Tuberculosis Screening With AeoNose and CAD4TB in Paraguayan Prisons
Phase: NA
Conditions: Volatile Organic Compounds, Radiography
Sponsor: Radboud University Medical Center

PriNose protocol / Tuberculosis screening with AeoNose and CAD4TB in Paraguayan prisons 
 
 
 
 
 
 
 
 
 
 
 
 
 
(PROTOCOL)  
Tuberculosis screening with AeoNose and CAD4TB in Paraguayan prisons 
 
PriNose Study 
December 2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PriNose protocol / Tuberculosis screening with AeoNose and CAD4TB in Paraguayan prisons 
 
 
 
PROTOCOL TITLE  
Tuberculosis screening with AeoNose and CAD4TB in prisons in Asunción, Paraguay 
Acronym: PriNose 
 
Protocol ID 
<include protocol ID> 
Short title 
PriNose study 
Version 
3.0 
Date 
22-11-2019 
Coordinating investigator / 
project leaders 
 
1.   Luis Gomez MD, Pulmonologist. INERAM, 
Paraguay, PhD candidate 
2. Fernando Cardenas MD, family doctor, Ministry 
of Justice, Dpto. de Salud Penitenciaria, Paraguay 
3. Claudia Pinanez,  MD, specialist in geriatric 
diseases, Ministry of Justice, Department of 
vulnerable populations, Paraguay 
Principal investigator(s) 
 
Cecile Magis-Escurra MD, PhD  
Specialist in respiratory diseases 
Radboud University Medical Centre- TB Expert 
Centre Dekkerswald, Department of Respiratory 
Diseases, Nijmegen/ Groesbeek, The Netherlands 
Co-investigators 
1. Dra. Sarita Aguirre,  MD, Respiratory 
disease specialist. Directora del Programa 
Nacional de Control de la Tuberculosis, 
Asunción, Paraguay.  
2. Dra. Carolina Aquino, Bioquímica 
Directora del Laboratorio Central de Salud 
Pública (LCSP), Asunción, Paraguay 
3. Dra Chynthia Diaz, Bioquimica,  Dpto. de 
Biología Molecular y Biotecnología, 
Instituto de Investigaciones en Ciencias de 
la Salud (IICS) 
4. Dr. Guillermo Sequera MD, ministry of 
Health, director of surveillance department 

PriNose protocol / Tuberculosis screening with AeoNose and CAD4TB in Paraguayan prisons 
 
 
 
5. Dr. Adrian Sanchez-Montalvá, Hospital 
Universitario Vall d’Hebron dpt. of 
infectious diseases, Barcelona, Espana 
Sponsor  
Radboud University Medical Centre  
(EUSAT-RCS consortium) 
Subsidising party 
European Commission (RISE, Marie Curie Horizon 
2020, grant number 823890) 
Radboud Institute for Health Sciences (RIHS) 
External advisors 
Dr. Michael Kimerling MD, PhD 
Independent TB consultant 
Paris, France 
 
 
 
Laboratory sites  
1. National Reference Lab (LCSP) 
2. Instituto de Investigaciones en Ciencias de 
Salud (IICS), laboratory of the National University 
of Asuncion (UNA) 
3. Laboratorio San Alfonso, barrio Tacumbú, 
Asuncion 
Study site 
State prison facilities in Paraguay  

PriNose protocol / Tuberculosis screening with AeoNose and CAD4TB in Paraguayan prisons 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Sponsor or legal representative 
Head of Department: 
Prof. Michel van den Heuvel MD, 
PhD 
Radboud University Medical Centre, 
Dept. of Respiratory Diseases 
Nijmegen, The Netherlands 
 
 
 
16-12-2019 
Principal Investigator 
Cecile Magis-Escurra MD, PhD 
Radboud University Medical Centre 
Nijmegen, The Netherlands 
 
 
12-12-2019 
Coordinating Investigator/Project 
leader 
Luis Gomez MD 
Respiratory Disease Specialist 
INERAM hospital 
Asuncion, Paraguay 
 
 
 
13-12-2019 
 
 

PriNose protocol / Tuberculosis screening with AeoNose and CAD4TB in Paraguayan prisons 
 
 
 
Co-investigators 
INERAM: 
Arturo Battaglia MD 
Cynthia Meza MD 
Gladys Molinas MD 
Nilza Martinez MD 
Rosarito Coronel MD 
 
PNCT: 
Cynthia Cespedes, nurse 
Ivon Montiel, nurse 
Patricia Ferreira, nurse 
Sarita Aguirre, MD Director of the National 
Program of Tuberculosis Control (PNCT), 
Asunción, Paraguay  
 
LCSP:  
Carolina Aquino, biochemist, and Director 
of the Central Laboratory of Public Health 
(LSCP), Asunción, Paraguay.  
Pilar Munoz, biochemist 
Geronimo Ortellado, biochemist 
Belén Jara, biochemist 
 
IICS:  
Chynthia Diaz, biochemist 
Diego Gonzalez, biochemist 
Prof. Graciela Russomando,  
 
Laboratory San Alfonso 
Dra Irma Lovera, biochemist and head of 
the laboratory 
 
 
 
 
 

PriNose protocol / Tuberculosis screening with AeoNose and CAD4TB in Paraguayan prisons 
 
 
 
 
Ministry of Health:  
Dr. Guillermo Sequera MD, Ministry of 
Health, director of surveillance department 
 
Ministry of Justice: 
Ever Velázquez, Director of Prison Health 
Fernando Cárdenas MD 
Claudia Piñánez MD 
Dra Romina Lombardo, Physician at 
Esperanza prison. 
Lic. Ruth Martínez, nurse at the prison of 
Tacumbú. 
Sr Isidro Arguello, Health agent in the 
prison of Tacumbú 
 
Hospital Universitario Vall d’Hebron, 
Barcelona 
Dr. Adrian Sanchez-Montalva, MD, 
Infectious disease specialist.  
Dr. Juan Espinosa, MD, Infectious disease 
specialist. 
 
 

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TABLE OF CONTENTS 
1. 
INTRODUCTION AND RATIONALE .............................................................................12 
2. 
OBJECTIVES ................................................................................................................15 
3. 
STUDY DESIGN ...........................................................................................................15 
4. 
STUDY POPULATION ..................................................................................................18 
4.1 
Population (base) ...................................................................................................18 
4.2 
Inclusion criteria .....................................................................................................19 
4.3 
Exclusion criteria ....................................................................................................19 
4.4 
Sample size calculation ..........................................................................................19 
5. 
INVESTIGATIONAL PRODUCT ....................................................................................22 
5.1 
Name and description of investigational  product(s) ...............................................22 
5.2 
Summary of findings from clinical studies ...............................................................22 
6. 
METHODS ....................................................................................................................23 
6.1 
Study parameters/endpoint ....................................................................................23 
6.1.1 
Case definitions: ..............................................................................................23 
6.1.2 
Main study parameter/endpoint .......................................................................26 
6.1.3 
Secondary study parameters/endpoints ..........................................................27 
6.1.4 
Other study parameters ...................................................................................27 
6.2 
Study procedures ...................................................................................................28 
6.3 
Collection of data ...................................................................................................29 
6.4 
Withdrawal of individual participants .......................................................................29 
6.5 
Replacement of individual participants after withdrawal or lost to follow-up ............29 
6.6 
Follow-up of participants withdrawn from treatment ................................................29 
6.7 
Premature termination of the study .........................................................................29 
7. 
SAFETY REPORTING ..................................................................................................30 
7.1 
Temporary halt for reasons of participant safety .....................................................30 
7.2 
AEs, SAEs and SUSARs ........................................................................................30 
7.2.1 
Adverse events (AEs) ......................................................................................30 
7.2.2 
Serious adverse events (SAEs) .......................................................................30 
7.2.3 
Suspected unexpected serious adverse reactions (SUSARs) . Fout! Bladwijzer 
niet gedefinieerd. 
7.2.4 
Follow-up of adverse events ............................................................................30 
7.2.5 
Data Safety Monitoring Board (DSMB) / Safety Committee .............................30 
8. 
STATISTICAL ANALYSIS .............................................................................................30 
8.1 
Primary study parameter(s) ....................................................................................31 
8.2 
Other study parameters ..........................................................................................32 
9. 
ETHICAL CONSIDERATIONS ......................................................................................32 
9.1 
Regulation statement .............................................................................................32 
9.2 
Recruitment and consent........................................................................................32 
9.3 
Benefits and risks assessment, group relatedness .................................................33 
9.4 
Compensation for injury .........................................................................................34 
9.5 
Incentives for participants .......................................................................................34 
10. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .............................34 

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10.1 Handling and storage of data and documents ........................................................34 
10.2 Amendments ..........................................................................................................34 
10.3 Annual progress report ...........................................................................................35 
10.4 Temporary halt and (prematurely) end of study report ............................................35 
10.5 Public disclosure and publication policy ..................................................................35 
11. STRUCTURED RISK ANALYSIS ..................................................................................35 
12. REFERENCES ..............................................................................................................35 
13. ADNEXES .....................................................................................................................37 
 
 
 
 
 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
AE 
Adverse Event 
AR 
Adverse Reaction 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CAD 
Computer Aided Detection 
CRF 
Case Report Form 
CXR 
Chest X-ray 
EDC 
Electronic Data Capture 
EU 
European Union 
TB 
Tuberculosis 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
IC 
Informed Consent 
MGIT 
Mycobacteria Growth Indicator Tube (BD Products) 
MREC  
Medical research ethics committee (MREC) 
NAAT 
Nucleic Acid Amplification Test, for example GeneXpert® 
PIF 
Participant Information Form 
PNCT 
Programa Nacional de Control de la Tuberculosis (National TB 
Program) 
PPL 
Personas Privadas de Libertad / people deprived of liberty,  prisoners 
POC 
Point-of-care 
PRONASIDA Programa Nacional de Control de VIH-sida (National HIV Program) 
Sponsor 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A 
party that provides funding for a study but does not commission it is 
not regarded as the sponsor, but referred to as a subsidising party. 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet 
Medisch-wetenschappelijk Onderzoek met Mensen 
WHO 
World Health Organization 
 
 
 

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SUMMARY 
Rationale: Despite being a curable disease, each day thousands of people die of 
tuberculosis (TB) worldwide. TB diagnosis is often delayed, increasing the likelihood of 
transmission. The aim of the World Health Organization (WHO) “End TB Strategy” is to end 
the global pandemic by 2035. To be able to succeed, better point-of-care (POC) tests are 
urgently needed to improve screening of high-risk populations. Prisons are recognized 
worldwide as high risk environments for the concentration, amplification and transmission of 
TB among prisoners and their communities outside. Paraguayan penal institutions are known 
to have very high incidence rates of active TB (3000-5000/100.000, according to Paraguayan 
Ministry of Justice). Two diagnostic tools for TB screening in high risk groups need additional 
validation: the AeoNose™, an ‘electronic nose device’ for breath sampling, and digital chest 
X-ray (CXR) with computer aided detection with CAD4TB® software. This study will 
systematically screen prisoners and its’ employees for TB, test the diagnostic performance of 
AeoNose™ and CAD4TB® and assess prison Mycobacterium tuberculosis epidemiology 
through several objectives: Objective: 1. Assessment of the sensitivity and specificity of the 
AeoNose™ and its utility for mass TB screening in high incidence settings. 2. Evaluation of 
the feasibility and utility of CAD4TB® digital CXR as a mass screening tool for TB. 3. 
Identification of factors that affect diagnostic accuracy of AeoNose™ and CAD4TB®. 4. 
Assessment of TB epidemiology in Paraguayan prisons and identification of mycobacterial 
strains. 5. Assessment of cost effectiveness of different screening algorithms. 6. 
Characterization of transmission dynamics and potential measures for transmission 
reduction. Study design: Diagnostic cohort study. Study population: Detainees (PPL) of 
Paraguayan penal institutions as well as their employees. Main study 
parameters/endpoints: 1. Number of TB cases diagnosed with GeneXpert® and/ or culture 
of Mycobacterium tuberculosis complex. 2. Number of TB cases correctly identified by 
AeoNose™. 3. Number of TB cases correctly identified by CAD4TB®. 4. Costs and 
effectiveness of different screening algorithms. 5. Mycobacterium tuberculosis complex 
strains identified with Whole Genome Sequencing. Nature and extent of the burden and 
risks associated with participation, benefit and group relatedness:  
Individual burden: all participants will perform one visit with a medical doctor for medical 
history, physical exam, digital X-ray (with CAD4TB®) and AeoNose sampling. Participants will 
be offered voluntary HIV testing and counseling. In case of presumptive TB (estimated 20-
30% of participants) two sputum samples will be taken, either spontaneous or saline-
induced. One sample is tested with GeneXpert and the other with liquid mycobacterial 
culture. Cases of presumptive TB with both negative GeneXpert® and culture results will be 
followed-up  after three and six months for repeat testing.  

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Individual and group health benefit: all participants will have a medical consultation and 
CXR. In case of an alternative diagnosis, the prison health service will be notified. People 
diagnosed in the study with active TB will be notified and referred to receive treatment 
through the  PNCT according to national guidelines. All participants will eventually benefit 
from less exposure to infectious TB cases through optimized infection control measures and 
treatment of infected individuals. The study will provide the PNCT with epidemiological data 
of this vulnerable group to optimize future TB control strategies.  
Long-Term Benefit:  
After the study, the Ministry of Health will have an accurate update on the incidence of TB in 
the three involved prisons. Based on this information, more effective strategies can be 
designed for TB elimination in Paraguayan prisons.  
The Ministry of Health (department of telemedicine - PNCT) will receive the mobile digital X-
ray machine and CAD4TB software that can be used throughout the country, especially in 
vulnerable groups and people living in remote areas.  
 
 

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1. INTRODUCTION AND RATIONALE 
Every day, over 4700 people die of tuberculosis (TB). The aim of the World Health 
Organization (WHO) “End TB Strategy” is to stop the global TB pandemic by 2035. (WHO, 
Geneva 2010) TB is a curable disease, but frequently remains undiagnosed, leading to 
ongoing transmission, infection and suffering. More accurate and affordable point-of-care 
(POC) tests are urgently needed that are suitable for the low resource settings in which TB is 
most prevalent. The pandemic is driven by transmission, which occurs not only from known 
TB patients but also from persons with unsuspected TB or ineffective TB treatment. The 
former WHO strategy of screening only symptomatic persons with (low-sensitive) sputum 
smear microscopy needs to be changed drastically as many TB cases remain undiagnosed.  
 
Target product profiles for new TB screening tools and diagnostics were devised stressing 
the high priority of a systematic screening test for active case finding. (WHO Consensus 
meeting report, Geneva 2014). New POC screening tests should rapidly identify high 
likelihood individuals to select them for subsequent laboratory tests that can confirm active 
disease. A cheap and rapid POC test can improve the efficacy and efficiency of screening 
algorithms by optimizing the use of expensive and time-consuming tests such as nuclear 
acid amplification tests (NAAT) or sputum mycobacterial culture. 
 
A promising POC diagnostic tool is the AeoNose™; an electronic nose device. (The eNose 
Company, Zutphen, the Netherlands). The AeoNose™ works with three metal oxide sensors 
which gather ‘smell prints’ of volatile organic compounds in the subject’s breath. The 
collected vectors are sent via internet to a central computer and then compared to a neural 
network of TB breath profiles. After establishing a validated neural network, new breath 
profiles can be analyzed within minutes. The device is battery-powered, handheld and 
lightweight, making it very suitable for POC use.  
The AeoNose™ showed promising results as a TB diagnostic tool in previous studies in 
Bangladesh and Paraguay. (Bruins, Tuberculosis 2013; Coronel Teixeira, J Infect 2017)  Like 
digital CXR and CAD, an electronic nose device could be implemented in a screening 
algorithm to “rule in” subjects for further testing. This could bring improvements of case 
finding and infection control, efficiency and reduced costs. Before the AeoNose™ can be 
brought to the market the technology needs to be further tested and the neural network 
needs to be validated in a large high-incidence population.  
 
 
Abnormalities corresponding with TB can be detected early on CXR, even in asymptomatic 
participants. The WHO now recommends to add CXR to screening algorithms (WHO, 

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Geneva 2013). Conventional X-ray technology is available even in many low-income 
countries, although imaging quality is often poor and systems need expensive X-ray films 
and skilled personnel to interpret results. Digital X-ray with computer-aided detection (CAD) 
has many advantages over conventional X-ray and is considered very promising for TB mass 
screening programs. A mobile digital X-ray laboratory with CAD has been developed and has 
already been used in several screening programs in high-incidence settings. (E.g. Philipsen, 
Sci Rep 2015) CXR is used to identify participants with presumptive TB and subsequently 
sputum is obtained for culture or a rapid molecular test (NAAT/GeneXpert®). Figure 1, 
adapted from Khan et al. (Eur Resp J 2017), illustrates the logistics of screening with CXR in 
either symptomatic participants (a) or mass screening in a vulnerable group or community 
(b).  
 
 
Figure 1. Screening in (a) symptomatic subjects or (b) a vulnerable group or community. 
Source: Khan et al. Eur Resp J 2017. 
 
Currently, there are three commercially available CAD systems to detect TB on CXR that 
show no significant differences in performances. (Qin et al., NatureResearch-scientific 
reports 2019) CAD4TB® by Delft Imaging Systems (Veenendaal, the Netherlands) was 

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developed in collaboration with the Diagnostic Image Analysis Group from Radboud 
University Medical Centre (Prof. dr. B. van Ginneken). The CAD4TB® software has already 
been adopted for screening purposes, including studies in prisons. The newest software 
version (version 6) performs as good as skilled human readers, with a high sensitivity of 95% 
and excellent negative predictive values (NPV) as well as specificity of 70% , depending on 
the selected threshold value of the output score. (Melendez, Int J TB Lung Dis 2018) Still, 
there is evidence lacking for WHO-endorsement for CAD as part of large screening 
programs. (Khan, Eur Resp J 2017) Most importantly, an optimal threshold value of the 
output score must be identified for different population and patient characteristics. 
Additionally, CAD needs to be compared against skilled human readers, and safety and 
ethical issues need to be addressed concerning the incidental detection of lung malignancies 
and other non-TB pathology. Finally, the large-scale implementation of such technology 
needs to be evaluated from a health-economic viewpoint. 
 
 
Setting: Prisons worldwide are high-risk environments for the concentration, amplification 
and further transmission of TB among prisoners, employees and the communities outside the 
prison. Sanitary conditions and healthcare services in prisons are often poor. Addressing the 
unmet healthcare needs of prisoners is crucial to achieve the goal of global tuberculosis 
control. (Kamarulzaman, Lancet 2016; Dara, Int J ID 2015) Paraguayan penal institutions 
also count with very high estimated incidence rates of active TB (3000-5000/100.000). 
Currently, since the assembly meeting in New York in 2018, reduction of TB incidence and 
transmission is a top priority for the Paraguayan Ministry of Health and the PNCT. In this 
study proposal, we aim to perform a mass TB screening in Paraguayan prisons using 
AeoNose™ and CAD4TB® and assess their utility for future screening algorithms. 
 
 

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2. OBJECTIVES 
Primary Objective:  
1. Assessment of the sensitivity and specificity of the AeoNose™ and its utility for mass 
TB screening. 
2. Evaluation of the feasibility and utility of CAD4TB® digital CXR as mass screening tool 
for TB in high incidence settings.  
3. Identification of factors that affect diagnostic accuracy of AeoNose™ and CAD4TB®.  
 
Secondary Objective(s): 
1. Assessment of TB epidemiology in Paraguayan prisons and identification of 
mycobacterial strains. 
2. Assessment of cost-effectiveness and cost-efficiency of mass screening programs 
integrating digital CXR with CAD4TB®, AeoNose™ or both techniques in conjunction. 
3. Characterization of transmission dynamics and potential measures for transmission 
reduction. 
 
3. STUDY DESIGN 
 
Diagnostic prospective cohort study: mass screening through conventional symptoms 
screening by a doctor, a physical exam and with testing of CAD4TB® and AeoNose™ as well 
as bacteriological testing of presumptive cases.  
  
An estimated 5000 individuals will be invited to participate. All prisoners and employees will 
be eligible for inclusion. The sample size is based on an estimated incidence of 3000-
5000/100.000 and a presumptive number of 150-250 active TB cases to be found in order to 
have enough cases to first train the neural network (with at least 500 - 1000 healthy 
individuals and a 100 active TB cases) and then perform the blind predictions on the rest of 
the participants to establish sensitivity and specificity of the AeoNoseTM.  
 
The study consists of one primary visit and, in case of suspicion of presumptive TB but no 
confirmed TB or alternative diagnosis, two follow-up visits after three and six months 
respectively. The estimated total study duration will be from February 2020  to May 2021. 
The study settingwill be the Buen Pastor, Esperanza and Tacumbú prisons in Asunción, 
Paraguay Required condition to perform the study in each of these three institutions is safety 
for the study team and also the guards and prisoners. In case of unstable conditions we will 
look for an alternative penal institution. 

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Start 
The entire prison population and all employees will be informed on the upcoming screening 
operation by prison authorities, prison health care workers and trained prison health 
promotors. Participants will be educated on the topic of TB and upcoming study through an 
awareness campaign before the start of the study. 
Visit 1.  
1. Participants will watch an animation (video) with voice-over in Spanish, Guarani or 
Portuguese with general information on TB and an explanation of the study 
procedures. Each participant will be offered to be tested voluntarily for HIV by 
PRONASIDA. An independent volunteer witness is present in the room to guarantee 
that the participant has understood the information correctly and is able to ask 
questions if necessary. Participants may then decide to sign informed consent using 
signature or fingerprint.  Individuals can also decline participation, or take time to 
consider their participation and return later. 
2. After providing informed consent, a study nurse will take vital signs (blood pressure, 
pulse and axillary temperature) and measure the participants weight, height, 
temperature, middle upper arm circumference, as well as ask for smoking habits, 
alcohol and/or drug. Co-morbidities, medication and food intake (4 hours prior to 
testing) will be recorded in the digital case record form. If the participant agrees a HIV 
rapid test will be performed using a finger prick blood sample. Participants with a 
positive result will be notified and referred to the national HIV program (PRONASIDA) 
for  final diagnosis and treatment according to national HIV guidelines.   
3. At the third station, a technician will perform the CXR and will process the digital 
image with CAD4TB™ software.  A TB likelihood output score is presented within 
minutes.  
4. At the fourth station, a technician will take a breath sample with an AeoNose™ device 
during 5 minutes in a room free of gasses and odours.  
5. At the fifth station, a doctor will first take a history of symptoms and will then examine 
the participant for signs of TB:  
o Healthy / ill  
o Cough (15 days or longer) 
o Sputum or haemoptysis 
o Weight loss 
o Fever (continuous or intermittent) 
o Night sweats 
o Chest pain 

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o Dyspnea / shortness of breath 
o Loss of appetite 
o Lymphadenopathy 
o Pulmonary auscultation 
o Abdominal distension 
o Anaemic conjunctivae 
Also the dental status of the participants will be assessed.  
The digital CXR image will be analysed by CAD4TB® and the doctor will interprete the 
CAD4TB® output score and check the CXR for alternative diagnosis. All the findings 
by the study doctor will be recorded in the digital case record form. At the end of this 
visit the doctor will decide on health status of the participant: either the participant is 
considered healthy or a presumptive TB case. 
For healthy participants the study ends after this 5th station. 
6. Presumptive TB cases (see 6.1.1 Case definitions) will be asked to produce two 
sputum samples outside, preferably in the open air. Sputum can be either be 
produced spontaneously or via sputum induction with nebulized  saline. Saline 
solution will be nebulized using a portable nebulizer. Sputum samples can deposited 
in standard containers. Samples will be stored in a cool box and transported to the 
laboratory the same day. Samples will then be processed and analysed by the 
National Reference Laboratory, Laboratory San Alfonso and IICS using GeneXpert® 
molecular testing and MGIT mycobacterial culture.  
7. All participants will be educated about the importance of seeking medical care if 
symptoms compatible with TB emerge at any time.  
8. In case a participant is passively identified with TB during the study period by the 
prison health services or PNCT, the study researchers will be notified. Clinical and 
microbiological data will be shared based on informed consent given by the 
participant.  
 
Follow-up of presumptive TB cases 
Participants who are considered presumptive cases of TB at Visit 1, but with negative 
sputum results or unable to produce sputum, will be followed-up after three and six months.  
 
Visit 2 and 3 (at three and six months after Visit 1) 
Study procedures are the same as Visit 1 steps 1 through 5. Participants will be asked for 
symptoms, have a physical exam, undergo CXR with CAD4TB® and AeoNose™ breath 
sampling. At visit 2 and 3, all participants will be asked to produce two sputum samples. In 
case of positive results for Mycobacterium tuberculosis, confirmed cases will be notified to 

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the PNCT. After visits 2 and 3 a decision will be made by the study team on the final health 
status of the participant (either considered healthy or a TB case). In case of a high suspicion 
of TB the prison health authorities and the PNCT may decide to treat this individual for active 
TB. The result of this anti-tuberculosis treatment will be communicated with the study team.  
 
Referral of TB cases and non-TB illness 
In case of a positive culture result or GeneXpert® with Mycobacterium tuberculosis, the 
participant will be informed and the PNCT will be notifiedthrough the prison authorities. 
GeneXpert® will provide a test result within 24 hours.  
 
In compliance with the current Paraguayan national TB treatment guidelines, established TB 
cases will  start anti-tuberculous therapy and will be followed-up by the PNCT and prison 
health service.  
 
For presumptive TB case with signs of severe illness but a negative GeneXpert® result, the 
PNCT and the prison health service will be notified. The prison health service and PNCT  
may decide whether it is justified to start empiric treatment for TB. This decision  will be 
made available to the investigators who will review these cases afterwards to categorize 
them according to the formal case definitions. 
 
If a participant presents to the study doctors with signs and symptoms compatible with 
severe illness, the prison health service will directly be notified in order to take an appropriate 
action. 
 
If the digital CXR shows pathological abnormalities not being  consistent with tuberculosis, 
the participant will be informed and referred to the prison health service.  
 
4. STUDY POPULATION 
4.1 Population (base)  
Persons deprived of liberty (PPL) adults (≥18 years) and personnel of the three 
penitentiary centers of Paraguay (Buen Pastor, Esperanza and Tacumbú prison) in 
Asunción. Both male and female (estimated 90% vs. 10%). Ethnicity is mostly mixed / 
Latin-American as well as indigenous from several different ethnicities. 

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4.2 Inclusion criteria 
1. All persons, either being a prisoner or employee, of the involved penal institutions after 
providing informed consent, including those with known active TB disease and/or 
currently on TB treatment.  
1. Exclusion criteria 
A potential participant who meets any of the following criteria will be excluded from 
participation in this study: 
1. Unable to exhale breath through the AeoNose™ during five minutes due to 
respiratory insufficiency 
2. Unable to stand in an upright position for the CXR 
3. Unable to communicate and comply with the instructions of the study team 
4.3 Sample size calculation 
Based on WHO definitions, mass screening includes every person in a vulnerable 
community. The combined prisons of Tacumbú, Esperanza, Buen Pastor house around 4700 
PPL. An additional 500 employees (estimated number, to be confirmed) who are in direct 
contact with the PPL will also be included. Based on calculations we expect to detect around 
200-240 cases of active tuberculosis in prisoners (3-6% of 4000 PPL) and 0-5 cases in 
employees (estimated 0.2% of 1000 employees). The number of expected cases in the latter 
group could be an underestimation as these people are in constant contact with PPL and 
have a higher risk than the general population of active TB.  
The study sample size is based on the need for at least 100 cases with active TB to build a 
robust AeoNose™ neural network (calibration model) and subsequently another 100-150 
cases to make a blind prediction (validation model). A systematic literature review of 
acceptability of TB screening among risk groups showed an average percentage of 
screening consent of 72% among prisoners. (range 18-100%, Mitchell et al. 2012) 
 

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5. INVESTIGATIONAL PRODUCT  
5.1 Name and description of investigational  product(s) 
- 
AeoNose™ (the eNose Company, Zutphen, the Netherlands): 
The Aeonose™  is a diagnostic device for exhaled-breath analysis. It is portable and 
requires a participant to breathe through the device for five minutes at tidal breathing. 
Volatile organic compounds (VOC) in the breath are captured by metal-oxide 
sensors. The unique ‘smell print’ of TB can be identified by an algorithm that is built 
on a de-blinded sample of the population in the calibration phase using an artificial 
neural network.  
- 
CAD4TB® (Delft Imaging, Veenendaal, the Netherlands):  
The CAD4TB® software analyses an X-ray image and detects  the many different 
abnormal structures that may be related to TB. The system has been trained in the 
detection of these abnormalities by applying machine learning to thousands of 
healthy and diseased X-rays from over 15 countries, through which it has become 
accurate and reliable. The output of the CAD4TB® software is a score between 0 and 
100, indicating the likelihood that the participant, above a threshold score, has 
possible, probable or definite (active) TB. The CAD4TB® software also outputs a 
‘heatmap’ – a colour-coded image of the lungs indicating regions of abnormality. 
 
5.2 Summary of findings from clinical studies 
- 
AeoNose™: In recent years we performed several studies to test the AeoNose™ as a 
POC screening test, as well as a biomarker of treatment response. In a pilot study in 
194 subjects in Bangladesh, a sensitivity of 93.5% and a specificity of 85.3% was de-
tected discriminating healthy controls from TB patients. (Bruins, Tuberculosis 2013) In 
a pilot study in 110 subjects in Asuncion, Paraguay, the AeoNose™ showed a sensi-
tivity of 88% and a specificity of 92%. (Coronel Teixeira, J Inf 2017)  The device 
showed ease of operation and no adverse effects were observed. A field study in 
2016 in an indigenous community in Paraguay (Puerto Casado) showed a specificity 
of 92% (IndiPaNose study, manuscript under revision), with no sensitivity calculated 
because of no actual active TB cases in the community. A pilot study assessing 
changes after initiation of therapy showed a fall in AeoNose™ signal correlating with 
treatment outcome (clinical signs and symptoms, CXR and sputum smear), demon-
strating its potential as a biomarker for treatment response. (Elnosur study, poster 
presentation ERS congress 2015). An AeoNose™ study in 327 Indonesian patients 
differentiating pulmonary TB from suspected pulmonary TB demonstrated a lower 
sensitivity and specificity of 78-85% and 42-55%, respectively. The study indicated 

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the need for a large calibration group for better pattern recognition. The authors con-
cluded that the AeoNoseTM is better suited for screening purposes than clinical diag-
nosis. (Saktiawati, PLOS One 2019). The utility of the AeoNose™ for screening pur-
poses was recently confirmed by researchers from Peru who demonstrated the possi-
bility of using the AeoNose™ to identify high-risk patient who should receive confirm-
atory TB testing as part of a FAST strategy (Find cases Actively, Separate safely, and 
Treat effectively). AeoNose™ demonstrated a sensitivity of 85-93% and specificity of 
63-73% in a population of 629 patients that underwent AeoNose™ analysis on admis-
sion to the hospital. (Nathavitharana et al., accepted abstract, The Union conference 
2019) 
 
- 
CAD4TB®: CAD4TB® has been widely adopted over the past years, currently analyz-
ing over 5000 CXR every day in clinics all over the world, including in prison facilities. 
The CAD4TB® software is going through continuous improvements. In a systematic 
review of CAD for detection of tuberculosis, version 1 and 3 of the software achieved 
AUC of 0.71-0.87 for sputum confirmed cases. (Pande, Int J TB Lung Dis. 2016) Ver-
sion 5 of the software demonstrated an AUC of 0.90 in 47510 images with the clinical 
decision to treat as reference. (Melendez, Int J TB Lung Dis. 2018) Sensitivity and 
specificity is dependent on the selected threshold score. Threshold scores achieving 
high sensitivity (≥95%) can be reached with moderate-to-high specificity, performing 
similarly to expert radiologists. Version 6 of CAD4TB® was released in 2018. In a vali-
dation data set of 5665 CXR images the software attained an AUC of 0.987 for radio-
logical reference and 0.885 for bacteriological reference (Murphy et al. Arxiv pre-print 
March 2019). CAD4TB® has already been tested as a triage tool to select presump-
tive TB cases for GeneXpert® testing. In a study in 388 South African presumptive TB 
cases, pre-screening with CAD4TB® version 3 led to an almost 50% reduction in cost 
per screened subject and cost per notified TB case, and more than doubled the num-
ber of subjects screened per day, at the expense of only a small number of missed 
cases. (Philipsen, Sci Rep 2015) These benefits were realized by quickly identifying 
TB negative subjects and reducing the total number of expensive and time-consum-
ing NAAT tests. 
 
6. METHODS 
6.1 Study parameters/endpoint 
6.1.1 Case definitions:  
Presumptive TB: 

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Any participant with symptoms or signs suggestive of TB as judged by the study doctor. 
 
AND / OR 
Any participant with CAD4TB output score above the threshold level (to be determined), 
or presenting with CXR abnormalities as judged compatible with TB by the study doctor.  
 
Respiratory symptomatic 
Any participant presenting with cough or phlegm of duration of more than 14 days. 
 
TB case:  
A participant in whom TB has been confirmed bacteriologically, or a participant who 
started treatment empirically and showed response to treatment after the initial phase of 
TB treatment, as documented by the PNCT.  
 
TB Case Classification 
1. 
A bacteriologically confirmed case of pulmonary TB is defined by either Gen-
eXpert® OR mycobacterial culture (MGIT™) positive for M. tuberculosis com-
plex  
2. 
A bacteriologically unconfirmed case is defined by: 
1. 
Not meeting definition for bacteriologically defined case 
2. 
Decision by the PNCT of the necessity for empiric treatment with anti-
TB chemotherapy 
 
A TB case is categorized by anatomical site of disease:  
3. 
Pulmonary TB (PTB): The disease affects the lung parenchyma. Pul-
monary TB is the most common form. 
4. 
Extrapulmonary TB (EPTB): The disease affects other sites including 
lymph nodes, pleurae, meningeal, pericardial, peritoneal, spinal, intesti-
nal, genitourinary, larynx, spine, bones and joints, and skin. 
5. 
A patient diagnosed with both pulmonary and extrapulmonary localisa-
tions of TB is classified as PTB + EPTB. 
 
Classification based on history of Previous Treatment (in accordance with Paraguayan 
National TB guidelines):  
Cases will be defined to whether or not the participant has previously received TB 
treatment.  

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- 
New treatment: A participant who has never had treatment for TB or who has 
taken anti-TB medicines (for treatment of active TB) for less than one month at 
time of enrolment. 
- 
Current treatment: A participant who is currently taking anti-TB medicines (for 
treatment of active TB) for one month or more at time of enrolment.  
- 
Previous treatment: further classified according to result of last treatment:  
▪ 
Relapse: A participant previously treated for TB, who has been de-
clared cured or has completed treatment up to the final stage of treat-
ment and currently diagnosed with a new episode of active TB, 
whether a reactivation or new episode of TB (re-infection) 
▪ 
Failure: A participant who has treatment failure at the end of his/her 
most recent treatment.  
▪ 
Lost to follow up: A participant declared lost to follow-up during his/her 
most recent treatment. 
▪ 
Other previously treated participants: All participants who have been 
previously treated but with unknown or undocumented treatment out-
come of the most recent treatment.  
▪ 
Unknown history of previous treatment: participants who relate having 
been treated but without documentation, only verbal reference. 
 
Classification based on patterns of drug resistance: 
- 
Monoresistant, polyresistant, MDR, XDR TB (in accordance with Para-
guayan National TB guidelines) 
 
Classification based on HIV status 
- 
Case of TB with HIV 
- 
Case of TB without HIV 
- 
Case of TB with unknown HIV status 
 
Bacillary load and Severity of TB: 
Bacillary load, as determined by: 
- 
GeneXpert® cycle time (Ct) value    
- 
MGIT™ culture time-to-positivity 
 
Radiological severity 
- 
CAD4TB score 
- 
Unilateral, bilateral, pleural, miliary 

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- 
Presence of cavitations 
- 
Percentage of lung affected as defined by a posteroanterior X-ray projection 
(<33%, 33-66%, >66%) 
 
- 
Clinical severity (TBscore, Wejse et al. 2008 and TBscoreII, Rudolf 2014) 
Severity according to TBscore 2008: Class I (0-5 points) 
- 
Class II (6-7 points) 
- 
Class III (8 or more points) 
Parameter 
Points assigned TBscore 
Points assigned TBscoreII 
Self-reported: 
 
 
Cough 
1 
1 
Haemoptysis 
1 
- 
Dyspnea 
1 
1 
Chest pain 
1 
1 
Night sweating 
1 
- 
Anaemic conjunctivae 
1 
1 
Tachycardia (>90/min) 
1 
-  
Positive finding at lung aus-
cultation 
1 
- 
Axillary temperature >37.0 
1 
- 
BMI <18 
1 
1 
BMI <16 
1 
1 
MUAC <220 
1 
1 
MUAC <200 
1 
1 
BMI: Body Mass Index; MUAC: Middle Upper Arm Circumference. 
6.1.2 Main study parameter/endpoint 
1. The total number of TB cases found by GeneXpert® and/ or culture of Mycobac-
terium tuberculosis complex.  
2. Number of TB cases correctly identified by AeoNose™. 
3. Number of TB cases correctly identified by digital X-ray with CAD4TB®. 
4. Factors affecting or influencing sensitivity and specificity of AeoNose™ and 
CAD4TB®, e.g. medication use, smoking and drugs, co-morbidities, BMI, dental 
status 

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6.1.3 Secondary study parameters/endpoints 
1. Costs of different (theoretical) screening algorithms using AeoNose™, CAD4TB® 
or both in conjunction.  
 
2. Signal differences and diagnostic performance of AeoNose™ and CAD4TB® in: 
a. HIV cases (and CD4 count if available) 
b. Patients with syphilis (and disease stage if available) 
c. Diabetes cases (insulin dependent or non-insulin dependent) 
d. Cases of EPTB 
 
3. Number of incidental CXR findings and the effect on CAD4TB® output score.  
 
4. Numbers of Mycobacterium tuberculosis complex bacterial strains identified. 
6.1.4 Other study parameters 
- 
Body weight (kilograms) 
- 
Height (centimetres) 
- 
Blood pressure and heart frequency 
- 
Body temperature (axillary) 
- 
Upper arm circumference 
- 
Date of birth 
- 
Place of birth 
- 
Place of residence 
- 
Gender (male/female) 
- 
Sexual orientation 
- 
Nationality  
- 
Level of education (none, primary school, secondary school, vocational, higher 
education, unknown) 
- 
Occupational history 
- 
Civil state (single, married, widowed, separated, divorced, unknown) 
- 
Ethnicity  
▪ 
If indigenous: specific indigenous ethnicity 
- 
Dental state (good / plaque / inflamed gums / broken or missing teeth) 
- 
Symptoms (including onset and intensity)  
▪ 
Cough 
▪ 
Sputum or haemoptysis 
▪ 
Weight loss 

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▪ 
Night sweats 
▪ 
Chest pain 
▪ 
Dyspnea 
▪ 
Other 
- 
Medical history and co-morbidities   
- 
Previous diagnosis/treatment for TB (year and duration of treatment in months) 
- 
Location in prison (cell, hallway, yard, others) 
- 
Date of current prison entry, previous prison entries 
- 
Medication (especially antibiotics)  
- 
Smoking  
- 
Drug use  
- 
Factors influencing breath signal: intake and timing of ingestion of food, drinks, 
cigarettes 
- 
CAD4TB® output score 
- 
Radiological severity of TB  
- 
AeoNose™ breath profiles 
- 
GeneXpert® cycle time (if positive) 
- 
MGIT™ culture time-to-positivity (if positive) 
- 
Clinical severity of TB (TBscore, Wejse et al. 2008 and TBscoreII, Rudolf 2014) 
- 
CXR abnormalities as described by study doctor (infiltrate, cavity, 
lymphadenopathy, mass lesion, pleural abnormality, etc.) 
6.2 Study procedures 
AeoNose™:  
Participants will be asked to produce a breath sample by tidal breathing through the 
AeoNose™ device for 5 minutes. In case of interruption (e.g. cough) the interval 
between stop and re-start will be recorded. Participants will use a nose clamp to avoid 
inhaling unfiltered air. The air sample collection will be performed under standardized 
conditions in a room free of gases with no small molecules (such as alcohol) that can 
influence the breath profile of the participant. The exhaled-breath signals  will be stored 
on the study computer and later sent to The eNose Company for analysis. 
 
Digital CXR and CAD4TB®:  
X-ray image will be taken using a digital X-ray system connected with CAD4TB® 
software. Participants will be asked to stand in front of the panel while one posterior-
anterior X-ray image is taken. Images will be processed by the CAD4TB® software. A 
TB likelihood output score is presented to the doctor within minutes. Study doctors will 
also view and interprete the digital CXR during the consultation. 
 
Sputum induction:  

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Participants with presumptive TB will be asked to produce two sputum samples. If 
necessary sputum can be induced using saline nebulisation sputum. The technique of 
sputum induction consists of inhaling an aerosol of saline over period of two minutes. 
This procedure will be preferably carried out in open air using a portable nebulizer and 
saline solution provided by the researchers. Participants are then instructed to 
expectorate in a standard sputum container. 
 
6.3 Collection of data 
Data collected during the screening will be recorded using electronic CRF’s on the 
Castor EDC (electronic data capture) platform. (Castor EDC, Amsterdam, the 
Netherlands) Data will be inserted immediately and stored securely in the Castor study 
database. Information will be linked to a unique study ID number and contain no 
personal identifying information. 
AeoNose™ breath profiles data, CXR images and CAD4TB® output will be securely 
stored and linked to the participant ID number.  
After the breath profiles have been shared with The eNose Company they will be 
stored indefinitely (anonymized and encrypted) on a server in Amsterdam, the 
Netherlands.  
 
6.4 Withdrawal of individual participants 
Participants can leave the study at any time for any reason if they wish to do so 
without any consequences. All participants included for follow-up will be educated 
about their presumptive diagnosis and encouraged to follow-up either inside or 
outside the prison. The investigator can decide to withdraw a participant from the 
study for medical reasons (e.g. hospitalization for a non-TB illness). 
6.5 Replacement of individual participants after withdrawal or lost to follow-up 
Not applicable  
6.6 Follow-up of participants withdrawn from treatment 
Not applicable 
6.7 Premature termination of the study 
Potential reason for (a temporarily interruption or) premature ending of the study are 
if the security of participants and researchers or quality of the study can not be 
guaranteed in the Paraguayan prison environment. In case the study is ended 
prematurely, the sponsor will notify the accredited MREC within 15 days, including 
the reasons for the premature termination. 
 

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7. SAFETY REPORTING 
7.1 Temporary halt for reasons of participant safety 
 
In accordance to section 10, subsection 4, of Dutch Medical Research Involving Human 
Participants Act (WMO), the sponsor will suspend the study if there is sufficient ground 
that continuation of the study will jeopardise either the study team or participants health or 
safety. The sponsor will notify the accredited MREC (Laboratorio Central de Salud 
Publica, LCSP, Asunción, Paraguay) without undue delay of a temporary halt including 
the reason for such an action. The study will be suspended pending a further positive 
decision by the accredited MREC. The investigator will take care that study participants 
are kept informed.  
In this study the safety risk is primarily the potential for aggressive or violent behaviour by 
PPL against other PPL, employees or members of the study team. The prison officials  
will ensure the safety of the study team and participants during the study.  
7.2 AEs, SAEs and SUSARs 
7.2.1 Adverse events (AEs) 
 
Adverse events are defined as an undesirable or harmful experience occurring to a 
participant during the study. All adverse events, whether or not considered related to 
study procedure, which are reported spontaneously by the participant or observed by 
the investigator or his staff will be recorded. Potential adverse events are not 
expected in this study.  
7.2.2 Serious adverse events (SAEs) 
There is no risk of (serious) adverse events involved in participation in this study.  
7.2.3 Follow-up of adverse events 
Not applicable 
7.2.4 Data Safety Monitoring Board / Safety Committee  
Because the study investigations do not carry a health risk for the participants no 
Data Safety Monitoring Board will be installed. 
 
8. STATISTICAL ANALYSIS 
 
All data will be analysed using SPSS software. (IBM, United States) The data of the 
participants will be analysed cross-sectional. Follow-up of participants with presumptive TB 
but negative microbiological testing will be completed until 6 months after Visit 1. After a 

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follow up period of 6 months a final diagnosis will be made (either healthy or TB case and the 
participant will be offered treatment if indicated). A p-value (two-sided) < 0.05 is considered 
to indicate a statistically significant difference in all comparisons. Multivariate analysis will be 
performed to analyse influences of different combinations of participant characteristics. 
8.1 Primary study parameter(s) 
 
Primary study parameters are the number of TB cases cases by CAD4TB® CXR analysis 
and AeoNose™ breath sampling. 
A binary assessment of the presence or absence of illness has four possible outcomes: 
true positives (TP), false positives (FP), true negatives (TN) and false negatives (FN). 
The relationship of the four outcomes is the basis of sensitivity and specificity analysis 
which determines the accuracy and predictive value of a diagnostic tool.  
 
True positive: A positive test result in a confirmed TB case. 
False positive: A positive test result with negative sputum results and no confirmed TB 
during follow-up. 
True negative: A negative test result with no confirmed TB during follow-up. 
False negative: A negative test result with confirmed TB during follow-up. 
 
Receiver operating characteristics (ROC) will be generated using an ROC package, and 
bootstrapping to generate confidence intervals in the ROC plot. T-testing or McNemar’s 
test will be used to compute P values when comparing the sensitivity of conventional 
screening and CAD4TB® and AeoNose™ at assumed levels of specificity. We will also 
use multivariate analysis to analyse influences of different combinations of participant 
characteristics.  
 
CAD4TB®: The collection of digital CXR images will be analysed and compared to 
presumptive and confirmed cases. The number of true/false positives and negatives will 
be used to calculate sensitivity and specificity as described above. The predictive value of 
the test will be analysed. 
 
AeoNose™: After the end of the study, a subset of inclusions will be used as ‘calibration 
set’ for the neural network. This set will be selected randomly and from participants 
throughout the whole study period to avoid bias from the AeoNose™ conditions over 
time, bias from the first volunteering participants who might be more ill than the others 
and bias from different penal institutions. To build a reliable calibration model we try to 
include at least 30-40% of all TB cases in the calibration set. After calibrating  the neural 

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network, the breath profiles of the remaining participants will be tested for validation of the 
calibration model. A proprietary ‘hybrid’ tucker-tensor decomposition is used to compress 
the breath signal data in four dimensions (TP, FP, TN and FN) using PARAFAC22 and 
TUCKER323 kernels. The software used is customized for this specific data set-up. The 
compressed data is thereafter used to build a classification model using an artificial 
neural network (ANN) and the 95% confidence interval is calculated using the Wilson 
score interval. The result of the ANN is a similarity indication and the chosen threshold 
value determines the ratio between the outcomes.  
8.2 Other study parameters 
 
Not applicable 
 
9. ETHICAL CONSIDERATIONS 
9.1 Regulation statement 
This study will be conducted according to the principles of the Declaration of Helsinki (8th 
revision, 2013) and in accordance with the Dutch Medical Research Involving Human 
Participants Act (WMO). Principles of Good Clinical Practice (GCP) will be adhered. The 
Medical Research Ethics Committee (MREC) of the National Reference Laboratory 
(LCSP) in Asuncion, Paraguay will approve the study. Permission to conduct the study 
has already been obtained from the prison authorities, the Paraguayan Ministry of 
Justice, and the Ministry of Health has expressed their special interest, emphasized the 
importance of the project and has called on their employees to cooperate with the project 
to let it succeed. The study is in accordance with the WHO Operational Guide on 
Tuberculosis Screening (2015) and Red Cross Guidelines for Control of TB in Prisons 
(2009). 
 
9.2 Recruitment and consent 
Participants will be recruited from employees and prisoners of three Paraguayan penal 
institutions:Tacumbú, Buen Pastor, Esperanza.  
 
The study will be integrated in a wider prison public health campaign which will inform on 
tuberculosis, how to recognize the disease, how to prevent transmission and how to seek 
care.  
 
During the study visit, participants will be informed about tuberculosis and the study 
procedures by an independent staff member who can answer questions. This staff 

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member will be trained to understand the study procedures and advantages and 
disadvantages of participation. An educational animation video with voice-over will be 
shown to all potential participants (Annex I). Participants will be informed of the test 
procedures and test results, including the potential benefits and harms of screening and 
the potential harms of not being screened. This information will be given in either Spanish, 
Guarani or Portuguese and adapted to a primary school educational level to be 
understood by all participants. The information will be presented in the presence of an 
independent witness, e.g. a prison or church volunteer.  The participant can confirm 
his/her agreement with a signature or fingerprint. 
 
At informed consent, participants will be asked for permission to exchange medical data 
between PNCT & PRONASIDA program and the study researchers. 
 
In case of presumptive TB the participant will be informed immediately. Sputum and HIV 
test results will be communicated with the highest priority. Patients will then be referred to 
the indicated national program (PNCT and/or PRONASIDA). 
In case of suspicion of other pathology, referral will be arranged to the  prison health service. 
See also the participant information form and informed consent form in Appendix. 
 
9.3 Benefits and risks assessment, group relatedness 
Prior to this study, none of the prisons perform the entry and/or annual TB screening rec-
ommended by WHO and ECDC (WHO, Geneva 2013; ECDC Stockholm/Lisbon 2018). 
TB cases are currently detected actively by trained prison health promotors and passively 
by seeking medical consultation due to symptoms. As TB often manifests with a pro-
longed period of mild or unspecific symptoms, many patients remain undiagnosed, un-
treated and contagious for a long period. Participation in the screening offers the partici-
pant an opportunity to be checked for TB. The mass screening offered in this study is an 
opportunity for the PNCT to comply with WHO recommendations for prison screening 
and thus improving the prisoners’ healthcare and reducing the continuous spread of TB 
within and from the prisons.  After this study the PNCT will possess accurate information 
on the prevalence of TB within Paraguayan prisons which can serve as the basis for fu-
ture policy and program enhancements. CAD4TB® analysis brings no burden to the par-
ticipant. Breath sampling with the AeoNose™ device is risk-free and a minimal burden for 
the participant. Group-relatedness is inherent in a mass screening study of a known high 
risk population. For an accurate assessment of the prevalence of TB in the prisons and 

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the utility of the CAD4TB® and AeoNose™ as a screening tool the entire group needs to 
be investigated without any inadvertent selection of participants. 
9.4 Compensation for injury 
Compensation for injury is not applicable as the study does not include any invasive 
procedures or other risk of injury. The sponsor requests the MREC for dispensation of the 
obligation to provide insurance for participants.  
9.5 Incentives for participants 
The participants will not receive an individual monetary compensation or equivalent for 
their participation. The only perceived incentive is to identify and address a potentially life-
threatening disease that can be transmitted to fellow staff or prisoners, family and 
associated communities. 
 
10. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
10.1 Handling and storage of data and documents 
Participant data will be handled confidentially and anonymously. For each participant a 
unique identification code will be used to link the study data to the participant’s personal 
characteristics. The personal data will be handled according to the European General Data 
Protection Regulation (GDPR). 
 
Data will be recorded using electronic CRF’s on the Castor EDC (electronic data capture) 
platform. (Castor EDC, Amsterdam, the Netherlands) Castor is compliant with all relevant 
regulations including ICH E6 Good Clinical Practice, GDPR, HIPAA, FDA 21 CFR Part 
11, ISO 27001, and ISO 9001. Castor is also dedicated to the FAIR principles for data 
storage. (https://www.force11.org) Data will be inserted immediately and stored securely 
on the Castor servers using a dedicated study computer. Information will be linked to a 
unique study ID number only known to the study investigators and contain no personal 
identifying information. AeoNose™ breath samples as well as digital CXR and CAD4TB® 
output will be stored securely on the study computer and linked to the unique study ID. All 
data will be stored for ten years.  
10.2 Amendments  
Amendments are changes made to the research after a favourable opinion by the 
accredited MREC has been given. All amendments will be submitted to the MREC for 
approval.  
A ‘substantial amendment’ is defined as an amendment to the terms of the MREC 
application, or to the protocol or any other supporting documentation, that is likely to 
affect to a significant degree: 
- 
The safety or physical or mental integrity of the participants of the trial; 

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- 
The scientific value of the trial; 
- 
The conduct or management of the trial; or 
- 
The quality or safety of any intervention used in the trial. 
 
All substantial amendments will be notified to the MREC and to the competent authority. 
Non-substantial amendments will not be notified to the accredited MREC and the 
competent authority, but will be recorded and filed by the sponsor.  
10.3 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the 
accredited MREC once a year. Information will be provided on the date of inclusion of the 
first participant, numbers of participants included and numbers of participants that have 
completed the trial, serious adverse events/ serious adverse reactions, other problems, 
and amendments.  
10.4 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited MREC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last patient’s last visit.  
The sponsor will notify the MREC immediately of a temporary halt of the study, including 
the reason of such an action.  
In case the study is ended prematurely, the sponsor will notify the accredited MREC 
within 15 days, including the reasons for the premature termination. 
Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited MREC.  
 
10.5 Public disclosure and publication policy 
The study will be registered at https://clinicaltrials.gov/, as well as 
https://eudract.ema.europa.eu/. Research data and results will be disclosed/published in 
the medical literature unreservedly (Open Access).  
 
11. STRUCTURED RISK ANALYSIS  
Participation in the study does not carry any health risk for the participant.  
 
12. REFERENCES 
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High-priority target product profiles for new tuberculosis diagnostics: report of a 
consensus meeting. Geneva: World Health Organization; April 2014 
- 
Systematic screening for active tuberculosis: Principles and Recommendations. 
Geneva: World Health Organization; 2013.  
- 
Philipsen RHHM, Sánchez CI, Maduskar P, Melendez J, Peters-Bax L, Peter JG, 
et al. Automated chest-radiography as a triage for Xpert testing in resource-
constrained settings: A prospective study of diagnostic accuracy and costs. Sci 
Rep 2015; 
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Khan FA, Pande T, Tessema B, Song R, Benedetti A, Pai M, et al. Computer-
aided reading of tuberculosis chest radiography: Moving the research agenda 
forward to inform policy. European Respiratory Journal. 2017 
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Melendez J, Hogeweg L, Sánchez CI, Philipsen RHHM, Aldridge RW, Hayward 
AC, et al. Accuracy of an automated system for tuberculosis detection on chest 
radiographs in high-risk screening. Int J Tuberc Lung Dis. 2018; 
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Marcel Bruins, Zeaur Rahimc, Albert Bosa, Wendy W.J. van de Sande, Hubert 
Ph. Endtz, Alex van Belkum. Diagnosis of active tuberculosis by e-nose analysis 
of exhaled air. Tuberculosis 2013.  
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Coronel Teixeira R, Rodríguez M, Jiménez de Romero N, Bruins M, Gómez R, 
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diagnose tuberculosis. J Infect. 2017; 
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Kamarulzaman A, Reid SE, Schwitters A, et al. Prevention of transmission of HIV, 
hepatitis B virus, hepatitis C virus, and tuberculosis in prisoners. Lancet 2016.  
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Tuberculosis control in prisons: current situation and research gaps. Masoud Dara 
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Pande T, Cohen C, Pai M, Ahmad Khan F. Computer-aided detection of 
pulmonary tuberculosis on digital chest radiographs: a systematic review. Int J 
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Saktiawati A, Stienstra Y, Subronto et al. Sensitivity and specificity of an 
electronic nose in diagnosing pulmonary tuberculosis among patients with 
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tuberculosis on chest radiographs: An evaluation of the CAD4TB v6 system. Pre-
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European Centre for Disease Prevention and Control, European Monitoring 
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finding of communicable diseases in prison settings. Stockholm and Lisbon: 
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Walzl G, McNerney R, du Plessis N, Bates M, McHugh TD, Chegou NN, et al. 
Tuberculosis: advances and challenges in development of new diagnostics and 
biomarkers. The Lancet Infectious Diseases. 2018; 
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Wejse C, Gustafson P, Nielsen J et al. TBscore: Signs and symptoms from 
tuberculosis patients in a low-resource setting have predictive value and may be 
used to assess clinical course. Scandinavian Journal of Infectious Diseases. 
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Rudolf F, The Bandim TBscore – reliability, further development, and evaluation 
of potential uses. Global Health Action; 2014 
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Qualitative/Quantitative Literature Metasynthesis, Mitchell et al. KNCV 
Tuberculosis Foundation; 2012  
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Guía Nacional para el manejo de la Tuberculosis. Ministerio de Salud Pública y 
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Guidelines for the controle of Tuberculosis in Prisons. International Committee of 
the Red Cross, Geneva, Switzerland; 2009 
 
13. ADNEXES 
I. 
Approval Ministry of Justice 
II. 
Approval Ministry of Public Health 
III. 
PIF  
IV. 
Case Record Form (CRF)