Evaluation of Host Biomarker-based Point-of-care Tests for Targeted Screening for Active TB
Conditions: Pulmonary Tuberculosis
Sponsor: Prof Gerhard Walzl

 
 
 
 
RIA Diagnostics 
 
 
2nd European & Developing Countries 
Clinical Trials Partnership Programme 
(EDCTP2) 
 
 
Grant Agreement Number DRIA2014-311 
 
 
 
 
Evaluation of host biomarker-based point-of-care 
tests for targeted screening for active TB 
 
 
Deliverable Nr. 2.1 
Study protocol 
 
Contractual delivery date: 
Month 3 (30/06/2016) 
 
Actual delivery date: 
Month 3 (30/06/2016) 
 
Responsible partner: 
P1: Stellenbosch University (SUN) 
 
 
 

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Deliverable number 
D2.1 
Deliverable title 
Study Protocol 
Nature 
R 
Dissemination level 
Co 
Work package number 
WP 2 
Work package leader 
SUN 
Author(s) 
Dr Elizna Maasdorp 
Keywords 
Study Protocol 
 
The research leading to these results has received funding from the EDCTP2 programme supported by 
the European Union under grant agreement No DRIA2014-311. 
 
The author is solely responsible for the content, which does not represent the opinion of the European 
Community or the EDCTP. The Community and the EDCTP are not responsible for any use that might be 
made of data appearing therein. 
 
 
 

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Deliverable 2.1, the study protocol has been achieved.   
 
A generic protocol was developed by the Stellenbosch University team for distribution to other sites.  
Other sites are to adapt this protocol according to local circumstances and requirements, for submission 
to their various regulatory bodies. 
 
The protocol has been circulated to all the sites on 5 May 2016. 
 
Please find attached the generic draft circulated. 
 
 

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Title: 
Evaluation of host biomarker-based point-of-care tests for targeted screening for 
active TB 
 
Acronym: 
Screen TB 
 
Protocol Number: to be allocated 
 
Sponsored by: 
The European & Developing Countries Clinical Trials Partnership (EDCTP) 
 
Principal Investigator: 
Prof Gerhard Walzl 
 
Version Number:  
0.3 
 
Date:   
05/05/2016

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Protocol Summary 
 
 
 
 
 
3 
Schematic of Study Design 
 
 
 
 
5 
1 
Key Roles 
 
 
 
 
 
 
6 
 
2 
Background Information and Scientific Rationale 
 
 
8 
2.1 
Background Information  
 
 
 
 
8 
2.2 
Scientific Rationale 
 
 
 
 
 
8 
2.3 
Potential Risks and Benefits 
 
 
 
 
10 
3 
Objectives 
 
 
 
 
 
 
10 
4 
Study Design 
 
 
 
 
 
 
11 
4.1 
Study Phases 
 
 
 
 
 
 
12 
4.2  
Evaluation of TB diagnosis 
 
 
 
 
12 
5 
Study Population  
 
 
 
 
 
14 
5.1 
Selection of the Study Population  
 
 
 
14 
5.2 
Inclusion/Exclusion Criteria 
 
 
 
 
14 
6 
Study Procedures/Evaluations 
 
 
 
 
14 
6.1 
Study Procedures 
 
 
 
 
 
14 
6.2 
Study Schedule  
 
 
 
 
 
18 
6.3 
Laboratory Evaluations 
 
 
 
 
 
20 
7 
Data Management Plan 
 
 
 
 
 
22 
7.1 
Data Collection  
 
 
 
 
 
22 
7.2 
Data Management 
 
 
 
 
 
22 
7.3 
Data Storage 
 
 
 
 
 
 
22 
8 
Assessment of Safety 
 
 
 
 
 
22 
9 
Clinical Monitoring 
 
 
 
 
 
23 
10 
Statistical Considerations  
 
 
 
 
23 
10.1 
Study Outcome Measures and Analysis 
 
 
 
23 
10.2 
Sample Size Considerations 
 
 
 
 
24 
11 
Ethical Considerations 
 
 
 
 
 
24 
11.1 
Subject Confidentiality 
 
 
 
 
 
24 
11.2 
Future Use of Stored Specimens 
 
 
 
 
24 
11.3 
Informed Consent Process 
 
 
 
 
25 
12 
Literature References 
 
 
 
 
 
25 
 

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Protocol Summary 
Title: 
Evaluation of host biomarker-based point-of-care tests for targeted screening for active TB 
(Screen TB) 
Population: A total of 800 people presenting at primary health care clinics with presumed active 
pulmonary tuberculosis, aged 18 to 70 years, male or female gender, will be recruited. They should be 
willing to give informed, written consent, including consent for HIV testing. They should have symptoms 
that could be compatible with active TB (cough > two weeks, plus at least one of the following: fever, 
weight loss, hemoptysis and night sweats). Participants should not have been on TB treatment for the 
past 90 days and should not have received immune suppressive therapy, be known with alcohol of drug 
abuse or have a haemoglobin level <10g/dl. HIV co-infection is not an exclusion criterion.  Participants 
will be recruited from primary health care clinics in Cape Town, South Africa, Windhoek in Namibia, 
Addis Ababa in Ethiopia, Banjul in The Gambia and Kampala in Uganda.  
Number of Sites: 
See Section 1 
Study Duration: 
3 years 
Subject Duration: 
6 months for TB cases, 2 months for non-TB cases 
Objectives: 
The overall objective of the study is to incorporate a six-marker serum signature into a multiplex UCP-
LFA format, referred to as TransDot, for finger-prick blood testing. The end point of the study is the 
accuracy (sensitivity and specificity) of the UCP-LFA TransDot test on finger-prick blood for active TB and 
will be prospectively compared against gold standard composite diagnostic criteria (GeneXpert, MGIT 
culture, TB sputum smear, CXR, TB symptom screen and response to TB treatment).  
Primary: 
• The primary outcome of interest will be accuracy, sensitivity and specificity of the TransDot finger-
prick test when compared with the composite gold standard tests. 
 
Secondary: 
• To evaluate the agreement between the POC TransDot test and laboratory based ELISAs first (both on 
serum), and subsequently between POC TransDot (on fingerprick blood) and laboratory based  TransDot 
(on serum).   

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• To investigate the utility of a TransDot test at month 2 and month 6 as a marker of treatment 
response. 
• To identify additional host marker signatures that can be utilized for future improvement of diagnostic 
tests in the TransDot format or other point-of care tests that might become available in the future  
• To collect appropriate additional host samples for future biomarker research.  
 
 
 

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Schematic of Study Design: 
 
 
 
 
 
 
 
10ml blood collected for serum 
optimization and validation (vs 
ELISA) of TransDot test at LUMC 
and later for lab-based TransDot 
at local site laboratory 
4 ml for QFN testing 
Study-specific biorepository:  
-16ml blood for PBMC 
-2.5ml for PAXgene 
-2 Salivettes (1ml saliva each) 
-3x1ml sputum 
-Urine-10ml 
No TB 
Probable TB 
Possible TB 
Definite TB 
First 500 recruited into training set 
300 Recruited into test set 
Fingerprick TransDot test performed at field site 
after symptom screen and clinical evaluation and 
before CXR  
4ml for QFN testing 
Study-specific biorepository:  
-10ml blood collected for serum  
-16ml blood for PBMC,  
-2.5ml for PAXgene 
-2 Salivettes (1ml saliva each) 
-3x1ml sputum 
-Urine- 10ml 
Week 2 Follow-up (all GeneXpert negative participants):  Medical history and examination, evaluation of 
treatment response to either antibiotics or TB treatment and further diagnostic examination as clinically 
Meets exclusion criteria 
Meets inclusion criteria 
Routine diagnostic tests: 
Sputum (3ml for  smear, 
GeneXpert 
and 
MGIT 
culture 
CXR 
HIV rapid test (finger prick) 
Urine pregnancy test for 
women 
Week 8 Follow-up (all participants):  
Participants not diagnosed with TB:  Repeat diagnostic assessment (medical history and examination, 
sputum GeneXpert and culture, CXR) if indicated according to study clinician.     
Participants on TB treatment:  Clinical treatment response assessment (medical history and examination , 
smear (1ml sputum), culture (1ml sputum).  Repeat TransDot test for investigation of its treatment response 
utility.  Draw 10ml blood for serum, 16ml for PBMC, 2.5ml for Paxgene, 2 salivettes, 10ml of urine, 5x 1ml of 
sputum for smear, culture and storage 
Exclude from study 
Continue routine care in local 
clinic 
Baseline visit: 
Medical history 
Clinical examination 
Informed consent 
Week 24 Follow-up (only TB case participants) :   Clinical treatment response assessment (medical 
history and examination , smear (1ml sputum),culture (1ml sputum), CXR).  Repeat TransDot test for 
investigation of its treatment response utility.   Draw 10ml blood for serum, 16ml for PBMC, 2.5ml for 
Paxgene, 2 salivettes, 10ml of urine, 5x 1ml of sputum for smear, culture and storage. 
Screening of TB suspects for study criteria 
Each participant final outcome classified by outcome classification committee (blinded to TransDot test results 
in test set participants) according to previously developed case definitions, incorporating GeneXpert, culture, 
CXR,  TB symptoms, response to antibiotics and TB treatment response.  This constitutes the composite gold 
standard test

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1. 
KEY ROLES 
Individuals 
Stellenbosch 
University 
Roles  
Name 
Qualification 
 
Principal Investigator 
Gerhard Walzl 
MBChB, MMed, 
FCP, PhD 
Sub-Investigators 
Novel Chegou 
PhD 
 
Stephanus Malherbe 
MBChB 
 
Elizna Maasdorp 
MBChB, MSc 
Quality Assurance manager 
Liesel Muller  
MSc 
Laboratory manager 
Bronwyn Smith 
MSc 
Research assistants 
Nonjabulo Makhoba 
BSc 
Study Clinician 
Justine Khoury 
MBChB 
Research nurses 
Shirley McAnda 
Dip Nursing 
Protocol Statistician 
Martin Kidd 
PhD 
Database Managers 
Gian van der Spuy 
PhD 
 
Kim Stanley 
MSc 
Safety Officer 
Stephanus Malherbe 
MBChB 
Collaborating Institutions 
 
Stellenbosch University, South Africa 
MRC, The Gambia 
University of Namibia, Windhoek, Namibia 
Makerere University School of Medicine, Kampala, Uganda 
Armauer Hansen Research Institute, Addis Ababa, Ethiopia 
Eurice, Germany 
London School of Hygiene and Tropical Medicine, London, UK 
Leiden University Medical Centre, Leiden, The Netherlands 
 
For information regarding this protocol, contact: 
 
Prof Gerhard Walzl 
Stellenbosch University Immunology Research Group 
DST/NRF Centre of Excellence for Biomedical TB Research/MRC Centre for TB Research 
Division of Molecular Biology and Human Genetics 

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Faculty of Medicine and Health Sciences 
Stellenbosch University  
 
PO Box 241, Cape Town, 8000 
Francie van Zijl Drive, Tygerberg, South Africa 
 
Tel:   +27 21 938 9158 
Fax: +27 21 938 9863 
E-mail: 
 
gwalzl@sun.ac.za

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2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE  
 
2.1  BACKGROUND INFORMATION 
Tuberculosis (TB) is a leading cause of death and places severe pressure on health care services of 
the developing world.  Despite the introduction of the highly sensitive and specific GeneXpert 
MTB/RIF (GeneXpert) test [1] with a potential turn-around time of two hours, many people in high 
TB prevalence areas still do not have access to efficient TB diagnostic services due to logistical 
constraints that plague these settings.  
Centralized laboratory placement of GeneXpert instruments necessitate transportation of sputum 
samples, which means additional clinic visits before initiation of treatment, often leading to loss to 
follow-up.   Twenty-six percent of patients with positive sputum microbiology are not started on 
treatment, partly due to the need for multiple health care visits [2]. The relatively high cost of both 
instrument and cartridges are also problematic. Liquid culture is even less accessible and takes up to 
42 days to deliver a result while sputum smear microscopy has a low sensitivity of 60% [3].  
Microbiological tests are less sensitive in human immune-deficiency virus (HIV) co-infection [4] and 
are less suitable in young children, in whom it can be difficult to obtain sputum.  The high burden of 
undiagnosed TB fuels ongoing transmission and poor treatment outcomes [5]. 
Although the policy of indiscriminate TB case finding by mobile mass chest X-rays (CXR) has not been 
successful and was abandoned decades ago, a recent World Health Organisation (WHO) document 
has revisited the concept of targeted CXR screening for active pulmonary TB, with strong screening 
recommendations for several high risk groups, including close contacts of TB cases, people living 
with HIV, residents of high TB prevalence areas or remote areas with poor access to health care [6]. 
CXR has high sensitivity (98%; 95% CI 95-100%) but lower specificity (75%, 95% CI 72-79%) [6]. 
Availability of X-rays in resource-limited settings is even less than for GeneXpert, requires skilled 
personnel, is relatively expensive (> 10 Euros/CXR) and is therefore not an optimal screening tool.  
A cost effective, rapid, inexpensive, point-of-care screening test with high sensitivity and high 
negative predictive value would identify people with a high likelihood for active TB and would 
prioritize them for testing with more expensive and technically or logistically demanding assays 
including GeneXpert or liquid culture, and thus facilitate cost-effective diagnostic work-up in 
resource-limited settings.   
2.2 
SCIENTIFIC RATIONALE 

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Host serum protein biomarkers that indicate a high likelihood for active TB disease represent 
attractive targets for integration into screening tests. The EDCTP 1-funded African European 
Tuberculosis Consortium (AE-TBC) has previously investigated host biosignatures in whole blood 
culture supernatants after overnight stimulation with Mycobacterium tuberculosis (MTB) specific 
proteins.  During this project >1,300 people from high TB prevalence areas (South Africa, Namibia, 
Malawi, The Gambia, Uganda and Ethiopia) with symptoms suggestive of active TB were recruited 
and worked up diagnostically with CXR, sputum culture, smear and GeneXpert. One third of the 
participants had confirmed active TB and 26% were HIV co-infected.   A no-go decision was taken 
regarding the whole blood MTB stimulated culture signature as low sensitivity (75 - 80%) with 
specificity of 86% was deemed insufficient for further development and as an overnight assay is not 
suitable for rapid point of care (POC) testing.  
However, a promising serum host inflammatory signature was identified during this project after 
investigation of more than 70 serum host inflammation markers, including acute phase proteins, T 
helper  cell 1, T helper cell 2 and regulatory cytokines, soluble cytokine receptors and growth factors. 
The original set of markers for screening were chosen according to the availability of multiplexed 
assays (Luminex platform) and their known roles in inflammation and represented a wide spectrum 
of markers with diagnostic potential. The most promising host serum protein signature was 
subsequently validated on 687 people from five African countries with suspected TB, regardless of 
HIV infection status or ethnicity, providing the basis for the follow-up work suggested herein. The 
six-analyte signature of C-reactive protein (CRP), Interferon gamma (IFN-γ), pre-albumin, 
complement factor H (CFH), apolipoprotein A1 and inducible protein 10 (IP-10) ascertained TB 
disease with a sensitivity of 89% (CI 78 – 95%) and specificity of 76% (CI 68 – 83%), (positive 
predictive value of 61%; negative predictive value of 94%) ([7], [8]).  Measurement of multiple 
markers and evaluation of their ratios offset a lack of disease specificity of individual markers. These 
performance characteristics would constitute a valuable screening test if converted into an 
inexpensive POC test and would prioritize individuals with a high likelihood of active TB for 
confirmatory testing by GeneXpert.   
During the AE-TBC project, partner Leiden University Medical Centre (LUMC) developed and 
validated a user-friendly lateral flow assay (LFA) for simultaneous detection of multiple host TB 
biomarkers (cytokines and antibodies) in blood or other body fluids [9, 10]. These LFAs are likely to 
assist in rapid TB diagnosis in low-resource settings and were tested in the AE-TBC field sites.  
Importantly, our test formats utilize novel, nano-sized upconverting phosphor (UCP) reporter 
particles as read-out [11, 12, 13]. The UCP-technology delivers flexibility with respect to sensitivity 
and robustness as the ultrasensitive fluorescent label is not hampered by common auto-

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fluorescence background. Moreover, the label does not fade, allowing UCP-test strips to be stored 
indefinitely for reanalysis. The LFA can also be modified for measurement of markers in fingerprick 
blood, which will further enhance its POC utility. 
The overall objective of the study is to incorporate the six-marker signature into an UCP-LFA format, 
the TransDot assay, enabling fingerprick blood testing. The end point of the study is the accuracy 
(sensitivity and specificity) of the UCP-LFA TransDot test on fingerprick blood for active TB and will 
be prospectively compared against composite gold standard diagnostic criteria of GeneXpert, 
mycobacterial growth indicator tube (MGIT) culture, TB sputum smear, CXR, TB symptom screen, 
response to antibiotics and response to TB treatment). 
2.3 
POTENTIAL RISKS AND BENEFITS 
2.3.1 
POTENTIAL RISKS 
Potential risks are limited to the minor risks associated with phlebotomy and the small risk of subject 
confidentiality being compromised.  As this study does not involve any intervention, we do not 
expect to add any additional risks other than the ones inherent to standard of care, to participants.   
2.3.2 
KNOWN POTENTIAL BENEFITS 
Participants will be investigated for tuberculosis more thoroughly than during standard of care and 
will therefore benefit from a more certain diagnosis.  They will also be followed up for 6 months and 
have additional investigations during follow up and at end of treatment, which will guide their 
further care.  They will be referred to the appropriate level of care for any incidental findings during 
investigation, like malignancy, which would most likely not have been found at that stage in other 
circumstances. 
3 
 OBJECTIVES 
Primary objective:   
To prospectively evaluate the accuracy (sensitivity and specificity) of the UCP-LFA TransDot 
fingerprick test (hereafter called TransDot) in a laboratory-free manner, based on the six marker 
serum biosignature as a screening test for active TB, as compared to a composite gold standard ( 
GeneXpert, MGIT culture,  chest X-ray, TB symptom screen, response to antibiotics and response to 
TB treatment) in a population of TB suspects from resource-poor, high TB incidence settings in South 
Africa, Uganda, The Gambia, Namibia and Ethiopia. 
Secondary objectives:   

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To evaluate the agreement between the TransDot test and laboratory based ELISAs first (both on 
serum), and subsequently between the TransDot (on fingerprick blood) and laboratory based  
TransDot (on serum).  
To investigate the utility of a TransDot test at month 2 and month 6 as a marker of treatment 
response. 
To identify additional host marker signatures that can be utilized for future improvement of 
diagnostic tests in the TransDot format or other point-of care tests that might become available in 
the future. 
To collect appropriate additional host samples for future biomarker research. 
4. 
STUDY DESIGN   
A non-interventional, diagnostic, prospective cohort design will be utilized, incorporating two 
separate sets of participants – overall a training set of 500 and a test set of 300 participants.  The 
purpose of the training set will be to optimize the TransDot test and Reader to excellent sensitivity 
(90% or above) and acceptable specificity (80%).  The test set will then be utilized to evaluate the 
TransDot test accuracy on fingerprick blood at the point-of-care site in a laboratory-free manner on 
a new prospective cohort of participants.   
Training set participants will be recruited and receive investigations for TB.  Blood samples will also 
be collected from them for performance of ELISAs and laboratory-based TransDot tests. These blood 
samples will be drawn at baseline, week 8 and week 24 at end of treatment for confirmed TB cases 
and at baseline and  for non-TB cases.  
Test set participants  will be recruited and receive investigations for TB.  A POC TransDot test will be 
performed on fingerprick blood at baseline, and at week 8 and week 24 in participants on TB 
treatment, as well as a laboratory based TransDot test on serum at baseline.   The week 8 and week 
24 TransDot tests will be used to investigate the test’s utility as an indicator of treatment response. 
Follow up of both sets of participants will be to week 24 for those found to have active TB, and week 
8 for those found to be non-TB cases.  There will be study visits at baseline, week 2 (GeneXpert 
negative participants only), week 8 and week 24 (participants on TB treatment only). 
The TransDot test will be evaluated against a composite gold standard in the form of previously 
developed case definitions, consisting of GeneXpert, MTB culture, CXR, TB symptoms, response to 
antibiotics and TB treatment response.  The case definitions will be applied to participant data by an 
outcome classification committee (see 4.2) who will remain blinded to the TransDot results of the 
test set, until after completion of all participants’ outcome classification.  

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The primary outcome of interest will be performance of the TransDot test as compared to the gold 
standard, measured by sensitivity and specificity.  
4.1 
STUDY PHASES 
The study will be conducted in three phases.  During the first phase a blood POC test using UCP-LFA 
TransDot [14] technology will be developed by LUMC, incorporating six serum markers, CRP, IFN-γ, 
pre-albumin, complement factor H, apolipoprotein A1 and IP-10.  During this phase optimal antibody 
pairs for the lateral flow strips will be evaluated. In the event of a lack of optimal test performance 
for individual markers, replacement markers will be selected according to ongoing marker evaluation 
at the respective sites.   
The TransDot test device technology will be optimized using serum from a subset of the first 500 
recruited participants.  These samples will be shipped to LUMC.   
During a second phase of the study, TransDot devices will be available in the African laboratories, to 
test the remaining training set participants’ serum locally.  The first two phases will aim to optimise 
the sensitivity and specificity of the TransDot test.    
The third phase will be the evaluation of the TransDot test in the field setting, by research nurses 
testing test set participants’ fingerprick blood.   
4.2 
EVALUATION OF TB DIAGNOSIS 
An outcome classification committee will classify each participant according to the standard case 
definitions below.  The committee will consist of clinician investigators from the participating sites.   
They will assign a classification of Definite TB, Probable TB, Possible TB or non-TB (see table below) 
to each participant, as soon as possible after the week 8 follow up time point.  Participants started 
on TB treatment at clinics will be followed up  for 6 months to evaluate treatment response. During 
phase 3 of the study, the committee will remain blind to the point of care TransDot test results on 
the test cohort, until outcome classification is complete. 

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The case definitions will be applied as shown in the table below:    
Pos = positive; neg = negative; con = contaminated; GR = good response; PR = poor response; NG = none given 
Category 
Culture  
GeneXpert 
AFB smear 
CXR 
Symptoms 
TB Treatment 
started by clinic 
TB Treatment 
response 
Response to antibiotic 
(AB), if prescribed 
Class 
A 
pos 
 
 
  
pos 
  
  
  
Definite 
B 
neg or con 
pos  (no TB in past year) 
 
 
pos 
 
 
 
Definite 
C 
neg or con 
neg or pos (TB in past year) 
pos 
pos 
pos 
yes 
GR 
 
Definite 
D 
neg or con 
neg or pos (TB in past year) 
pos 
neg  
pos 
yes  
GR  
  
Probable 
E 
neg or con 
neg or pos (TB in past year) 
neg 
pos 
pos 
yes 
GR 
 
Probable 
F 
neg or con 
neg or pos (TB in the past year) 
neg 
pos 
pos 
No 
 
PR 
Possible 
G 
neg or con 
pos (TB in past year) 
neg 
neg 
pos 
yes 
GR 
 
Possible  
H 
neg 
neg 
neg 
pos 
pos 
no 
 
GR 
No TB 
I 
neg 
neg  
neg 
neg  
pos 
no 
 
NG 
No TB 
J 
Not done or con 
Not done 
Not done 
 
 
 
 
 
Exclude  
K 
Not done or con 
pos (TB in past year) 
pos or neg 
neg or not done 
pos 
yes 
PR 
 
Exclude  
L 
Not done or 
contam 
Pos TB in past year 
pos or neg 
neg or not done 
pos 
no 
NG 
 PR 
Exclude 
Definitions: 
TB symptoms or signs: symptoms volunteered or specifically elicited by interviewer and signs objectively observed by examiner that include     
Cough ≥ 2 weeks;  Fever (subjective, present ≥ 2 weeks);  Weight loss (significant, unintentional loss of  ≥ 10% of body weight in last 6 months. If no previous weight available, the patient’s judgement 
of weight loss will be used);  Haemoptysis (blood stained to blood clots);  Wasting (clinical finding of examining health care professional) ;  Night sweats (drenching of sleeping clothes or bedding, not 
related to environment);  Adenopathy (clinical finding of significantly enlarged lymph nodes by examining health care professional), of particular significance in HIV co-infected individuals;  Other: may 
be considered in exceptional cases and on individual basis by case review team and include shortness of breath and chest pain 
Sputum culture positive: liquid or solid agar positive with confirmed speciation for M. tuberculosis complex 
CXR compatible with active TB: CXR pattern regarded by research clinician as typical for adult TB (any of the following: cavitation, diffuse upper lobe or mid-zone infiltrates, including air-space nodules or 
clustered nodules, consolidation in upper or mid zones, unilateral or bilateral lymph node enlargement, particularly in HIV co-infected participants, pleural effusion). CXR pattern not typical for adult TB (diffuse 
infiltrates in lower lobe, miliary type infiltrate) may be included after consultation of the outcome classification committee.  
TB treatment response: baseline symptom improvement of ≥50%, weight gain, sputum smear or culture conversion, CXR improvement after at least two months of TB treatment. 
- Failed antibiotic (AB) treatment: No response of symptoms to a 7 day, broad spectrum oral or intravenous antibiotic 

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5. 
STUDY POPULATION 
5.1 
SELECTION OF THE STUDY POPULATION 
Adult participants with presumed active TB disease will be recruited in South Africa, the Gambia, 
Uganda, Namibia and Ethiopia.  Each site will recruit approximately 160 participants with suspected 
TB, but faster recruiting sites may recruit more and slower recruiting sites fewer, until the desired 
overall total of about 800 participants is reached.  In South Africa, up to 300 participants will be 
recruited from primary health care clinics in Cape Town.  
Patients presenting to the health care facility with symptomatic pulmonary disease and a high 
likelihood of having tuberculosis will be enrolled and followed up for outcome classification.  
5.2 
INCLUSION/EXCLUSION CRITERIA 
Inclusion criteria 
1. 
Symptoms suggestive of TB disease:  cough for more than two weeks, fever, malaise, 
weight loss, night sweats, haemoptysis, chest pain, loss of appetite. 
2. 
Willingness to give consent to take part in the study. 
3. 
Willingness to undergo HIV testing or be willing to have their HIV infection status 
disclosed to the study field workers.   
4. 
Eighteen years or older and aged 70 years or younger.   
Exclusion criteria 
1. 
Permanent residence in study area for less than 3 months or with no permanent 
address.     
2. 
Pregnancy or breastfeeding.   
3. 
HB<9g/l 
4. 
On TB treatment currently or in the last ninety days.    
5. 
HIV positive patients currently on INH prophylaxis, or in the last ninety days. 
6. 
Known quinolone or aminoglicozide antibiotic use reported in the past 60 days. 
Patients who had previous TB and patients with extra-pulmonary TB in addition to pulmonary TB or 
other concomitant diseases will not be excluded.   Patients with drug resistance detected on 
GeneXpert or culture will not be excluded from the primary analysis.  Both HIV positive and HIV 
negative individuals will be enrolled.   
6 
STUDY PROCEDURES/EVALUATIONS 
6.1 
STUDY PROCEDURES 

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Patients who present to a primary health care clinic with pulmonary symptoms and who are 
presumed to have TB disease, will be approached to participate in the study by research recruiters 
or nurses.    Sputum results from the National Health Laboratory System may also be accessed to 
identify patients who were investigated for TB. 
Participants will be recruited into either the training or the test cohort, with the test cohort 
recruitment starting after the training cohort has completed the enrolment of 500 participants.  
Participants will be required to attend the baseline visit, a week 2 visit (to review results and 
antibiotic response in those not already diagnosed with TB at this point), a week 8 visit (all 
participants, to assess TB treatment response in TB cases and to assess alternative diagnosis or 
resolution of symptoms in non-TB cases) and a week 24 visit (TB participants only, to assure TB was 
treated successfully).   
Willing and eligible patients will be informed about the study by a research nurse, guided by the 
informed consent document, and asked to sign the document, after being given the opportunity to 
ask questions.   The participant will receive a copy of the informed consent form, while the original 
consent form will be filed in the regulatory file and another copy will be filed in the participant’s file. 
Each recruited participant will be assigned a unique study ID number by the recruiting nurse.  
Personal and demographic information on each participant will be collected by the nurse using an 
electronic case report form on a tablet computer.  Information such as name, address, telephone 
numbers, other contact person, age, sex, ethnicity, language and employment will be collected and 
uploaded to a secure, password protected and centralized database hosted at Stellenbosch 
University.   
Standardized TB symptoms case report forms and medical history case report forms will be 
completed for all participants at each visit by the research nurse and recorded electronically via the 
tablet into the database.  Information collected will include TB symptoms presence, severity and 
duration, previous TB episodes and treatment outcome, other medical conditions, medication use, 
HIV status (if known), previous surgery, family history, smoking history, alcohol consumption and 
recreational drug usage.   
A directed physical examination will be performed at each visit and recorded in the physical 
examination case report form via the tablet into the database. 
Sputum for smear, GeneXpert and TB culture will be collected at the baseline, week 8 and week 24 
visits by asking the participant to cough and collect expectorated sputum in a sputum container, 
while seated at a sputum collection booth, which is fitted with a high-efficiency particulate 
arrestance (HEPA) filter for infection control purposes.   

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CXRs will be performed on all participants at baseline.  At week 8 and 24 only those who were 
diagnosed on CXR, where the radiographic treatment response pattern is required and those in 
whom a follow-up CXR is ordered by a clinician for clinical reasons, will have follow-up x-rays.     
Any clinical information or test results that could potentially impact a participant’s clinical care, will 
be shared with the participant’s local clinic. 
Rapid HIV tests will be performed on all participants with previously negative or unknown HIV status, 
after pre-test counselling.  Results will be given to participants, along with post-test counselling.  A 
negative result will be considered negative, while a positive result will be confirmed by a second 
rapid test from a different manufacturer.  Incongruent results will finally be confirmed by an HIV 
Elisa.    Newly diagnosed HIV infected participants will receive a CD4 count and be referred to their 
local clinic for possible antiretroviral treatment. 
Blood, sputum, urine and saliva samples will be collected at baseline, week 8 and 24, and 
additionally at week 2 if clinically indicated for TB diagnosis. 
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Sample types and investigations per timepoint
Sample collection and Investigations performed 
 
Participant group and Time Point 
Baseline 
Week 8 
Week 24 
Sample type 
Processing and analysis destination 
Sample 
volume 
All 
TB arm 
Non-TB arm 
TB arm 
Whole blood in Serum 
Seperator Tube (SST) 
Serum separation for ELISA, Luminex, TransDot optimisation and laboratory-based evaluation, 
storage in study specific bio-repository 
10 ml 
Yes 
Yes 
Yes 
Yes 
Whole blood in Sodium 
Heparin tube 
Peripheral  blood mononuclear cell isolation for future biomarker discovery work 
2 x 8 ml = 
16ml 
Yes 
Yes 
No 
Yes 
Whole blood 4 QFN 
tubes 
Quantiferon Gold test 
4 x 1ml = 
4ml 
Yes 
No 
No 
No 
Whole blood in PAXgene 
tube 
Future whole blood RNA signature discovery work 
2.5ml 
Yes 
Yes 
No 
Yes 
Fingerprick blood 
HIV rapid test 
1 to 2 
drops 
Yes 
No 
No 
No 
Whole blood in EDTA 
tube 
CD4 count 
2 ml 
New HIV 
diagnosis only 
No 
No 
No 
Fingerprick blood 
POC TransDot test in Test set participants 
1 to 2 
drops 
Yes (Test set) 
Yes (Test set) 
No 
Yes (Test set) 
TOTAL BLOOD VOLUME 
Total volume over 24 weeks  for TB arm:  89.5 ml (+2 ml for CD4 in some) 
Total volume over 8 weeks for non-TB arm:  42.5 ml (+ 2ml for CD4 in some) 
 
32.5ml (+ 2ml 
for CD4 in 
some) 
28.5 ml 
10 ml 
28.5 ml 
Sputum 
AFB smear 
1 ml 
Yes 
Yes 
Only if clinically 
indicated 
Yes 
Sputum 
GeneXpert 
1 ml 
Yes 
No 
Only if clinically 
indicated 
No 
Sputum 
Culture 
1 ml 
Yes 
Yes 
Only if clinically 
indicated 
Yes 
Sputum 
Future sputum-based diagnostic or treatment response investigations, including GeneXpert 
Ultra and new culture techniques 
2 x 1ml for 
storage 
Yes 
Yes 
No 
Yes 
Induced sputum 
(hypertonic saline 
nebulisation) 
Diagnostic or Future sputum-based diagnostic or treatment response investigations, including 
GeneXpert Ultra and new culture techniques 
Up to 5ml 
as 
necessary 
If not enough 
sputum 
spontaneously 
If not enough 
sputum 
spontaneously 
No 
If not enough 
sputum 
spontaneously 
Saliva from two salivettes 
chewed 
Stored for future biomarker discovery work 
2 x 1ml  
Yes 
Yes 
No 
Yes 
Urine 
Stored for future biomarker work 
10 ml 
Yes 
Yes 
No 
Yes 
Urine 
Urine pregnancy test in women 
Dipstix 
Yes, women 
No 
No 
No 
CXR 
Diagnosis and treatment response evaluation 
 
Yes 
Only if 
indicated for 
treatment 
response 
Only if clinically 
indicated 
Only if 
indicated for 
treatment 
response 

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Fingerprick TransDot test 
For test set participants, serum will be collected for local laboratory based TransDot tests. POC 
TransDot tests will also be performed on fingerprick blood by the research nurse in a standardized 
manner across sites, at the baseline visit and before any results from the composite gold standard 
tests are available.  A drop of blood will be applied to the test strip and the test strip will be 
submitted to the reader.  The reported result will be recorded in the case report form.  Agreement 
between the laboratory based and point of care TransDot tests will be calculated.  Study 
investigators will be blinded to the TransDot test results until the final outcome classification has 
been completed for each participant.  TransDot test results will also not be shared with clinic staff 
and no clinical decisions will be made based on TransDot test results. 
A TransDot test will also be done  at weeks 8 and 24 to investigate the utility of the test for 
monitoring treatment response as a secondary outcome.   It will be compared to week 8 and 24 
treatment outcomes as defined by sputum results, radiology, where applicable and TB symptoms.    
It is expected that approximately one third of the participants will have a final diagnosis of active TB, 
whereas the non-TB cases will have a range of conditions including viral and bacterial lower respiratory 
tract infections and non-infectious conditions like chronic obstructive pulmonary disease.  
The decision to treat for TB and the administration of treatment will remain the responsibility of the 
national healthcare system, although all relevant results to aid the decision will be shared with clinic staff.  
Referrals to the appropriate level of care for any accidental discoveries of other conditions or positive HIV 
results will also be done by the research team.   
Participants at Stellenbosch University will receive R150 for participation in the study to compensate for 
travel expenses and loss of hours at work.   
6.2  
STUDY SCHEDULE: 
Baseline visit (all participants) 
Obtain written informed consent. 
Assign unique study participant ID. 
Conduct TB symptoms questionnaire. 
Obtain relevant medical history. 
Perform directed physical examination. 
Collect sputum samples for smear (except if results are obtainable from the government clinic), GeneXpert, 
MGIT culture and storage.   
Perform chest x-ray. 

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Perform pre- and post-test counselling for HIV test, except in participants who disclose their known positive 
status to study nurses.   
Collect 10ml blood for serum separation for laboratory based TransDot testing (Training and Test cohort). 
Collect 4ml for Quantiferon (QFN) Gold test 
Collect 16ml of blood for peripheral blood mononuclear cell (PBMC) isolation. 
Collect 2.5ml of whole blood into PAXgene tubes for the study-specific biorepository. 
Collect 2ml of saliva into the study-specific biorepository. 
Collect 10ml urine into the study-specific biorepository. 
Perform POC TransDot test and record the result (Test cohort). 
Assure that all participants with symptoms of TB have attended the government clinic to obtain routine TB 
investigations. 
Week 2 visit (only participants not diagnosed with TB by the baseline visit GeneXpert or sputum smear) 
Conduct TB symptoms questionnaire and judge whether any symptoms improved or resolved on course of 
antibiotics for GeneXpert or smear negative patients. 
Week 8 visit (all participants) 
Conduct TB symptoms questionnaire. 
Obtain relevant medical history. 
Perform directed physical examination. 
Perform CXR (only when indicated for treatment response). 
Collect sputum samples for TB sputum smear (except if smear result is available from the government 
clinic), MGIT culture and storage (only participants on TB treatment or those for whom it is clinically 
indicated).    
Collect 10 ml blood for serum separation for laboratory based TransDot testing (Training cohort). 
Collect 16ml of blood for PBMC isolation (participants on TB treatment only). 
Collect 10ml urine into the study-specific biorepository (participants on TB treatment only). 
Perform POC TransDot test and record the result (Test cohort participants on TB treatment only). 
Week 24 visit (only participants who started TB treatment) 
Conduct TB symptoms questionnaire. 
Obtain relevant medical history. 
Perform directed physical examination. 
Collect sputum samples for TB sputum smear (except if smear result is available from the government 
clinic), MGIT culture and storage.  
Perform induced sputum if inadequate sputum volumes are available.   
Perform CXR (only when indicated for treatment response). 

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Collect 10 ml blood for serum separation for laboratory based TransDot testing (Training and Test cohort). 
Collect 16ml of blood for PBMC isolation. 
Collect 2.5ml PAXgene tube for study-specific biorepository. 
Collect 2ml saliva for study-specific biorepository. 
Collect 10ml urine into the study-specific biorepository. 
Perform TransDot test and record the result (Test cohort). 
6.3 
LABORATORY EVALUATIONS 
6.3.1 
LABORATORY EVALUATIONS/ASSAYS 
Sputum will be collected according to standard protocols for GeneXpert (1ml), mycobacterial growth 
indicator tube (MGIT) (1ml) and performance of direct smears for acid-fast bacilli (1ml), if smear results 
cannot be accessed from department of health facilities. Any remaining sputum will be used either for 
cryostorage for the study-specific biorepository (we will aim for 2x1ml aliquots) or performance of new 
microbiological tests, including GeneXpert Ultra and the detection of extracellular mycobacterial 
deoxyribonucleic acid (DNA). We will share the findings of our routine diagnostic tests with the clinics, who 
will remain responsible for treatment decisions and management. 
Any future use of sputum samples will be restricted to detection of mycobacterial or host molecules in the 
context of TB diagnostic or biomarker research.  
6.3.2 
SPECIMEN COLLECTION, PREPARATION, HANDLING AND SHIPPING 
Serum (approximately 5ml) will be obtained through centrifugation of 10ml of whole blood. Serum aliquots 
of 500µl each will be stored at minus 80 °C until either shipment to Leiden University Medical Centre for  
direct comparison between TransDot tests and ELISA or Luminex for host markers or investigation by 
multiplex cytokine array technology or ELISA for host markers with diagnostic potential. 
Peripheral blood mononuclear cells will be isolated through the Histopaque/Ficoll Density Media method 
and stored in liquid nitrogen until further use. The future experiments will be aimed at detection of surface 
markers or intracellular markers on different immune cells to investigate differences between active TB 
cases and non-TB cases and to track changes during treatment.  
PAXgene tubes will be stored at minus 80 °C until use. RNA will subsequently be extracted to investigate 
the gene expression profile of immune cells during active TB and during treatment to compare this to non-
TB cases. There have been recently described gene expression biosignatures with promise as diagnostic 
tests but such signatures will require validation in different cohorts. 
Sputum aliquots for future use, will be stored raw and in Trizol at minus 80 °C. 

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Saliva will be stored at minus 80 °C until use. We have previously found promising diagnostic biosignatures 
in saliva that are made up of host inflammatory markers and these observations require validation in larger 
studies. 
Urine (10ml) will be stored at minus 80 °C for future biomarker investigations. 
6.3.2.1 INSTRUCTIONS FOR SPECIMEN PREPARATION, HANDLING AND STORAGE 
Specimens will be prepared, handled and stored according to our established SOPs.  Serum will be 
transported at room temperature, saliva, PAXgene tubes and sodium heparin tubes (for peripheral blood 
mononuclear cell isolation) on ice.  
All sample tubes will only be labelled with study-specific participant identifiers (bar codes) and will not 
contain any personal identifiers. Similarly, tubes for storage will only be marked by unique study and 
participant-specific codes and not with personal identifiers. The identities of participants can only be linked 
back to the names of individuals through the consent form, which contains the link between personal 
identifiers and study ID. Informed consent documents and source documentation will be stored in the 
regulatory file and participant files in access controlled, locked cabinets at the study site.  
6.3.2.2 SPECIMEN SHIPMENT 
Shipment of a total of 374 serum samples from each site (a projected recruitment of 160 participants per 
site of which one third (53) have active TB with 3 time points for TB cases and 2 for non-TB cases) to LUMC 
will take place. Shipments will occur at approximately 6-monthly intervals and each shipment will contain 
the samples obtained during the preceding 6-month period. Shipments might also contain fewer samples if 
the aims to align TransDot assay performance with ELISA or Luminex have been achieved. Shipment will be 
done according to International Air Transport Association (IATA) approved procedures and through 
accredited shippers of biohazardous materials and all Material Transfer Agreements, export and import 
permits will be in place. Shipments will be temperature controlled. Appropriate contact details of senders 
and receivers of shipments will be noted on shipping documents.  
7.   
DATA MANAGEMENT PLAN 
7.1   
DATA COLLECTION 
Participant information will be collected directly into electronic case report forms by research nurses using 
tablet computers, equipped with the Research Electronic Data Capture (REDCap) application for this 
purpose.  X-ray report forms will be captured in an electronic case report form in REDCap by the study 
clinician and laboratory test result reports will similarly be captured by laboratory personnel.    
All reports form part of source documentation and will be filed in participant folders. 

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Outcome classification conducted by the clinical committee will be done using standard outcome 
classification forms, which will be captured into an electronic case report form in the database.    
7.2   
DATA MANAGEMENT 
Information captured into electronic case report forms, is uploaded in real time to a secure, password 
protected database, hosted by Stellenbosch University.   The database will be managed by the database 
manager  of the study team, who will oversee regular data checks for missing data and data validation.   
7.3   
DATA STORAGE 
Informed consent documents and source documentation, including clinical result reports, will be stored in 
the regulatory file and participant files in access controlled, locked cabinets at the study site.  Records will 
be kept for a minimum of 15 years, in compliance with local and international policies.    
A daily automated back-up of the database onto an off-site back-up server will done.  In addition, a 2 
weekly manual back-up of the data onto an external hard drive will be done by the database managers. 
8.   
ASSESSMENT OF SAFETY  
An adverse event(AE) is any untoward medical or psychological occurrence in a human research 
participant, including any abnormal laboratory finding, symptom or disease, and which does not necessarily 
have a causal relationship with the research or any risk associated with the research.  It also includes any 
event that can affect research participants or data integrity negatively, or that has the potential to impact 
negatively on members of the research team, or on the project as a whole, and that is deemed significant 
by the investigator.    
A serious adverse event (SAE) is an adverse event that, in the view of either the investigator or sponsor, is 
life-threatening or results in death, hospitalisation, persistent or significant disability, a birth defect or 
requires intensive intervention to prevent permanent impairment or any of the previously mentioned 
outcomes. 
AEs/SAEs can be considered related or unrelated to study procedures.  As this is not an interventional 
study, we do not expect clinical AEs or SAEs to be related to any study procedures.  All SAEs will be reported 
in a standardized way. 
Additional reporting of adverse events to the EDCTP will be done according to their reporting requirements. 
All SAEs will be reported for the whole study period and even after the study has been completed, if the 
study team becomes aware of SAEs potentially related to study procedures. The principal investigator 
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the study regulatory file. Additionally, SAEs will be reported to the Stellenbosch University Human Research 
Ethics Committee within 7 calendar days.   
9.   
CLINICAL MONITORING 
 As this study is not a clinical trial, no clinical regulatory organisation will be involved in monitoring.  We will 
comply to any monitoring requirements from the EDCTP. 
10. 
STATISTICAL CONSIDERATIONS 
10.1 
STUDY OUTCOME MEASURES AND ANALYSIS 
The main outcomes of interest are sensitivity and specificity of the point of care TransDot test during phase 
3 of this study.  Sensitivity will be calculated as the proportion of true positives (as determined by the 
composite gold standard of TB case definitions), who test positive on the TransDot test.  Specificity will be 
calculated as the proportion of true negatives who test negative on the TransDot test.  
For the primary analysis, the true positives as determined by the composite gold standard of TB case 
definitions, will consist of the Definite and Probable TB classes.  The Possible TB class will be excluded as 
their diagnosis may involve a significant amount of uncertainty.  This group will be included in a secondary 
analysis to show the TransDot test’s performance in this uncertain group of participants. 
Secondary outcomes of interest are positive and negative predictive values of the TransDot test, which will 
be calculated as the proportion of participants with positive or negative tests who are positive or negative 
according to case definitions, respectively.   
 
Sensitivity, specificity, positive and negative predictive values will be reported with 95% confidence 
intervals.   
Reproducibility of the test will be determined between laboratory based tests and point of care tests, as 
well as between the point of care tests conducted in the different countries, using the Kappa statistic for 
agreement. 
10.2 
SAMPLE SIZE CONSIDERATIONS 
95% Confidence intervals (CI) for sensitivity and specificity evaluate how precisely sensitivity and specificity 
have been estimated. The width of CI is dependent on the sample size. 100 TB cases in the final point of 
care test evaluation (test set), would result in a CI of 85 to 95% for a target sensitivity of 90% (as obtained 
in the AE-TBC study), based on the binomial distribution. As we expect that about one third of those 
screened for TB, will be diagnosed with TB, we will aim to recruit 300 participants into this test set.  In order 
to provide for the training set that will be utilized during phase 1 and 2 of this study, we will recruit 500 
participants into the training set, for a total of 800 participants recruited for this study. 

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11.   
ETHICAL CONSIDERATIONS 
 
11.1   SUBJECT CONFIDENTIALITY  
Results obtained from the analysis of data obtained in this study may be submitted for publication by the 
investigators.  No personal identifying information will be included in publications. All study documentation 
will be identified by the unique study IDs assigned to participants with no personal identifiers of the 
participants.   All records will be stored in locked cabinets. Electronic database access is password protected 
and any data exported and shared for analysis purposes from the database, will contain only study IDs and 
no personal identifiers.  
 
No data will be shared with parties not involved in the study through data collection, monitoring, analysis 
or sponsoring of the study.  New ethical approval will be obtained for any data sharing beyond the scope of 
this project. 
11.2 
 FUTURE USE OF STORED SPECIMENS 
Some of the participants’ serum samples will be shipped to LUMC for testing in their laboratories, using 
techniques not available in the African laboratories.  Up to 5ml of serum (from 10ml blood), 2.5ml blood in 
PAXgene tubes, peripheral blood mononuclear cells from 8ml of blood and 2ml saliva per participant per 
visit will also be stored for future use, not directly related to this project but concerning diagnostic or 
treatment response aspects of TB.  The samples will be stored at the sites where they were collected and in 
the case of the South African component of the study at the Stellenbosch University Immunology Research 
Group laboratories.  However, up to 5x1ml serum aliquots from up to 500 participants will be stored at 
LUMC for optimisation of TransDot assays. Participants will have the opportunity during the informed 
consent process to decline the storage of their samples for future use, or to specify whether they want to 
restrict future use to tuberculosis related research or are willing to make their samples available for any 
biomedical research.  The samples from subjects who decline consent for storage and future use will be 
destroyed at the end of the study.    
11.3 
INFORMED CONSENT PROCESS 
Obtaining informed consent is a process rather than an event and discussions to ensure participant 
understanding and continued willingness to participate should take place at every study visit.   
No trial activities will take place before the informed consent form has been signed by a participant or 
before the respective ethical review boards have given permission for the study. A research nurse will 
discuss the purpose, duration, procedures, alternatives, risks and benefits of the study with potentially 
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study with family members before signing the consent form.  The nurse conducting the discussion will also 
ask the participant questions  to assess the participant’s understanding.  
People who are not able to give informed consent for themselves, will not be included in this study.  At 
Stellenbosch University the informed consent discussion will be conducted in English or Afrikaans, 
according to the preference of the participant.  In the case of Xhosa speaking participants, an interpreter 
could be used to translate English or Afrikaans to Xhosa.  Informed consent forms will be available in 
English, Afrikaans and Xhosa.  The language used will be documented in the participant file.  In the case of a 
participant who cannot read and write, an independent witness will be present during the discussion and 
will sign in addition to the participant’s thumbprint.  This will also be documented in the participant file.   
The participant may withdraw consent at any stage and will be informed that withdrawal will not influence 
their routine clinical care. 
A participant may also be withdrawn from the study at the discretion of the study investigators if their 
further participation is somehow deemed to be a risk to their safety or if lack of the participant’s 
cooperation compromises the quality of the study. 
12. 
LITERATURE REFERENCES 
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2. Systematic screening for active tuberculosis. Principles and recommendations. WHO/HTM/TB/2013.04 
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3. Kivihya-Ndugga LEA, Van Cleeff MRA, Githui WA, Nganga LW, Kibuga DK, Odhiambo JA, Klatser PR.  A 
comprehensive comparison of Ziehl-Neelsen and fluorescence microscopy for the diagnosis of tuberculosis 
in a resource-poor urban setting.  Int J Tuberc Lung Dis. 2003: 7(12):1163–1171. PMID:14677891. 
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