onto the Truelab UnoDx 
PCR machine, which detects the presence of MTB DNA, and provides and 
automated result as either MTB-detected, MTB not detected or indeterminate 
result. For MTB positive results, another aliquot of the same DNA extracted is 
then transferred (reflex) to the Truenat MTB-RIF Dx chip. 
 
To comprehensively evaluate the performance of the Truenat MTB assays and 
RIF assay, prior studies have been conducted as part of a three-phase/stage 
approach: i) a validation of the Truenat MTB assays and RIF assay diagnostic 
performance on frozen samples and ii) an operational assessment of the 
assays performance. This approach will allow for a rapid, direct assessment of 
assay performance against characterized specimens from diverse clinical sites 
(Africa, Eastern Europe and Asia) to confirm adequate performance of the 
Truenat MTB assays and RIF assay prior to the large clinical evaluation. The 
current trial corresponds to iii) the evaluation trial to confirm the diagnostic 
accuracy and to ensure that the performance characteristics will be consistent 
in the sites of intended use in a geographical diverse population (microscopy 
centre level) as well as patient important outcome will be assessed during this 
trial.  
 
 Benefit/Risk Assessment 
Knowledge gained from this trial may benefit society by improving TB diagnosis. 
Trial participants may directly benefit from the trial because they will be 
provided with a higher standard of TB diagnostic care than may be routinely 
available to them. 

 
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Given the minimal risks associated with this trial and the potential benefits to 
society and individuals, the benefits outweigh the aggregated risks. As for any 
clinical trial, there is a possibility of unknown and unforeseen risk; that 
possibility is small for this trial. If unforeseen risks are recognized during the 
trial, then FIND, trial partners, IRBs/ethics committees, and participants must 
be provided with relevant information. 
 
2 Trial Objectives and Endpoints 
Table 1. Trial objectives and endpoints 
Objectives 
Endpoints 
1. Primary 
 
 
1.1. Estimate diagnostic accuracy of the 
Truenat assays (MTB and MTB 
Plus) for Mycobacterium 
tuberculosis (MTB) detection among 
individuals undergoing evaluation 
for pulmonary TB, overall and per 
sample, separately for smear-
positive and smear-negative TB 
samples using a culture reference 
standard. 
 
1.2. Estimate diagnostic accuracy of the 
Truenat MTB-RIF Dx assay for RIF 
resistance detection among 
individuals undergoing evaluation 
for pulmonary TB and DR TB, using 
phenotypic/genotypic drug 
susceptibility testing (DST)1. 
 
1.1.1. Sensitivity of the Truenat MTB and 
MTB Plus assays (including 
analysis by smear-status and by 
sample). 
1.1.2. Specificity of the Truenat MTB and 
MTB Plus assays (including 
analysis by sample). 
 
 
 
1.2.1 Sensitivity of the Truenat MTB-RIF 
Dx assay 
 
1.2.2 Specificity of the Truenat MTB-RIF 
Dx assay 
 
2. 
Secondary 
 
 
2.1. Compare the diagnostic accuracy of 
the Truenat assays (MTB and MTB 
Plus) and MTB-RIF Dx assay to that of 
Xpert MTB/RIF, using a reference 
standard of culture for TB diagnosis 
 
2.1.1. Sensitivity of the Truenat MTB and 
MTB Plus assays and MTB-RIF Dx 
assay compared to Xpert MTB/RIF 
                                            
1 All samples that exhibit RIF resistance, as determined by either phenotypic DST or Truenat RIF, will 
be sent for DNA sequencing to determine the mutations responsible for drug resistance. A subset of 
drug sensitive samples, of approximately matching sample size, will be randomly selected for 
sequencing, to act as a comparator sequence 

 
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and phenotypic/genotypic DST for 
detection of RIF resistance. 
 
2.2. Assess patient important outcomes, 
including time to detection of TB and 
RIF resistance.  
2.1.2. Specificity of the Truenat MTB and 
MTB Plus assays and MTB-RIF Dx 
assay compared to Xpert MTB/RIF 
 
2.2.1. Patient important outcomes (see 
details in section Patient important 
outcomes under Statistical analysis 
plan) 
 
 
 
3. Trial Design 
3.1. 
General Design 
This will be a prospective, multicentre, diagnostic accuracy trial in which the 
performance of an investigational rapid molecular diagnostic test (index test) 
on sputum samples (Truenat MTB assays and RIF assay) will be assessed in 
India, using solid and liquid culture as reference standard for the diagnosis of 
TB and MGIT SIRE as reference standard for the detection of RIF resistance. 
 
3.2. 
Scientific Rationale for Trial Design 
Subjects undergoing evaluation for TB and DR will be enrolled after informed 
consent is obtained. Results of the investigational Truenat assays will not be 
used for clinical care and will not be provided to clinicians or participants.  
Laboratory technicians performing Truenat testing will be blinded to the results 
of conventional tests results.  HIV testing or information about the status will be 
obtained in order to provide estimates of test performance in the important sub-
group of HIV-infected cases with TB/DR symptoms, and also since HIV testing 
is considered standard of care for TB cases. 
 
3.3. 
End of Trial Definition 
A participant is considered to have completed the trial if he/she has completed 
the last visit.  The end of the trial is defined as the date of the last visit of the 
last participant in the trial globally. 
 
4. Trial Population and Eligibility  

 
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Trial population: Adults with presumptive tuberculosis disease. 
Trial/sample size:  Target enrolment for the multicentre trial is 1,666 
participants enrolled. 
Setting: In India, a total of 9 DMC will represent the intended settings of use 
including urban, peri-urban/hilly, tribal and rural sites with low and high 
throughput laboratories in India. Based on these characteristics the following 
numbers of presumptive TB cases, including smear-positive cases, are 
anticipated per DMC type (Table 2). 
 
Table 2. Types of designated microscopy centres in India 
S.No 
DMC type 
Nr. subjects with TB 
symptoms with sputum 
examination/month* 
% Smear-positive 
among subjects with 
TB symptoms/month* 
1 
Low volume DMC (in health 
posts/UPHC) 
40 
 (30 - 50) 
2% - 3% 
2 
High volume DMC (in UPHC) 
120 
 (100 - 150) 
6% - 9% 
3 
High volume DMC (in Tertiary 
care/hospitals for chest 
diseases or TB) 
300 
 (200 - 500) 
10% - 12% 
*Under routine conditions.  
 
Participating DMC have been selected based on the following requirements: 
o Smear microscopy must be available 
o Xpert MTB/RIF must not be available 
o At least 10 hours of electricity available (Trueprep Auto 10h per 
charge for 16 runs; Truelab UnoDx 4h per charge for 8-10 runs) 
o At least 10 sputum samples collected per week, i.e. ~2 samples per 
day  
o Have easy access to a reference laboratory with capacity to perform 
reference standard tests to ensure timely and high quality results 
 
Given the important role of the private sector in India, 1 private laboratory will 
be included in the trial which is an MDR reference hospital. 
Additionally, 3 other global sites will be setup in South America, Eastern 
Europe and Asia/Africa to achieve a wider geographic variation. These sites 
will be outpatient TB clinics at district or regional health facilities.  
In order to meet enrolment targets (n= 1,666 total enrolled participants: India 
will enrol n = 1,110; other global sites will enrol n = 556). 

 
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All sites will need to be able to attain informed consent and obtain four sputum 
samples per patient for index and reference tests (as per sample flow in 
Figure 2). Participating centres must also have sufficient space to 
accommodate the Truenat instruments. 
Location: Trial sites in India are listed in Table 3. Global sites will be 
determined among sites in South America, Eastern Europe and Asia/Africa. 
 
Table 3. List of trial sites in India 
Site name 
Location 
Madhupura Urban Health Centre, Ahmedabad 
Urban 
CHC Chhala, Ahmedabad 
Rural 
PHC Kuha, Ahmedabad 
Rural 
Kamrup Metro, Guwahati 
Hilly/Peri-urban 
Sonapur District Hospital, Guwahati 
Tribal belt 
Railway Hospital, Guwahati 
Hilly/Peri-urban 
Ayanavaram, Chennai 
Urban 
Villiwakkam, Chennai 
Peri-urban 
Thanthai Periyar, Chennai 
Urban 
PD Hinduja Hospital, Mumbai* 
Urban 
*Private sector 
Prospective approval of protocol deviations to recruitment and enrolment 
criteria, also known as protocol waivers or exemptions, is not permitted. 
 
4.1. 
Inclusion Criteria  
 
• Age 18 years or above; 
• Clinical suspicion of pulmonary TB (including cough ≥2 week and at 
least 1 other symptom typical of TB); 
• Willingness to provide 3 sputum specimens at enrolment; 
• Willingness to have a trial follow-up visit approximately 2 months after 
enrolment; 
• Provision of informed consent. 
 
Two groups of participants will be enrolled.  Namely a “Case Detection Group” 
and a “Drug Resistant Group”.   
 

 
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Case Detection Group:  Participants are eligible to be included only if 
they meet all the conditions above.  
 
Drug Resistant TB Group:  In addition to the criteria of the Case 
Detection Group, participants should also meet the following conditions: 
• Non-converting pulmonary TB cases (category I and 
category II failures)  
 
4.2. 
Exclusion Criteria 
 
Participants are excluded from the trial in case of:  
 
Case Detection Group:   
• Receipt of any dose of TB treatment within 60 days prior 
to enrolment (even if within last two days only).  
 
Drug Resistant TB Group:   
• Receipt of any dose of MDR-TB treatment within 60 days 
prior to enrolment (even if within last two days only).  
 
 
4.3. 
Lifestyle Considerations  
None 
 
4.4. 
Screen Failures  
Screen failures are defined as participants who consent to participate in the trial 
but are not subsequently entered in the trial. A minimal set of screen failure 
information is required to ensure transparent reporting of screen failure 
participants to meet the Consolidated Standards of Reporting Trials 
(CONSORT) publishing requirements and to respond to queries from regulatory 
authorities. Minimal information includes demography, screen failure details, 
eligibility criteria, and any serious adverse event (SAE). 
Early exclusions 
Incomplete sputum sample set 
Participants who have provided consent and who are enrolled, but who do not 
provide a total of 3 sputum specimens of sufficient volume will be classified as 
early exclusions; each will be removed from the trial and a new trial subject 
enrolled.  Participants who are classified as early exclusions will be referred to 
the appropriate local health service for TB evaluation and care.  If positive for 

 
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M. tuberculosis, results of conventional microbiological tests (e.g., sputum 
smear microscopy, mycobacterial cultures, and Xpert MTB/RIF) will be reported 
to appropriate local health authorities in accordance with local public health 
reporting regulations. Trial data for participants who are classified as early 
exclusions will not be used for any final analyses. 
Individuals who do not meet the criteria for participation in the trial (screen 
failure) may not be rescreened.   
 
 
5. Trial Intervention 
Trial Intervention is defined as any investigational intervention(s), marketed 
product(s), or medical device(s) intended to be used with a trial participant 
according to the trial protocol. Nevertheless in the current trial, the results of the 
intervention i.e. Truenat MTB, MTB Plus and RIF will not inform patient care 
decisions. 
 
5.1. 
Medical Devices 
The devices and diagnostic tests manufactured for sponsor use in this trial are: 
• TruePrep Auto 
• Truenat MTB chip 
• Truenat MTB Plus chip 
• Truenat UnoDx 
• Truenat MTB-RIF Dx chip 
Other medical devices (not manufactured by or for sponsor) to be used in this 
trial are: 
• GeneXpert IV 
• MGIT 960 
Instructions for medical device use are provided in the Manual of Procedures. 
Any medical device incidents, including those resulting from malfunctions of the 
device, must be detected, documented and reported by the investigator 
throughout the trial (see section 9 Safety and Incident Reporting). 
 
5.2. 
Preparation/Handling/Storage/Accountability 

 
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The investigational products are intended for the detection of MTB in patient 
samples as well as the detection of MTB mutations associated with resistance 
to Rifampicin. The investigational products will be strictly accounted for, 
including receipt and inventory, storage, use during the trial, and return or 
disposal, as detailed in the Manual of Procedures provided by FIND. 
Acquisition:  Procurement of the investigational products will be done through 
FIND, which will coordinate shipments from the manufacturer. It is the 
responsibility of each trial site to maintain an updated inventory of the trial 
materials and to inform FIND immediately if additional materials are required. 
Storage:  Procedures for product storage and disposal will be described in the 
Manual of Procedures. Briefly, investigational products will be checked for 
quality at the time of receipt at the site. Investigational products will be stored 
according to the manufacturer’s instructions; expired or unused investigational 
products will be destroyed with documentation. The investigator or designee 
must confirm appropriate temperature conditions have been maintained during 
transit for all devices, reagents and samples received and any discrepancies 
are reported and resolved before use of the trial intervention. 
Test handling and performance:  Testing using the investigational products 
will be performed according to the manufacturer’s instructions outlined within 
the Manual of Procedures. Only participants enrolled in the trial may have 
samples evaluated using the medical devices described herein, and only 
authorized site staff may process samples and perform the diagnostic test. All 
trial medical devices, reagents and samples must be stored in a secure, 
environmentally controlled, and monitored (manual or automated) area in 
accordance with the labelled storage conditions with access limited to the 
investigator and authorized site staff. 
The investigator, institution, or the head of the medical institution (where 
applicable) is responsible for trial intervention accountability, reconciliation, and 
record maintenance (ie, receipt, reconciliation, and final disposition records). 
Import permits:  It is expected that most countries will require import permits 
for receiving the investigational materials.  Local sites are responsible for 
making import permit applications in a timely manner.   
Quality control check for incoming shipments:  Upon arrival of each new 
shipment of assays, the sites will conduct and document an incoming quality 
check following the Manual of Procedures. New lots may only be used after this 
quality check is successfully passed. 

 
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Local procurement:  Sites are responsible for assessing their needs and 
procuring any supplies, reagents and kits needed for the trial that are locally 
available in order to include these costs in the trial budget.  
 
5.3. 
Minimization of Error and Bias 
5.3.1. Patient selection  
Spectrum bias will be avoided by enrolling a consecutive series of subjects and 
using a cross-sectional trial design. Enrolment will be based on clearly defined 
eligibility criteria, targeting patients suspected to have TB as defined by 
WHO12, thus representing future target populations. Descriptive statistics on 
patient characteristics and estimates of diagnostic accuracy stratified by site, 
smear and HIV status to further ensure the validity and generalizability of trial 
results will be provided.  
 
5.3.2. Index test 
The risk of review bias is minimal. Interpretation of results from the index tests 
does not require interpretation by the end-users and is based on pre-defined 
and automatically implemented thresholds. Further, results will become 
available and will be recorded before those of the reference standard (culture 
or DST). Where other rapid molecular assays are performed on the same 
sample, laboratory staff members will be instructed to record results 
independently of other test results. 
 
5.3.3. Reference standard  
Each participating laboratory will undergo an on-site laboratory evaluation to 
ensure standardized and high-quality performance of culture across sites (see 
also section Quality Assurance). Results from reference standard testing will 
be recorded blinded to index test results, eliminating the risk of review bias. 
Presence of MTB complex will be confirmed for all patients from a positive 
culture using MPT64 identification test and/or Line Probe Assay. 
A reference standard of MTB culture (using two MGIT and two LJ cultures) will 
be used for TB detection. For RIF resistance detection a composite reference 
standard of phenotypic and genotypic RIF DST by sequencing will be used (as 
detailed in section Case definitions). This will allow for a high confidence in the 
interpretation of Truenat RIF resistant assay results. RIF sequencing will be 
done at a central laboratory for standardisation purposes (where possible). 
 

 
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5.3.4. Flow and timing  
The planned patient and sample flow includes testing of three samples per 
patient with the index test and reference standard and there is thus little risk of 
disease progression bias. Manual mixing or splitting of decontaminated sample 
(pellet) together with random allocation of pellet aliquots (i.e. pipetting order) 
will also ensure that neither the index test nor the reference standard test are 
given an artificial advantage by virtue of being used on a higher quality sample. 
 
5.3.5. Handling of indeterminate results  
Samples for which the index test is indeterminate or cultures are contaminated 
will be excluded from the main accuracy analyses but reported separately. 
Results based on initial testing only as well as those obtained upon repeat-
testing (for indeterminate results, where possible) will be presented. 
 
5.3.6. Blinding Procedure 
This is an open-label trial. The results of the Truenat MTB and RIF assays are 
generated automatically. Nevertheless, there is a potential bias of laboratory 
tests that are subject to operator’s interpretation such as smear microscopy and 
LJ culture.  
• Different operators will be assigned to Xpert MTB/RIF and smear 
microscopy. Similarly for MGIT and LJ culture. 
• Technicians will be instructed to record results independently of other 
test results. 
 
6. Discontinuation of Trial Intervention and Participant 
Discontinuation/Withdrawal  
 
6.1. 
Participant Discontinuation/Withdrawal from the Trial  
• A participant may be withdrawn at any time at the discretion of the 
investigator for safety, behavioural, compliance, or administrative reasons. 
• Participants who withdraw will be referred to the appropriate local health 
service for TB evaluation and care. If positivity for MTB is confirmed 
(through sputum smear microscopy, mycobacterial cultures or Xpert 
MTB/RIF), results will be reported to appropriate local health authorities in 
accordance with local public health reporting regulations. 

 
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• If the participant withdraws consent for disclosure of future information, 
FIND may retain and continue to use any data collected before such a 
withdrawal of consent. 
• If a participant withdraws from the trial, he/she may request destruction of 
any samples taken and not tested, and the investigator must document this 
in the site trial records. 
• See Manual of Procedures for data to be collected at the time of trial 
discontinuation and follow-up. 
 
6.2. 
Lost to Follow Up 
A participant will be considered lost to follow-up if he or she repeatedly fails to 
return for scheduled visits and is unable to be contacted by the trial site.  
The following actions must be taken if a participant fails to return to the clinic 
for a required trial visit: 
• The site must attempt to contact the participant and reschedule the missed 
visit as soon as possible and counsel the participant on the importance of 
maintaining the assigned visit schedule and ascertain whether or not the 
participant wishes to and/or should continue in the trial. 
• Before a participant is deemed lost to follow up, the investigator or designee 
must make every effort to regain contact with the participant. These contact 
attempts should be documented in the participant’s medical record. 
• Should the participant continue to be unreachable after 3 attempts at 
different time points, he/she will be considered to have withdrawn from the 
trial.  
 
7. Trial Procedures 
• Trial procedures and their timing are summarized in the SOA. Protocol 
waivers or exemptions are not allowed. 
• Adherence to the trial design requirements, including those specified in the 
SOA, is essential and required for trial conduct. 
• Eligibility criteria must be completed and reviewed to confirm that potential 
participants meet the requirements. The investigator will maintain a 
screening log to record details of all participants screened and to confirm 
eligibility or record reasons for screening failure, as applicable.  
• Procedures conducted as part of the participant’s routine clinical 
management (e.g. chest X-ray) and obtained before signing of the ICF may 
be utilized for screening or baseline purposes provided the procedures met 
the protocol-specified criteria and were performed within the time frame 
defined in the SOA. 

 
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7.1. 
Trial Workflow 
Following initial screening, individuals will be referred to trial personnel for 
additional information and to review eligibility (if initial assessment was done by 
non-trial clinicians). If appropriate, samples will be obtained following the 
informed consent process. Information on signs/symptoms (duration of cough, 
fever, haemoptysis, night sweats and weight loss) and TB history (previous TB 
episodes, dates of treatment, treatment outcome) will be obtained. Information 
on TB history will be critical given the potential for molecular tests to detect DNA 
from nonviable, non-intact bacilli in patients with recent history of TB10. 
 
7.2. 
Efficacy Assessments 
See section 10.4 Efficacy Analyses. 
 
7.3. 
Safety Assessments 
See Section 9, Safety and Incident Reporting. 
 
7.4. 
Health Economics 
The data collected may be used to conduct exploratory economic analyses and 
a specific study protocol will be prepared. 
 
8. Specimen Collection, Handling, Transport and Storage 
Participants will be asked to provide a total of four sputum samples at enrolment 
and over 2 days. The intent is for all samples to be collected before the participant 
starts any form of TB treatment, unless the participant is part of the Drug Resistant 
TB group.  
Each specimen should be of 2 ml or greater in volume.  On Day 1, each participant 
will be asked to submit two spot sputa (S1 and S2, approximately 30-60min apart) 
after enrolment.  Participants will be given a labelled sputum pot and instructions 
for use, and instructed to collect a second sputum (S3) the next morning (Day 2) 
before going to the clinic. At the clinic participants will be asked to provide a third 
spot sputum (S4).  In the event that a participant fails to return on Day 2, S3 and 
S4 may be collected a maximum of 7 days after enrolment, provided that no TB 
treatment has been initiated (the Case Detection group). 

 
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HIV testing will be offered to all participants as part of the trial procedures unless 
any one or more of the following are present:  
• written results of a positive HIV antibody test; 
• written results of a positive HIV viral load; 
• documentation in the medical record of positive HIV status by a treating 
clinician; 
• immediate/verifiable documentation of HIV negativity within the preceding 
one month.  
HIV testing can be performed using any test method approved by local health 
authorities.  Depending on the test method used, this test will require approximately 
1 ml of saliva (rapid oral test), or up to approximately 5 ml of blood. Participants 
refusing to undergo HIV testing will not be excluded from the trial. 
 
8.1. 
Reference Standard Test and Index Test Procedures 
At each site, as per laboratory flow (Figure 2), standard diagnostic algorithms 
will be followed according to national guidelines and policies of each 
participating country.  If national guidelines cannot be fulfilled using study-
specific samples and test results, then an additional spot sputum should be 
collected.   
 
Figure 2. Laboratory workflow 
 
Report conventional results back to Clinic  
NALC-NaOH
Decontamination
Sputum 2
Day 1 (spot & spot)
500µl
Resuspend pellet in 
2ml PBS (final vol)
MGIT SIRE
Store: leftover sputum, pellet, ELUTE , 
MTB+ and NTM+ culture isolates; 
Store: positive chips and cartridges
Truenat MTB 
& MTB plus
Day 2 (morning & spot)
200µl
MGIT
LJ
Xpert
500µl
Sputum 3
500µl
MGIT SIRE
200µl
MGIT
LJ
Store: leftover sputum, pellet, ELUTE , 
MTB+ and NTM+ culture isolates; 
Store: positive chips and cartridges
Reference laboratory
Truenat RIF
If MTB-pos
Microscopy 
centre
smear
Testing of discrepant cases
Testing of discrepant cases
≥2 ml
Resuspend pellet in 
2 ml PBS (final vol)
Sites:  All
Microscopy 
centre
If MTB-pos
If MTB-pos
≥2 ml
Reference laboratory
* Sputum 4 not required in PNG or PD Hinduja Hospital
Sputum 4*
≥2 ml
NALC-NaOH
Decontamination
Sputum 1
Xpert
Truenat RIF
If MTB-pos
1ml raw
≥2 ml
Truenat MTB 
& MTB plus
Truenat MTB 
& MTB plus
Truenat RIF
If MTB-pos
Use 500µl 
for Truenat
POOL SPUTA AND HOMGENIZE
1.5ml
2ml
500µl
500µl
smear

 
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Study spot Sputum 1 (S1) will be collected on Day 1. Approximately 30-60 
minutes later, a second spot sputum (S2) will be collected and sent directly to 
the reference laboratory.  
Participants will be given a labelled sputum pot and instructions on how to use 
it to collect an early-morning sputum (S3), and asked to bring this back to the 
clinic the next day (Day 2), and provide another spot sputum (S4). 
 
On Day 1, smear microscopy will be performed on Sputum S1 and S2 in the 
reference laboratory or, if required, in the microscopy centre.  In the reference 
lab, S1 and S2 will be pooled together and homogenised using glass beads 
and a vortex. Homgenised sputa will be further split:  1.5ml will be used for 
analysis on raw sputa, and at least 2ml used for NALC-NaOH decontamination. 
Briefly, DNA will be extracted independently from i) raw sputum and ii) 
decontaminated pellet, by the Trueprep Auto device and tested by both the 
Truenat MTB and the MTB Plus chips, both of which are read by the Truelab 
UnoDx real-time PCR analyser.  All DNA samples testing positive by the MTB 
assay will be subsequently tested by the Truenat MTB-RIF Dx assay (reflex) 
which is also read by Truelab UnoDx analyser.  
Xpert assays will be performed on the same raw and decontaminated samples. 
MGIT and LJ culture will be performed on only on the decontaminated sample. 
 
On Day 2, morning sputum S3 will be returned to the clinic in a labelled sputum 
pot and the participant will be asked to provide spot sputum S4.  S3 wil be sent 
to the reference laboratory and a second round of MGIT and LJ culture will be 
performed on decontaminated sample. As for S1 and S2, each positive culture 
will be identified for MTB complex using MPT64 identification test and/or Line 
Probe Assay. MGIT SIRE will be used to determine the phenotypic DST for RIF. 
All samples that exhibit RIF resistance, as determined by either phenotypic DST 
or Truenat RIF, will be sent for DNA sequencing to determine the mutations 
responsible for drug resistance.  A subset of drug sensitive samples, of 
approximately matching sample size, will be randomly selected for sequencing, 
to act as a comparator sequence. 
 
Spot sputum S4 will be processed in the microscopy centre:  500ul raw sputum 
will be used for DNA extraction by Trueprep Auto and MTB detection by the 
Truenat assays.  Any MTB-positive samples will be subsequently tested by the 
Truenat MTB-RIF Dx assay (reflex).   
The intended objective is to test the Truenat assay in the setting of use (i.e. 
microscopy centre).  If the microscopy centre has an Xpert machine (or if the 

 
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microscopy centre and the reference lab are the same), then the Truenat assay 
need only be performed once along side Xpert (on Day 1). 
 
All leftover pellet, eluate and culture isolates will be stored as per the Manual 
of Procedures for further testing in case of discordant results e.g. result of 
Truenat RIF differs from that of phenotypic DST or Xpert MTB/RIF.   
Importantly, laboratory technicians performing smear microscopy will be 
blinded to the results of Truenat MTB assays and other conventional test 
results. Moreover, the results of the Truenat MTB assays and Truenat RIF will 
not be reported back to the clinic i.e. will not be used for patient management. 
 
8.2. Trial supplies and incoming quality check 
• A list of materials for Truenat MTB and RIF testing is provided in Appendix 
1. 
• Income Quality Check (IQC) procedures are described in the Manual of 
Procedures. This will be conducted upon arrival of each new shipment of 
assays. New lots may only be used after this quality check is successfully 
passed. 
 
8.3. 
Other Trial Procedures 
8.3.1. Assessment of patient important outcomes 
The following will be assessed as patient important outcomes using the 
Truenat MTB assays and RIF assay compared to routine tests 
• Time to detection of TB and susceptibility to RIF. 
• Yield over smear microscopy and Xpert MTB/RIF. 
• Upfront diagnosis of RIF (reflex on all Truenat MTB or MTB Plus 
positive versus current standard of smear-positive reflexed to 
Xpert MTB/RIF based on risk factors). 
See Patient important outcomes under Statistical analysis plan. 
8.3.2. Participant follow-up 
A follow-up visit at Day 56 (+/- 14 days) post-enrolment will be conducted 
to a subset of participants in order to collect additional information on the 
TB status. 
• Culture-negative, Truenat MTB (and/or MTB plus) and Xpert 
discordant cases. During the prospective assessment, Truenat 
results will not be provided to clinicians or participants or used for 

 
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decision-making. Thus participants who are Xpert-negative but 
Truenat-positive at enrolment (“discordant”) will not be treated on 
the basis of Truenat results. All culture negative cases with 
discrepant Truenat and Xpert results will undergo a follow-up visit 
performed at Day 56 (+/-14) post-enrolment to assess: 
o Interval medication history, including TB treatment and 
clinical evolution. 
o An additional spot sputum will be obtained for smear 
microscopy and culture (LJ and MGIT) provided the patient 
was not started on therapy and is able to provide a 
spontaneously produced sputum sample. 
This will aid in the identification of patients who would be diagnosed 
(in the absence of Truenat MTB assays being available for decision 
making) on clinical grounds. 
• Cases Detection group with negative results. The first 267 
patients who are negative on all tests (approximately 20%) will be 
followed at Day 56 (+/-14) post-enrolment to assess: 
o Interval medication history, including TB treatment and 
clinical evolution 
o An additional spot sputum will be obtained for smear 
microscopy and culture (LJ and MGIT) provided the 
pulmonary symptoms persist and the patient was not started 
on therapy 
The purpose of this follow-up visit is to identify the participants in this 
subset who are diagnosed/initiated on treatment on clinical grounds 
and those that are missed completely. 
A follow-up visit is not required for participants who are started on 
treatment (based on Xpert or culture results). 
8.3.3. Analysis of discordant results 
Given that the sensitivity and specificity of culture and culture-based DST for 
Rifampicin is not perfect, misclassification of samples by these reference 
standards may introduce bias into the sensitivity and specificity estimates for 
the Truenat assays and underestimate their diagnostic performance. 
In order to address this bias, cases with discordant results (and a subset of 
cases with concordant results) will be further assessed following a Manual of 
Procedures and described separately in the final trial report. The same number 
of non-discordant cases will be tested to avoid introduction of additional bias.  
8.4. 
Genetics 

 
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Besides sequencing of M. tuberculosis to identify mutations recognized to be 
associated with drug resistance, no other genetic tests are evaluated in this 
trial. 
8.5. 
 Biomarkers 
Biomarkers are not evaluated in this trial. 
 
9. Safety and Incident Reporting 
Given the nature of this trial i.e. diagnostic accuracy and comparison of diagnostic 
solutions, the probability of an AE and SAE to be associated with the investigational 
products is exceedingly rare. 
Safety reporting is therefore limited in scope to events associated with the 
collection of samples and those that occur at participating laboratories using the 
investigational medical device i.e. TruePrep Auto and Truenat UnoDx. For the 
purposes of this trial only fatal SAEs and medical device incidents fulfilling the 
definition of AE or SAE (see section 9.2 Medical Device Incidents) will be reported. 
The definitions can be found in Appendices 2 and 3. 
The investigator and any qualified designees are responsible for detecting, 
documenting, and recording events that meet the definition of an AE or SAE and 
remain responsible for following up all AEs that are serious, considered related to 
the trial intervention or trial procedures, or that caused the participant to 
discontinue the trial. If the AE meets the definition of a SAE it must be reported to 
FIND regardless if associated with and of the investigational diagnostic devices. 
 
9.1. 
Time Period for Collecting SAE Information  
Relevant AE or SAEs will be collected from the signing of the informed consent 
form (ICF) until the follow-up visit. 
Fatal SAEs will be recorded and reported to FIND or designee within 24 hours, 
as indicated in Appendix 2. The investigator will submit any updated SAE data to 
FIND within 24 hours of it being available. 
Investigators are not obligated to actively seek SAEs after conclusion of the trial 
participation. 

 
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The method of recording, evaluating, and assessing causality of the SAE and the 
procedures for completing and transmitting SAE reports are provided in 
Appendix 2. 
9.1.1. Reporting and Follow up of SAEs 
Further information on follow-up procedures is given in Appendix 2. 
9.1.2. Regulatory Reporting Requirements for SAEs 
• Prompt notification by the investigator to FIND of a fatal SAE is essential so 
that legal obligations and ethical responsibilities towards the safety of 
participants and investigational medical device operators are met.  
• FIND has a legal responsibility to notify both the local regulatory authority 
and other regulatory agencies about the safety of a trial intervention under 
clinical investigation. FIND will comply with country-specific regulatory 
requirements relating to safety reporting to the regulatory authority, 
Institutional Review Boards (IRB)/Independent Ethics Committees (IEC), 
and investigators. 
• An investigator who receives an investigator safety report describing a fatal 
SAE or other specific safety information (e.g. summary or listing of SAEs) 
from FIND will review and then file it in the ISF and will notify the IRB/IEC, 
if appropriate according to local requirements. 
9.2. 
Medical Device Incidents (including Malfunctions) 
Medical devices are being provided for use in this trial for the purpose of 
evaluating diagnosis of TB and detecting RIF resistance. In order to fulfil 
regulatory reporting obligations worldwide, the investigator is responsible for 
the detection and documentation of events meeting the definitions of incident 
or malfunction that occur during the trial with such devices.  
The definition of a Medical Device Incident can be found in Appendix 3. 
NOTE: Incidents fulfilling the definition of an AE/SAE (e.g. any event that 
occurs as a result of insufficient or inadequate instructions for use, 
installation, or operation, or any malfunction of the investigational device, as 
well as any event resulting from operator error(s), intentional misuse of the 
investigational medical device or protocol violation) will follow the processes 
outlined above and in Appendix 3 of the protocol. 

 
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9.2.1. Time Period for Detecting Medical Device Incidents 
Medical device incidents or malfunctions of the device that result in an 
incident will be detected, documented, and reported during all periods of 
the trial in which the medical device is used.  
Medical device incidents meeting the definition of AE or SAE will be 
reported to FIND or designee within 24 hours, as indicated in Appendix 
3. The investigator will submit any updated SAE data to FIND within 
24 hours of it being available. 
If the investigator learns of any incident at any time after a participant 
has been discharged from the trial, and such incident is considered 
reasonably related to a medical device provided for the trial, the 
investigator will promptly notify FIND. 
The method of documenting Medical Device Incidents is provided in 
Appendix 3. 
9.2.2. Follow-up of Medical Device Incidents 
• All medical device incidents involving an AE will be followed and 
reported in the same manner as other AEs (see Appendix 2).This 
applies to all participants, including those who discontinue trial 
intervention as well as medical device operators. 
• The investigator is responsible for ensuring that follow-up includes 
any supplemental investigations as indicated to elucidate the nature 
and/or causality of the incident. 
• New or updated information will be recorded on the originally 
completed form with all changes signed and dated by the 
investigator. 
9.2.3. Reporting of Medical Device Incidents to Sponsor 
• 
Medical device incidents will be reported to FIND within 24 hours 
after the investigator determines that the event meets the protocol 
definition of a medical device incident. 
• 
The Medical Device Incident Report Form will be sent to FIND by 
email. 
• 
The same individual will be the contact for the receipt of medical 
device reports and SAE. 

 
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9.2.4. Regulatory Reporting Requirements for Medical Device 
Incidents 
• 
The investigator will promptly report all incidents occurring with any 
medical device provided for use in the trial in order for FIND to fulfil 
the legal responsibility to notify appropriate regulatory authorities 
and other entities about certain safety information relating to medical 
devices being used in clinical studies. 
• 
The investigator, or responsible person according to local 
requirements (e.g. the head of the medical institution), will comply 
with the applicable local regulatory requirements relating to the 
reporting of incidents to the IRB/IEC. 
 
 
10. 
Statistical Considerations  
This trial is designed to assess diagnostic accuracy of the index test compared to 
culture reference standard, and to assess the diagnostic accuracy of RIF 
resistance detection compared to a reference standard of phenotypic drug 
susceptibility testing. Further this trial will compare diagnostic accuracy and RIF 
detection of the index tests to that of Xpert MTB/RIF. As such, the trial has been 
designed to capture a sufficient number of smear negative TB cases to be able to 
provide estimates of sensitivity with a confidence interval width of approximately 
20%.  
This section provides an overview of the data analysis strategy and methodology. 
A detailed Statistical Analysis Plan (SAP) will be written before the start of 
recruitment. 
10.1. 
Statistical Hypotheses   
Primary endpoint 1.1 is to assess the diagnostic accuracy of the Truenat MTB 
assays for MTB detection among individuals undergoing evaluation for 
pulmonary TB, overall and per sample, separately for smear-positive and 
smear-negative TB samples using a culture reference standard. 
Primary endpoint 1.2 will estimate diagnostic accuracy of the Truenat RIF 
assay for RIF resistance detection among individuals undergoing evaluation 
for pulmonary TB and DR TB, using a reference standard of 
phenotypic/genotypic DST i.e. MGIT SIRE and sequencing for RIF resistance. 
Secondary endpoints will compare the diagnostic accuracy of the Truenat MTB 
assays and RIF assay to that of Xpert MTB/RIF, using a reference standard of 
culture for TB diagnosis and MGIT SIRE and sequencing for detection of RIF 

 
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resistance.  Additionally, the trial will assess patient important outcomes, 
including: i) time to detection of TB and RIF resistance, ii) yield over smear 
microscopy and Xpert MTB/RIF and iii) upfront diagnosis of RIF (reflex testing 
on all Truenat MTB or MTB Plus positive versus current standard of smear-
positive reflexed to Xpert MTB/RIF based on risk factors).  
10.2. 
Sample Size Determination 
The sample size was chosen to achieve high confidence in the accuracy 
estimates for MTB-detection and RIF resistance detection for the overall multi-
country trial for which the Indian trial is one of the countries.  
Based on an expected sensitivity of Truenat MTB plus for detection of TB 
among smear-negative/culture-positive cases of 67% (based on preliminary 
data), 80 smear-negative/culture-positive cases would be required to achieve a 
total width of the 95% confidence interval of 20% (95%CI: 57 to 77). Assuming 
a TB prevalence of 20% and a prevalence of smear-negative/culture-positive 
cases among TB cases of 30%, the total number of subjects to be enrolled 
would be 1,333. To account for losses, this is inflated by 20%, yielding a final 
sample size of 1,666 participants under investigation for TB overall. 
In India, two thirds of these trial participants will be recruited i.e. n = 1,110 thus 
67 smear-negative/culture-positive cases and a 95% confidence interval of 
21% could be achieved (95%CI 55 to 79). 
The other one-third of enrolled participants (n = 556) will be recruited in three 
other countries in order to provide geographic variation. 
The secondary objective of determining diagnostic accuracy of RIF resistance 
by Truenat MTB is based upon an expected Truenat RIF sensitivity of 95% with 
a confidence interval of 10% (90-100%), requiring n=37 RIF-resistant 
participants detected.  Assuming a prevalence of 20% culture-positive TB cases 
detected across all presumed TB cases, and 2.8% RIF resistance amongst all 
culture-positive TB cases and 12% prevalence of RIF resistance amongst TB 
retreatment cases, we predict 1,542 re-treatment patients would thus need to 
be enrolled.  While the prevalence of culture-positive TB cases may be higher 
if enrolment is conducted at a drug-resistance TB referral clinic, we 
conservatively accept that this may not be the case.  As such, we may not reach 
sufficient sample size to allow analysis of secondary objectives in this trial. 
Detection of RIF resistance will be continually monitored throughout the trial, 
and any possible shortfall will be supplemented with a future sub-study, if 
needed, using confirmed RIF-resistant sputum samples from the FIND 
specimen bank of cryopreserved samples.  We will place an enrolment cap of 
200 enrolled participants in the drug resistance group for the entire trial, in order 
to not undermine the primary objective of enrolment of smear-negative culture 

 
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positive TB cases through inadvertent over-enrolment of DR cases who are 
more likely to be smear-positive culture-positive.       
10.3. 
Populations for Analyses 
For purposes of analysis, the following participant populations are defined: 
Table 4. Populations for analyses 
Population 
Description 
Enrolled  
All participants who sign the ICF 
Evaluable 
All participants with a culture positive or culture negative results.  
Contaminated cultures will be excluded.  Contamination is defined as 
below: 
Smear-positive 
≥ 1 positive smear (inclusive of scanty positive smears). 
Smear positives with only negative or contaminated cultures will be 
excluded from analysis. 
Culture-positive  
≥ 1 LJ and/or MGIT culture growth confirmed MTB complex. 
Cross-Contamination: A single LJ culture with ≤ 20 colonies or a single 
MGIT culture with MTB growth ≥28 days per patient will be excluded from 
analysis. 
Culture-negative 
At least 2 LJ or MGIT have no culture growth after >56 days and >42 days 
Contaminated 
culture 
LJ: Cultures completely overgrown by bacterial or fungal contaminations 
within 3 weeks (discarded). In case of mixed cultures, isolated MTB 
colonies transferred to new LJ tube (repeat culture). 
MGIT: Instrument positivity tests for confirmation of MTB complex 
negative.  
2 contaminated cultures will lead to exclusion of this patient from analysis 
unless other criteria for culture-positivity/negativity are met 
Xpert-positive 
MTB positive on Xpert® MTB/RIF. 
Xpert-negative 
MTB negative on Xpert® MTB/RIF. 
Xpert-indeterminate  Any indeterminate, error, or inability to produce a result from a single 
Xpert® MTB/RIF run. 
Non-TB case 
Smear-negative, Xpert-negative and culture-negative and not started on 
TB treatment on the basis of clinical criteria. 
NTM: Specimens with growth of mycobacteria other than MTB complex 
only. 
Clinical TB case 
Any participant who tests smear-negative, Xpert-negative, culture-negative 
but is started on TB treatment on the basis of clinical criteria. 

 
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Phenotypic RIF 
resistant 
Culture-positive and growth for RIF on MGIT SIRE DST testing.  
Phenotypic RIF 
sensitive 
Culture-positive and no growth for RIF on MGIT SIRE DST testing 
Genotypic RIF 
resistant 
Sequencing identifies mutations recognized to be associated with RIF-
resistance (defined based on consultation with WHO prior to analysis) 
Genotypic  RIF 
sensitive 
Sequencing identifies no mutations recognized to be associated with RIF-
resistance (defined based on consultation with WHO prior to analysis) 
Composite 
reference standard 
RIF resistant 
If phenotypic DST shows sensitivity but sequencing identifies mutations 
recognized to be associated with resistance, the composite reference 
standard will be considered RIF-resistant. 
If phenotypic DST shows resistance but sequencing does not identify 
mutations to be associated with resistance, the composite reference 
standard will be considered RIF-resistant (as mutations will be assumed 
outside of the region sequenced). 
Safety 
All participants assigned to trial intervention. 
 
 
10.4. 
Statistical Analysis Plan 
The statistical analysis plan (SAP) will be developed and finalized before 
database lock and will describe the participant populations to be included in 
the analyses, and procedures for accounting for missing, unused, and spurious 
data. This section is a high-level summary of the planned statistical analyses 
of the primary and secondary endpoints.  
 
10.5. 
Efficacy Analyses 
Table 5. Statistical analysis methods 
Endpoint 
Statistical Analysis Methods 
Primary 
Sensitivity and specificity, and ANOVA comparison of Likelihood Ratio 
test.   
Secondary 
Difference in sensitivity and specificity between each index assay and 
Xpert with 95%CI around the difference using methods appropriate for 
paired data (e.g. Tango score CI) 
 
10.5.1. General Approach 
For each test, estimates of sensitivity and specificity will be derived together with 
95% confidence intervals. Estimates will be calculated on the overall dataset and 
by subgroups defined below. 

 
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10.5.2. Analysis of Primary Outcomes 
Analyses of the diagnostic accuracy of the index test will be done according to 
the case definitions. Table 6 summarizes the way diagnostic tests results are 
usually reported when comparing the index test results to a reference standard. 
Based on the definitions in the table, the following values are defined: 
Sensitivity = TP / (TP + FN) 
Specificity = TN / (FP + TN) 
Table 6. Definition of classification metrics 
 
Case definition 
Test result 
 
TB 
Non-TB 
Total 
Detected 
TP 
FP 
TP + FP 
Not detected 
FN 
TN 
FN + TN 
Total 
TP + FP 
FP + TN 
All 
Abbreviations: TB=true positive, FP=false positive, FN=false negative, TN=true negative 
Only patients with uncontaminated culture results and without indeterminate test 
results will be included in the analysis of performance characteristics for MTB 
detection. Similarly, such patients will contribute to the analysis of the 
performance characteristics for RIF detection. For each point estimate, a 95% 
confidence interval will be derived based on Wilson score. Missing data and 
invalid results will be reported in the descriptive tables, and will not be imputed. 
10.5.3. Analysis of Secondary Outcomes 
• Accuracy of Truenat MTB assays and RIF assay compared to Xpert 
MTB/RIF. 
The same parameters and sub-groups as for the analysis of primary 
outcomes will be determined for Xpert MTB/RIF and compared to those of 
Truenat MTB assays and RIF assay side by side. 
• Patient-important outcomes 
o The median time to detection of TB using the Truenat MTB assays will 
be determined and compared to that of smear, Xpert MTB/RIF, liquid 
and solid cultures. Furthermore, the median time to detection of RIF 
resistance using the Truenat RIF assay will be determined and 
compared to that of phenotypic DST. 
o The number of additional TB (culture-positive) cases detected by the 
Truenat MTB assays compared to smear and Xpert MTB/RIF. 
o The number of RIF-resistant cases detected using the Truenat RIF 
assay compared to those detected following the current standard of 
smear-positive reflexed to Xpert MTB/RIF. 

 
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10.5.4. Descriptive statistics  
Descriptive statistics tables will be generated to summarize the characteristics 
of the participants. The number of participants included and excluded will be 
reported. Among the included participants, the information will be broken down 
by site, gender, age group, HIV status, history of TB and enrolment group (Case 
Detection Group and Drug Resistant Group). Results will be reported either in 
absolute numbers (e.g. number of subjects in a group) or summarized by IQR, 
percentage. 
10.5.5. Additional sub-group analyses  
The trial outcomes defined will be also evaluated on the following 
subpopulations: 
• Sensitivity for MTB detection:  
o Per-patient: Truenat MTB assays on S1 and S2 against culture status 
(based on cultures from S2 and S3) 
o Per-sample processing method: Truenat MTB assays on S1 against 
culture result on S2 and Truenat MTB assays on S2 against culture 
result on S2 (same specimen) 
o By smear status  
o By HIV status (where available) 
o On unprocessed (S1) vs decontaminated specimen (S2) 
o After repetition (if result invalid) 
o Compared to Xpert MTB/RIF on S2 (based on culture from S2 and S3) 
• Specificity for MTB detection: 
o By TB history  
o By prior TB treatment (> 60 days ago) 
o Compared to Xpert MTB/RIF (based on cultures from S2 and S3) 
• Sensitivity and specificity for rifampin resistance detection:  
o Against phenotypic DST 
o Compared to Xpert MTBR/RIF on S2 (based on DST from S2 and S3) 
• Intermediate/invalid/erroneous results 
o For MTB detection 
o For RIF resistance detection 
• Quantitative ability (based on assay cycle threshold (Ct) values) 
o In comparison to culture (positivity and time to positivity, grade for solid 
culture) 
o In comparison to smear (positivity and grade) 
Additional subgroups will be defined and specified in the final SAP. 

 
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10.5.6. Planned Interim Analyses  
No interim analysis is planned.  Continued data monitoring will be performed 
(see section 12 Data handling and record keeping). 
 
10.6. 
Safety Analyses 
Not applicable. Given the nature of this trial i.e. diagnostic accuracy and 
comparison of diagnostic solutions, the probability of an AE and SAE to be 
associated with the investigational products is exceedingly rare. Therefore, no 
safety analysis will be conducted. However, AE and SAE will be reported as per 
Section 9 Safety and incident reporting.  
 
10.7. 
Independent Data Monitoring Committee (IDMC) 
Not applicable. Based on FIND internal procedures, an IDMC will be 
established only for trials where participants are at greater than minimal risk e.g. 
invasive techniques for specimen collection or use of index test results to inform 
patient care. 
 
11. 
 Regulatory and Ethical Considerations   
11.1. 
Regulatory and Ethics Approvals 
This trial will be conducted in accordance with the protocol and with the 
following: 
• Consensus ethical principles derived from international guidelines 
including the Declaration of Helsinki  
• Applicable ICH Good Clinical Practice (GCP) Guidelines 
• Applicable laws and regulations 
The protocol, protocol amendments, ICF and other relevant documents (eg, 
advertisements) must be submitted to an IRB/IEC by the investigator and 
reviewed and approved by the IRB/IEC before the trial is initiated.  
• Any amendments to the protocol will require IRB/IEC approval before 
implementation of changes made to the trial design, except for changes 
necessary to eliminate an immediate hazard to trial participants.  
• The investigator will be responsible for the following: 

 
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o Providing written summaries of the status of the trial to the IRB/IEC 
annually or more frequently in accordance with the requirements, 
policies, and procedures established by the IRB/IEC 
o Notifying the IRB/IEC of SAEs or other significant safety findings as 
required by IRB/IEC procedures 
o Providing oversight of the conduct of the trial at the site and 
adherence to requirements of ICH guidelines, the IRB/IEC, the WHO 
Good Clinical Laboratory Practice (GCLP), European regulation 
536/2014 for clinical studies (if applicable), and with applicable 
national regulations. 
 
11.2. 
Financial Disclosure 
Investigators and sub-investigators will provide FIND with sufficient, accurate 
financial information as requested to allow FIND to submit complete and 
accurate financial certification or disclosure statements to the appropriate 
regulatory authorities. Investigators are responsible for providing information on 
financial interests during the course of the trial and for 1 year after completion 
of the trial. 
 
11.3. 
Informed Consent Process 
• The investigator or his/her representative will explain the nature of the trial to 
the participant or his/her legally authorized representative and answer all 
questions regarding the trial.  
• Prospective participants must be informed that their participation is voluntary 
and will be required to sign and date a statement of informed consent that meets 
the requirements of local regulations and/or ICH guidelines where applicable, 
and the IRB/IEC or trial centre. 
• The medical record must include a statement that written informed consent was 
obtained before the participant was enrolled in the trial and the date the written 
consent was obtained. The authorized person obtaining the informed consent 
must also sign the ICF. 
• Illiterate patients must provide a thumbprint on the ICF and the ICF must be 
signed and dated by an impartial witness. 
• Participants must be re-consented to the most current version of the ICF(s) 
during their participation in the trial.  
 
11.4. 
Data Protection 

 
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• Participants will be assigned a unique identifier by FIND. Any participant 
records or datasets that are transferred to FIND will contain the identifier only; 
participant names or any information which would make the participant 
identifiable will not be transferred.  
• The participant must be informed that his/her personal trial-related data will be 
used by FIND in accordance with local data protection law. The level of 
disclosure must also be explained to the participant.  
• The participant must be informed that his/her medical records may be examined 
by Clinical Quality Assurance auditors or other authorized personnel appointed 
by FIND, by appropriate IRB/IEC members, and by inspectors from regulatory 
authorities. 
 
11.5. 
Subject Confidentiality 
The Principal Investigator will be responsible for the recording of all subject’s 
personal details, screening number and unique trial number in the Subjects 
Identification List. To ensure confidentiality, this list must be kept in a place, with 
access restricted to authorized trial staff. All laboratory specimens, including 
stored specimens, as well as trial reports, data collection tools, and 
administrative forms will be identified by a using the patient’s unique trial 
number. Names will not be used on any of these documents. All local databases 
will be secured with password-protected access systems. The Investigator must 
ensure anonymity of the patient and ensure that all documents are anonymised 
before being transmitted to FIND. 
 
11.6. 
Other ethical considerations 
11.6.1. Use of stored specimens:   
Sputum volume after processing as well as Trueprep used chips will be 
stored temporarily in case further testing is deemed to be necessary. 
Samples will be labelled with a trial identifier; the specimens will be 
linked to results of other mycobacteriology tests, the final trial diagnosis 
(e.g. non-TB case, culture-positive TB case, clinical TB case, 
Indeterminate) of the participant. Specimens will be stored temporarily 
at the sites. Further testing may be required at a Reference Laboratory 
or specialized research laboratories. An export permit will be obtained 
accordingly if required. Moreover, after trial completion leftover well-
characterised specimens could be used for future research of product 
development as long as trial participants have agreed to this during the 
initial visit. 

 
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11.6.2. Protocol deviations  
Protocol deviations occur when there is non-adherence to the Protocol 
and includes Informed Consent, enrolment, and other occurrences of 
non-adherence to the Protocol. Protocol deviations should be sent to the 
local IRB/IEC per the local IRB/IEC guidelines. Any protocol deviation 
that meets reporting requirements of the local IRB/IEC will also be 
reported with the same timeliness to the FIND project manager.  
 
11.6.3. Termination of the Trial 
Given the nature of the trial, termination due to safety or other reasons 
is not anticipated. However, should the trial be terminated, participants 
will be referred to the appropriate local health service for TB evaluation 
and care. If positive for MTB, results of conventional microbiological tests 
(i.e., sputum smear microscopy and mycobacterial cultures) will be 
reported to appropriate local health authorities in accordance with local 
public health reporting regulations. 
11.6.4. Site Performance and Data Submission 
In the event that one site does not meet enrolment targets within the trial 
timelines, the trial will go forward at the other sites and the investigators 
will proceed with data submission as per trial plan.  
 
 
12. 
Data Handling and Record Keeping 
• All participant data relating to the trial will be recorded on printed or electronic 
CRF unless transmitted to FIND or designee electronically (e.g., laboratory 
data). The investigator is responsible for verifying that data entries are accurate 
and correct by physically or electronically signing the CRF.  
• The investigator must maintain accurate documentation (source data) that 
supports the information entered in the CRF.  
• The investigator must permit trial-related monitoring, audits, IRB/IEC review, 
and regulatory agency inspections and provide direct access to source data 
documents.  
• FIND or designee is responsible for the data management of this trial including 
quality checking of the data.  

 
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• Trial monitors will perform ongoing source data verification to confirm that data 
entered into the CRF by authorized site personnel are accurate, complete, and 
verifiable from source documents; that the safety and rights of participants are 
being protected; and that the trial is being conducted in accordance with the 
currently approved protocol and any other trial agreements, ICH GCP, and all 
applicable regulatory requirements.  
12.1. 
Source Data and Source Documents 
• Source documents provide evidence for the existence of the participant and 
substantiate the integrity of the data collected. Source documents are filed at 
the investigator’s site and will include testing logs, registers, screening forms, 
laboratory forms, operating characteristics surveys, and data collected through 
direct patient interviews. 
• Data reported on the CRF or entered in the eCRF that are transcribed from 
source documents must be consistent with the source documents or the 
discrepancies must be explained. The investigator may need to request 
previous medical records or reports if available.  
• The investigator/institution should maintain adequate and accurate source 
documents and trial records that include all pertinent observations on each of 
the site’s trials subjects.  Source data should be attributable, legible, 
contemporaneous, original, accurate and complete. Changes to source data 
should be traceable, should not obscure the original entry and should be 
explained if necessary. 
 
12.2. 
Data to be captured 
Demographic data, treatment and testing records, clinical status, comparator 
and reference test results, index test results and selected operational and 
environmental data will be collected using paper and/or eCRF and transmitted 
electronically to FIND using the OpenClinica. 
 
12.3. 
Data Management 
FIND is responsible for the data management for the trial, including the setup 
of the database, programming, editing and range checking. Trial site staff will 
be responsible for entering trial data from a paper CRF into OpenClinica within 
10 working days of a participant’s visit or the availability of results.  The site will 
be provided with individual password-protected accounts to access 
OpenClinica, following a training session given by FIND. Data entry training will 
be provided by FIND, either on site or remotely sharing screen through Skype 

 
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or any other similar system. OpenClinica provides an audit trail system 
recording all data entries/changes and queries between FIND and the site.  
No information concerning the trial or the data generated from the trial will be 
released to any unauthorized third party without prior written approval of FIND 
and manufacturers. 
  
12.4. 
Data Archiving 
Records and documents, including signed ICFs, pertaining to the conduct of 
this trial must be retained by the investigator for 10 years after trial completion 
unless local regulations or institutional policies require a longer retention period. 
No records may be destroyed during the retention period without the written 
approval of FIND. No records may be transferred to another location or party 
without written notification to FIND. 
 
 
13. 
Quality Management  
Quality Control and Quality Assurance systems with written Standard Operating 
Procedures will be established to ensure that the trial is conducted and the data 
are generated, documented and reported in compliance with this protocol, good 
clinical practice (GCP), good clinical laboratory practice (GCLP) and applicable 
regulatory requirements. Data should be collected according to trial specific 
Data Management plan. 
 
13.1. 
Quality Control 
Quality control will be applied to each stage of data handling to ensure that all 
data are reliable and have been processed correctly.  
Risk Based Monitoring will be done and the trial will be monitored remotely 
(centrally) as well as on-site. Further details on monitoring visits will be included 
in the Monitoring Plan.  
 
13.2. 
Quality assurance  

 
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FIND will conduct clinical and laboratory assessments at the DMC, selected private 
laboratories and reference laboratory sites prior to trial start. On-site visits will 
include observation of routine procedures, assessment of quality management 
systems as well as review of historical data such as quality indicators (including 
but not limited to smear- and culture-positivity, smear-positive/culture-negative, 
Xpert-positive/culture-negative rates). Following the initial assessments and 
implementation of any recommendations, on-site training will be conducted by 
FIND (see section Training plan and piloting of laboratory and data collection 
procedures).  
The FIND Clinical Trial Standard will be implemented to verify the clinical and 
laboratory quality prior to initiation of the trial and the data quality, applicable 
regulatory documentation, and subject safety throughout the trial. 
The trial co-investigators are responsible for conducting routine quality assurance 
and quality control activities to internally monitor trial progress and protocol 
compliance in addition to procedures in the FIND Manual of Procedures.  The co-
investigators will ensure that all trial personnel are appropriately trained and 
applicable documentations are maintained on site.  
 
13.3. 
Training plan and piloting of laboratory and data 
collection procedures 
The principal investigators should ensure that all persons assisting with the trial 
are adequately informed about the protocol, the investigational product(s) and 
their trial-related duties and functions.  
The principal investigators are responsible for supervising any individual or 
party to whom the investigator delegates trial-related duties and functions 
conducted at the trial site.  The principal investigators should ensure this 
individual or party is qualified to perform those trial-related duties and functions 
and should implement procedures to ensure the integrity of the trial-related 
duties and functions performed and any data generated.  All key trial staff shall 
be trained and certified in GCP and certificates provided to FIND. 
On-site training on all relevant trial procedures that are not part of routine care 
will be provided by FIND to the relevant staff, i.e. the trial coordinator at each 
site, the data entry personnel, the staff at sputum collection sites and the DMC 
and reference laboratory staff. The training will include clinical and laboratory 
aspects with focus on hands-on training on the Truenat MTB assays and RIF 
assay. Proficiency to perform the Truenat MTB assays and RIF assay will be 
assessed using a proficiency testing tool designed for this trial. 

 
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13.4. 
Trial and Site Closure 
FIND designee reserves the right to close the trial site or terminate the trial at any 
time for any reason at the sole discretion of FIND. Trial sites will be closed upon 
trial completion. A trial site is considered closed when all required documents and 
trial supplies have been collected and a trial-site closure visit has been performed. 
The investigator may initiate trial-site closure at any time, provided there is 
reasonable cause and sufficient notice is given in advance of the intended 
termination. 
Reasons for the early closure of a trial site by FIND or investigator may include but 
are not limited to: 
• Failure of the investigator to comply with the protocol, the requirements of the 
IRB/IEC or local health authorities, FIND's procedures, or GCP guidelines 
• Inadequate recruitment of participants by the investigator 
• Discontinuation of further trial intervention development 
 
 
14. 
Work plan & timeline 
Trial preparation activities start with IRB approval. Trial initiation followed by 
enrolment will last for 32 weeks and details of the trial timeline are shown below.  
 
Table 7. Trial timeline 
Phase 
Activity 
Duration 
Estimated timeline 
Preparation 
IRB approval 
10 weeks 
Q3-2018 
 
Training 
3-4 days 
Q4-2018 
Implementation 
Enrolment 
32 weeks 
Q4-2018 to Q2-2019 
 
Follow-up 
8 weeks 
Q3-2019 
 
Laboratory testing (enrolment, 
FU, discordant cases) 
26 weeks 
Q4-2019 
Reporting 
Data analysis 
3 weeks 
Q4-2019 
 
Report write up 
4 weeks 
Q4-2019 
 
 

 
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15. 
 Publication Policy  
• The results of this trial may be published or presented at scientific meetings. 
Details are provided in the Trial Agreement (e.g. Memorandum of 
Understanding, Collaboration Agreement).  
• FIND will comply with the requirements for publication of trial results. In 
accordance with standard editorial and ethical practice. 
• Authorship will be determined by mutual agreement and in line with 
International Committee of Medical Journal Editors authorship requirements. 
 
 
16. 
References 
1. 
World 
Health 
Organization. 
Global 
tuberculosis 
report 
2015. 
2015 
DOI:10.12987/yale/9780300190298.001.0001. 
2 Dye C, Watt CJ, Bleed DM, Hosseini SM, Raviglione MC (2005) Evolution of 
tuberculosis control and prospects for reducing tuberculosis incidence, prevalence, 
and deaths globally. JAMA 293: 2767–2775. 
3 Bruchfeld J, Aderaye G, Palme IB, Bjorvatn B, Kallenius G, Lindquist L. Sputum 
concentration improves diagnosis of tuberculosis in a setting with a high prevalence 
of HIV. Trans R Soc Trop Med Hyg 2000; 94: 677. 
4 Perkins MD, Cunningham J. Facing the crisis: improving the diagnosis of 
tuberculosis in the HIV era. J Infect Dis 2007; 196: S15. 
5 Steingart KR, Ramsay A, Pai M. Optimizing sputum smear microscopy for the 
diagnosis of pulmonary tuberculosis. Expert Rev Anti Infect Ther 2007; 5: 3277. 
6 World 
Health 
Organization. 
Global 
Tuberculosis 
Control, 
2010. 
http://whqlibdoc.who.int/publications/2010/9789241564069_eng. pdf (accessed 
March 18, 2011). 
7   Truenat MTB: Chip-based Real Time PCR test for Mycobacterium tuberculosis. 
Package 
Insert: 
http://molbiodiagnostics.com/packinserts/Truenat%20MTB%20packinsert.pdf 
(accessed October 7, 2016). 
8 Nikam C, Jagannath M, Narayanan MM, et al. Rapid diagnosis of Mycobacterium 
tuberculosis with Truenat MTB: a near-care approach. PLoS One 2013; 8: e51121. 
9   Pai M. Promoting affordable and quality tuberculosis testing in India. J Lab 
Physicians 2013; 5(1): 1-4. 

 
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10 Theron G, Venter R, Calligaro G, et al. Xpert MTB/RIF Results in patients with 
previous tuberculosis: can we distinguish true from false positive results? Clin Infect 
Dis. 2016; 62: 8. 
11 Steingart, KR, Schiller I, Horne DJ, et al., Xpert(R) MTB/RIF assay for pulmonary 
tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev, 2014. 
1: p. CD009593. 
 
 
17. 
Appendices 
Appendix 1: Supply requirements for Truenat testing 
Appendix 2: Safety Definitions and Reporting  
Appendix 3: Incident Definitions and Reporting 
Appendix 3: Protocol Amendment Summary 
 
 

 
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Appendix 1:  Supply requirements for Truenat testing 
Item 
Trueprep Auto Sample Prep  
Truelab Uno DxAnalyzer 
Truenat MTB chip 
Truenat MTB Plus chip 
Truenat MTB-RIF Dx chip 
Additional Trueprep Supplies 
Trueprep Auto Sputum sample prep kit (cartridges, reagents) 
Additional Truenat Supplies 
Truenat Universal Control Kit 
Precision micropipettes (fixed volume) including micropipette tips 
Additional Truelab Supplies 
Truelab AC adapter 
Other Supplies not provided  
        - 70% isopropyl alcohol 
        - Gloves 
        - Paper towel 
        - Squash bottles 
        - Discard bin for waste (dry) 
        - Discard bin for waste (wet) 
        - 0.5% sodium hypochlorite solution 
        - Mask 
        - Gown 
        - Timer 
All the Molbio instruments and reagents will be supplied including package inserts and user manuals 
 
 
 

 
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Appendix 2:  Safety Definitions and Reporting 
AE Definition 
• 
An AE is any untoward medical occurrence in a patient or clinical trial participant, temporally 
associated with the use of trial intervention, whether or not considered related to the trial 
intervention. 
• 
NOTE: An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease (new or exacerbated) temporally associated with 
the use of trial intervention. 
 
SAE Definition 
a. 
Results in death 
b. 
Is life-threatening 
The term 'life-threatening' in the definition of 'serious' refers to an event in which the participant 
was at risk of death at the time of the event. It does not refer to an event, which hypothetically 
might have caused death, if it were more severe. 
c. 
Requires inpatient hospitalization or prolongation of existing hospitalization 
In general, hospitalization signifies that the participant has been detained (usually involving at 
least an overnight stay) at the hospital or emergency ward for observation and/or treatment that 
would not have been appropriate in the physician’s office or outpatient setting. Complications that 
occur during hospitalization are AEs. If a complication prolongs hospitalization or fulfils any other 
serious criteria, the event is serious. When in doubt as to whether “hospitalization” occurred or 
was necessary, the AE should be considered serious. 
Hospitalization for elective treatment of a pre-existing condition that did not worsen from baseline 
is not considered an AE. 
d. 
Results in persistent disability/incapacity 
• 
The term disability means a substantial disruption of a person’s ability to conduct normal life 
functions. 
• 
This definition is not intended to include experiences of relatively minor medical significance 
such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, and accidental 
trauma (eg, sprained ankle) which may interfere with or prevent everyday life functions but 
do not constitute a substantial disruption. 
e. 
Is a congenital anomaly/birth defect 
f. 
Other situations: 
• 
Medical or scientific judgment should be exercised in deciding whether SAE reporting is 
appropriate in other situations such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize the 
participant or may require medical or surgical intervention to prevent one of the other 
outcomes listed in the above definition. These events should usually be considered serious. 

 
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Examples of such events include intensive treatment in an emergency room or at home for 
allergic bronchospasm, blood dyscrasias or convulsions that do not result in hospitalization, 
or development of drug dependency or drug abuse. 
 
SAE Reporting to FIND via an Electronic Data Collection Tool 
• 
The primary mechanism for reporting an SAE to FIND will be the electronic data collection 
tool. 
• 
If the electronic system is unavailable for more than 24 hours, then the site will use the paper 
SAE data collection tool (see next section). 
• 
The site will enter the SAE data into the electronic system as soon as it becomes available. 
• 
After the trial is completed at a given site, the electronic data collection tool will be taken off-
line to prevent the entry of new data or changes to existing data. 
• 
If a site receives a report of a new SAE from a trial participant or receives updated data on a 
previously reported SAE after the electronic data collection tool has been taken off-line, then 
the site can report this information on a paper SAE form (see next section) or to the 
[X/medical monitor/SAE coordinator] by telephone. 
• 
Contacts for SAE reporting can be found in the Manual of Procedures. 
 
SAE Reporting to FIND via Paper CRF 
• 
E-mail transmission of the SAE paper CRF is the preferred method to transmit this 
information to FIND. 
• 
In rare circumstances and in the absence of facsimile equipment, notification by telephone is 
acceptable with a copy of the SAE data collection tool sent by overnight mail or courier 
service. 
• 
Initial notification via telephone does not replace the need for the investigator to complete 
and sign the SAE CRF pages within the designated reporting time frames. 
• 
Contacts for SAE reporting can be found in the Manual of Procedures. 
 
 
 
 

 
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Appendix 3:  Incident Definition and Reporting 
Medical Device Incident Definition 
• 
A medical device incident is any malfunction or deterioration in the characteristics and/or 
performance of a device as well as any inadequacy in the labeling or the instructions for use 
which, directly or indirectly, might lead to or might have led to the death of a 
participant/user/other person or to a serious deterioration in his/her state of health. 
• 
Not all incidents lead to death or serious deterioration in health. The nonoccurrence of such a 
result might have been due to other fortunate circumstances or to the intervention of health 
care personnel. 
 
It is sufficient that: 
• An incident associated with a device happened. 
 
AND 
 
• The incident was such that, if it occurred again, might lead to death or a 
serious deterioration in health. 
A serious deterioration in state of health can include any of the following: 
• Life-threatening illness 
• Permanent impairment of body function or permanent damage to body 
structure 
• Condition necessitating medical or surgical intervention to prevent one of the 
above  
• Foetal distress, foetal death, or any congenital abnormality or birth defects 
 
Medical Device Incident Documenting 
• 
Any medical device incident occurring during the trial will be documented in the participant’s 
medical records, in accordance with the investigator’s normal clinical practice, and on the 
appropriate form of the CRF. 
• 
For incidents fulfilling the definition of an AE or an SAE, the appropriate AE/SAE CRF page will 
be completed as described in Appendix 3. 
• 
The CRF will be completed as thoroughly as possible and signed by the investigator before 
transmittal to FIND or designee. 
• 
It is very important that the investigator provides his/her assessment of causality (relationship 
to the medical device provided by FIND) at the time of the initial SAE report and describes any 
corrective or remedial actions taken to prevent recurrence of the incident. 
• 
A remedial action is any action other than routine maintenance or servicing of a medical device 
where such action is necessary to prevent recurrence of an incident. This includes any 
amendment to the device design to prevent recurrence. 
 
 

 
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Appendix 4: Protocol Amendment Summary  
 
The Protocol Amendment Summary of Changes Table is located directly before the 
Table of Contents (TOC). 
Amendment Number 1 (India only).   
 
Overall Rationale for the Amendment 
Since the first submission of the trial protocol to the Ethics Committee in India, updates 
were needed given that: i) new results on Truenat became available, ii) further 
assessment of trial risks and revision of mitigation strategies including enrolment 
targets to determine the performance of the Truenat MTB-RIF Dx assay, adoption of 
the Common Protocol Template (TransCelerate). 
 

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