Multicentre Clinical Trial to Assess the Performance of the Xpert MTB/XDR Assay for INH- and Second-line Resistance Detection
Conditions: Tuberculosis, Pulmonary, Tuberculosis, Multidrug-Resistant
Sponsor: Foundation for Innovative New Diagnostics, Switzerland

                                                                                                             CONFIDENTIAL 
 
Xpert MTB/XDR Clinical Evaluation 
Protocol #1.1  
 
Version [1.1]; Date [05 NOV 2018] 
[Page 1 of 55]  
 
 
STUDY PROTOCOL 
 
Phase II: Multicentre Clinical Study to 
Assess the Performance of the Xpert 
MTB/XDR Assay for INH- and Second-
line Resistance Detection 
 
Xpert MTB/XDR Clinical Evaluation   
 
Version: v1.1 
 
Date: 05 NOV 2018 
 
Sponsor Name: FIND 
 
Funder: FIND (KFW & NL) 
 
Disease Programme: TB 
 
Regulatory Agency Identifying Number(s):  
Clinicaltrials.gov Registration Number: 
NCT03728725 
 
 
 
Foundation for Innovative New Diagnostics 
Campus Biotech 
Chemin des Mines 9 
1202 Geneva 
Switzerland 
T: +41 (0)22 710 05 90 
F: +41 (0)22 710 05 99 
 
 
 
 

 
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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The information contained in this document, especially unpublished data, is the property 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.  

 
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Table of Contents 
Institutions/Partners Involved in the Trial .......................................................................... 6 
Signature Page (Sponsor) ................................................................................................... 8 
Statement of Hinduja Hospital and Medical Research Centre Principal Investigator ......... 10 
Statement of Phthisiopneumology Institute Principal Investigator ................................... 11 
Statement of Wits Health Consortium Principal Investigator ............................................ 12 
Statement of National Institute of Tuberculosis and Respiratory Diseases Principal 
Investigator ..................................................................................................................... 13 
Protocol History/Amendment Summary* ........................................................................ 14 
List of Abbreviations and Acronyms ................................................................................. 15 
Protocol Synopsis ............................................................................................................. 17 
Project Timeline ............................................................................................................... 19 
1 
Introduction ......................................................................................................... 20 
 
Study Rationale ............................................................................................................ 20 
 
Potential Risks and Benefits ......................................................................................... 20 
 
Background .................................................................................................................. 20 
2 
Study Objectives and Endpoints ............................................................................ 21 
3 
Study Design ......................................................................................................... 22 
 
General Design ............................................................................................................. 23 
 
Scientific Rationale for Study Design ............................................................................ 23 
 
Study Setting ............................................................................................................... 23 
 
End of Study Definition ................................................................................................ 24 
4 
Study Population and Eligibility ............................................................................ 24 
 
Inclusion Criteria: ......................................................................................................... 24 
 
Exclusion Criteria: ........................................................................................................ 25 
 
Early Exclusions ............................................................................................................ 25 
 
Participant Discontinuation/Withdrawal from Study ................................................... 25 
5 
Study Intervention ................................................................................................ 26 
 
in vitro Diagnostics ....................................................................................................... 26 
 
Preparation/Handling/Storage/Accountability ............................................................. 26 
5.2.1 
Acquisition ...................................................................................................................... 26 
5.2.2 
Handling ......................................................................................................................... 27 
5.2.3 
Storage ........................................................................................................................... 27 
5.2.4 
Accountability ................................................................................................................. 27 
 
Minimization of Error and Bias ..................................................................................... 27 
5.3.1 
Patient Selection ............................................................................................................. 27 
5.3.2 
Index Test ....................................................................................................................... 28 
5.3.3 
Reference Standard and Comparators ........................................................................... 28 
5.3.4 
Laboratory Methods ....................................................................................................... 29 
6 
Study Procedures .................................................................................................. 29 
6.1.1 
Sample Selection ............................................................................................................ 29 
6.1.2 
Study Workflow .............................................................................................................. 30 

 
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7 
Specimen Collection, Handling, Transport and Storage ......................................... 31 
 
Reference Standard Test and Index Test Procedures .................................................... 31 
 
Genetics ....................................................................................................................... 32 
 
Biomarkers .................................................................................................................. 32 
8 
Safety and Incident Reporting ............................................................................... 32 
8.1.1 
Time Period for Collecting SAE Information ................................................................... 33 
8.1.2 
Reporting and Follow up of SAEs .................................................................................... 33 
8.1.3 
Regulatory Reporting Requirements for SAEs ................................................................ 33 
8.1.4 
IVD Incidents (including Malfunctions) ........................................................................... 34 
8.1.5 
Time Period for Detecting IVD Incidents ........................................................................ 34 
8.1.6 
Follow-up of IVD Incidents ............................................................................................. 34 
8.1.7 
Reporting of IVD Incidents to Sponsor ........................................................................... 34 
8.1.8 
Regulatory Reporting Requirements for Medical Device Incidents ............................... 35 
9 
Statistical Considerations ...................................................................................... 35 
 
Sample Size Determination .......................................................................................... 35 
 
Statistical Analysis Plan ................................................................................................ 36 
9.2.1 
Exclusion Criteria for Diagnostic Performance Analysis ................................................. 37 
9.2.2 
Definitions for Diagnostic Performance Analysis ........................................................... 37 
9.2.3 
Additional Characteristics Assessed ............................................................................... 38 
9.2.4 
Efficacy Analyses ............................................................................................................. 38 
 
Planned Interim Analyses ............................................................................................. 38 
9.3.1 
Data Monitoring Committee .......................................................................................... 39 
10 
Regulatory and Ethical Considerations .................................................................. 39 
 
Regulatory and Ethics Approvals .................................................................................. 39 
 
Informed Consent Process ........................................................................................... 40 
 
Data Protection ............................................................................................................ 40 
 
Other Ethical Considerations ........................................................................................ 40 
11 
Data Handling and Record Keeping ....................................................................... 41 
 
Source Data and Source Documents ............................................................................. 41 
 
Data Management ....................................................................................................... 42 
12 
Quality Management ............................................................................................ 43 
 
Quality Control ............................................................................................................ 43 
 
Quality assurance ........................................................................................................ 44 
 
Study and Site Closure ................................................................................................. 44 
13 
Publication Policy ................................................................................................. 44 
14 
References ............................................................................................................ 46 
15 
Appendices ........................................................................................................... 48 
Appendix 1: Safety Definitions and Reporting .......................................................................... 48 
Appendix 2: Incident Definition and Reporting ......................................................................... 48 
Appendix 3: Protocol Amendment Summary Table .................................................................. 48 
Appendix 4: Sample Requirement to Achieve Target Sample Size ............................................. 48 
Appendix 5: Definitions of Diagnostic Test Results ................................................................... 48 
Appendix 1:  Safety Definitions and Reporting ................................................................. 49 
Appendix 2:  Incident Definition and Reporting ................................................................ 51 
Appendix 3: Protocol Amendment Summary Table .......................................................... 52 

 
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Appendix 4: Sample Requirement to Achieve Target Sample Size ..................................... 53 
Appendix 5: Definitions of Diagnostic Test Results ........................................................... 54 
 
 
 

 
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Institutions/Partners Involved in the Trial 
The main institutions/organizations/partners are listed below. More information can be found 
in the Study Contact List for the names, telephone, e-mail address, title and role on the study. 
Organization/Institution/Company/Partner 
Role in the Study 
FIND, Geneva, Switzerland 
Sponsor 
Hinduja Hospital and Medical Research Centre, 
Mumbai, India 
Principal investigator 1 
Phthisiopneumology Institute, Chisinau, Moldova Principal investigator 2 
Wits Health Consortium, Johannesburg, South 
Africa 
Principal investigator 3 
National Institute of Tuberculosis and 
Respiratory Diseases, New Delhi, India 
Principal investigator 4 
Cepheid 
IVD Manufacturer 
 
 
FIND 
Contact person: Claudia Denkinger 
claudia.denkinger@finddx.org 
 
Campus Biotech 
Chemin des Mines 9 
1202 Geneva 
Switzerland 
 
Cepheid 
Contact person: Nova Via 
nova.via@cepheid.com 
 
904 E Caribbean Drive 
Sunnyvale, CA 94089 
United States   
 
Hinduja Hospital and Medical Research Centre 
Contact person: Camilla Rodrigues 
dr_crodrigues@hindujahospital.com 
 
Veer Savarkar Marg 
Mahim, Mumbai 
Maharashtra 400016, India 
 

 
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Phthisiopneumology Institute 
Contact person: Valeriu Crudu 
valeriu.crudu@pas.md 
 
13 Varnav Str  
MD-2025 Chisinau, Moldova 
 
National Health Laboratory Services 
Contact person: Professor Wendy Stevens 
Wendy.stevens@nhls.ac.za 
 
31 Princess of Wales Terrace 
Parktown 
2193 
 
National Institute of Tuberculosis and Respiratory Diseases 
Contact person: Manpreet Bhalla 
m.bhalla@nitrd.nic.in 
 
Sri Aurobindo Marg, Near Qutub Minar, Seth Sarai,  
Mehrauli, New Delhi,  
Delhi 110030, India 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Terms of references and nature of agreements are available from the sponsor on request. 

 
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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 study as described and 
ensure adherence to GCP/GCLP, the principles outlined in the Declaration of Helsinki 
and applicable regulatory requirements. 
 
HEAD OF TB PROGRAMME 
Name: Claudia Denkinger 
Institution: FIND 
 
Signature: 
 _____________________ 
 
Date:_______________ 
 
 
 
 
 
 
 
 
DD/MMM/YYYY 
HEAD OF CLINICAL & REGULATORY AFFAIRS 
Name:  Jennifer Kealy 
Institution:  FIND 
 
Signature: 
 _____________________ 
 
Date:_______________ 
 
 
 
 
 
 
 
 
DD/MMM/YYYY 
PROJECT MANAGER   
Name: Sophia Georghiou 
Institution: FIND 
 
Signature: 
 ______________________ 
 
Date:_______________ 
 
 
 
 
 
 
 
 
DD/MMM/YYYY 
TRIAL MANAGER   
Name: Adam Penn-Nicholson 
Institution: FIND 
 
Signature: 
 ______________________ 
 
Date:_______________ 
 
 
 
 
 
 
 
 
DD/MMM/YYYY 
TRIAL STATISTICIAN 
Name: Stefano Ongarello 
Institution: 
 
Signature: 
 _______________________ 
 
Date:_______________ 
 
 
 
 
 
 
 
 
DD/MMM/YYYY 

 
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DATA MANAGER 
Name: Aurélien Macé 
Institution: FIND 
 
Signature: 
 _______________________ 
 
Date:_______________ 
 
 
 
 
 
 
 
 
DD/MMM/YYYY 

 
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Statement of Hinduja Hospital and Medical Research Centre 
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 study 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 study 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 the sponsor. Monitoring and auditing by the 
sponsor, 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 study protocol may not be disseminated or 
discussed with a third party without the express consent of the sponsor.   
 
Name of Principal Investigator: ________________________ 
(Print) 
Site: _____________________________ 
 
 
Signature: _________________________ 
        Date: ____________________ 
 
 
 
 
 
  
 
              DD/MMM/YYYY 

 
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Protocol #1.1  
 
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Statement 
of 
Phthisiopneumology Institute 
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 study 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 study 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 the sponsor. Monitoring and auditing by the 
sponsor, 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 study protocol may not be disseminated or 
discussed with a third party without the express consent of the sponsor.   
 
Name of Principal Investigator: ________________________ 
(Print) 
Site: _____________________________ 
 
 
Signature: _________________________ 
        Date: ____________________ 
 
 
 
 
 
  
 
              DD/MMM/YYYY 

 
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Statement of Wits Health Consortium 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 study 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 study 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 the sponsor. Monitoring and auditing by the 
sponsor, 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 study protocol may not be disseminated or 
discussed with a third party without the express consent of the sponsor.   
 
Name of Principal Investigator: ________________________ 
(Print) 
Site: _____________________________ 
 
Signature: _________________________ 
        Date: ____________________ 
 
 
 
 
 
  
 
              DD/MMM/YYYY 
 
 

 
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Statement of National Institute of Tuberculosis and 
Respiratory Diseases 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 study 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 study 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 the sponsor. Monitoring and auditing by the 
sponsor, 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 study protocol may not be disseminated or 
discussed with a third party without the express consent of the sponsor.   
 
Name of Principal Investigator: ________________________ 
(Print) 
Site: _____________________________ 
 
Signature: _________________________ 
        Date: ____________________ 
 
 
 
 
 
  
 
              DD/MMM/YYYY 
 
 

 
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Protocol History/Amendment Summary* 
 
Version number Release date 
Comments 
1.0 
15 October 2018 
Initial version (updated timeline from 15 Sep 2018 
version) 
1.1 
05 November 2018 
Added ETH (ethionamide) to MGIT DST testing, 
throughout, and updated objectives to reflect testing. 
Revised study flow language and figure to better 
illustrate Xpert MTB/RIF screening steps as part of 
routine diagnostic testing.  As such, sputum collection 
volume need only be >3ml 
1.1 
05 February 2019 
Changed NHLS to Wits Health Consortium throughout. 
Added Wendy Stevens name as WHC PI. 
 
 
*Refer to Appendix 3 for Protocol Amendment History 
 

 
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List of Abbreviations and Acronyms  
 
Abbreviation/acronym 
Meaning 
AE 
Adverse Event 
AMK 
Amikacin 
BSL 
Biosafety Level 
CAP 
Capreomycin 
CRF 
Case Report Form 
DMC 
Data Monitoring Committee 
DR-TB 
Drug-resistant TB 
DST 
Drug Susceptibility Testing 
eCRF 
Electronic Case Report Form 
ETH 
Ethionamide 
GCP 
Good Clinical Practice 
GCLP 
Good Clinical Laboratory Practice 
GDP 
Good Documentation Practice 
ICF 
Informed Consent Form 
ICH 
International Council on Harmonisation 
IDMC 
Independent Data-Monitoring Committee 
IEC 
Independent Ethics Committee 
INH 
Isoniazid 
IRB 
Institutional Review Board 
ISF 
Investigator Site File 
ISO 
International Organization for Standardization 
IUO 
Investigation Use Only 
IVD 
in vitro Diagnostic 
KAN 
Kanamycin 
LOD 
Limit of Detection 
LPA 
Line Probe Assay 
MDR 
Multidrug-resistant 

 
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MTB 
Mycobacterium tuberculosis 
NGS 
Next Generation Sequencing 
NTP 
National TB Programme 
QA 
Quality Assurance 
QC 
Quality Control 
QMS 
Quality Management System 
RA 
Regulatory Authority 
RBM 
Risk Based Monitoring 
RM 
Risk Management 
RIF 
Rifampicin 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SNRL 
Supranational Reference Laboratory 
SOP 
Standard Operating Procedure 
TB 
Tuberculosis 
TMF 
Trial Master File 
WHO 
World Health Organization 
XDR 
Extensively Drug-resistant 
 
 
 

 
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Protocol Synopsis 
 
Title 
Phase II: Multicentre Clinical Study to Assess the Performance of the Xpert MTB/XDR 
Assay for INH- and Second-line Resistance Detection 
Short title  
Xpert MTB/XDR Clinical Evaluation 
Protocol version and 
date 
v1.1 05-NOV-2018 
Background and 
rationale 
Evidence of the clinical diagnostic accuracy and operational characteristics of the 
Xpert MTB/XDR assay is needed to comprehensively evaluate Xpert MTB/XDR 
validity and inform global and national policy decision-making. 
Objectives 
Primary objectives  
1.1 Estimate the diagnostic accuracy of the Xpert MTB/XDR assay for INH and ETH 
resistance detection  
1.2 Estimate the diagnostic accuracy of the Xpert MTB/XDR assay for fluoroquinolone 
resistance detection  
1.3 Estimate the diagnostic accuracy of the Xpert MTB/XDR assay for second-line 
injectable resistance detection  
1.4 Assess Xpert MTB/XDR technical performance, including non-determinant rates, 
ease of use and other systems operational characteristics. 
Secondary objectives  
2.1 Assess additional Xpert MTB/XDR performance characteristics, including direct 
performance versus performance on cultured samples, performance between sites, by 
smear result, by gene target and compared to Hain MTBDRplus and MTBDRsl 
Study design 
Multicentre, cross-sectional diagnostic accuracy study 
Study sites/setting 
Clinical sites with high rates of drug-resistant TB in India, Moldova and South Africa 
Study population  
Patients with pulmonary TB symptoms and at least one DR-TB risk factor will be 
screened by Xpert MTB/RIF or Ultra. Patients with a clear TB-positive and RIF-
resistant or RIF-sensitive result by Xpert MTB/RIF or Ultra and who consent to study 
procedures will be tested by Xpert MTB/XDR. An anticipated 284 TB-positive and 316 
additional RIF-resistant patients will be enrolled in this study. 
Eligibility criteria  
Screening criteria: 
• 
Age 18 years and above 
• 
Symptoms suggesting pulmonary TB, i.e. persistent cough (generally ≥2 
weeks or as per local definition of TB suspect) and at least one risk factor for 
DR-TB  
Inclusion criteria: 
• 
A clear TB-positive and RIF-resistant or -sensitive result by Xpert MTB/RIF or 
Xpert MTB/RIF Ultra  
• 
Provision of Informed Consent 
• 
Production of an adequate quantity (³3mL) of sputum 
Primary endpoints 
1.1 Sensitivity and specificity estimates for INH and ETH resistance detection 
1.2 Sensitivity and specificity estimates for fluoroquinolone resistance detection  
1.3 Sensitivity and specificity estimates for second-line injectable resistance detection  

 
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1.4 Assessment of Xpert MTB/XDR technical performance  
Secondary endpoints 
2.1 Assessment of additional Xpert MTB/XDR performance characteristics 
Study duration  
10 weeks for IVD testing, with an additional 15 weeks to complete culture, DST, and 
NGS 
Time schedule 
IRB/IEC Approval:  
June-December 2018 
Shipment: 
 
January-March 2019 
Training: 
 
March 2019 
All Testing: 
 
April-September 2019 
Data Cleaning: 
 
September-October 2019 
Preliminary Analysis:  
October 2019 
GCP statement 
This study will be conducted in compliance with the protocol, the Declaration of 
Helsinki, ICH-GCP, ISO 13485: 2016 (if applicable) as well as all national legal and 
regulatory requirements. 

 
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Project Timeline 
 

 
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1 Introduction 
FIND and partners intend to address the need for a multi- and extensively drug-
resistant tuberculosis (M/XDR-TB) diagnostic solution for patients in settings with a 
high burden of drug-resistant tuberculosis (DR-TB) though the development, 
evaluation and introduction of an Xpert MTB/XDR assay.  
 
 
 Study Rationale 
Evidence of the clinical diagnostic accuracy and operational characteristics of the 
Xpert MTB/XDR assay is needed to comprehensively evaluate assay validity in 
settings of intended use and inform global and national policy decision-making. 
 
 Potential Risks and Benefits 
This study has no known medical risks for participants. Sample inclusion in this study 
will not affect TB or M/XDR-TB patient routine care and treatment. No clinical 
decisions will be made from Xpert MTB/XDR results. The possibility of unknown or 
unforeseen risk from specimen collection and diagnostic testing is minimal. 
Knowledge gained from this study may benefit patient communities by improving 
DR-TB diagnosis. Given the minimal risks associated with this study and the 
potential benefits to society and individuals, the benefits outweigh the aggregated 
risks.  
If unforeseen risks are recognized during the study, then FIND, study partners, 
Institutional Review Boards (IRBs)/Independent Ethics Committees (IECs), and 
participants must be provided with relevant information. 
 
 Background 
In recent years, tuberculosis (TB) control efforts have been complicated by the rise 
and spread of MDR-TB, or TB that is resistant to the first-line drugs isoniazid (INH) 
and rifampicin (RIF), and XDR-TB, or MDR-TB that has developed additional 
resistance to a fluoroquinolone and any of the injectable compounds [amikacin 
(AMK), kanamycin (KAN) and/or capreomycin (CAP)]1,2. The rapid diagnosis and 
appropriate treatment of M/XDR-TB is essential to prevent significant morbidity, 
mortality and further transmission of disease. For treatment of uncomplicated MDR-
TB, the World Health Organization (WHO) recently endorsed a 6-9 month treatment 
regimen, thereby replacing conventional 18-24 month regimens3. The 
fluoroquinolones and second-line injectables are key components of the 6-9 month 
regimen, and so it is necessary to rule-out resistance to these compounds prior to 
treating patients with the shorter regimen.  

 
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The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is an integrated, automated, 
cartridge-based system for MDR-TB diagnosis that uses the GeneXpert instrument 
platform. WHO confirmed evidence to support the widespread use of the Xpert 
MTB/RIF assay in 2010 and the assay has since been widely used in TB programs4, 
but it is only capable of identifying Mycobacterium tuberculosis (Mtb) and detecting 
RIF resistance, and so it does not provide the comprehensive resistance profile 
necessary to determine if the shorter TB treatment regimen is appropriate for certain 
patients. Conventional diagnosis of TB drug resistance relies upon the slow growth 
of Mtb in solid or liquid media, only then followed by resistance testing to determine 
phenotypic drug resistance. These conventional drug susceptibility testing (DST) 
methods can take several weeks to yield results5-6, require significant laboratory 
infrastructure and training, and are potentially biohazardous. In view of the 
inadequacy of these conventional tests for resistance detection, the development of 
rapid tests for INH and second-line resistance detection has become a research and 
implementation priority. 
Rapid diagnostics for second-line drug resistance detection identify mutations in the 
gyrA and gyrB genes7, associated with fluoroquinolone resistance, and rrs and eis 
gene regions, associated with second-line injectable resistance (with eis promoter 
mutations exclusively associated with KAN resistance)8. Recently, a novel 
GeneXpert cartridge was developed to detect mutations occurring in these genes, as 
well as in the katG and inhA gene regions, associated with INH resistance9. A 
Research Use Only (RUO) version of the Xpert MTB/XDR cartridge showed 
promising performance for INH and second-line resistance detection in a clinical 
evaluation study (sensitivity 92.7-98.1%; specificity 94.3-99.6%)10. The assay 
chemistry has since been improved and additional gene targets have been added to 
the assay to improve the detection of INH resistance (i.e. fabG1 and ahpC).  
Evidence of assay accuracy from an external laboratory validation using a well-
characterized set of Mtb strains as well as from large-scale, multicentre clinical 
studies is needed to confirm the validity of the most recent Xpert MTB/XDR assay for 
INH and second-line resistance detection, and to recommend its use in diverse 
clinical settings. The focus of this protocol is the multicentre clinical evaluation. 
 
2 Study Objectives and Endpoints 
Objectives 
Data Collected 
Endpoint 
Primary 
 
 
1.1 Estimate the diagnostic 
accuracy of the Xpert MTB/XDR 
assay for INH and ETH resistance 
detection, for a clinically diverse set 
of TB samples, with phenotypic DST 
and katG, fabG1, ahpC and inhA 
284 Xpert MTB/RIF TB-
positive and 316 RIF-
resistant clinical samples 
tested by Xpert MTB/XDR, 
phenotypic DST and NGS 
 
1.1 Sensitivity and specificity 
estimates for INH and 
ETH resistance detection 
 
 

 
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sequencing results as a composite 
reference standard. 
1.2 Estimate the diagnostic 
accuracy of the Xpert MTB/XDR 
assay for fluoroquinolone 
resistance detection, for a clinically 
diverse set of TB samples, with 
phenotypic DST and gyrA and gyrB 
sequencing results as a composite 
reference standard. 
1.3 Estimate the diagnostic 
accuracy of the Xpert MTB/XDR 
assay for second-line injectable 
resistance detection, for a clinically 
diverse set of TB samples, with 
phenotypic DST and rrs and eis 
sequencing results as a composite 
reference standard. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.2 Sensitivity and specificity 
estimates for 
fluoroquinolone 
resistance detection 
 
 
1.3 Sensitivity and specificity 
estimates for second-line 
injectable resistance 
detection 
 
1.4 Assess Xpert MTB/XDR 
operational aspects and ease of 
use, including Xpert MTB/XDR non-
determinant rates. 
Completion of laboratory 
CRFs for all samples and 
completion of user-
appraisal questionnaires by 
at least two operators per 
site at two different 
timepoints (after training 
and after study completion) 
1.4 Assessment of Xpert 
MTB/XDR operational 
aspects and ease of use  
 
Secondary 
 
 
2.1 Assess additional Xpert 
MTB/XDR performance 
characteristics: 
o 
Direct performance versus 
performance on cultured 
samples 
o 
Performance between sites 
o 
Performance by smear 
result 
o 
Performance by gene target 
o 
Performance compared to 
Hain MTBDRplus/sl 
o 
Performance by patient HIV 
status 
o 
Performance by patient pre-
treatment status 
o 
Performance as reflex test 
to Ultra or MTB/RIF assay 
Smear, culture, NGS, 
MTBDRplus, MTBDRsl and 
two Xpert MTB/XDR results 
(direct from sputum and 
from cultured strain) for 
each clinical sample tested 
 
2.1 Assessment of additional 
Xpert MTB/XDR 
performance 
characteristics 
 
 
3 Study Design 

 
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 General Design 
Multicentre clinical diagnostic accuracy study. 
 
 Scientific Rationale for Study Design 
The current study is part of a two-phase project. 
- 
Phase 1 (the current study) is an analytical study to validate and expand upon 
the Xpert MTB/XDR analytical data that the manufacturer (Cepheid, Sunnyvale, 
California) has compiled for internal validation, CE-IVD marking and/or other 
regulatory purposes. 
- 
Phase 2 is a clinical study to determine the diagnostic accuracy at intended 
settings of use.  
The two phases will provide complementary pieces of evidence to address the study 
objectives. 
The main purpose of this clinical study is to confirm accuracy estimates from the 
external laboratory validation and ensure that Xpert MTB/XDR diagnostic 
performance characteristics will be consistent at sites of intended use. Assay 
performance when testing sputum samples (direct testing) and and cultured isolates 
(indirect testing), assay performance compared to the Hain MTBDRplus and 
MTBDRsl (Hain Lifesciences GmbH) line probe assays (LPAs), and assay error and 
failure rates and device operational characteristics will also be assessed during this 
phase of the study.  
In order to address clinical study objectives quickly and efficiently, this study will first 
assess Xpert MTB/XDR performance for Xpert MTB/RIF MTB-positive samples from 
patients prospectively enrolled in the study. Focusing the study on Xpert MTB/RIF 
MTB-positive samples is in line with the intended use of the assay as a reflex, or 
follow-up, test to any Xpert MTB/RIF or Ultra MTB-positive result. This will also allow 
for an assessment of assay performance for INH resistance detection among all 
MTB-positive samples including INH mono-resistant, as this population might be 
different from a largely RIF-resistant population. However, to ensure numbers are 
adequate for an assessment of Xpert MTB/XDR assay performance for second-line 
resistance detection, once sufficient MTB-positive, RIF-sensitive samples have been 
tested, a subset of solely Xpert RIF-resistant samples will be tested until enrolment 
targets are met (see section 9.1 and Appendix 4: Sample Requirement to Achieve 
Target Sample Size). 
 
 Study Setting 
Study sites will be geographically diverse and representative of regional epidemics 
(i.e. TB and resistance prevalence, HIV co-infection, lineage distribution). 
Laboratories will represent the intended setting of use for the Xpert MTB/XDR assay, 

 
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i.e. laboratories that routinely process a large number of DR-TB samples and 
wherein second-line drug resistance may be suspected. Site preference will be given 
to countries/sites that are able to be early adopters (in terms of both political will and 
financial means) and sites with strong National TB Programme (NTP) relationship 
and global reach in order to gain visibility and encourage buy-in to facilitate national 
and global policy uptake. Participating sites must also have a sufficiently high 
throughput of TB clinical samples, and have access to phenotypic DST and NGS 
testing to allow for the efficient assessment of Xpert MTB/XDR assay performance. 
 
Regional distributions and country options (to be confirmed): 
• Africa (South Africa) 
• Asia (India) 
• Eastern Europe (Moldova) 
 
 End of Study Definition 
Phase II will be considered completed once all in vitro diagnostic (IVD) testing has 
completed and all culture, DST and NGS results have been returned for at least 284 
TB-positive and 316 additional RIF-resistant clinical samples, and all user appraisal 
questionnaires have been completed at the three clinical sites. 
 
4 Study Population and Eligibility 
Individuals who have one or more risk factors for DR-TB presenting to participating 
centres will be screened by Xpert MTB/RIF or Ultra. Those who test MTB-positive by 
Xpert MTB/RIF or Ultra at the study sites will be asked to participate. Once at least 
360 MTB-positive patients have been tested, an additional subset of Xpert MTB-
positive, RIF-resistant patients (n=400) will also be asked to participate in this study. 
Individuals will be recruited at outpatient clinic settings and inpatient hospital 
settings. Individuals will be asked by non-study clinicians or staff if they would be 
interested in participating in the study. Interested individuals will be referred to study 
personnel for additional information and screening, if appropriate. HIV-positive 
individuals and HIV-negative individuals will be included in this study.  
Prospective approval of protocol deviations to recruitment and enrolment criteria, 
also known as protocol waivers or exemptions, is not permitted. 
 
 Inclusion Criteria:  
Patients meeting the following criteria will be screened by Xpert MTB/RIF or Xpert 
MTB/RIF Ultra:  

 
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• Age 18 years or above; 
• Symptoms suggesting pulmonary TB, i.e. persistent cough (generally ≥3 
weeks or as per local definition of TB suspect), and at least one of the 
following: 
o Previously received >1 month of treatment for a prior TB episode or 
o Failing TB treatment with positive sputum smear or culture after 
≥3 months of a standard TB treatment or 
o Had close contact with a known drug-resistant TB case or 
o Newly diagnosed with MDR-TB within the last 30 days or 
o Previously diagnosed with MDR-TB and failed TB treatment with 
positive sputum smear or culture after ≥3 months of a standard MDR-
TB treatment regimen 
Patients meeting the above criteria will be screened by Xpert MTB/RIF or Xpert 
MTB/RIF Ultra. TB patients meeting the following criteria will be included in the 
study: 
• A clear Mtb-positive and RIF-resistant or RIF-sensitive result by Xpert 
MTB/RIF or Xpert MTB/RIF Ultra 
• Provision of informed consent; 
• Production of an adequate quantity (³3mL) of sputum 
 
 Exclusion Criteria:  
Participants will be excluded from the study if informed consent is not provided. 
 
 Early Exclusions 
Participants who have provided consent and who are enrolled, but who do not 
provide an adequate quantity of sputum at the start of the study, will be classified as 
early exclusions; each will be removed from the study and a new study 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 Mtb, results 
of conventional microbiological tests (e.g., sputum smear microscopy, mycobacterial 
cultures, and/or Xpert MTB/RIF) will be reported to appropriate local health 
authorities in accordance with local public health reporting regulations. Study data for 
participants who are classified as early exclusions will not be used for any final 
analyses. 
 
 Participant Discontinuation/Withdrawal from Study 
A participant may be withdrawn at any time at the discretion of the investigator for 
safety, behavioural, compliance, or administrative reasons. 

 
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If the participant withdraws consent for disclosure of future information, the sponsor 
may retain and continue to use any data collected before such a withdrawal of consent. 
If a participant withdraws from the study, he/she may request destruction of any 
samples taken and not tested, and the investigator must document this in the site study 
records. 
See Schedule of Assessments for data and/or samples to be collected at the time of 
study discontinuation and follow-up and for any further evaluations that need to be 
completed. 
 
5 Study Intervention 
Study Intervention is defined as any investigational intervention(s), marketed 
product(s), or medical device(s) intended to be used with a study participant 
according to the study protocol. 
 
 in vitro Diagnostics 
The IVD manufactured for FIND use in this study is the Xpert MTB/XDR assay. The 
Investigational Use Only (IUO) product is intended for the detection of Mtb and 
mutations associated with INH, ETH and second-line drug resistance. Likewise, the 
10-color GeneXpert system provided for this study should only be used for testing 
IUO cartridges and study samples. The investigational product and 10-color system 
will be strictly accounted for, including receipt and inventory, storage, use during the 
trial, and return or disposal, as detailed in the Study Manual for this study. 
Other IVDs (not manufactured for FIND) obtained for use in this study are: 
• Xpert MTB/RIF assay and/or the Xpert MTB/RIF Ultra assay 
• Hain MTBDRplus assay 
• Hain MTBDRsl assay 
Instructions for use of these assays will be provided in the SOPs for this study. 
Any IVD incidents, including those resulting from malfunctions of the IVD, must be 
detected, documented and reported by the investigator throughout the study (see 
section 8.1.4). 
 
 Preparation/Handling/Storage/Accountability 
5.2.1 Acquisition 
Procurement of the Xpert MTB/XDR assays will be done through FIND, which will 
coordinate shipments from the manufacturer. It is the responsibility of each 
laboratory to maintain an updated inventory of the study materials and to inform 
FIND immediately if additional materials are required. 

 
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It is expected that the respective countries will require import permits for receiving 
the investigational materials. The laboratory is responsible for obtaining relevant 
import permits in a timely manner. The sponsor and IVD manufacturer may support 
the laboratory to obtain import permits. Upon arrival of each new shipment of Xpert 
assays or reagents, the laboratory 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. 
 
5.2.2 Handling 
The investigator or designee must confirm appropriate temperature conditions have 
been maintained during transit for all Xpert MTB/XDR assays received and any 
discrepancies are reported and resolved before use of the Xpert MTB/XDR assay. 
Only those participants enrolled in the study may have their samples tested by the 
Xpert MTB/XDR assay, and only authorized and trained staff may operate the Xpert 
MTB/XDR assay. Testing using the investigational product will be performed 
according to the manufacturer’s instructions outlined within the SOP. 
 
5.2.3 Storage 
Procedures for product storage and disposal will be described in the SOP. All Xpert 
MTB/XDR assays must be stored in a secure, environmentally controlled, and 
monitored (manual or automated) area in accordance with the labeled storage 
conditions with access limited to the investigator and authorized site staff. 
 
5.2.4 Accountability 
The investigator, institution, or the head of the medical institution (where applicable) 
is responsible for Xpert MTB/XDR accountability, reconciliation, and record 
maintenance (i.e., receipt, reconciliation, and final disposition records). 
Further guidance and information for the final disposition of unused study 
interventions are provided in the SOP. 
 
 Minimization of Error and Bias 
5.3.1 Patient Selection 
A consecutive series of Xpert MTB/RIF MTB-positive patients (n=284) will be 
enrolled in this study, followed by an additional, consecutive series of Xpert MTB/RIF 
RIF-resistant patients (n=316) between the three countries with participating clinical 
sites. Enrolment will be based upon clearly defined eligibility criteria. 

 
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Analyses will be conducted for relevant sub-groups of the enrolled TB patient 
population (i.e. smear status for TB, drug-resistance profile, mutations detected, 
etc.), which may be considered to be indicators of disease severity. 
 
5.3.2 Index Test 
The Xpert MTB/XDR assay detects Mtb mutations associated with INH, ETH and 
second-line drug resistance direct from sputum. In line with this intended use, clinical 
samples will be directly tested by the Xpert MTB/XDR assay. However, the assay will 
also be used to test the culture isolate. This additional Xpert MTB/XDR assay testing 
will allow for a comparison of performance and non-determinant rates between 
testing directly from the sample and testing from the culture isolate. It will also 
support resolution of potential discordant LPA and NGS results from the initial Xpert 
MTB/XDR test result, as these tests will also be performed from the culture isolate 
(discordance may arise as a result of infections with a mix of different strains, with 
only one strain being grown out in culture). Furthermore, as all assays will be 
performed on the same culture, there is also no risk of any assay being tested on a 
different quality sample. 
Review bias will be avoided in this study as the index test requires no manual 
interpretation by the operator.  All results are automatically generated by the 
GeneXpert instrument and will be directly recorded by the operator on the laboratory 
case report form (CRF).   
 
5.3.3 Reference Standard and Comparators 
There is little risk of reference standard bias in this study as a composite reference 
standard of phenotypic DST and NGS will be used for resistance detection (Table 1). 
The use of both phenotypic and genotypic information will ensure high confidence in 
the reference standard, as certain mutations (e.g. many eis promoter mutations) are 
known to confer only low-levels of phenotypic drug resistance, and so they may not 
be detected by phenotypic DST at only one critical concentration. Furthermore, all 
molecular assays evaluated in this study detect resistance-mutations in similar gene 
regions, and so all mutations that are detectable by the Xpert MTB/XDR assay are 
generally detected by NGS and/or the Hain LPAs and vice versa.  
 
Table 1. Composite reference standard for diagnostic performance analysis 
 
Composite Reference Standard 
Drug of Interest 
NGS of Reference Gene(s) 
MGIT960 Phenotypic DST 
Isoniazid 
katG, fabG1, inhA promoter, 
ahpC promoter 
Isoniazid 
Ethionamide 
inhA promoter 
Ethionamide 

 
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Fluoroquinolones 
Moxifloxacin 
Levofloxacin 
 
gyrA, gyrB 
gyrA, gyrB 
 
Moxifloxacin 
Levofloxacin 
Second-line Injectables 
Amikacin 
rrs 
Amikacin 
Kanamycin 
rrs, eis promoter 
Kanamycin 
Capreomycin 
rrs 
Capreomycin 
 
5.3.4 Laboratory Methods 
The participating sites will undergo an on-site laboratory and clinical evaluation 
during the site initiation visit to ensure standardized and high-quality performance of 
molecular methods. 
  
6 Study Procedures 
Study procedures and their timing are summarized in the Schedule of Assessments. 
Protocol waivers or exemptions are not allowed. 
Adherence to the study design requirements, including those specified in the Schedule 
of Assessments, is essential and required for study conduct. 
All screening evaluations must be completed and reviewed to confirm that potential 
participants meet all eligibility criteria. 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., 
blood count) 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 Schedule of Assessments. 
 
6.1.1 Sample Selection 
Up to two sputum samples (³3mL) will be collected from consecutive, prospectively-
enrolled patients with defined risk factors for DR-TB and a valid Xpert MTB/RIF or 
Xpert MTB/RIF Ultra result. Depending upon enrolment rates, study timelines may 
be accelerated by also using existing, well-characterized frozen TB clinical (sputum) 
samples collected during the course of routine clinical care and TB surveillance in 
the sites of intended use. These samples will only be used if recruitment or 
enrolment is slow at one or more sites.  At the 4-week study time point, recruitment 
rates will be assessed to determine whether this use of additional samples will be 
necessary. 
 

 
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6.1.2 Study Workflow 
Study flow is shown in Figure 1. All available Xpert MTB/RIF TB-positive samples will 
be tested in this study (i.e. consecutive patient samples) until sample size targets are 
met. All molecular assays will be performed in accordance with SOPs provided by 
the manufacturers. Specimen handling and processing by routine tests will be 
carried out according to NTP local policies and standards.  
Samples will first be tested by Xpert MTB/RIF or Ultra, as part of routine diagnostic 
procedures.  This Xpert MTB/RIF step will act as a screening tool for trial enrolment. 
Participants testing positive for MTB by Xpert MTB/RIF will be asked to volunteer for 
the Xpert MTB/XDR trial.  This (testing of specimens from TB-positive samples) is in 
line with the intended use of the XDR assay as an add-on test to the Xpert MTB/RIF 
assay.  
 A staged enrolment will proceed:  Stage 1 will enrol 360 MTB-positive participants, 
irrespective of RIF resistance/sensitivity.  Once enrolment is complete, Stage 2 will 
enrol 400 MTB-positive RIF-resistant participants (see section 9.1 and Appendix 4: 
Sample Requirement to Achieve Target Sample Size). This staging is designed to 
better evaluate Xpert MTB/XDR sensitivity for second-line resistance detection. 
In order to efficiently assess the diagnostic performance of the Xpert MTB/XDR 
assay, participants will be asked to provide >3ml of sputum (either a single sputum 
or two consecutively collected and pooled sputa).  Samples will be homogenized and 
split for testing.  
The Xpert MTB/XDR assay and acid-fast bacilli smear will be performed on the direct 
sample. 2ml of sputum will be decontaminated and used for MGIT and LJ culture. 
Following direct testing, phenotypic MGIT DST will be performed for all culture-
positive samples for INH, RIF, fluoroquinolone (moxifloxacin and levofloxacin), AMK, 
ETH (ethionamide), KAN and CAP. Cultured samples will also undergo subsequent 
molecular testing by LPA and targeted NGS of relevant gene regions (katG, inhA, 
fabG1, ahpC, gyrA, gyrB, rrs, and eis) and another Xpert MTB/XDR assay. NGS will 
be performed from the culture isolate to obtain sequencing reads of high quality. 
 
Figure 1. Phase II sample flow 

 
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All diagnostic test results (smear, Xpert MTB/XDR on direct sample, MGIT, LJ, Xpert 
MTB/XDR on culture isolate, MTBDRplus, MTBDRsl, DST and NGS results) will be 
recorded by the laboratory technicians for each site on the laboratory CRF. 
 
7 Specimen Collection, Handling, Transport and Storage 
Clinical (sputum) samples from enrolled patients will be used in this study. Patients 
will be asked to provide up to two sputum samples for testing after providing 
informed consent for the study. Sputum samples from the same patient will be 
pooled and used directly for acid-fast bacilli smear, Xpert MTB/XDR and MGIT and 
LJ culture. 
Appropriate laboratory staff at each site reference laboratory are responsible for 
receiving the samples, storing the samples and conducting all testing in line with 
BSL3 requirements. All leftover samples must be stored until study conclusion and 
completion of all analyses, in case any additional or repeat testing is requested by 
the sponsor. 
 
 Reference Standard Test and Index Test Procedures 
Xpert XDR 
Assay
Xpert XDR 
Assay
NGS 
Sequencing
MTBDRsl
DST
MGIT + LJ
MTBDRplus
FM Smear
≥3ml
700uL
700uL
2mL
NALC-NaOH
Decontamination
Resuspend pellet in 2mL PBS
50uL
If MTB-pos
↳Stage 1.  MTB-pos, then
↳Stage 2.  MTB-pos RIF-resistant only
Routine testing
# ≥ 3ml of sputum is required for the trial.  This can be achieved through an individual sputum or pooling of serially 
collected sputa, homogenized with glass beads.
Homgenized Sputum#
Xpert MTB/RIF 
or Ultra*
Enrolment
Routine Sputum 
Collect Trial Sputum 

 
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A composite reference standard of phenotypic DST and NGS will be used for 
resistance detection to ensure high confidence in the reference standard for this 
study. Phenotypic MGIT DST and targeted NGS will be performed on the culture 
isolate for each clinical sample. MGIT DST will be done on site to determine culture 
drug-susceptibility to INH, ETH, moxifloxacin, levofloxacin, AMK, KAN and CAP at 
the revised WHO critical concentrations15. Targeted NGS of the gene regions 
included in the Xpert MTB/XDR assay will be performed on DNA extracted from the 
cultured sample, which will be sent to a SNRL for NGS, or sequenced locally using 
primers specific for the gene regions included in the assay. The output for both 
phenotypic DST and NGS results are automated, and require no manual operator 
interpretation.  Testing results, when obtained, will be input onto the laboratory CRF. 
The index test will be performed on both the direct clinical sample as well as the 
culture isolate, to ensure high Xpert MTB/XDR diagnostic performance direct from 
sputum and to provide a basis for comparison if any discordants are seen between 
the initial Xpert MTB/XDR result and the phenotypic DST and additional molecular 
test results (NGS and LPA). The output for the Xpert MTB/XDR assay is automated 
and does not require manual interpretation.  As with the reference test, all Xpert test 
results will noted on the laboratory CRF. 
 
 Genetics 
No patient genetic information will be collected, analysed or recorded in this study. 
The proposed genetic sequencing and molecular analysis protocols use only TB 
pathogen-specific sequences and will be limited to analysis of M. tuberculosis 
bacteria genomes. 
 
  Biomarkers 
Biomarkers are not evaluated in this study. 
 
8 Safety and Incident Reporting 
The definitions of an adverse event (AE) and serious adverse event (SAE) can be 
found in Appendix 1: Safety Definitions and Reporting. 
AE will be reported by the participant spontaneously (or, when appropriate, by a 
caregiver, surrogate, or the participant's legally authorized representative).  
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 AEs that are serious, considered related to the 
study intervention or study procedures, or that caused the operator to discontinue 

 
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use of the Xpert MTB/XDR assay or participants to discontinue their participation in 
the Xpert/MTB XDR Clinical Evaluation. 
 
8.1.1 Time Period for Collecting SAE Information  
All SAEs will be collected from the signing of the informed consent form (ICF) OR 
start of Xpert MTB/XDR testing until the end of the Xpert MTB/XDR Clinical 
Evaluation at the time points specified in the SOP. 
All SAEs will be recorded and reported to the sponsor or designee within 24 hours, 
as indicated in Appendix 2: Incident Definition and Reporting. The investigator will 
submit any updated SAE data to the sponsor within 24 hours of it being available. 
Investigators are not obligated to actively seek SAEs after conclusion of the study 
participation. However, if the investigator learns of any SAE, including a death, at 
any time after a participant has been discharged from the study, or the IVD operators 
have completed IVD testing, and he/she considers the event to be reasonably 
related to the study intervention or study participation, the investigator must promptly 
notify the sponsor. 
The method of recording, evaluating, and assessing causality of the SAE and the 
procedures for completing and transmitting SAE reports are provided in Appendix 1: 
Safety Definitions and Reporting. 
 
8.1.2 Reporting and Follow up of SAEs 
All SAEs will be followed until resolution, stabilization, the event is otherwise 
explained, or the participant is lost to follow-up (as defined in Section Error! 
Reference source not found.).  Further information on follow-up procedures is 
given in Appendix 2: Incident Definition and Reporting) 
 
 
8.1.3 Regulatory Reporting Requirements for SAEs 
Prompt notification by the investigator to the sponsor of a SAE is essential so that 
legal obligations and ethical responsibilities towards the safety of participants and 
the safety of a study intervention under clinical investigation are met.  
The sponsor has a legal responsibility to notify both the local regulatory authority and 
other regulatory agencies about the safety of a study intervention under clinical 
investigation. The sponsor will comply with country-specific regulatory requirements 
relating to safety reporting to the regulatory authority, IRB/IEC, and investigators. 
An investigator who receives an investigator safety report describing a SAE or other 
specific safety information (e.g., summary or listing of SAEs) from the sponsor will 
review and then file it in the Investigator Site File (ISF) and will notify the IRB/IEC, if 
appropriate according to local requirements. 

 
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8.1.4 IVD Incidents (including Malfunctions) 
IVDs are being provided for use in this study for investigative purposes. 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 study with such devices.  
The definition of an IVD Incident can be found in Appendix 2: Incident Definition and 
Reporting. 
NOTE: Incidents fulfilling the definition of an AE/SAE will also follow the processes 
outlined above and in Appendix 1: Safety Definitions and Reporting of the protocol. 
 
8.1.5 Time Period for Detecting IVD Incidents 
IVD incidents or malfunctions of the device that result in an incident will be detected, 
documented, and reported during all periods of the study in which the IVD is used.  
If the investigator learns of any incident at any time after a participant has been 
discharged from the study, and such incident is considered reasonably related to a 
IVD provided for the study, the investigator will promptly notify the sponsor. 
The method of documenting IVD Incidents is provided in Appendix 2. 
 
8.1.6 Follow-up of IVD Incidents 
All IVD incidents involving an AE will be followed and reported in the same manner 
as other AEs (see Section 8.1.2). This applies to all participants, including those who 
discontinue study intervention. 
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. 
 
8.1.7 Reporting of IVD Incidents to Sponsor 
IVD incidents will be reported to the sponsor within 24 hours after the investigator 
determines that the event meets the protocol definition of a IVD incident. 
The Medical Device Incident Report Form will be sent to the sponsor by email. If 
email is unavailable, then the form should be faxed to the sponsor at +41 (22) 710 05 
99. 
The Trial Manager will be the contact for the receipt of IVD and SAE. 

 
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8.1.8 Regulatory Reporting Requirements for Medical Device Incidents 
The investigator will promptly report all incidents occurring with any IVD provided for 
use in the study in order for the sponsor 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. 
 
9 Statistical Considerations  
A detailed statistical analysis plan will be prepared separately and only some key 
elements are presented here. 
 
 Sample Size Determination 
To achieve the targeted precision for accuracy estimates, we estimate that 760 
samples be tested for this study, with a final 284 MTB-positive and an additional 316 
RIF-resistant samples tested by Xpert MTB/XDR and all reference standards and 
comparators (assumptions provided in Appendix 4), i.e. a total of 600 specimen 
results available for analysis. The desired precision for the accuracy estimates was 
chosen to achieve high confidence in the estimates for Xpert MTB/XDR resistance 
detection when considering evidence from both the ‘external laboratory validation’ 
(phase I) and the clinical trial (phase II). Sample size is detailed below in Table 2.   
 
Table 2. Sample size 
 
 
Clinical trial 
(TB samples) 
 
 
n 
Point estimate (95%CI)
 * 
Total width of CI 
* 
INH 
Sensitivity 
395 
90% 
 (86, 93) 
7% 
Specificity 
205 
98% 
(95, 99) 
4% 
FQs 
Sensitivity 
93 
90% 
(82, 95) 
13% 
Specificity 
507 
98% 
(96, 99) 
3% 

 
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AMK 
Sensitivity 
34 
90% 
(74, 97) 
23% 
Specificity 
566 
98% 
(96, 99) 
3% 
KAN 
Sensitivity 
71 
90% 
(80, 96) 
16% 
Specificity 
529 
98% 
(96, 99) 
3% 
CAP 
Sensitivity 
34 
90% 
(74, 97) 
23% 
Specificity 
566 
98% 
(96, 99) 
3% 
* 95%CI based on Wilson’s score method (as recommended by Newcombe et al. as well as CLSI/FDA) using 
continuity correction. 
 
 Statistical Analysis Plan 
The statistical analysis plan (SAP) will be developed and finalized before database 
lock and will describe the samples to be included in the analyses and procedures for 
accounting for missing, unused, and spurious data. This section is a summary of the 
planned statistical analyses of the primary and secondary endpoints. 
Primary analyses of outcome data will be done using the composite reference 
standard. Primary analyses will be focused on estimating accuracy (clinical 
sensitivity and specificity) for INH and second-line drug resistance detection: 
• Clinical Sensitivity: proportion positive by composite reference standard 
(phenotypic DST and NGS) that are detected as positive by index test (Xpert 
MTB/XDR) 
• Clinical Specificity: proportion negative by composite reference standard 
(phenotypic DST and NGS) that are detected as negative by index test (Xpert 
MTB/XDR) 
For simple proportions (sensitivity and specificity), 95% confidence intervals will be 
computed using the Wilson score method. Primary diagnostic accuracy analyses will 
be carried out across all study sites. All TB samples that generate both Xpert 
MTB/XDR and composite reference standard results will contribute to the analysis of 
the performance characteristics for INH- and second-line resistance detection. If 
discordances are observed between the Xpert MTB/XDR results from testing sputa 
and the culture isolate, results from the culture isolate will be considered in these 
analyses. 
Primary analyses will also evaluate assay operational characteristics, which will be 
captured through direct observation and user-appraisal questionnaires. Primary 
assessments of Xpert MTB/XDR operational characteristics will evaluate assay 
characteristics compared to Hain MTBDRplus/sl assays, including: 

 
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• Time to first result 
• Ease of use 
• Failure and invalid rate 
These assessments of Xpert and LPA characteristics will be descriptive, and any 
differences will be further explored by sample type (i.e. smear- and culture-result, 
patient HIV- and treatment statues, etc.).  Assessments of platform operational 
characteristics will be based upon information gathered during the clinical study, as 
well as information provided by the manufacturers. 
 
9.2.1 Exclusion Criteria for Diagnostic Performance Analysis 
Samples meeting any of the following criteria in the course of the study will be 
excluded from the primary analyses of Xpert MTB/XDR accuracy:  
• no valid result for NGS for relevant gene region or Xpert MTB/XDR, 
• smear-positive/culture negative,  
• single positive culture with ≤20 colonies (LJ) or time to positivity >28 days 
(MGIT),  
• culture positive but no MTB complex identification available,  
• specimens with growth of mycobacteria other than MTB complex only 
 
9.2.2 Definitions for Diagnostic Performance Analysis 
 
TEST RESULT 
DESCRIPTION 
Phenotypic Drug*-
resistant 
Culture-positive and growth for Drug* in conventional DST testing.  
Phenotypic Drug*-
sensitive 
Culture-positive and no growth for Drug* in conventional DST testing 
Genotypic Drug*-
resistant 
NGS identifies mutations recognized to be associated with resistance 
(defined based on consultation with WHO prior to analysis) 
Genotypic Drug*-
sensitive 
NGS identifies no mutations recognized to be associated with 
resistance (defined based on consultation with WHO prior to analysis) 
Composite reference 
standard Drug*-resistant 
If Phenotypic Drug*-sensitive but NGS identifies mutations recognized 
to be associated with Drug* resistance for the respective gene regions, 
the composite reference standard will be considered Drug*-resistant. 
If Phenotypic Drug*-resistant but NGS does not identify mutations 
recognized to be associated with Drug* resistance for the respective 
gene regions, the composite reference standard will be considered 
Drug*-resistant (as mutations will be assumed outside of the region 
sequenced). 

 
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* Drug: INH, ETH, FQ (moxifloxacin and levofloxacin for phenotypic DST), AMK, KAN or CAP 
 
9.2.3 Additional Characteristics Assessed 
Secondary analyses will investigate assay diagnostic performance between 
important subgroups, including: Xpert MTB/XDR direct versus indirect performance 
(on direct versus cultured samples), performance between sites, by smear result, by 
gene target, performance compared to Hain MTBDRplus and MTBDRsl assays, 
performance by reflex test to Xpert MTB/RIF or Ultra result, and performance by 
patient HIV and pre-treatment status. Detailed definitions for diagnostic test results 
are given in Appendix 5: Definitions of Diagnostic Test Results. 
 
9.2.4 Efficacy Analyses 
Endpoint 
Statistical Analysis Methods 
Primary 
• Sensitivity  
• Specificity 
• Data summaries 
Secondary 
• Chi-squared 
• Sensitivity 
• Specificity 
• Positive Predictive Value 
• Negative Predictive Value 
 
 Planned Interim Analyses  
Interim analyses for the phase II clinical trial will be led by the Trial Manager and 
conducted by the Trial Statistician. Preliminary data will be analysed once for every 
200 clinical samples tested. Briefly, once 200 clinical samples have been tested by 
Xpert MTB/XDR both directly on the clinical sample and on the culture isolate and 
have returned NGS results for all drugs tested, an interim analysis will be conducted. 
The diagnostic sensitivity and specificity of the Xpert MTB/XDR assay will be 
estimated against NGS for each drug evaluated, including for important sub-groups 
as detailed in section 2.  
Data will be unmasked for this analysis, and results will serve as an early indicator of 
Xpert MTB/XDR accuracy in the clinical trial.  If performance estimates fall outside of 
the presented confidence intervals for resistance detection (Table 2), the Data 
Monitoring Committee (DMC) will review all data for accuracy and consistency. 
Composite reference 
standard Drug*-sensitive 
If Phenotypic Drug*-sensitive and NGS shows either no mutations or 
only mutations that are not associated with Drug* resistance for the 
respective gene regions. 

 
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Additionally, the sponsor may conduct a monitoring visit to one or more sites to 
ensure adherence to SOPs and effective data management. 
 
9.3.1 Data Monitoring Committee 
The DMC for Phase II of the Xpert MTB/XDR evaluation will consist of the Head of 
TB Programme, Head of Clinical & Regulatory Affairs, Trial Manager, Data Manager, 
and the Data Entry Clerks at each study site. The head of the DMC will be the Data 
Manager, who will help to train site staff on the use of the database system and 
perform periodic data checks to ensure that data is collected, managed and reported 
clearly, securely and accurately throughout the project. The Data Entry Clerks at 
each site will perform first- and second-data entry and report any issues to the Trial 
Manager and Data Manager, who will work together with the Data Entry Clerks to 
resolve any data issues. The Head of Clinical & Regulatory Affairs will serve as an 
advisor to the DMC. 
 
10  Regulatory and Ethical Considerations   
 Regulatory and Ethics Approvals 
This study 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 study is initiated.  
• Any amendments to the protocol will require IRB/IEC approval before 
implementation of changes made to the study design, except for changes 
necessary to eliminate an immediate hazard to study participants.  
• The investigator will be responsible for the following: 
o Providing written summaries of the status of the study 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 study 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. 

 
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 Informed Consent Process 
The investigator or his/her representative will explain the nature of the study to the 
participant or his/her legally authorized representative and answer all questions 
regarding the study.  
Participants must be informed that their participation is voluntary. Participants or their 
legally authorized representative will be required to sign and date a statement of 
informed consent that meets the requirements of the IRB/IEC or study centre.  
There must be evidence that written informed consent was obtained before the 
participant was enrolled in the study and ample time was given to participant to 
consent. The date the written consent was obtained (as well as the time, ideally) must 
be recorded. The authorized person obtaining the informed consent must also sign 
and date the ICF. 
Illiterate participants must provide a thumbprint on the ICF and the ICF 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 study.  
A copy of the ICF(s) must be provided to the participant or the participant’s legally 
authorized representative.  
 
 Data Protection 
Participants will be assigned a unique identifier by the sponsor. Any participant records 
or datasets that are transferred to the sponsor 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 study-related data will be used 
by the sponsor 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 the 
sponsor, by appropriate IRB/IEC members, and by inspectors from regulatory 
authorities. 
 
  Other Ethical Considerations 
Following completion of study procedures, any remaining samples will be stored 
temporarily in case further testing for this study is deemed to be necessary. Samples 
will be labelled with a study identifier and date of collection; the specimens will be 
linked to results of other mycobacteriology tests, the final study diagnosis for each 

 
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sample (e.g. drug-resistant, drug-sensitive, indeterminant for each drug compound). 
Samples will be stored temporarily (up to 2 years) at the sites. Further testing may 
be required at a Reference Laboratory or specialized research laboratories abroad. 
An export permit will be obtained accordingly when possible. 
In the event that one site does not meet sample processing targets within the study 
timelines, the study will go forward at the other sites and the investigators will 
proceed with data submission as per study plan.  
 
11 Data Handling and Record Keeping 
All participant data relating to the study will be recorded on printed or electronic CRF 
unless transmitted to the sponsor 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 study-related monitoring, audits, IRB/IEC review, and 
regulatory agency inspections and provide direct access to source data documents.  
The sponsor or designee is responsible for the data management of this study 
including quality checking of the data.  
Study monitors will perform 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 study is being conducted in accordance with the currently approved protocol 
and any other study agreements, ICH GCP, and all applicable regulatory 
requirements.  
Records and documents, including signed ICFs, pertaining to the conduct of this 
study must be retained by the investigator for 2 years after study 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 the 
sponsor. No records may be transferred to another location or party without written 
notification to the sponsor.  
 
 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 may include: 
• Xpert MTB/XDR test results (electronic) 
• Xpert MTB/RIF test results (electronic) 

 
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• Xpert MTB/RIF Ultra test results (electronic) 
• Hain MTBDRplus and MTBDRsl test strips 
• NGS results (electronic records) 
• Manually-collected data such as lab requisitions, lab registers, and 
laboratory worksheets (AFB smear log, DST worksheets, QC records, etc.) 
• Data generated from additional, automated instruments; e.g., MGIT 
unloaded positive, negative, and DST reports 
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), depending on the study.  
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.   
 
 Data Management 
For the clinical study, de-identified sample CRF data will consist of participant 
medical history, clinical examination, and prior laboratory testing results when 
available. All clinical CRF notes will be transferred to the eCRF by Data Entry Clerks. 
All laboratory results (smear, culture, DST, molecular results) will be recorded by 
laboratory technicians directly on the laboratory CRF. All Xpert testing will generate 
electronic records of test results. All electronic results will be stored electronically on 
password-protected computers at the site. Data will also be transcribed into the CRF. 
LPA testing will generate results on paper test strips. These test strips will be 
interpreted by the operator and input into the laboratory CRF. All LPA test strips will 
be retained by the site as source documents. Laboratory CRFs will be transferred 
from paper CRFs to eCRFs by Data Entry Clerks at the sites and uploaded to the 
Openclinica study database. The OpenClinica Community v3.13 database is 
password controlled and restricted. FIND will ultimately be responsible for data 
management. 
Electronic double-data entry must be conducted by two on-site Data Entry Clerks. 
Data entry training will be provided by FIND, either on site or remotely. Sites will use 
a password-protected web-based data-entry tool which has the advantage that study 
monitors will have continuous access to the electronic data. Electronic data will be 
backed up daily. 
Where possible, first data entry will be done as soon as a result becomes available 
(real-time). Second data entry will only be done by study staff after all sample results 

 
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(culture, NGS, Xpert) are available. The Principal Investigator of each site will review 
all data entered for the site’s samples.  
Details relating to data entry procedures and timelines will be provided to the sites in 
a data management user manual. 
Study monitors will conduct regular checks to see that all paper and electronic 
records are consistent and accurate. A priority during monitoring visits will be to 
ensure correctness of electronic data entries in comparison to source data. 
Additional details relating to data entry procedures and timelines will be provided to 
the sites in the data management SOP. 
 
12 Quality Management  
Quality Management for this study consists of Quality Control activities, training and 
capacity building provided by FIND (or designee) to the investigational sites and 
laboratories, as well as the use of Standard Operating Procedures, Work Instructions, 
Tools and Templates.   
 
Training on the protocol, GCP and the use of the IVDs and laboratory tests will be 
provided by FIND.  A Laboratory Manual which describes all of the sample testing 
procedures will be provided by FIND prior to the commencement of the study.  
Training on the EDC system will be provided by FIND Data Management prior to first 
participant enrolment.  
 
 Quality Control 
Quality control should be applied to each stage of data handling to ensure that all 
data are reliable and have been processed correctly. The investigational site is 
responsible for performing regular Quality Control checks on the data they generate. 
Additionally, the sponsor will perform risk based monitoring of this study, and 
associated Quality Control checks, as described in the Monitoring Plan. 
QC will be applied to each stage of data handling to ensure that all data are reliable 
and have been processed correctly: 
• Laboratory staff will be trained in data collection and entry 
• All source documentation will be filed and preserved  
• All assay results will be recorded onto paper laboratory CRFs immediately 
following testing 
• All data will be double entered from the paper CRF to the eCRF by two on-site 
staff 
• Electronic data will be backed up daily 
• The Co-investigator will review all data entered for the site’s samples 

 
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• The Trial Manager and Data Manager will perform periodic data checks to 
ensure all paper and electronic records are complete and input data are 
consistent and accurate 
 
 Quality assurance  
As part of routine QA, the sponsor (FIND) or designee may conduct an audit of the 
investigational site. Monitoring visits may include, but are not limited to, review of 
regulatory files, laboratory reports, and protocol compliance. Study monitors will 
meet with co-investigators to discuss any problems and actions to be taken and 
document visit findings and discussions. 
FIND will provide training on data collection and entry, implement quality control 
procedures related to the data entry system and generate data quality control checks 
that will be run on the database. Any missing data or data anomalies will be 
communicated to the laboratory for clarification and resolution. The laboratory will 
provide direct access to all source data/documents for the purpose of monitoring and 
auditing by the sponsor by local and regulatory authorities, if requested. 
 
 Study and Site Closure 
The sponsor designee reserves the right to close the study site or terminate the 
study at any time for any reason at the sole discretion of the sponsor. Study sites will 
be closed upon study completion. A study site is considered closed when all required 
documents and study supplies have been collected and a study-site closure visit has 
been performed. 
The investigator may initiate study-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 study site by the sponsor 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, the sponsor's procedures, or GCP 
guidelines 
• Inadequate recruitment of participants by the investigator 
• Discontinuation of further study intervention development 
 
13 Publication Policy  
Authorship will be determined by mutual agreement and in line with International 
Committee of Medical Journal Editors authorship requirements, as described in the 

 
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publication policy section of the contractual agreement. Briefly, results of this study 
will be submitted for publication in peer reviewed journals as soon as feasible upon 
completion of the study. Appropriate attributions (i.e. co-authorship or 
acknowledgement regarding the generation of data as the case may be) will be 
made in all publications related to this study. Authorship determination will be in 
accordance with the guidelines provided by the International Committee of Medical 
Journal Editors.  
Before a paper or abstract is submitted for publication, the other party shall be 
provided thirty (30) days to review the proposed publication or disclosure to assure 
that Proprietary/Confidential Information is protected and to prevent, at its sole 
discretion, any publication of the party’s own Proprietary/Confidential Information.  In 
the event that Cepheid desires to protect any of the information disclosed in the 
proposed publication, then FIND agrees to delay publication by an additional 60 days 
or such other reasonable amount of time as required to allow Cepheid to make any 
applicable filings.  
 

 
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14 References 
1 World Health Organization. Global tuberculosis report 2015. 2015 
DOI:10.12987/yale/9780300190298.001.0001. 
2 World Health Organization. Report: Multidrug-resistant tuberculosis (MDR TB). 
1998 http://www.who.int/tb/publications/mdrtb-report/en/ 
3 WHO treatment guidelines for drug-resistant tuberculosis: 2016 update. 2016, 
World Health Organization: Geneva, Switzerland. 
4 Boehme, C.C., et al., Feasibility, diagnostic accuracy, and effectiveness of 
decentralised use of the Xpert MTB/RIF test for diagnosis of tuberculosis and 
multidrug resistance: a multicentre implementation study. Lancet, 2011. 
377(9776): p. 1495-505. 
5 Heifets LB, Cangelosi GA. Drug susceptibility testing of Mycobacterium 
tuberculosis: a neglected problem at the turn of the century. Int J Tuberc Lung Dis 
1999; 3: 564–581. 
6 Dinnes J, Deeks JJ, Kunst H, et al. A systematic review of rapid diagnostic tests 
for the detection of tuberculosis infection. Health Technol Assess 2007; 11: 1–
196. 
7 Avalos E, Catanzaro D, Catanzaro A, et al. Frequency and geographic distribution 
of gyrA and gyrB mutations associated with fluoroquinolone resistance in clinical 
Mycobacterium tuberculosis isolates: a systematic review. PLoS ONE 2015; 10: 
e0120470. 
8 Georghiou SB, Magana M, Garfein RS, et al. Evaluation of genetic mutations 
associated with Mycobacterium tuberculosis resistance to amikacin, kanamycin 
and capreomycin: a systematic review. PLoS ONE 2012; 7: e33275. 
9 World Health Organization. The use of molecular line probe assay for the 
detection of resistance to second-line anti-tuberculosis drugs. 2013 
http://apps.who.int/iris/bitstream/10665/78099/1/WHO_HTM_TB_2013.01.eng.pdf
?ua=1 
10. Xie YL, Chakravorty S, Armstrong DT, et al. Evaluation of a Rapid Molecular 
DrugSusceptibility Test for Tuberculosis. New Eng J Med 2017; 377: 1043-54. 
11 Chakravorty S, Roh SS, Glass J, et al. Highly multiplexed, ten-color detection of 
isoniazid, fluoroquinolone, amikacin and kanamycin resistant tuberculosis in an 
automated assay suitable for point-of-care use. J Clin Microbiol 2016; doi: 
10.1128/JCM.01771-16 

 
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12 Seifert M, Catanzaro D, Catanzaro A, et al. Genetic mutations associated with 
isoniazid resistance in Mycobacterium tuberculosis: a systematic review. PLoS 
ONE 2015; 10: e0119628. 
13 Denkinger C, Dolinger D, Miotto P, Rodwell T. Single Nucleotide Polymorphisms 
association with drug resistance in Mycobacterium tuberculosis complex - 
Systematic Review. 2013. Report. 
14 Tessema B, Nabeta P, Valli E, et al. FIND Tuberculosis Strain Bank: a resource 
for researchers and developers working on tests to detect Mycobacterium 
tuberculosis and related drug resistance. J Clin Microbiol 2017; 
doi:10.1128/JCM.01662-16 
 
15 Technical Expert Group Meeting Report: critical concentrations for drug 
susceptibility testing for TB medicines.  Geneva: World Health Organization; 2017 
(WHO/HTM/TB/2017.22). Licence: CC BY-NC-SA 3.0 IGO. 
 
 
 

 
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15 Appendices 
Appendix 1: Safety Definitions and Reporting  
Appendix 2: Incident Definition and Reporting 
Appendix 3: Protocol Amendment Summary Table 
Appendix 4: Sample Requirement to Achieve Target Sample 
Size 
Appendix 5: Definitions of Diagnostic Test Results 
 
 
 

 
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Appendix 1:  Safety Definitions and Reporting 
 
Adverse Event (AE) Definition 
• 
An AE is any unfavorable and unintended sign (including abnormal laboratory finding), 
symptom or disease temporally associated with the use of an investigational product, 
whether or not related to the investigational product.   
 
Serious Adverse Event (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 (e.g., 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. 
Examples of such events include intensive treatment in an emergency room or at home for 
allergic bronchospasm or convulsions that do not result in hospitalization, or development of 
drug dependency or drug abuse. 

 
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SAE Reporting to FIND  
• 
The SAE Report must be sent to the FIND Head of Program and Trial Manager via e-mail, 
marked High Priority, with a follow up call to ensure receipt.  
• 
Initial notification via telephone does not replace the need for the investigator to complete 
and sign the SAE Report within the designated reporting time frames. 
• 
Contacts for SAE reporting can be found on the front of the protocol. 
 
 
 
 
 

 
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Appendix 2:  Incident Definition and Reporting  
Medical Device/IVD Incident Definition 
• 
A medical device incident is any malfunction or deterioration in the characteristics and/or 
performance of a device or IVD 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  
• Fetal distress, fetal death, or any congenital abnormality or birth defects 
 
Medical Device Incident Documenting 
• 
Any medical device incident occurring during the study 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 medical device incidents fulfilling the definition above, complete the SAE Report Form. 
• 
It is very important that the investigator provides his/her assessment of causality (relationship to 
the medical device provided by the sponsor) 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 3: Protocol Amendment Summary Table 
 
The Protocol Amendment Summary of Changes Table for the current amendment is 
located directly before the Table of Contents (TOC). 
Amendment [amendment number]: ([date]) 
 
Overall Rationale for the Amendment 
[Rationale] 
Section # and Name 
Description of Change 
Brief Rationale 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 

 
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Appendix 4: Sample Requirement to Achieve Target 
Sample Size 
  
MTB+ group* 
Additional RIF-R group 
TOTAL 
Samples Tested 
100% 
360 
100% 
400 
760 
Invalid Xpert Ultra results 
1% 
4 
1% 
4 
8 
Culture-contaminated 
10% 
36 
10% 
40 
76 
Molecular-test non-determinant* 
10% 
36 
10% 
40 
76 
Included** 
79% 
284 
79% 
316 
600 
INH-resistant 
29% 
82 
99% 
313 
395 
INH-sensitive 
71% 
202 
1% 
3 
205 
FQ-resistant 
5% 
14 
25% 
79 
93 
FQ-sensitive 
95% 
270 
75% 
237 
507 
AMK/CAP-resistant 
2% 
6 
9% 
28 
34 
AMK/CAP-sensitive 
98% 
278 
91% 
288 
566 
KAN-resistant 
1% 
3 
21.5% 
68 
71 
KAN-sensitive 
99% 
281 
78.5% 
248 
529 
 
*Assume 23% of TB-positive samples are RIF-R based upon previous studies and data from these 
regions11,13. 
 
**We assume 29% INH resistance, 5% FQ resistance and 1-2% second-line injectable (AMK, KAN 
and CAP) resistance among TB-positive patients based upon previous studies and data from these 
regions11,13. Testing 284 TB-positive specimens (based on Xpert MTB/RIF and Ultra-testing) would 
then be expected to yield 82 INH-resistant but only 14 FQ-resistant specimens and 3-6 second-line 
injectable-resistant specimens (limiting factor in sample size calculations). This allows for 
determination of Xpert MTB/XDR performance for INH resistance detection as a reflex test to any TB-
positive result, but is not sufficient for determination for determination of Xpert MTB/XDR performance 
for second-line resistance detection. To efficiently get to a sufficient number of second-line resistant 
samples, we will supplement this by testing 316 additional RIF-resistant samples. To account for 
contaminated cultures and invalid/non-determinant Xpert MTB/RIF or Ultra, NGS and Xpert MTB/XDR 
results, we would inflate these numbers by 21%, leading to a total of 360 TB-positive and 400 
additional RIF-resistant samples, i.e. a total of 760 patient samples tested in this study. 
 
 

 
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Appendix 5: Definitions of Diagnostic Test Results 
 
TEST RESULT 
DESCRIPTION 
Smear-positive 
≥ 1 positive FM smear (inclusive of scanty positive smears) using 
WHO/IUATLD grading. 
Culture-positive  
LJ and/or MGIT culture growth-confirmed MTB complex. 
Culture-negative 
LJ or MGIT have no culture growth after >56 days and >42 days 
Contaminated culture 
LJ: Culture 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). 
NGS INH-sensitive 
Targeted NGS of katG, fabG1, ahpC and inhA does NOT detect any 
mutations associated with INH resistance. 
NGS INH-resistant 
Targeted NGS of katG, fabG1, ahpC and inhA detects at least one 
mutation associated with INH resistance. 
NGS ETH-sensitive 
Targeted NGS of inhA does NOT detect any mutations associated with 
INH resistance. 
NGS ETH-resistant 
Targeted NGS of inhA detects at least one mutation associated with 
INH resistance. 
NGS FQ-sensitive 
Targeted NGS of gyrA and gyrB does NOT detect any mutations 
associated with FQ resistance. 
NGS FQ-resistant 
Targeted NGS of gyrA and gyrB detects at least one mutation 
associated with FQ resistance. 
NGS AMK-sensitive 
Targeted NGS of rrs does NOT detect any mutations associated with 
AMK resistance. 
NGS AMK-resistant 
Targeted NGS of rrs detects at least one mutation associated with AMK 
resistance. 
NGS KAN-sensitive 
Targeted NGS of rrs and eis does NOT detect any mutations associated 
with KAN resistance. 
NGS KAN-resistant 
Targeted NGS of rrs and eis detects at least one mutation associated 
with KAN resistance. 
NGS CAP-sensitive 
Targeted NGS of rrs does NOT detect any mutations associated with 
CAP resistance. 
NGS CAP-resistant 
Targeted NGS of rrs detects at least one mutation associated with CAP 
resistance. 
MTBDRplus INH-
sensitive 
No detection of katG or inhA mutations associated with INH resistance 
by line probe assay (WT probe hybridization, no MUT probes 
hybridized). 
MTBDRplus INH-
resistant 
Detection of katG or inhA mutations associated with INH resistance by 
line probe assay (MUT probe hybridization or absence of WT probe 
hybridization). 

 
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* Drug: INH, ETH, FQ (moxifloxacin and levofloxacin for phenotypic DST), AMK, KAN or CAP 
**Xpert non-determinants: result during initial run or after repeat following an invalid result 
 
 
 
MTBDRsl FQ-sensitive 
No detection of gyrA or gyrB mutations associated with FQ resistance 
by line probe assay (WT probe hybridization, no MUT probes 
hybridized). 
MTBDRsl FQ-resistant 
Detection of gyrA or gyrB mutations associated with FQ resistance by 
line probe assay (MUT probe hybridization or absence of WT probe 
hybridization). 
MTBDRsl AMK-sensitive 
No detection of rrs mutations associated with AMK resistance by line 
probe assay (WT probe hybridization, no MUT probes hybridized). 
MTBDRsl AMK-resistant 
Detection of rrs mutations associated with AMK resistance by line probe 
assay (MUT probe hybridization or absence of WT probe hybridization). 
MTBDRsl KAN-sensitive 
No detection of rrs or eis mutations associated with KAN resistance by 
line probe assay (WT probe hybridization, no MUT probes hybridized). 
MTBDRsl KAN-resistant 
Detection of rrs or eis mutations associated with KAN resistance by line 
probe assay (MUT probe hybridization or absence of WT probe 
hybridization). 
MTBDRsl CAP-sensitive 
No detection of rrs mutations associated with CAP resistance by line 
probe assay (WT probe hybridization, no MUT probes hybridized). 
MTBDRsl CAP-resistant 
Detection of rrs mutations associated with CAP resistance by line probe 
assay (MUT probe hybridization or absence of WT probe hybridization). 
Xpert MTB/XDR Drug*-
sensitive 
Valid result with Drug* resistance NOT detected by Xpert MTB/XDR. 
Xpert MTB/XDR Drug*-
resistant 
Valid result with Drug* resistance detected by Xpert MTB/XDR. 
Xpert MTB/XDR Drug*-
indeterminant  
Valid result with Drug* resistance indeterminant. 
Xpert MTB/XDR non-
determinant** 
No valid run result by Xpert MTB/XDR.