Two-site Laboratory-based Diagnostic Accuracy and Feasibility Study of the Xpert MTB/XDR Assay for Detection of Isoniazid, Fluoroquinolone, Ethionamide and Second-line Injectable Anti-tuberculosis Drug Resistance
Conditions: Tuberculosis, Resistance Bacterial, Diagnoses Disease
Sponsor: Foundation for Innovative New Diagnostics, Switzerland

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 1 of 37 
Study Protocol 
Two-site laboratory-based diagnostic accuracy and feasibility study of 
the Xpert MTB/XDR assay for detection of isoniazid, fluoroquinolone, 
ethionamide and second-line injectable anti-tuberculosis drug 
resistance 
Short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Registration number: 
Study sponsor: Foundation for Innovative New Diagnostics (FIND) 
Study coordinator: University of Cape Town (UCT) 
Protocol version: 1.1
Date: 16 September 2020

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 2 of 37 
Study partners 
Sponsor representative 
Foundation for Innovative New Diagnostics (FIND) 
Samuel G Schumacher 
Email: Samuel.Schumacher@finddx.org 
Address: Chemin des Mines 9, Campus Biotech, 1202 Geneva, Switzerland 
FIND 
Eva Junyent Sallares 
Email : eva.junyent@finddx.org 
FIND 
Adam Penn-Nicholson 
Email: Adam.Penn-Nicholson@finddx.org 
FIND 
Statistician 
Stefano Ongarello 
E-mail : Stefano.Ongarello@finddx.org
Study coordinator 
University of Cape Town (UCT) 
Helen Cox 
Email: helen.cox@uct.ac.za 
Address: Falmouth Building, Anzio Road, Observatory, Cape Town, 7935, South Africa 
Study sites 
UCT 
Site investigator: Helen Cox 
Wits Health Consortium (Pty) Ltd (WHC) 
Site investigator: Lesley Scott 
Email: lesley.scott@wits.ac.za 
Address: 31 Princess of Wales Terrace, Parktown, Johannesburg, 2193 
Other partners 
Ospedale San Raffaele SRL (OSR) 
Daniela Cirillo 
Email: cirillo.daniela@hsr.it 
Address: Via Olgettina 60, 20132 Milano, Italy 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Ludwig – Maximilians – Universitaet Muenchen (LMU) 
Katharina Kranzer 
Email: Katharina.Kranzer@lshtm.ac.uk 
Address: Geschwister Scholl Platz 1, 80539 Munich, Germany, 
Swiss Tropical and Public Health Institute (Swiss TPH) 
Klaus Reither 
Email: klaus.reither@swisstph.ch 
Address: Socinstrasse 57, 4051 Basel, Switzerland 
African Society for Laboratory Medicine (ASLM) 
Nqobile Ndlovu 
Email: nndlovu@aslm.org 
Address: Nega City Mall, 8th Floor, Joseph Tito Street, Kirkos Subcity, Kebele 08, Addis Ababa, Ethiopia 
University of Heidelberg 
Claudia Denkinger 
Email: Claudia.Denkinger@uni-heidelberg.de 
Address: Im Neuenheimer Feld 672324, 69120 Heidelberg 
Co-investigators 
UCT 
Adrian Brink 
Email: adrian.brink@uct.ac.za 
UCT 
Chad Centner 
Email: chadc@nis.za 
UCT 
Mark Nicol 
Email: Mark.Nicol@uwa.edu.au 
WHC 
Anura David 
Email: anura.david@witshealth.ac.za 
OSR 
Eliza Tagliani 
Email: tagliani.elisa@hsr.it 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 4 of 37 
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.  

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 5 of 37 
Signature page (sponsor) 
Protocol title: Two-site laboratory-based diagnostic accuracy and feasibility study of the Xpert MTB/XDR 
assay for detection of isoniazid, fluoroquinolone, ethionamide and second-line injectable anti-tuberculosis 
drug resistance 
Protocol number: TB042 
We, the undersigned, have developed, reviewed and approved this protocol, including appendices. We will 
supervise and coordinate the clinical trial according to the principles outlined in the Declaration of Helsinki 
and Good Clinical Practice and in compliance with applicable regulatory requirements. The signatures 
below confirm that the signatories have reviewed and approved this document which shall govern the 
conduct of the specified research study. 
SPONSOR: 
Foundation for Innovative New Diagnostics (FIND) 
_________________________________  _________________________________  _________________________________ 
Name 
 
 
 
     Signature 
 
 
         Date 
STUDY COORDINATOR: 
University of Cape Town (UCT) 
_________________________________  _________________________________  _________________________________ 
Name 
 
 
 
     Signature 
 
 
         Date 
STUDY SITES: 
University of Cape Town (UCT) 
_________________________________  _________________________________  _________________________________ 
Name 
 
 
 
     Signature 
 
 
         Date 
Wits Health Consortium (Pty) Ltd (WHC) 
_________________________________  _________________________________  _________________________________ 
Name 
 
 
 
     Signature 
 
 
         Date 
STUDY PARTNERS: 
Ludwig-Maximilians-Universitaet Muenchen (LMU) 
_________________________________  _________________________________  _________________________________ 
Name 
 
 
 
     Signature 
 
 
         Date 
Helen Cox
12 Feb 2021
Helen Cox
12 Feb 2021
Samuel Schumacher
12 Feb 2021
Lesley Scott
25020221

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 6 of 37 
Ospedale San Raffaele SRL (OSR) 
_________________________________  _________________________________  _________________________________ 
Name 
 
 
 
     Signature 
 
 
         Date 
Swiss Tropical and Public Health Institute (Swiss TPH) 
_________________________________  _________________________________  _________________________________ 
Name 
 
 
 
     Signature 
 
 
         Date 
University of Heidelberg 
_________________________________  _________________________________  _________________________________ 
Name 
 
 
 
     Signature 
 
 
         Date 
African Society for Laboratory Medicine (ASLM) 
_________________________________  _________________________________  _________________________________ 
Name 
  Signature 
  Date 
Nqobile Ndlovu
12 February 2021
Klaus Reither 
 
 
 
 
 
 
 
25.Feb 2021
Claudia Denkinger 
12. Feb 2021
Daniela Maria Cirillo
25 Feb 2021
Daniela Maria Cirillo
Daniela Maria Cirillo
25 Feb 2021
25 Feb 2021

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Table of contents 
 
Study partners 
2 
Sponsor representative 
2 
Study coordinator 
2 
Study sites 
2 
Other partners 
2 
Co-investigators 
3 
Confidentiality statement 
4 
Signature page (sponsor) 
5 
Table of contents 
7 
Protocol history / amendment summary 
9 
List of abbreviations and acronyms 
10 
Protocol synopsis 
11 
Background 
14 
Aims and objectives 
15 
Endpoints 
17 
Primary endpoint 
17 
Secondary endpoints 
17 
Study design 
19 
Diagnostic accuracy study 
19 
Reference standard 
19 
Standard of care comparator 
19 
Time-to-result study 
19 
Study sites 
19 
UCT: Green Point TB Laboratory, Cape Town 
19 
WHC: Port Elizabeth Hospital Complex, Port Elizabeth 
20 
Study population and setting 
20 
Eligibility criteria 
20 
Sample size 
21 
Minimisation of error and bias 
23 
Study intervention 
24 
In vitro diagnostics 
24 
Study procedures 
25 
Xpert MTB/XDR 
25 
SOC comparator 
26 
Reference standard 
27 
Blinding procedure 
29 
Statistical analysis 
30 
Analysis populations 
30 
Descriptive statistics 
30 
Analysis of endpoints 
30 
Missing values 
31 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Sample size 
31 
Data management 
32 
Ethical considerations 
33 
Safety assessments 
34 
Covid-19 considerations 
35 
End of study definition 
36 
References 
37 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 9 of 37 
Protocol history / amendment summary 
Version number 
Release date 
Comments 
1.0 
9 July 2020 
Initial version 
1.1
16 September 2020
,QFOXGeV institutional review 
committee VXJJeVteG eGLtV LQ 
reG on pages 23 and 27

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 10 of 37 
List of abbreviations and acronyms 
CRF: Case report form 
FIND: Foundation for Innovative New Diagnostics 
IT: Index test 
ITT: Intended to test 
IUO: Investigational use only 
IVD: In vitro diagnostic 
LMU: Ludwig-Maximilians-Universitaet Muenchen 
LPA: Line probe assay 
MGIT: Mycobacterial Growth Indicator Tube (Bactec liquid culture) 
Mtb: Mycobacterium tuberculosis 
MDR-TB: Multidrug-resistant tuberculosis 
NHLS: National Health Laboratory Service 
OSR: Ospedale San Raffaele SRL 
PP: Per protocol 
RS: Reference standard 
SR: Sample Reagent (Xpert MTB/RIF Ultra) 
SOC: Standard of care 
UCT: University of Cape Town 
WHC: Wits Health Consortium (Pty) Ltd 
WHO: World Health Organization 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Protocol synopsis 
Title 
Two-site laboratory-based diagnostic accuracy and feasibility study of the Xpert 
MTB/XDR assay for detection of isoniazid, fluoroquinolone, ethionamide and 
second-line injectable anti-tuberculosis drug resistance 
Short title 
TB-CAPT MTB/XDR Study 
Code 
TB042 
Protocol version 
and date 
Version 11, 16 September 2020
Background and 
rationale 
The WHO-endorsed Cepheid Xpert MTB/RIF and Xpert MTB/RIF Ultra cartridge-
based molecular assays have been rolled out widely in South Africa and 
elsewhere, providing rapid TB diagnosis as well as rifampicin susceptibility testing. 
However, knowledge of susceptibility to isoniazid and second-line anti-tuberculosis 
drugs is critical to selecting the most effective treatment regimen for rifampicin-
resistant and multidrug-resistant tuberculosis (MDR-TB), and for preventing 
generation of further resistance. With high burdens of rifampicin-resistant- and 
MDR-TB in South Africa and other countries, new second-line susceptibility tests 
that are rapid, scalable and affordable need to be evaluated and considered for 
implementation. 
The Cepheid Xpert MTB/XDR cartridge, which runs on the same platform as Xpert 
MTB/RIF Ultra, has been developed to detect additional resistance to isoniazid, 
fluoroquinolones and second-line injectable anti-tuberculosis drugs and provides 
results within 2 hours and on primary samples. An evaluation of the the Xpert 
MTB/XDR assay is currently underway in clinical settings in South Africa, India and 
Moldova. The TB-CAPT MTB/XDR Study will add further diagnostic accuracy and 
feasibility data to the evidence base for the Xpert MTB/XDR assay.  
Study design 
Two-site laboratory-based diagnostic accuracy and feasibility study 
Index test 
The Cepheid Xpert MTB/XDR assay performed directly on routine patient 
respiratory samples which have tested positive for rifampicin-resistant 
Mycobacterium tuberculosis (Mtb) by Xpert MTB/RIF Ultra 
Reference 
standard 
The primary analysis will be based on a composite reference standard comprising 
phenotypic susceptibility testing and whole genome sequencing performed on 
cultured isolates. For the composite reference standard, samples will be defined 
as “resistant” if they are resistant by either phenotypic susceptibility testing or 
whole genome sequencing, and “susceptible” if they tested susceptible by both 
methods. Isolates will be obtained from the second or follow-up routine specimen 
Comparator 
Secondary analyses will compare the accuracy of Xpert MTB/XDR to that of 
standard of care (SOC) susceptibility testing (comprising the Hain MTBDRplus and 
MTBDRsl line probe assays and selected phenotypic susceptibility testing as per 
the South African national diagnostic algorithm) 
Primary 
objective(s) 
To estimate the diagnostic accuracy of Xpert MTB/XDR for detection of isoniazid, 
fluoroquinolone, ethionamide and second-line injectable drug resistance on patient 
samples that test positive for rifampicin-resistant Mtb on Xpert MTB/RIF Ultra 
using a composite reference standard 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Secondary 
objective(s) 
1.
To estimate the diagnostic accuracy of Xpert MTB/XDR for detection of
isoniazid, fluoroquinolone and second-line injectable drug resistance
compared to phenotypic susceptibility testing and whole genome sequencing
separately
2.
To estimate the diagnostic accuracy of Xpert MTB/XDR for detection of each
resistance-conferring mutation as identified by whole genome sequencing
3.
To compare the diagnostic accuracy of Xpert MTB/XDR to the accuracy of the
SOC comparator for the detection of isoniazid, fluoroquinolone and second-
line injectable drug resistance against the composite reference standard
4.
To compare the proportion of participants for whom valid susceptibility results
become available based on Xpert MTB/XDR to that of SOC
5.
To determine time-to-result for isoniazid, fluoroquinolone and second-line
injectable drug susceptibility testing, comparing Xpert MTB/XDR to SOC line
probe assays
6.
To evaluate the feasibility of performing Xpert MTB/XDR on residual specimen
previously processed for Xpert MTB/RIF Ultra (the “Sample Reagent [SR]-
sputum mix”) under routine conditions
Primary 
endpoints 
(outcomes) 
Sensitivity and specificity of Xpert MTB/XDR vs. the composite reference standard 
for detection of: 
•
Isoniazid
•
Fluoroquinolone
•
Ethionamide
•
Second-line injectable drug resistance
Secondary 
endpoints 
(outcomes) 
1.
Sensitivity and specificity of Xpert MTB/XDR compared to phenotypic
susceptibility testing and whole genome sequencing separately
2.
Sensitivity and specificity of Xpert MTB/XDR for detection of individual
resistance-conferring mutations
3.
Sensitivity and specificity of Xpert MTB/XDR compared to that of SOC vs. the
composite reference standard for detection of:
o
Isoniazid
o
Fluoroquinolone
o
Second-line injectable drug resistance
4.
Proportion of participants for whom valid susceptibility testing results become
available based on Xpert MTB/XDR assay or based on SOC testing
5.
Median time-to-result comparing Xpert MTB/XDR to SOC (line probe assays)
6.
Feasibility endpoints:
o
Proportion of participants with sufficient residual SR-sputum mix (≥2
ml) available to perform Xpert MTB/XDR
o
Median period between preparation of SR-sputum mix and start of
Xpert MTB/XDR test
o
Proportion of specimens with non-determinate Xpert MTB/XDR
results, separately for each drug class and combined for all, and
comparing SR-sputum mix stored according to manufacturer
recommendations (at 2-8⁰C for ≤4 hours or 25-35⁰C for ≤2.5 hours)
and those exceeding manufacturer limits for operational reasons
Study sites 
•
UCT: National Health Laboratory Service (NHLS) Green Point TB Laboratory,
Cape Town, South Africa (≥500 Xpert MTB/RIF Ultra tests performed daily)
•
WHC: NHLS Port Elizabeth Hospital Complex TB Laboratory, Port Elizabeth,
South Africa (≥400 Xpert MTB/RIF Ultra tests performed daily)

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Study 
population 
Respiratory specimens testing rifampicin-resistant Mtb positive on Xpert MTB/RIF 
Ultra 
Eligibility 
criteria 
Inclusion: 
•
Ultra-positive, rifampicin-resistant respiratory specimen (expectorated sputum,
induced sputum, tracheal aspirates and bronchoalveolar lavage)
Exclusion for all endpoints: 
•
Residual SR-sputum mix not retained
•
Patient specimen previously included in the study
Exclusion for diagnostic accuracy and time-to-result endpoints: 
•
Volume of residual SR-sputum mix for Xpert MTB/XDR is <2 ml
•
Xpert MTB/XDR unsuccessful
•
No second / follow-up specimen received
•
Second or follow-up specimen culture-negative, contaminated or not available
for reference standard testing
•
Reference standard uninterpretable
Biostatistics 
Analysis populations 
•
Intended to test (ITT): all samples intended to be tested
•
Per protocol (PP): all samples with valid test results for the index test and
reference standard
Analysis of primary endpoint: 
Sensitivity and specificity will estimated together with their 2-sided 95%-Wilson-
Score-intervals. 
Sample size 
For diagnostic accuracy and time-to-result study: 
N=220 UCT 
N=100 WHC  
Total N=320 
For feasibility study: 
N=373 UCT 
N=380 WHC 
Total N=753 
Study duration 
38 weeks (estimated 2 rifampicin-resistant specimens/site/day) 
Time schedule 
July 2020: Submit for departmental and ethical review 
July 2020: Submit to NHLS, Department of Health (local and provincial) for 
approval 
October 2020: First patient in  
July 2021: Last patient In 
September 2021: Reference standard results available for all participants  
Study flow 
See Figure 2 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Background 
The WHO-endorsed Cepheid Xpert MTB/RIF and Xpert MTB/RIF Ultra cartridge-based molecular assays 
have been rolled out widely in South Africa and elsewhere,1 providing rapid tuberculosis diagnosis as well 
as rifampicin susceptibility testing. However, knowledge of susceptibility to isoniazid and second-line anti-
tuberculosis drugs is critical to selecting the most effective treatment regimen for rifampicin-resistant and 
multidrug-resistant tuberculosis (MDR-TB), and for preventing generation of further resistance.2 
The Cepheid Xpert MTB/XDR cartridge has been developed to detect additional resistance to isoniazid, 
fluoroquinolones and second-line injectable anti-tuberculosis drugs. This rapid, cartridge-based real-time 
PCR assay, which runs on the same platform as Xpert MTB/RIF and Xpert MTB/RIF Ultra, provides results 
within 2 hours and on primary samples,3,4 thereby having clear advantage over the WHO-endorsed Hain 
Lifescience Genotype MTBDRplus and Genotype MTBDRsl assays which are technically complex and 
perform better on cultured isolates especially in smear-negative cases. Other strategies like phenotypic 
testing are hampered by the long delay to get results, the need for specialised testing centers, technical 
demands and reliability. Next generation sequencing is currently expensive, too complex to be implemented 
widely and requires DNA from cultured isolates. With high burdens of rifampicin-resistant- and MDR-TB in 
South Africa and other countries,5,6 new second-line susceptibility tests that are rapid, scalable and 
affordable need to be evaluated and considered for implementation. 
In a feasibility study, the prototype of the Xpert MTB/XDR assay showed moderate sensitivity against 
phenotypic susceptibility testing to detect isoniazid resistance (83%), although this result may have been 
biased by the reference method utilised (critical concentration) and sensitivity was higher when compared 
to sequencing (98%).3 The sensitivity of MTBDRplus for detection of phenotypic isoniazid resistance is 
estimated to be 90%.7 Sensitivity of Xpert MTB/XDR for the detection of fluoroquinolone resistance was 94-
97%,3 comparing favourably with the sensitivity of MTBDRsl (93%).8 An evaluation of the final version of 
the Xpert MTB/XDR assay is currently underway in clinical settings in South Africa, India and Moldova.9  
The TB-CAPT MTB/XDR Study will add further diagnostic accuracy and feasibility data to the evidence 
base for the Xpert MTB/XDR assay. 

Aims and objectives 
The aim of the study is to evaluate the Xpert MTB/XDR cartridge for diagnostic accuracy, and to evaluate 
the feasibility of performing Xpert MTB/XDR on residual respiratory specimens previously processed for 
Xpert MTB/RIF Ultra (the “Sample Reagent [SR]-sputum mix”) under routine conditions 
The primary objective is to estimate the diagnostic accuracy of the Xpert MTB/XDR assay for the detection 
of resistance to: 
a.
Isoniazid
b.
Fluoroquinolones
c.
Ethionamide
d.
Second-line injectable anti-tuberculosis drugs
in respiratory specimens positive for rifampicin-resistant Mycobacterium tuberculosis (Mtb) by Xpert 
MTB/RIF Ultra using a composite reference standard. 
The composite reference standard comprises phenotypic susceptibility testing and whole genome 
sequencing (WGS) performed on culture isolates. For the composite reference standard, samples will be 
defined as “resistant” if they are resistant by either phenotypic susceptibility testing or WGS and 
“susceptible” if they tested susceptible by both methods. 
The secondary objectives are to: 
1)
Estimate the diagnostic accuracy of Xpert MTB/XDR for detection of resistance to isoniazid,
fluoroquinolones and second-line injectable anti-tuberculosis drugs compared to:
a.
Phenotypic susceptibility testing alone
b.
WGS alone
2)
Estimate the diagnostic accuracy of Xpert MTB/XDR for detection of individual resistance-
conferring mutations as determined by WGS
3)
Compare the diagnostic accuracy of Xpert MTB/XDR to the accuracy of standard of care (SOC)
susceptibility testing (comprising Hain MTBDRplus and MTBDRsl line probe assays [LPA]s and
selected phenotypic testing according to the national diagnostic algorithm) against the composite
reference standard
4)
Compare the proportion of participants for whom valid1 susceptibility testing results become
available based on Xpert MTB/XDR to the proportion of participants for whom valid susceptibility
testing results become available based on SOC testing
5)
Determine the time-to-result of Xpert MTB/XDR and compare to that of LPAs performed as SOC
6)
Evaluate the feasibility of performing Xpert MTB/XDR on residual SR-sputum mix under routine
conditions:
a.
Determine the proportion of participants with sufficient residual SR-sputum mix (≥2 ml)
available to perform Xpert MTB/XDR
b.
Determine the median period between processing for Xpert MTB/RIF (preparation of SR-
sputum mix) and the start of the Xpert MTB/XDR test
c.
Determine the non-determinate rate of Xpert MTB/XDR:
i.
For each drug separately and combined for all
ii.
Comparing specimens stored according to manufacturer recommendations (at 2-
8⁰C for ≤4 hours or 25-35⁰C for ≤2.5 hours) and those exceeding those limits for
operational reasons
iii.
Correlated with semi-quantitation of bacterial load on Xpert MTB/RIF Ultra
1 Valid result defined as a non-indeterminate result for all three anti-tuberculosis drug classes tested 
Protocol short title: TB-CAPT MTB/XDR Study  
Protocol number: TB042 
Protocol Version: v1.1  
 
 
 
 
 
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Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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iv.
By sub-group (sufficient specimen, smear status, age group)
7)
Exploratory analyses:
a.
Diagnostic accuracy by sub-group
b.
Diagnostic accuracy by rifampicin susceptibility result concordance between Xpert
MTB/RIF Ultra and LPA
c.
Detection of heteroresistance for each anti-tuberculosis drug class, comparing Xpert
MTB/XDR to WGS
d.
Description of discordance between phenotypic testing and WGS
e.
Description 
of 
discordance 
between 
testing 
methodologies 
by 
presence 
of
heteroresistance

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Endpoints 
Primary endpoint 
Sensitivity and specificity of Xpert MTB/XDR assay vs. the composite reference standard for detection of 
resistance to: 
1.
Isoniazid
2.
Fluoroquinolones
3.
Ethionamide
4.
Second-line injectable anti-tuberculosis drugs
Secondary endpoints 
1)
Sensitivity and specificity of Xpert MTB/XDR for detection of resistance to:
a.
Isoniazid
b.
Fluoroquinolones
c.
Second-line injectable anti-tuberculosis drugs
compared to phenotypic anti-tuberculosis drug susceptibility testing and WGS separately 
2)
Sensitivity and specificity of Xpert MTB/XDR vs. WGS for detection of mutations in the following gene
targets:
a.
inhA promoter
b.
katG
c.
gyrA
d.
gyrB
e.
eis
f.
rrs
3)
Sensitivity and specificity of Xpert MTB/XDR compared to that of SOC testing against the composite
reference standard for detection of resistance to:
a.
Isoniazid
b.
Fluoroquinolones
c.
Second-line injectable anti-tuberculosis drugs
4)
Proportion of participants for whom valid test results become available based on:
a.
Xpert MTB/XDR
b.
SOC testing:
i.
LPAs performed directly on specimen (“direct”)
ii.
LPAs performed on culture isolates (“indirect”)
5)
Median time-to-result:
a.
Separately for each drug class, and comparing:
i.
Xpert MTB/XDR
ii.
SOC testing:
1.
Direct LPAs
2.
Indirect LPAs
6)
Feasibility endpoints:
a.
Proportion of participants with sufficient residual SR-sputum mix (≥2 ml) available to perform
Xpert MTB/XDR

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 18 of 37 
b.
Median period between preparation of SR-sputum mix and start of Xpert MTB/XDR test
c.
Proportion of specimens with non-determinate MTB/XDR results:
i.
Combined for all
ii.
Separately for each drug class
iii.
Separately for each target gene
iv.
Comparing specimens stored according to manufacturer recommendations (at 2-8⁰C
for ≤4 hours or 25-35⁰C for ≤2.5 hours) and those exceeding those limits for operational
reasons
v.
By subgroup:
1.
Sufficient specimen (yes/no)
2.
Smear status
3.
Age group
7)
Exploratory endpoints:
a.
Sensitivity and specificity of Xpert MTB/XDR assay for detection of resistance to each anti-
tuberculosis drug class by subgroup:
i.
Sufficient specimen (yes/no)
ii.
Smear status
iii.
Age group
b.
Sensitivity and specificity of Xpert MTB/XDR assay for detection of resistance to each anti-
tuberculosis drug class by rifampicin susceptibility result concordance between Xpert MTB/RIF
Ultra and LPA
c.
Agreement between Xpert MTB/XDR and WGS for the detection of heteroresistance for each
anti-tuberculosis drug class
d.
Description of discordance between phenotypic testing and WGS
e.
Description of discordance between testing methodologies by presence of heteroresistance

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Study design 
Study description 
This is a 2-site laboratory-based diagnostic accuracy and feasibility study. Residual SR-sputum mix 
remaining from samples which have tested positive with rifampicin-resistant Mtb on Xpert MTB/RIF Ultra 
will be submitted for Xpert MTB/XDR (the index test). A second or follow-up specimen, submitted routinely 
either together with the first specimen (Cape Town) or after the rifampicin-resistant result (Port Elizabeth), 
will undergo Bactec MGIT culture routinely. Should the MGIT culture be positive, it will be used for the 
reference standard tests. 
Reference standard 
The composite reference standard comprises phenotypic susceptibility testing and WGS performed on 
culture isolates from the second or follow-up specimen. For the composite reference standard, samples will 
be defined as “resistant” if they are resistant by either phenotypic susceptibility testing or WGS, and 
“susceptible” if they tested susceptible by both methods. This reference standard has been chosen as it is 
the same standard used in the previous diagnostic accuracy study. 
Standard of care comparator 
The SOC comparator will comprise susceptibility testing for isoniazid, fluoroquinolones and second-line 
injectable anti-tuberculosis drugs using MTBDRplus and MTBDRsl LPAs either directly on specimens, or if 
unsuccessful, on culture isolates, and phenotypic testing, according to the South African national algorithm 
(Figure 3). 
Time-to-result study 
Time-to-result (in hours) will be defined as per Table 1 and compared for each drug class separately. 
Table 1. Definition of time-to-result endpoints in the TB-CAPT MTB/XDR Study 
Start point 
End point 
SOC 
Xpert MTB/XDR 
Registration-to-first 
second-line anti-
tuberculosis drug 
susceptibility result 
Time at which first 
specimen is registered 
in the laboratory 
Time at which first LPA (direct 
or indirect) susceptibility result 
is entered on the laboratory 
information system 
Time at which Xpert MTB/XDR 
resulted on the GeneXpert / or 
entered on the laboratory 
information system if feasible 
Rifampicin-resistant 
result-to-first 
second-line anti-
tuberculosis drug 
susceptibility result 
Time at which the 
rifampicin-resistant 
result is reported on the 
laboratory information 
system 
Study sites 
UCT: Green Point TB Laboratory, Cape Town 
Green Point laboratory is a centralised National Health Laboratory Service (NHLS) tuberculosis diagnostic 
laboratory located in Green Point, Cape Town, Western Cape, South Africa. The laboratory receives 
specimens from about 420 facilities in the Western Cape, including secondary hospitals and primary health 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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clinics. The laboratory performs >500 Ultra tests daily. It has facilities for MGIT culture, Hain MTBDRplus 
and MTBDRsl LPAS performed directly or indirectly on culture isolates. The laboratory is SANAS-accredited 
and has ongoing collaboration with UCT and Stellenbosch University. There is a dedicated microbiologist 
on site. 
WHC: Port Elizabeth Hospital Complex, Port Elizabeth 
The Port Elizabeth National Health Services Laboratory is a high-throughput tuberculosis diagnostic 
laboratory based in Port Elizabeth, Eastern Cape, South Africa. It services approximately 69 primary health 
care facilities and hospitals in the Eastern Cape.  The laboratory performs 400-500 Ultra tests daily. The 
laboratory also performs MGIT culture, first- and second-line line probe assays performed directly from 
specimens and from culture isolates and first and second-line phenotypic drug susceptibility testing. The 
laboratory is SANAS accredited with a dedicated pathologist on site. 
Study population and setting 
The study population will comprise patients from any of the laboratory’s referral facilities who test positive 
for rifampicin-resistant Mtb by Xpert MTB/RIF Ultra testing of respiratory specimens. Respiratory specimens 
are expectorated sputum, induced sputum, tracheal aspirates and bronchoalveolar lavage, but exclude 
gastric washings and nasopharyngeal aspirates. 
For patients with two or more respiratory specimens, only the first will be utilised. 
Eligibility criteria 
Inclusion: 
●
Mtb-positive, rifampicin-resistant respiratory specimen identified on Xpert MTB/RIF Ultra
Exclusion: 
●
Residual SR-sputum mix not retained or not found
●
Patient previously included in the study
Exclusion for diagnostic accuracy and time-to-result endpoints: 
●
Insufficient residual SR-sputum mix remaining for Xpert MTB/XDR (<2 ml)
●
Xpert MTB/XDR unsuccessful
●
No second / follow-up specimen received
●
Second / follow-up specimen culture-negative, contaminated or not available
●
Reference standard uninterpretable (phenotyping or WGS)
o
Where phenotypic susceptibility testing results are uninterpretable, specimens will still be
included in WGS comparison
o
Where WGS results are uninterpretable, specimens will still be included in phenotypic
susceptibility testing comparison

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Figure 1. Eligibility flow in the TB-CAPT MTB/XDR Study. RR = rifampicin-resistant, SR = Xpert MTB/RIF Ultra 
Sample Reagent, MGIT = Mycobacterial Growth Indicator Tube, DST = drug-susceptibility testing, WGS = whole 
genome sequencing  
Sample size 
320 specimens will be included in the diagnostic accuracy study. The sample size is dictated by the 
resources allocated to the project and it is expected that the data generated will be combined in meta-
analyses with other studies currently underway. The sample size calculation regards the uncertainty of 
estimation of sensitivity among reference standard (RS)-positives and specificity among RS-negatives, 
respectively, expressed by the width of the 2-sided 95% confidence intervals. For technical reasons, the 
Pearson-Clopper-CI have been used in sample size considerations. The differences compared to 
the Wilson-Score-CI planned to be used in statistical analysis are negligible. Within sample size 
considerations, the widths of 95%-Pearson-Clopper-intervals for 70%, 80% and 90% sensitivity (or 
specificity) were dependent on prevalence of resistance to a specific anti-tuberculosis drug (11-50%) and 
sample sizes of 320, 500 and 750. The width ranges from more 30% (70% sensitivity at a prevalence (for 
D+) of 11% among 320 patients) down to 4.7% (90% spec at a prevalence (for D-) of 89% among 750 
patients. 
Table 2 shows expected prevalence6 of resistance to anti-tuberculosis drug class and the related number 
of resistant and susceptible specimens among 320, 500 and 750 participants together with confidence 
intervals for proportions of 70%, 80% and 90%.  

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Table 2. Widths of 95%-Pearson Clopper confidence intervals dependent on prevalence of resistance and diagnostic 
accuracy for a total sample size of 320 
Type of 
resistance 
Resistant 
Prev. 
Measure 
D+ or D- among 
320 
500 
750 
N 
lCL 
uCL 
N 
lCL 
uCL 
N 
lCL 
uCL 
Isoniazid 
resistance 
yes 
61% 
n (Prev.) 
196 
305 
458 
70% Sens 
137 63.0% 76.3% 214 64.5% 75.1% 321 65.6% 74.2% 
80% Sens 
157 73.7% 85.4% 244 75.1% 84.3% 366 76.0% 83.6% 
90% Sens 
176 84.9% 93.8% 275 86.1% 93.1% 412 86.9% 92.6% 
no 
39% 
n (1-Prev.). 125 
195 
293 
70% Spec 
88 
61.2% 77.9% 137 63.0% 76.3% 205 64.4% 75.2% 
80% Spec 
100 71.9% 86.6% 156 73.7% 85.4% 234 75.0% 84.4% 
90% Spec 
113 83.4% 94.6% 176 84.9% 93.8% 264 86.0% 93.2% 
Second-line 
injectable 
resistance 
yes 
15% 
n (Prev.) 
48 
75 
113 
70% Sens 
34 
54.9% 82.5% 
53 
58.3% 80.0% 
79 
60.6% 78.3% 
80% Sens 
38 
65.9% 90.2% 
60 
69.2% 88.4% 
90 
71.4% 86.9% 
90% Sens 
43 
77.6% 96.9% 
68 
80.9% 95.7% 102 82.9% 94.9% 
no 
85% 
n (1-Prev.) 
272 
425 
638 
70% Spec 
190 64.2% 75.4% 298 65.4% 74.3% 447 66.3% 73.5% 
80% Spec 
218 74.7% 84.6% 340 75.9% 83.7% 510 76.7% 83.0% 
90% Spec 
245 85.8% 93.3% 383 86.7% 92.7% 574 87.4% 92.2% 
Fluoroquinolone 
resistance  
yes 
11% 
n (Prev.) 
36 
55 
83 
70% Sens 
25 
52.3% 84.2% 
39 
56.1% 81.6% 
58 
58.9% 79.6% 
80% Sens 
29 
63.2% 91.5% 
44 
67.0% 89.6% 
66 
69.8% 88.0% 
90% Sens 
32 
75.2% 97.5% 
50 
78.9% 96.4% 
75 
81.3% 95.6% 
no 
89% 
n (1-Prev.). 285 
445 
668 
70% Spec 
200 64.3% 75.3% 312 65.5% 74.2% 468 66.4% 73.5% 
80% Spec 
228 74.9% 84.5% 356 76.0% 83.6% 534 76.8% 83.0% 
90% Spec 
257 85.9% 93.2% 401 86.8% 92.6% 601 87.5% 92.2% 
Ethionamide 
resistance 
yes 
50% 
n (Prev.) 
160 
250 
375 
70% Sens 
112 62.3% 77.0% 175 63.9% 75.6% 263 65.1% 74.6% 
80% Sens 
128 73.0% 85.9% 200 74.5% 84.8% 300 75.6% 83.9% 
90% Sens 
144 84.3% 94.2% 225 85.6% 93.4% 338 86.5% 92.8% 
no 
50% 
n (1-Prev.) 
160 
250 
375 
70% Spec 
112 62.3% 77.0% 175 63.9% 75.6% 263 65.1% 74.6% 
80% Spec 
128 73.0% 85.9% 200 74.5% 84.8% 300 75.6% 83.9% 
90% Spec 
144 84.3% 94.2% 225 85.6% 93.4% 338 86.5% 92.8% 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 23 of 37 
D+ =presence of resistance, D- =absence of resistance, Prev=prevalence, y=yes, n=no, lCL=lower confidence level, 
uCL=upper confidence level  
Red and green: widest and narrowest interval  
Due to rounding errors, deviations of +/-1 case could occur for total N. 
It is expected that a proportion of specimens will not be available for the diagnostic accuracy study 
because of an invalid Xpert MTB/XDR result (~5%), unavailability of a second or follow-up specimen 
and negative or contaminated culture. An additional 433 specimens will be required giving a final total of 
N=753 (Table 3). 
Table 3. Number of specimens required to undergo Xpert MTB/XDR to achieve final sample size of N=320 for 
diagnostic accuracy study, and anticipated enrolment period 
UCT 
WHC 
Total 
% 
N 
% 
N 
N 
Total specimens required 
373 
380 
753 
  Available for feasibility study 
100 
373 
100 
380 
753 
  Not available for diagnostic accuracy and time-to-result study 
41 
153 
71 
280 
433 
  Insufficient specimen remaining for Xpert MTB/XDR 
1 
4 
1 
4 
  Xpert MTB/XDR indeterminate 
5 
19 
5 
19 
  No second / follow-up specimen 
10 
37 
40 
152 
  Culture-negative or contaminated 
20 
75 
20 
76 
  MGIT not available 
5 
19 
5 
19 
  Available for diagnostic accuracy and time-to-result study 
59 
220 
29 
100 
320 
Enrolment period  
(@ 2 rifampicin-resistant specimens/day) 
37 weeks 
38 weeks 
Minimisation of error and bias 
Consecutive, unselected samples with rifampicin-resistant MTB detected on Xpert Ultra testing in the 
routine laboratory will be used. These will include samples from a wide variety of facility types, geographical 
areas and patient/clinical backgrounds. Patients will only be excluded if they have been included previously, 
and not for any patient related factors. The study population will therefore be directly representative of the 
future target population and context of the Xpert MTB/XDR test. 
Reference standard phenotyping, WGS and downstream bioinformatics analysis will be performed blinded 
to the index test result, which will have been performed in a different laboratory several weeks or months 
earlier. 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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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 isoniazid, 
ethionamide, fluoroquinolone and second-line injectable 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. 
Any IVD incidents, including those resulting from malfunctions of the IVD, must be detected, documented 
and reported by the investigator throughout the study 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 25 of 37 
Study procedures 
Respiratory specimens submitted routinely from healthcare facilities in the laboratory’s referral area will be 
used for study procedures (Figure 2). Residual SR-sputum mix from the first specimen will be used for 
Xpert MTB/XDR, and the second or follow-up specimen for the reference standard and SOC comparator. 
Xpert MTB/XDR 
Respiratory specimens routinely received by the laboratory for Xpert MTB/RIF Ultra will be processed as 
per routine (non-study) standard operating procedures: 
1.
Add SR at a ratio of 2:1 SR:sputum to unprocessed sputum
2.
Vortex for 10 s
3.
Incubate at room temperature for 10 m
4.
Vortex for 10 s
5.
Incubate at room temperature for 5m
6.
Transfer 2 ml of SR-sputum mix to the Xpert MTB/RIF Ultra cartridge
7.
Load Xpert MTB/RIF Ultra cartridge on GeneXpert instrument
Under routine non-study conditions, the residual SR-sputum mix is discarded after it has been used for 
Xpert MTB/RIF Ultra. In the CAPT-TB MTB/XDR Study, these samples will be not be discarded 
immediately, but set aside for possible Xpert MTB/XDR later: 
1.
After transferring SR-sputum mix to Xpert MTB/RIF Ultra cartridge, set residual SR-sputum mix
aside at room temperature
2.
Review Xpert MTB/RIF Ultra results on the GeneXpert after 2 hours and note laboratory numbers
of rifampicin-resistant samples
3.
Remove rifampicin-resistant SR-sputum samples from the batch that was set aside in (1)
4.
Transfer 2 ml of SR-sputum mix to the Xpert MTB/XDR cartridge
5.
Load Xpert MTB/XDR cartridge on GeneXpert instrument
6.
Discard remaining SR-sputum mix samples
7.
If operational factors preclude performing (4) and (5) immediately after the rifampicin-resistant
result is reported on the GeneXpert instrument:
a.
Place rifampicin-resistant SR-sputum samples in a refrigerator (4℃)
b.
The next morning, rifampicin-resistant specimens will be removed from the refrigerator and
brought to room temperature
c.
Continue (4) to (6)

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Figure 2. Specimen flow in the TB-CAPT MTB/XDR Study with both routine (green) and study (yellow) processes 
included. SR = Sample Reagent, Rif-S = rifampicin-susceptible, Rif-R = rifampicin-resistant,  LPA = line probe assay, 
MGIT= Mycobacterial Growth Indicator Tube, Pheno = phenotypic drug susceptibility testing, WGS = whole genome 
sequencing 
SOC comparator 
A second or follow-up specimen is sent routinely according to national guidelines for any rifampicin-resistant 
case of tuberculosis. In Cape Town, the second specimen is “front-loaded” / sent at the same time as the 
first specimen. In PE, the follow-up specimen will be sent routinely when the patient is brought back for the 
rifampicin-resistant result. The second or follow-up specimen undergoes routine MTBDRplus and MTBDRsl 
LPA testing, directly on the specimen (“direct”), and, if direct testing is unsuccessful, on the BACTEC MGIT 
culture isolate (“indirect”), with or without supplementary phenotypic testing as required (Figure 3). The 
finalised NHLS result will count as the SOC comparator. 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Figure 3. Routine NHLS workflow for second / follow-up specimen received from patients with rifampicin-resistant 
results on Xpert MTB/RIF Ultra testing of the first specimen. Testing for rifampicin, fluoroquinolones and second-line 
injectable anti-tuberculosis drugs are included in the algorithm. Testing for bedaquiline and linezolid, and others, are 
also performed routinely in the NHLS. Ultra = Xpert MTB/RIF Ultra, FLQ = fluoroquinolone, INJ = injectables, RIF = 
rifampicin, INH = isoniazid, MGIT = Mycobacterial Growth Indicator Tube, R = resistant, S = susceptible 
Reference standard 
The primary analysis will be based on a composite reference standard comprising phenotypic susceptibility 
testing and WGS performed on culture isolates. Cultured isolates will be obtained from routine MGITs 
performed on the second or follow-up specimen. For the composite reference standard, samples will be 
defined as “resistant” if they are resistant by either phenotypic susceptibility testing or WGS and 
“susceptible” if they tested susceptible by both methods. 
The reference standard will be performed on the second / follow-up specimen received for participants with 
rifampicin-resistant Mtb result on Xpert MTB/RIF Ultra testing of the first specimen (Figure 4). 
Phenotypic susceptibility testing will be performed on the second / follow-up specimen, if culture positive, 
using Bactec MGIT and testing the following anti-tuberculosis drugs at the stated concentrations: 
•
Isoniazid @ 0.1 µg/ml
•
Levofloxacin @ 1.0 µg/ml
•
Kanamycin @ 2.5 µg/ml
A proportion of phenotypic testing will be done routinely by the NHLS laboratory according to the national 
testing algorithm. :Kere pKeQRt\pLF teVtLQJ LV QRt LQGLFDteG DFFRrGLQJ tR tKe QDtLRQDO DOJRrLtKm teVtLQJ 
will be performed at a local research laboratory with appropriate expertise )LJXre . In summary, non-
SOC phenotypic testin will be performed in the these scenarios: MDR-TB (isoniazid), levofloxaxin-resistant 
MDR-TB (levofloxacin) and all isolates (kanamycin). The %DFteF 0*,7 pODtIRrm will be utilised at all sites 
DQG testing ZLOO tKereIRre be VtDQGDrGLVeG 1RQS2& teVtLQJ ZLOO QRt be repRrteG tR the clinical service.

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 28 of 37 
Isolates of Mtb from MGIT culture of the second / follow-up specimen will be inactivated and shipped to 
Ospedale San Raffaele SRL (OSR) in Milan, Italy for WGS to detect resistance-conferring mutations. 
Appropriate bioinformatic pipelines and anti-tuberculosis drug resistance databases will be utilised. 
Figure 4. Reference standard workflow in the TB-CAPT MDR/XDR Study. Rif-R = rifampicin-resistant, SOC = 
standard-of-care, MGIT = Mycobacterial Growth Indicator Tube, DST = drug susceptibility testing, WGS = whole 
genome sequencing, NGS = next generation sequencing 
Figure 5. Phenotypic susceptibility testing in the TB-CAPT MTB/XDR study. Blue = routine procedure, purple = study 
procedure. RR = rifampicin-resistant, FL = first-line, SL = second-line, LPA = line probe assay, DST = drug 
susceptibility testing, FLQ = fluoroquinolones, pheno = phenotypic 
Xpert RR-TB
100%
FL LPA
INH R
80%
Pheno INH DST
Study
INH S
20%
Pheno INH DST 
Routine 
(0.1ug/ml)
SL LPA
FLQ S
85%
Pheno FLQ DST
Routine (Levo)
FLQ R
15%
Pheno FLQ DST 
Study
Phenotypic Kan 
DST
Study

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Blinding procedure 
The index test and reference standards will be performed independently and at different times on different 
samples by different operators in separate laboratories. The index test will be performed on the first sample 
in the routine laboratory and the reference standard on the second sample in either a different part of the 
routine laboratory or in a research laboratory. Each specimen will be assigned a separate laboratory 
number and the report issued separately. 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 30 of 37 
Statistical analysis 
A full Statistical Analysis Plan will be developed prior to conclusion of the study. A brief overview of planned 
statistical analyses is provided here. 
Analysis populations 
•
Intended to test (ITT): all samples intended to be tested
•
Per protocol (PP): all samples with valid test results for the index test and reference standard
Descriptive statistics 
Continuously scaled variables are described by mean, standard deviation, median, minimum and 
maximum. Categorical scaled variables are described by absolute and relative frequencies. 
Analysis of endpoints 
This is a study to estimate diagnostic accuracies, thus hypotheses are not established. 
Diagnostic accuracy study 
Estimation of sensitivity and specificity 
The following 2x2 table is analysed: 
Table  4. Contingency table for analysis of diagnostic accuracy 
RS 
Positive 
Negative 
Total 
IT 
Positive 
TP = a 
FP = b 
a+b 
Negative 
FN = c 
TN = d 
c+d 
Total 
a+c 
b+d 
a+b+c+d 
RS=reference standard, IT=index test, TP=true positive, FP=false positive, FN=false negative, TN=true negative 
The diagnostic accuracy measures are calculated according the following formulas: 
Table 5. Formulas and rules to calculate diagnostic accuracy measures 
Measure 
Formula 
Confidence interval 
Sensitivity 
a/(a+c) 
2-sided 95%-Wilson-Score-CI
Specificity 
d/(b+d) 
2-sided 95%-Wilson-Score-CI
Comparison of diagnostic accuracies 
More details will be provided in the Statistical Analysis plan 
Further endpoints / feasibility study 
Other endpoints are evaluated by means of descriptive statistics; estimates are presented with 95%-
confidence interval. Time-to-result analyses will include calculation of medians and Kaplan-Meier analyses. 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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More details will be provided in the Statistical Analysis plan. 
Missing values 
No imputation 
Sample size 
See page 21 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Data management 
Data will be entered into case report forms or obtained from the laboratory information system. 
Data management will be co-ordinated by Ludwig-Maximilians-Universitaet Muenchen (LMU) and FIND. 

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
 Page 33 of 37 
Ethical considerations 
This study will address an urgent need for rapid susceptibility testing in rifampicin-resistant and MDR-TB, 
which currently takes days, weeks or months to complete, and often results in long delays in the initiation 
of or change to appropriate treatment.10 We are evaluating a test that could reduce that turnaround time to 
hours, with potential knock-on effects on more rapid treatment initiation and reduction in development / 
transmission of resistance. Therefore, the potential benefits of this study to patient care and society are 
substantial. 
Because rifampicin resistance is relatively rare and difficult to predict, prospective enrolment of patients 
would take too long and be too resource intensive to undertake. Laboratory-based screening would enable 
identification of patients with drug resistance and therefore eligibility for second-line testing. However, the 
centralised tuberculosis laboratory receives patient samples from a geographically wide referral area – 
locating and recalling these patients for enrolment in the study would be impractical. Furthermore, the Xpert 
MTB/XDR test is currently an investigational assay and therefore not reportable or able to be used to impact 
patient care. The proposed study procedures would not affect the usual care of patients as we will utilise 
specimens that would ordinarily be discarded. Similarly, the reference standard tests (next generation 
sequencing) are non-routine tests not currently utilised for patient care in South Africa. These tests will be 
performed post hoc with patient identifiers removed. 
For these reasons, we are proposing a waiver of informed consent for this diagnostic accuracy and 
feasibility study. 
The study poses no risks or benefits to participants. Participants will still receive standard of care testing 
according to the national algorithm and only leftover samples will be utilised.  
This study will be conducted according to the ethical principles set forth in the Declaration of Helsinki, ICH-
GCP, and local regulatory requirements as applicable. All source documents will be available for review by 
relevant institutional review committees when requested.  

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Safety assessments 
The study will comply with all biosafety precautions appropriate to the study procedures, including the 
preparation of primary specimens for Xpert MTB/XDR and handling of culture isolates for the reference 
standard. Study procedures will be performed in laboratories accredited for safe handling of these specimen 
types, and according to the laboratories’ safety protocols. All testing will be performed by appropriately 
trained and accredited personnel. Study staff will be monitored for TB symptoms, including cough, fever, 
weight loss and night sweats.  

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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Covid-19 considerations 
Study procedures will comply with all UCT, WHC, NHLS and FIND Covid-19 safety policies in effect at the 
time of implementation. A Covid-19 Risk Management Plan will be submitted separately. No patient contact 
is required for the study.  

Protocol short title: TB-CAPT MTB/XDR Study 
Protocol number: TB042 
Protocol Version: v1.1  
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End of study definition 
Participation in this laboratory-based study will start on Xpert MTB/XDR testing of the first specimen and 
end with finalisation of the reference standard testing results from the second specimen. There are no 
specific stopping rules on an individual participant level. The study will end when the reference standard 
tests for the last sample included are finalised. The study will be stopped if any information becomes 
available with regard to any risk to participants or study personnel. Should Xpert MTB/XDR be approved 
and implemented nationally, patient results will be reported to the clinical service.  

Protocol short title: TB-CAPT MTB/XDR Study 
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Protocol Version: v1.1  
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References 
1.
Cazabon D, Suresh A, Oghor C, et al. Implementation of Xpert MTB/RIF in 22 high tuberculosis
burden countries: are we making progress? Eur Respir J 2017; 50(2).
2.
WHO. WHO consolidated guidelines on drug-resistant tuberculosis treatment. 2019.
3.
Xie YL, Chakravorty S, Armstrong DT, et al. Evaluation of a Rapid Molecular Drug-Susceptibility
Test for Tuberculosis. N Engl J Med 2017; 377(11): 1043-54.
4.
Chakravorty S, Roh SS, Glass J, et al. Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and
Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-
Care Use. J Clin Microbiol 2017; 55(1): 183-98.
5.
WHO. Global Tuberculosis Report. 2019.
6.
Ismail NA, Mvusi L, Nanoo A, et al. Prevalence of drug-resistant tuberculosis and imputed
burden in South Africa: a national and sub-national cross-sectional survey. Lancet Infect Dis 2018; 18(7):
779-87.
7.
Nathavitharana rifampicin-resistant, Cudahy PG, Schumacher SG, Steingart KR, Pai M, Denkinger
CM. Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis: a
systematic review and meta-analysis. Eur Respir J 2017; 49(1).
8.
Tagliani E, Cabibbe AM, Miotto P, et al. Diagnostic Performance of the New Version (v2.0) of
GenoType MTBDRsl Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable
Drugs: a Multicenter Study. J Clin Microbiol 2015; 53(9): 2961-9.
9.
Xpert MTB/XDR Clinical Evaluation Trial. 2019. https://clinicaltrials.gov/ct2/show/NCT03728725.
10.
Cox H, Dickson-Hall L, Ndjeka N, et al. Delays and loss to follow-up before treatment of drug-
resistant tuberculosis following implementation of Xpert MTB/RIF in South Africa: A retrospective cohort
study. PLoS Med 2017; 14(2): e1002238.