The Impact of Alcohol Consumption on Tuberculosis Treatment Outcomes
Phase: NA
Conditions: Tuberculosis, Alcohol Consumption, Treatment Adverse Effect
Sponsor: Boston Medical Center

 ____________________________________________________________________________________________ 
i 
 
The Impact of Alcohol Consumption on TB Treatment 
Outcomes 
 
Sponsored by: 
National Institute of Allergy and Infectious Diseases (NIAID) 
 
Principal US Investigator: Karen R. Jacobson, MD, MPH 
Assistant Professor of Medicine 
Section of Infectious Diseases 
Boston University School of Medicine 
801 Massachusetts Ave. 
Crosstown 2nd Floor 
Boston, MA 02118 
karen.jacobson@bmc.org 
617-414-5213 
 
Principal South African Investigator: Tara Carney, Ph.D. 
Specialist Scientist 
Alcohol, Tobacco and Other Drug Research Unit 
South African Medical Research Council 
Francie Van Zyl Drive 
Parow 
tara.carney@mrc.ac.za 
27 21 938 0326 
 
DMID Protocol Number:  
16-0043 
 
Keywords: 
Tuberculosis, alcohol, treatment outcomes 
 
Version Number: 8.4                 6 September 2022 
 
 
Please note: TRUST-II referred below is not a new project, but rather a continuation and 
extension of study activities from TRUST-I, as well as the inclusion of a new study activity which 
is included in the protocol. 
 
NCT02840877 
 
Statistical Plan pages  39-43 
 
 

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ii 
Statement of Compliance 
 
This study will be conducted in compliance with the protocol, International Conference on 
Harmonisation Good Clinical Practices E6 (ICH-GCP) and the applicable regulatory 
requirements. 
• 
U.S. Code of Federal Regulations applicable to clinical studies (45 CFR 46 – Protection 
of Human Subjects). 
• 
Completion and certification of the Human Subjects Protection Training at all 
participating institutions or offered equivalent that meet the requirements of NIH Policy 
“Required Education In the Protection Of Human Research Participants” (Notice OD-00-
039), Revision Date: August 25, 2000. 
• 
ICMR Ethical Guidelines for Biomedical Research on Human Subjects and Guidelines 
for Good Clinical Laboratory Practices. 
 

 ____________________________________________________________________________________________ 
iii 
SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
We, Drs. Jacobson and Myers, have read the Department of Health: Ethics in health research: 
principles, processes and structures, second edition, 2015, the Guidelines for Good Practice in 
the Conduct of Clinical Trials with Human Participants in South Africa, Second Edition, 2006, 
Department of Health, Pretoria, South Africa (where applicable), and the Declaration of Helsinki 
(2013) and have prepared this proposal with due cognisance of its content. Furthermore we will 
adhere to the principles expressed when conducting this proposed research project. 
 
Site Investigator:* 
 
Signed: 
 
Date: 
6 September 2022 
 
Name: Karen Jacobson, MD, MPH 
Principal US Investigator 
Title:   Assistant Professor of Medicine 
           Section of Infectious Disease 
           Boston University School of Medicine (BUSM) 
           And Boston Medical Center  (BMC) 
 
 
 
 
 
Date: 
 6 September 2022 
 
Name: Tara Carney, PhD* 
            Principal South African Investigator 
Title:    Specialist Scientist 
            Alcohol tobacco and Other Drug Research Unit 
            South African Medical Research Council 
 
 
 
 
 

 ____________________________________________________________________________________________ 
iv 
Statement of Compliance ............................................................................................................. i 
Signature Page ........................................................................................................................... ii 
List of Abbreviations .................................................................................................................... v 
Protocol Summary ..................................................................................................................... vi 
 
1 
Key Roles ........................................................................................................................... 1 
2 
Background Information and Scientific Rationale................................................................ 4 
2.1 
Background Information .......................................................................................... 4 
2.2 
Rationale ................................................................................................................ 4 
2.3 
Potential Risks and Benefits ................................................................................... 8 
2.3.1 
Potential Risks ............................................................................................ 8 
2.3.2 
Known Potential Benefits........................................................................... 11 
3 
Objectives ........................................................................................................................ 12 
4 
Study Design .................................................................................................................... 13 
5 
Study Population .............................................................................................................. 15 
5.1 
Selection of the Study Population ......................................................................... 15 
5.2 
Inclusion/Exclusion Criteria ................................................................................... 17 
6 
Study Procedures/Evaluations .......................................................................................... 20 
6.1 
Study Procedures ................................................................................................. 20 
6.2 
Laboratory Evaluations ......................................................................................... 23 
6.2.1 
Laboratory Evaluations/Assays ................................................................. 23 
6.2.2 
Special Assays or Procedures ................................................................... 24 
6.2.3 
Specimen Shipping ................................................................................... 25 
7 
Study Schedule ................................................................................................................ 27 
7.1 
Pre-Screening ....................................................................................................... 27 
7.2 
Screening ............................................................................................................. 27 
7.3 
Enrollment/Baseline Visit (Treatment initiation visit) .............................................. 28 
7.4 
Weekly Visits (Treatment Weeks 1-12) ................................................................. 28 
7.5 
Daily Assessments (Treatment Months 1-6) ......................................................... 28 
7.6 
Single PK/PD Visit (In Treatment Week 4 – Treatment Week 8 window) .............. 28 
7.7 
Monthly Follow-Up Visits (Treatment Month 1-6) .................................................. 29 
7.8 
Post-Treatment Follow-Up (Post-treatment Months 3, 6, 9, 12) ............................ 29 
7.9 
Criteria for Discontinuation or Withdrawal of a Participant (or a Cohort), if 
applicable ............................................................................................................. 30 
8 
Assessment of Outcome Measures .................................................................................. 31 
8.1 
Specification of the Appropriate Outcome Measures ............................................ 31 
8.1.1 
Primary Outcome Measures ...................................................................... 31 
8.1.2 
Secondary Outcome Measures ................................................................. 31 
9 
Safety assessment and reporting ..................................................................................... 33 
9.1 
Definition of Adverse Event (AE) ........................................................................... 33 
9.2 
Definition of Serious Adverse Event (SAE) ........................................................... 33 
9.3 
Reporting Procedures ........................................................................................... 34 
9.3.1 
Serious Adverse Event Detection and Reporting ....................................... 34 

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v 
9.3.2 
Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings ........................................................ 34 
9.3.3 
Type and Duration of the Follow-up of Participants After Adverse Events . 34 
10 
Clinical Monitoring Structure ............................................................................................. 35 
10.1 
Site Monitoring Plan .............................................................................................. 35 
11 
Statistical Considerations ................................................................................................. 36 
11.1 
Study Outcome Measures .................................................................................... 36 
11.2 
Sample Size Considerations ................................................................................. 37 
11.3 
Participant Enrollment and Follow-Up ................................................................... 38 
11.4 
Analysis Plan ........................................................................................................ 39 
12 
Access to Source Data/Documents .................................................................................. 42 
13 
Quality Control and Quality Assurance ............................................................................. 43 
14 
Ethics/Protection of Human Participants ........................................................................... 44 
14.1 
Ethical Considerations .......................................................................................... 44 
14.2 
Declaration of Helsinki .......................................................................................... 44 
14.3 
Institutional Review Board .................................................................................... 44 
14.4 
Informed Consent Process ................................................................................... 44 
14.4.1 Informed Consent Process ........................................................................ 46 
14.5 
Exclusion of Women, Minorities, and Children (Special Populations) .................... 46 
14.6 
Participant Confidentiality ..................................................................................... 46 
14.7 
Future Use of Stored Specimens .......................................................................... 47 
15 
Data Handling and Record Keeping ................................................................................. 48 
15.1 
Data Management Responsibilities ....................................................................... 48 
15.2 
Data Capture Methods .......................................................................................... 48 
15.3 
Types of Data ....................................................................................................... 48 
15.4 
Timing/Reports ..................................................................................................... 49 
15.5 
Study Records Retention ...................................................................................... 49 
15.6 
Protocol Deviations ............................................................................................... 49 
16 
Publication Policy ............................................................................................................. 49 
17 
Literature References ....................................................................................................... 51 
 
SUPPLEMENTS/APPENDICES 
A: Study Schedule 
 

 ____________________________________________________________________________________________ 
vi 
AE 
Adverse Event 
AFB 
Acid Fast Bacilli 
ALT 
Alanine Transaminase 
AST 
Aspartate Transaminase 
AUC 
Area Under the Curve 
AUDIT 
Alcohol Use Disorders Identification Test 
BMC 
Boston Medical Center 
BUSM 
Boston University School of Medicine 
CBC 
Complete Blood Count 
CDC 
Community Day Centre 
CES-D 
Center for Epidemiologic Studies Depression Scale 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CRF 
Case Report Form 
CXR 
Chest X-Ray 
DMID 
Division of Microbiology and Infectious Diseases 
DOTS 
Directly observed therapy, short course 
DUDIT 
Drug Use Disorders Identification Test 
EMB 
FG 
Ethambutol 
Focus Group 
FTND 
Fagerstrom Test for Nicotine Dependence  
FWA 
Federal-Wide Assurance 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HR 
Hazard Ratio 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IDI 
INH 
Individual Interview 
Isoniazid 
IRB 
Institutional Review Board 
MDR TB 
Multidrug resistant tuberculosis 
MGIT 
Mycobacteria Growth Indicator Tube 
MIC 
Minimum inhibitory concentration 
MOP 
Manual of Procedures 
MTB 
Mycobacterium tuberculosis 
N 
Number (typically refers to participants) 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, DHHS 
NIH 
National Institutes of Health 
OCRA 
Office of Clinical Research Affairs, DMID, NIAID, NIH, DHHS 
OHRP 
Office for Human Research Protections 
ORA 
Office of Regulatory Affairs, DMID, NIAID, NIH, DHHS 
PD 
Pharmacodynamics 
PETH 
Phosphatidylethanol 

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vii 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
PZA 
Pyrazinamide 
RIF 
Rifampicin 
REC 
Research Ethics Committee 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAMRC 
South Africa Medical Research Council 
SOP 
Standard Operating Procedure 
SU 
Stellenbosch University 
TB 
Tuberculosis 
TLFB 
Timeline Follow Back 
TTP 
Time to positivity 
UCT 
WGS 
University of Cape Town 
Whole Genome Sequencing 
WHO 
World Health Organization 

 ____________________________________________________________________________________________ 
viii 
Title: 
The impact of alcohol consumption on TB treatment outcomes 
 
Population: 
450 participants (male and female, ages 15 years and older, HIV infected 
and uninfected) initiating treatment for drug susceptible TB disease in the 
Worcester CDC or Avian Park Clinic in Worcester, South Africa. We 
expect 30% to be HIV positive.  
 
Number of Sites: 
 
• 
Worcester Community Day Centre (CDC), Avian Park Clinic and 
Brewelskloof Hospital, Worcester, South Africa 
• 
Ukwanda Rural Clinical School, Worcester, South Africa 
• 
Boston Medical Center/Boston University School of Medicine, 
Boston, MA  
• 
South Africa Medical Research Council (SAMRC), Tygerberg, 
South Africa 
• 
University of Cape Town, Cape Town, South Africa 
• 
Stellenbosch University, Cape Town, South Africa 
 
Study Duration: 
12 years (including time for manuscript preparation) 
Budget duration:  
10 years 
 
Participant Duration: 
18 months 
 
Objectives: 
 
Aim 1. To (i) examine the associations between problem alcohol use and TB treatment 
outcomes, and (ii) demonstrate that these associations persist independent of adherence to TB 
treatment among HIV-infected and uninfected participants.  
Hypothesis 1a: Poor TB treatment outcomes, measured as delayed time-to-culture 
conversion, are associated with problem alcohol use after controlling for non-adherence. 
Hypothesis 1b: End organ damage due to chronic alcohol use, rather than effects of 
acute ingestion, will be the strongest predictor of poor treatment outcomes independent 
of adherence. 
 
Aim 2. To evaluate the effect of problem alcohol use on the PK/PD of TB drugs among HIV 
uninfected participants.  
Hypothesis 2a: Persons with problem alcohol use will have altered peak drug 
concentration (Cmax) and area under the curve (AUC) due to effects on drug absorption 
and metabolism.  
Hypothesis 2b: These PK changes will be associated with delayed culture conversion 
and higher treatment failure/relapse rates; and/or with increased toxicity resulting in 
regimen changes or death. 
 
Aim 3. We will use existing samples and data and continue to collect samples and data to (A) 
evaluate Mtb diversity in host, its dynamics overtime and in a specific set of drug resistance, 
drug tolerance, virulence and immune regulator genes, for evidence of directional and 
diversifying selection. We will (B) also evaluate how Mtb diversity and genes under selection 
associate with time-to culture conversion (three consecutive weeks of negative growth) and 

 ____________________________________________________________________________________________ 
ix 
negative treatment outcomes, adherence, HIV, diabetes mellitus (DM), and substance use. We 
will (C) leverage MIC and sequence data from TRUST. We will combine these with a large 
public Mtb MIC and WGS dataset enriched for high-level antibiotic resistance generated by 
studies that include the NIAID funded Harvard TB Centers of Excellence for Translational 
Research (CETR). We will train an in silico MIC predictor and probe interactions between 
mutations and the Mtb lineage on a genome-wide scale. The current TRUST investigators, as 
well as Dr. Maha Farhat, Harvard Medical School will oversee this aim.  
Hypothesis 3a: We expect to see a clear signal of selective sweeps on candidate 
tolerance and resistance genes in host. By extrapolation from other pathogenic bacteria 
we expect selective forces on Mtb immune-regulator genes to generate more complex 
structural variation, possibly in a pattern of in host diversification. Immune selective 
forces are expected to be attenuated in HIV co-infected patients and potentially also in 
patients with problem alcohol use.   
Hypothesis 3b: It is possible that retreatment patients will have different rates of 
pathogen diversity or a higher rate of mixed strain infections. Based on preliminary data, 
we suspect participants with viable and resilient pathogen populations to have a higher 
degree of genetic diversity at baseline or manifest persistent diversity or selective 
sweeps of resistance variants overtime. We expect these same participants to be at 
much higher risk for delayed culture conversion and negative treatment outcomes. 
Hypothesis 3c: The primary outcome of this aim is generating an in silico MIC model with 
error metrics for predicting in vitro MIC and binary DST, and hazard ratios for association 
with delays in culture conversion. This model can be used to predict MICs from WGS for 
new observations and will capture a large number of genetic variants and the effect of 
hetero-resistance. 
 
Aim 4. A) To compare rates of dysglycemia (both hyperglycemia and hypoglycemia) in people 
living with HIV (PLWH) and HIV-uninfected persons receiving TB treatment in order to assess 
changes in blood glucose levels from study enrollment by HIV status and how alcohol use 
mediates the relationship; and B) to assess the role stress, inflammation and alcohol 
consumption play in relation to blood sugar levels in PLWH and HIV-uninfected individuals and 
to assess epigenetic modifications at DNA sites known to be involved in TB risk and neutrophil, 
monocyte, T and B cell function. 
 
Hypotheses: This will be preliminary data to assess if there are differences in these 
specific stress and inflammatory markers post TB treatment amongst PLWH and HIV-
uninfected individuals and potentially identify underlying mechanism(s) causing 
abnormal blood sugar levels, specifically the impact of alcohol use. The proposed work 
will allow us to assess the relationship between HIV and dysglycemia and how it is 
mediated by alcohol use in a cohort of TB patients at a more granular level. The pilot 
data will provide the necessary evidence for a larger prospective study powered to 
evaluate the underlying mechanism(s) in this relationship. 
 
Overview of Study Design: 
 
We will recruit microbiologically-confirmed, pulmonary TB patients in Worcester, South Africa, 
and follow them over an 18-month period. Patients will complete an interviewer-administered 
questionnaire on their alcohol use and other health-related behaviors, including tobacco and 
illicit drug use, and their recent alcohol use will be confirmed using a biomarker 
(phosphatidylethanol). We will collect chest radiographs, sputum smears and culture, and blood 
samples to compare the biology of treatment response in patients with and without problem 
alcohol use. During the 6-month treatment period, we will use smart mobile-phone technology to 

 ____________________________________________________________________________________________ 
x 
document daily drug adherence by trained community workers and will collect serial measures 
of alcohol intake and serial sputa isolates to assess treatment response, and record TB drug 
side effects. In addition, we will perform intensive PK/PD studies of isoniazid, rifampin, 
ethambutol, and pyrazinamide in 200 HIV-seronegative patients. We will follow the full cohort for 
12 months post-treatment to examine long-term TB outcomes, including relapse and death. 
Between the 9 months and 18 months post-treatment, we will collect up to 30 mL of blood to 
assess the changes in blood glucose levels and to conduct the inflammatory marker analyses 
outlined in Aim 4. 
 
We will also expand the TRUST-I cohort to include participants with retreatment TB (defined as 
received TB treatment in the prior 2 years, some who have completed a full 6 months and some 
who will be interrupters). This will allow the comparison of retreatment and new TB, enrich for 
delayed culture conversion and negative treatment outcomes (specifically failure, relapse, or 
death), and allow for direct-from-sputum sequencing and bacterial burden measurement on 
fresh sputum samples. We will sequence 1-3 isolates from previously and newly enrolled 
participants. 
 
Schematic of Study Design: 
 

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1 
Qualitative Component: 
 
We will also conduct a combination of focus group discussions with a subset of 
participants who participated in the larger study and individual interviews with 
TB/substance use professionals in order to assess participants’ perceptions of the 
relationship between alcohol use and TB disease and the reasons for changes in 
alcohol consumption during illness and throughout TB treatment. We also aim to assess 
the readiness of participants who consume alcohol to partake in reduction or cessation 
programs during TB treatment.  

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2 
1 KEY ROLES 
Division of Microbiology and Infectious Diseases (DMID):  
 
Program Officer 
Robin M. Mason  
Respiratory Disease Branch/DMID/NIAID  
5601 Fishers Lane, Room 8E34 
Rockville, MD 20892-9825 
Phone:  (240) 627-3310  
Fax:   (301) 496-8030  
E-mail:  rmason@niaid.nih.gov  
Medical Officer 
Mamodikoe Makhene 
Medical Officer, Division of Microbiology and Infectious Diseases 
National Institute of Allergy and Infectious Diseases 
5601 Fishers Lane, 8E24 
Rockville, MD 20892 
Phone: 240-627-3309 
Fax: 301-496-8030 
mmakhene@niaid.nih.gov 
 
Study Sites:  
 
Boston University (BU) and Boston Medical Center (BMC) 
801 Massachusetts Avenue 
Boston, MA 02118, USA 
 
Medical Research Council of South Africa (SAMRC) 
Alcohol, tobacco and Other Drug Research Unit 
Tyberberg, South Africa 7505 
 
Stellenbosch University (SU) 
DST/NRF Centre of Excellence for Biomedical Tuberculosis Research 
US/SAMRC Centre for Tuberculosis Research 
Tygerberg, South Africa 7505 
 
University of Cape Town (UCT) 
Division of Clinical Pharmacology 
Observatory, Cape Town, South Africa 7925 
 
Harvard Medical School (HMS) 
Department of Biomedical Informatics 
10 Shattuck Street 
Boston, MA 02115 
Avian Park Clinic 
Pelikaan Street, 
Worcester, South Africa 6850 
 

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3 
Worcester Community Day Centre (CDC) 
Sugget Street 
Worcester, South Africa 6850 
 
Brewelskloof Hospital (BKH) 
Haarlem Street 
Worcester, South Africa 6850 
 
Ukwanda Rural Clinical School – Stellenbosch University (Ukwanda) 
Durban Street 
Worcester, South Africa 6850 
 
Project Roles: 
 
Study Investigator 
Institution 
Role 
Email address 
Karen Jacobson 
Boston Medical Center 
BMC PI 
karen.jacobson@bmc.org 
Tara Carney 
SAMRC 
Local/Subcontract PI 
tara.carney@mrc.ac.za 
C. Robert 
Horsburgh 
Boston University 
Subcontract PI 
rhorsbu@bu.edu 
Helen McIlleron 
University of Cape Town 
Subcontract PI 
helen.mcilleron@uct.ac.za 
Robin Warren 
Stellenbosch University 
Subcontract PI 
rw1@sun.ac.za 
Charles Parry 
SAMRC 
SAMRC Co-Investigator 
charles.parry@mrc.ac.za  
Bronwyn Myers 
SAMRC/Curtin University 
SAMRC Co-Investigator    bronwyn.myers@mrc.ac.za 
Laura White 
Boston University 
BU Biostatistician 
lfwhite@bu.edu  
Maha Farhat 
Harvard Medical School 
HMS PI (for Aim 3) 
Maha_Farhat@hms.harvard.edu  
 

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4 
1 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
1.1 
Background Information 
An urgent need exists globally to identify modifiable drivers of poor TB treatment outcomes.  
TB is the leading cause of death due to an infectious disease globally. Despite recent declines 
in TB incidence, approximately 8.6 million new TB cases occurred in 2012.1 The slight global 
decline in incidence conceals regional variations; particularly the African and European regions 
did not halve 1990 mortality levels by 2015. South Africa is the third highest TB burden country, 
with an estimated TB incidence of 1,003 cases per 100,000 population, and has failed to 
achieve 85% treatment success for smear positive pulmonary TB cases.2 In South Africa’s 
Western Cape Province, where this study will be based, TB is the leading cause of natural 
death.3 In Cape Town, approximately 15% of TB cases result in treatment failure and 26% of the 
total TB burden are re-treatment cases, which is higher than elsewhere on the African 
continent.4 Understanding drivers of TB vulnerability and poor treatment response, both at the 
individual and population level, is essential for TB control – even more so with the recent rise of 
drug resistant TB strains. 
 
Problem alcohol use in vulnerable populations is a key driver of poor TB treatment response, 
both globally and in South Africa. In high-income countries, non-communicable diseases, 
including diabetes and alcohol use disorders, are thought to be more significant causes of 
immunological impairment in TB patients than HIV due to their greater prevalence.5 In high-HIV 
burden countries like South Africa, excess alcohol consumption may synergistically with HIV 
weaken the immune system, leading to more progression to active TB disease and poorer TB 
treatment response.6 A report from the United States National Tuberculosis Surveillance 
System, 1997-2012, found that 15% of all TB cases and 25% of US-born TB cases had 
documented excess alcohol use.7 Patients with excess alcohol use were more likely to have 
smear positive disease, delays in culture conversion, and higher mortality while on TB 
treatment. Better understanding of the relationship between alcohol and TB is needed to guide 
treatment of these patients in order to both improve individual outcomes and decrease 
transmission to others. Problem alcohol use, defined as a volume or pattern of alcohol 
consumption that results in adverse health events, is on the increase in low- and middle income 
countries, including South Africa.8 Overall, 10% of TB deaths globally have been attributed to 
alcohol as a risk factor.9 This number is estimated to be even higher in South Africa, with 23% of 
male and 7% of female TB deaths attributable to alcohol.8 Similarly in China and India, the two 
highest burden TB countries, 11-13% of male TB deaths are attributable to alcohol,9 making 
findings from this study potentially applicable to many other high TB burden settings globally. 
1.2 
Rationale 
Alcohol’s large attributable risk for TB deaths in South Africa is likely due in part to South 
Africa’s having one of the highest levels of alcohol consumption per drinker globally and one of 
the highest rates of heavy episodic drinking.8 The nationally representative South African Stress 
and Health Study found that 20% of adults in the Western Cape Province were likely to develop 
a substance use disorder in their lifetime, with alcohol being the most common abused 
substance.10 Problem alcohol use is especially prevalent in the rural farming regions of this 
province, where this study will be conducted. Upwards of one-third to close to 60% of men and 
one-third to close to 50% of women in these communities self-report heavy episodic drinking 
and screen positive for problem drinking using standardized screening tools.11-13 

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5 
 
There is a critical need for more effective TB treatment strategies for patients with a history of 
problem alcohol use. Alcohol use is greatly overrepresented among active TB patients. 
Screening patients with problem alcohol use in an urban setting for TB infection revealed rates 
of active TB that were 28-fold greater than those of age-matched residents.14 A meta-analysis 
reported a three-fold increased risk of active TB in individuals who consume more than 40g 
ethanol/day or who have an alcohol use disorder.15 In addition, retrospective studies from many 
countries have consistently reported that patients with problem alcohol use have worse TB 
treatment outcomes, including higher rates of treatment failure (defined as persistently smear 
positive after 4 months of therapy), relapse, and death compared to patients who do not 
consume alcohol.16-20 Alcoholic patients in Brazil had a 2.8-fold increased odds of treatment 
failure compared to nonalcoholics.17 Patients admitted to hospitals in Russia for treatment of 
chronic or relapsing TB had 3 times higher rates of alcohol use disorders compared to patients 
newly infected with TB.21 A 2.7-fold increased risk of death during TB treatment was reported in 
Russia for patients with problem alcohol use compared to those without, and there was a 4.4-
fold increased risk of death during TB treatment in US patients with reports of excessive alcohol 
use compared to those without.22,23 All of these studies demonstrate worse treatment outcomes 
in patients with problem alcohol use, supporting the need for more effective, safer, and better 
tolerated TB treatment strategies for this vulnerable population. 
 
A major knowledge gap is the degree to which poor treatment outcomes in alcohol-abusing 
patients are due to noncompliance alone. Problem alcohol use impacts on retention in care and 
adherence to daily TB treatment. Poor medication adherence and increased loss to follow-up 
from TB care have been documented for patients consuming alcohol regularly in several 
countries, including Russia, Brazil, India, Estonia, and in PI Jacobson’s studies of multidrug 
resistant (MDR) TB patients in Worcester, South Africa, where patients reporting alcohol use 
had a 2.1-fold higher rate of loss to follow-up compared to patients with no alcohol use 
history.18,24-27 A systematic review found a pooled odds ratio of 3.0 (95% CI: 1.8-5.0) for alcohol 
dependence as a loss to follow-up predictor.9 A study in India, in which there was a high 
prevalence of diabetes, low body mass index (BMI), and cavitary disease, reported that drinking 
alcohol during treatment for MDR TB was the only factor independently associated with 
unsuccessful outcomes.17 Although those who drank during treatment missed more doses than 
those who did not, which likely contributed to the poor outcomes, it is noteworthy that some 
alcohol-consuming patients who were adherent still had unsuccessful treatment. Yet there has 
been no research to identify reasons (beyond adherence) for these poorer outcomes among 
patients with problem alcohol use. A key barrier to understanding the persistent biologic effect of 
alcohol on TB disease is inadequate data on adherence, including detailed data on daily 
adherence (or number of missed doses of medication).18,22,24,25 Research combining better 
approaches to alcohol ascertainment and adherence monitoring is needed to advance 
understanding of the pathways by which alcohol use and TB disease interact. 
 
Recent studies suggest that much of the increased rate of treatment failure in TB patients may 
be due to microbiologic failures not directly attributable to nonadherence. In recent hollow-fiber 
studies, in which degrees of noncompliance were modelled, poor adherence was associated 
with microbiologic failure only when nonadherence exceeded 60%, and acquired drug 
resistance, defined as loss of susceptibility to one of the TB drugs, never occurred with 
nonadherence to a 4-drug regimen.28 Similarly, a meta-analysis concluded that studies with 
lower loss to follow-up rates did not differ significantly in microbiologic failure, acquired drug 
resistance, or relapse compared to studies with higher loss to follow-up rates.29 The authors 
theorized that microbiologic failures emerge when patients are consistently under-dosed or due 
to PK and microbial variability. Thus, although it is important to develop systems that will help 

 ____________________________________________________________________________________________ 
6 
patients who use alcohol to stay in treatment, the higher rates of poor microbiological outcomes 
may also be due to the impact of alcohol use on PK and bacterial susceptibility or other 
mechanisms driving treatment response, that need to be better understood. 
 
Evaluation of sources of PK variation can facilitate optimization of TB treatment regimens by 
identifying avoidable sources of variation and risk factors for low or high drug concentrations in 
patients. Several studies have shown the importance of optimal TB drug concentrations on the 
rate of kill of MTB in hollow fiber systems, in animal models of TB, and in patients.30-32 Clinical 
studies have also shown low drug concentrations to be associated with poorer outcomes in 
patients receiving combination therapy.33-35 In one study, patients with problem alcohol use had 
a significant decrease in the absorption and increase in the metabolism of the first-line TB drug 
isoniazid (INH), which led to lower maximum concentrations of the drug and a shorter half-life.36 
Researchers have reported increased acetylation in rat liver cells in the presence of ethanol, 
leading to speculation that alcohol enhances the activity of N-acetyltransferase, which also 
catalyzes the metabolism of INH, potentially leading to an increased metabolism rate.37 Problem 
alcohol use has also been associated with acquisition of drug resistant TB (primarily INH and 
rifampicin (RIF) resistance), which may reflect a failure to attain optimal drug levels. In 
Indonesia, MDR TB was found to be associated with excessive alcohol use, and TB patients 
admitted to hospitals in Russia were found to have an 8-fold increased likelihood of TB drug 
resistance if they had a history of an alcohol use disorder.21,38 A detailed analysis of the PK of all 
4 TB drugs in patients with problem alcohol use and their impact on PD will allow for improved 
understanding of which TB drugs are most affected in this population. This information will allow 
for more appropriate, increased dosing of specific drugs, drug substitutions for individual drugs 
that are consistently performing poorly, and potential extension of the intensive or continuation 
phases of therapy in this at-risk population. 
 
Evidence supports the additional biologic impact of chronic alcohol use on TB outcomes. 
Attention has been focused on the need to stop or decrease acute alcohol ingestion during TB 
treatment, but the lower rates of cure may also be due to chronic sequelae of years of alcohol 
use. Alcohol ingestion interferes with anatomic barriers that protect the lung from infection, 
impairs the actions of cytokines such as tumor necrosis factor alpha, and inhibits the expression 
of growth factors such as lung granulocyte colony stimulating factor.39 Functional activities of 
macrophages, including mobilization, adherence, phagocytosis, superoxide production, and 
microbicidal activity, are inhibited by alcohol.39 Alcohol-induced deficits in alveolar macrophage 
function diminish the capacity of these cells to eliminate invading pathogens. Harmful effects of 
chronic alcohol use may be of particular importance in TB where more than 90% of inhaled 
mycobacteria are normally destroyed by alveolar macrophages.400 Bacilli not killed by alveolar 
macrophages survive and proliferate intracellularly. Studies have shown that exposure to 
alcohol enhances intracellular growth of mycobacteria in human macrophages.41,42 These 
immune deficits could also be exacerbated by concomitant nutritional deficiencies or tobacco 
use that are often associated with chronic alcohol use.43,44 Chronic alcohol exposure may 
suppress cytokine production, which has an essential role in cellular communication, activation, 
proliferation, migration, and regulating inflammation and other healing mechanisms.45 Utilizing a 
pulmonary route for MTB infection in a mouse model, researchers found fewer CD4+ cells in the 
lungs of mice chronically exposed to alcohol, one likely reason for their poorer containment of 
the mycobacterial challenge compared to non-alcohol consuming mice.46 This inability to 
increase the CD4+ population in response to pulmonary infections is at least partially due to 
diminished IL-2 production and lymphocyte proliferation in regional lymph nodes. Additionally, 
granuloma formation was abnormal in the alcohol-consuming mice. These authors also found a 
diminished capability of the CD4+ cells to secrete IFN-γ when stimulated with MTB-infected 
macrophages. These findings suggest mechanisms that may be responsible for compromise of 

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7 
the ability to contain and effectively clear TB infection in patients with chronic alcohol exposure, 
independent of medication adherence.  
 
Aim 3. In clinical trial settings with close patient observation, approximately 5% of drug 
susceptible (DS) TB patients relapse after standard 6-month therapy, and ~20% relapse after 4-
months of short course therapy even when treated with fluoroquinolones (FLQ). TB patients can 
benefit greatly if we could identify signs of poor response earlier and adjust therapy intensity 
accordingly. Host factors including treatment adherence, drug absorption and metabolism, HIV 
infection, alcohol use and disease severity are associated with poor treatment response. 
Bacterial factors measurable at the microbiological level including bacterial burden and drug 
resistance (DR), are also important predictors of treatment response. Currently, a strong, rapid 
metric to monitor treatment response does not exist. In the United States, the Center for 
Disease Control (CDC) recommends a monthly sputum specimen for AFB smear and 
mycobacterial culture until two consecutive specimens are negative (culture convert) to monitor 
treatment response and identify individuals failing therapy. Patients with delays in time-to-
culture conversion (into the third treatment month and beyond) are at higher risk for poor 
treatment outcomes including treatment failure, relapse or death. Mtb culture is expensive, 
biohazardous and labor intensive, taking weeks to yield a result. These delays mean potential 
opportunities to modify treatment and avoid failure are missed. In some settings, the 
combination of low resources to perform culture and delays in culture results has led TB 
programs to collect samples for repeat smear acid fast bacilli (AFB) alone, to culture at even 
later intervals after treatment initiation, or to perform no microbiological treatment monitoring at 
all. These monitoring gaps are compounded by the one-size fits all approach to drug susceptible 
TB treatment i.e. that all patients receive standard first-line four drug therapy at standardized 
doses. If a metric were available to indicate which patients need early adjustment of the 
standard first-line therapy, or which patients would have good outcomes with shortened duration 
of therapy, this would be a major step forward toward more precision TB care and could reduce 
additional bacterial resistance generation.   
 
Note: Aim 3 will be evaluated by TRUST-II, which is the extension of our existing study (TRUST-
1).  
 
Aim 4. Research suggests that the hosts immune response to active TB and/or HIV disease 
results in a prolonged state of systemic inflammation which can have negative metabolic effects. 
Advances in HIV treatment have resulted in patients living longer, largely due to antiretroviral 
therapy (ART). This has resulted in PLWH experiencing increases in lifestyle-associated 
diseases, specifically diabetes. Current global diabetes disease burden is estimated at 415 
million, which is projected to hit 642 million by 2040 (over 60% increase), with sub-Saharan 
Africa (SSA) bearing the brunt of this, and South Africa at the forefront. Diabetes is currently the 
2nd leading cause of death in South Africa, behind TB. The current estimated prevalence of 
diabetes in PLWH on ART in South Africa is around 5%, while pre-diabetes is estimated at 15%, 
although this is considered an underestimate as the majority of people do not have access to 
proper screening, diagnostics or treatment for diabetes. As such, there is a need for larger 
longitudinal studies to enhance the understanding of risk factors (including alcohol use) for 
diabetes among PLWH in low- and middle-income settings. South Africa’s high burden of HIV 
and TB and the emerging diabetes epidemic, highlight the importance of investigating these 
associations. The interaction of these three diseases complicates clinical management of 
PLWH, potentially increasing the risk of morbidity and mortality. 
 
We hypothesize that PLWH are at a decreased risk of hyperglycemia, but increased risk of 
hypoglycemia compared to their HIV-negative counterparts and that the relationship is mediated 

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8 
by alcohol use. Preliminary data from the TRUST study collected at enrollment suggests that 
PLWH may have a decreased risk of diabetes and hyperglycemia compared to their HIV-
uninfected counterparts. Although the numbers are small, preliminary data also suggest that 
PLWH had a higher percentage of patients with hypoglycemia (HbA1c <4.5%) compared to 
those HIV-uninfected. These preliminary data in addition to the conflicting results in the literature 
warrant the need for further investigation of this relationship. Further, we hypothesize that 
systemic inflammation from infectious diseases (HIV and TB) increases a patient’s risk of 
dysglycemia, which is modified further by alcohol use, as research suggests that the hosts 
immune response to active TB and/or HIV disease results in a prolonged state of systemic 
inflammation which can have negative metabolic effects. 
1.3 
Potential Risks and Benefits 
1.3.1 Potential Risks 
 
Risks specific to study participation are low. The study procedures that can cause physical harm 
are:  
 
• 
Chest x-ray: Standard chest x-rays will be performed if not already done by the South 
Africa TB program (chest x-rays are standard on TB patients at diagnosis in the United 
States). There is always a slight risk of damage to cells or tissue from being exposed to 
any radiation, including the low levels of radiation used for x-rays. However, the risk of 
damage from the standard chest X-ray is usually very low compared with the potential 
benefits of the test. Given the minor risk associated with radiation exposure, we will not 
perform chest X-rays on any women who are pregnant. 
 
• 
Blood collection through venipuncture: There is very little risk associated with 
venipuncture. Some patients may experience minor discomfort, minimal bleeding, a 
bruise or swelling at the site of puncture. In rare cases patients may experience bleeding 
at the site of puncture, fainting or feeling light-headed, hematoma, localized infection, 
multiple punctures to locate veins. Any area where the skin anesthetic will be applied 
may become red and/or irritated. There are no known alternatives to venipuncture. 
Separate sterile equipment will be used on all participants. Emergency services will be 
available in the event of adverse events. The study nurses will be trained and 
experienced in phlebotomy prior to implementation of the study.  
• 
Strict airborne respiratory precautions will be taken when patients are providing sputum 
samples to prevent secondary exposure of clinical staff to infectious droplets. 
Adequacy of Protection against Risks 
 
Recruitment and Informed Consent: Recruitment will be open to all eligible participants, and 
every effort will be made to enroll a representative and unbiased sample of participants. 
Potential participants will be provided with an accurate and fair description of the risks or 
discomforts and the anticipated benefits of the research study, as outlined in 45 CFR 46, 
subpart A, as well as the International Conference on Harmonization – Good Clinical Practice 
(ICH-GCP) guidelines and the South African guidelines for Good Clinical Practice (GCP), 
second edition. Eligible participants identified through a structured pre-screening process who 
are recruited for screening will meet with a trained research assistant who will describe the 

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9 
purpose, goals, investigator and participant responsibilities and conduct of the study. The 
description will emphasize the design of the study, objectives and participant expectations, the 
voluntary participation, and confidentiality. It will be made clear that participation in this study 
does not affect the routine healthcare they will receive. Only participants with signed or marked 
consents will be enrolled. Separate consent will be obtained from parent/legal guardian for 
children under the age of 18 in addition to consent from minor children age 15-17 years. For 
recruitment into the focus groups, participants from the main cohort who have consented to 
future contact and who reported alcohol use during the main study will be contacted using the 
locator form they filled out (phone number, address) by phone or community tracking to assess 
eligibility and interest in participating in a focus group. Scheduled participants will be consented 
on the day of the focus group before any study procedures begin. For the key informant in depth 
interviews we will identify eligible participants within our wide network of substance use and TB 
professionals in Worcester and ask them (phone or email) if they are interested in participating 
in an in depth interview. They will be consented before any study procedures take place. Upon 
approval of Aim 3 (TRUST II), participants who are actively enrolled in TRUST will be re-
consented with amended consent forms indicating the future use of their samples for Whole 
Genome Sequencing of the TB bacteria. We will also contact participants who completed their 
time in the study and are no longer enrolled in TRUST to ask them if they are interested in 
signing a new consent form that allows for the use of their stored specimens for the purposes 
described in Aim 3. For Aim 4, participants who are currently enrolled will be re-consented, with 
the consent containing an option to opt-in or out of the additional 30 mL blood draw. For 
participants who have finished the study (completed their final 12 month post-treatment visit) but 
are within the window of 18 months post-treatment end are eligible for the 30 mL blood draw. 
We will contact these participants to see if they are interested in this component of the study. If 
they are interested, they will attend the study site and we will perform informed consent with a 
consent form specific to this 30 mL blood draw and its uses. We will also have a separate, DNA 
specific consent form, which describes the use of a small amount of the 30 mL blood to be used 
for epigenetic analyses. Participants can choose not to consent for the epigenetic work, in which 
case we will not use any of their blood for these DNA analyses. 
 
Waiver of Informed Consent: A waiver of informed consent is requested in exception 
circumstances described below to obtain and analyze bacterial (Mycobacterium tuberculosis) 
DNA extracted from previously enrolled participant sputum for the Aim 3 analyses with the goal 
of understanding how TB bacteria responds to TB therapy.  
 
Justification for the waiver of consent is described below.  
  
• The research involves no more than minimal risk to subjects.  
  
The proposed study is minimal risk and has abundant mechanisms in place to ensure protection 
of patient confidentiality. The risk of breach of confidentiality is minimized through rigorous 
ethics training for staff and protection of participant data as described below. Samples will be 
labeled with a Specimen ID. The laboratory conducting analyses on these samples will have no 
access to participant data linked to that Specimen ID. 
  
• Waiving the requirements for informed consent will not adversely affect the rights and welfare 
of study subjects  
  
The samples the investigators are requesting access to under this waiver are already-collected  
sputum samples and thus pose no physical risk to participants. No human DNA or other 
biomarkers will be obtained from the samples. Only bacterial DNA will be extracted and 

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10 
sequenced. The DNA sequencing has no impact on individual participants. Results will not be 
reported in any way that identifies individuals and will therefore not adversely affect participants’ 
rights and welfare.  
  
• The research could not be carried out practicably without the waiver or alteration.  
  
In order to accurately study, determine, and propose a metric that could indicate which TB 
patients need early adjustment of the standard first-line therapy, or which patients would have 
good outcomes with shortened duration of therapy, which would be a major step forward to 
more precision TB care and could reduce additional bacterial resistance generation, we require 
as close to complete TB bacteria whole genome sequencing data during the study period as 
possible. Each bacterial DNA whole genome sequence obtained from TRUST specimens will be 
critical to informing the model. This model will then be further informed by epidemiologic 
information provided by consented participants. We will make every attempt to reach and 
obtain consent from our participants for this aim of the study; however, the waiver or 
alteration is critical for analyzing specimens from participants who may have passed 
away, moved, or are otherwise unreachable. 
  
• If the research involves using identifiable private information or identifiable biospecimens, the 
research cannot practicably be carried out without using such information or biospecimens in an 
identifiable format.   
 
All samples are labeled with a Specimen ID. Any third party contracted for sequencing of the TB 
bacteria will be fully blinded to any additional participant data except for the Specimen ID. 
  
• Whenever appropriate, the subjects or legally authorized representatives will be provided with 
additional pertinent information after participation.  
  
The investigators do not anticipate the TB bacteria whole genome sequencing to generate 
information that will be pertinent to study participants. There are no plans for providing 
additional information to participants regarding the study procedures occurring under waiver of 
informed consent, including results.   
 
Protection against Risk:  
Confidentiality: Using study identification numbers as identifiers on case report forms, clinical 
specimens, and computerized databases containing study information will minimize risks to 
breach of confidentiality. Personal and medical information regarding study participants will not 
be released to anyone other than authorized study personnel, the National Institutes of Health – 
United States and/or its designee, the IRB at BMC/BU, or the applicable Research Ethics 
Committee(s) (REC) at the Medical Research Council of South Africa or University of Cape 
Town, without written permission of the participant. Study records will be secured in locked file 
cabinets at the South African site and electronic files will be secured on password protected 
computers. The study database will be maintained on REDCap. This database will be 
electronically encrypted using secure socket layering (SSL) encryption technology and will 
reside on a secure, password and firewall protected server at Boston University Medical Center 
(BUMC). All data stored on the database will only be identifiable through a study participant ID 
number. For programmatic purposes, participants will be entered into the mobile DOTS system 
by both participant ID and first name and first initial of surname. The first name and first initials 
of the surname will be entered into the DOTS system so that DOTS workers can address the 
participant by their name, and also ask to speak to the participant by name (rather than study 
number) when calling them. Communications with the sites (clinical and laboratory) will use only 

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11 
Study ID number to identify participant records. All study documents, including patient records, 
laboratory data, and correspondence will be retained by the investigator for at least five years 
after the last participant has reached the final study data point. 
 
Safety: Routine procedures will be performed by experienced, trained and qualified nursing staff 
employed by this study. Study-related questionnaires will be administered in a culturally 
sensitive manner by trained research assistants who are fluent in both English and Afrikaans 
languages. Our preliminary work in the Worcester region has highlighted that all patients 
receiving care at the Worcester CDC will be Afrikaans or English speaking. TB treatment staff 
employed at these facilities are also Afrikaans and English speaking, with patients who are only 
comfortable in isiXhosa being treated at the nearby Empilsweni clinic. Necessary training from 
an experienced investigator will be conducted with field staff. Participants will receive TB 
treatment as standard of care in this region. Patients with MDR TB, although excluded from this 
study, will also continue to receive standard care for their MDR TB disease. Potential 
psychological harm associated with alcohol abuse assessments will be minimized through the 
use of well-trained health educators, the provision of a health information card on ways of 
minimising alcohol harms and the option of referral to a local alcohol treatment center or the 
clinic's social worker for further assessment and referral. Alcohol use among minors will not be 
reported to parents as this may increase risk of harms. Pregnant women will not be included in 
this study at enrollment and, therefore, will not be exposed to the additional risk associated with 
chest x-rays. If a woman becomes pregnant during the study period she will not be censured as 
there is no additional risk to her being in this cohort after the initial x-ray. 
 
Adverse Event Monitoring: Serious adverse events associated with the conduct of the study will 
be reported to the Ethics Committees in Boston and South Africa and the NIH in accordance 
with DHHS and international guidelines within 48 hours of awareness of their occurrence. Due 
to the laboratory/epidemiologic focus of the study, few serious adverse events are anticipated in 
this study. 
1.3.2 Known Potential Benefits 
Potential benefits of the proposed research to the study population include: 
 
•  
Participants will be followed with intensive monitoring for adherence measures 
which has the potential to decrease loss to follow-up from care. 
• 
Participants will receive close monitoring for 12 months after TB treatment 
completion for disease relapse or reinfection. Early recognition of relapse will 
allow for appropriate and timely treatment that may prevent further transmission 
to close contacts. 
•  
Participants who report substance use will be offered a referral to a local 
substance abuse treatment service and/or the social worker associated with the 
TB clinic. Uptake of this referral will be voluntary. Monitoring the uptake of these 
referrals is beyond the scope of this cohort study. To minimise risk of harms 
associated with continued alcohol use while receiving TB treatment, all patients 
will be advised not to drink while on TB treatment and will receive an alcohol 
information handout that will outline the potential risks of alcohol use while 
receiving TB treatment. 
 

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12 
2 
OBJECTIVES 
This prospective investigation of the relation between problem alcohol use and TB treatment will 
provide the basis for identifying novel interventions to improve cure rates in TB patients with co-
morbid problem alcohol use. New strategies for addressing the complex interactions between 
these diseases could include risk profiling to identify patients with problem alcohol use, 
innovative medication dosing, medication substitutions, changes in adherence monitoring, and 
extending length of treatment. 
 
Aim 1: To (i) examine the associations between problem alcohol use and TB treatment 
outcomes, and (ii) demonstrate that these associations persist independent of adherence to TB 
treatment among HIV infected and uninfected participants.  
 
Aim 2: To evaluate the effect of problem alcohol use on the PK/PD of TB drugs among HIV-
uninfected participants. 
 
Aim 3: Interrogate MTB isolates through DNA sequencing/drug susceptibility testing, to identify 
a metric(s), that could predict which patients need early adjustment of the standard first-line TB 
therapy, or which patients would have good outcomes with shortened duration of therapy. 
 
Aim 4: To assess differences in rates of dysglycemia in PLWH and HIV-uninfected persons and 
to assess the role inflammation plays in relation to blood sugar levels in PLWH and HIV-
uninfected persons and how alcohol use modifies these relationships with the goal of 
understanding the risk factors for diabetes among PLWH in low- and middle-income settings. 
We will also perform epigenetic analyses to test genes that may be associated with the body’s 
response to tuberculosis (TB) infection, HIV infection, and response to treatment. 
 
Qualitative Component Goals:  
 
Using qualitative research methods, we aim to assess: 
  
1) Participants’ and key stakeholders’ perceptions of the relationship between alcohol use 
and TB disease and the reasons for changes in alcohol consumption during illness and 
throughout  treatment and  
 
2) Readiness of participants who consume alcohol to partake in reduction or cessation 
programs during TB treatment. 
 

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13 
3 
STUDY DESIGN 
We will employ a longitudinal, repeated measures, observational cohort design (see Figure: 
Schematic Study Design above).  
 
For Aim 1, we will recruit 438 patients who are initiating drug susceptible pulmonary TB 
treatment according to South African treatment guidelines. Immediately following study 
enrollment, participants will complete an interviewer-administered questionnaire, a urine drug 
test will be administered, and blood will be drawn for biologic tests, including a dried blood spot 
for phosphatidylethanol (PEth) alcohol biomarker testing, and SARS-CoV-2 serology. 
Contingent upon funding, we will also draw blood for biomarker evaluation at baseline. Results 
from the baseline blood tests will be provided back to TB Program. We will collect sputum for 
mycobacterial culture and MTB whole genome sequencing (WGS) and we will extract clinical 
data on previous TB disease, Xpert MTB/RIF result, HIV status, and general physical health 
from treatment records. Minimum inhibitory concentrations (MICs) for 13 drugs (INH, RIF, 
rifabutin, ethambutol (EMB), amikacin, kanamycin, levofloxacin, moxifloxacin, ethionamide, 
linezolid, delaminid, bedaquiline and clofazimine) will be determined on baseline sputum 
isolates. MICs for INH will be performed while the participant is still on the intensive phase of 
treatment and results will be provided back to TB Program. Participants will then continue on 
standard of care delivered by the national TB program through their clinic: participants will 
receive standard TB therapy; receive monthly follow up appointments at the clinic where their 
national TB program DOTS worker cards will be checked; produce sputum samples to check for 
treatment response (AFB smear conversion) at 2 and 5 months; and be verified at 6 months as 
cured or failed therapy. We will compare the national TB program DOTS worker card to our own 
DOTS system to assess congruence between documented adherence. 
 
In addition to this standard of care, for Aim 1 of the research study, the research study DOTS 
workers will observe and document TB medication ingestion 5 days/week and have participants 
self-report weekend compliance. Participants will provide weekly sputum samples during the 
first 12 treatment weeks to measure time to positivity (TTP) to assess rate of sterilization/culture 
conversion as well as smear conversion. We will conduct follow up interviews every month for 
the 6 treatment months in which a brief interviewer-administered questionnaire (alcohol use and 
TB medication side effects) will be done and data on participants’ weight and AFB smear when 
available (2 and 5 months) will be extracted from their medical records. Contingent upon 
available funding, participants may provide blood for repeat biomarker evaluation at 
approximately 2 weeks and at 6 months of treatment to see how transcriptomic profiles (i.e., 
RNA transcripts produced by the host genome) change with treatment and if those changes are 
modified by a history of problem alcohol use. Also contingent upon funding, blood may be drawn 
for repeat PEth testing at month 3 and month 6 of treatment. If full funding is acquired, we will 
perform these additional procedures on all additionally-recruited patients who agree to these 
procedures. If less than full funding is available, we will enroll a convenience sample 
representative of the range of alcohol use and TB disease presentation that we have in our 
project. Participants will also provide a sputum specimen at their 5 month study visit to validate 
smear results from the medical record. If there is a positive side effect screen at any 
appointment, blood will be drawn to test for complete blood count (CBC), alanine transaminase 
(ALT), aspartate transaminase (AST) and creatinine (Cr). These lab results will be given to TB 
Program. For the research study, participants will also be interviewed at 3, 6, 9, 12 months post-
treatment and screened for any re-occurrence of TB symptoms. Participants with a positive TB 
symptom screen will be referred back to the clinic for microbiologic assessment, and we will 
extract the results of these assessments from their clinical records. If participants have a 

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14 
positive TB symptom screen, a positive smear and/or culture from the TB clinic, and/or are 
restarted on treatment, we will collect an additional specimen to allow for comparison of strains 
between the baseline and relapse infections in order to differentiate relapse from reinfection. 
Contingent upon funding, blood will be drawn at 12 months post-treatment to assess for PEth. In 
total, patients will be enrolled in the study for 18 months.  
 
For Aim 2, we will enroll a subgroup of 200 HIV-negative patients from our original cohort to 
participate in a single 8-hour TB drug PK testing during treatment weeks 4-8. We will follow 
standard procedures for collecting, storing and transporting samples, successfully used in our 
previous studies.47-49  
 
For Aim 3, MTB WGS and MICs will be conducted on previously enrolled and newly enrolled 
participants who undergo the same procedures outlined in Aim 1 above.  
 
For Aim 4, we will collect up to 30 mL of blood from participants between 9 months and 18 
months post-treatment. For those who have finished they study but are still within the window of 
18 months post-treatment, we will contact the individuals to see if they are interested in 
participating in this additional research and attend the study clinic for one blood draw of up to 30 
mL. These participants will be consented with a separate consent form specific for these 
purposes. With the blood samples, we will conduct HbA1c, blood glucose and stress and 
inflammatory biomarker analyses. Using a small amount of blood, we will perform epigenetic 
tests to analyze, and with the subject’s separate consent, genes that may be associated with 
the body’s response to tuberculosis (TB) infection, HIV infection and response to treatment 
 
For the qualitative component, we will enroll a random subgroup of up to 48 participants from the 
main study who use alcohol to participate in one of six focus groups lasting up to 1.5 hours each. 
We aim to have up to eight participants per group, and we will oversample for each group as 
previous research has indicated that not all eligible participants will be available on the day of the 
focus groups. In addition, we will also interview up to 12 TB or substance use professionals to 
better understand how they communicate about alcohol use to newly diagnosed TB patients and 
their perceptions on the impact of alcohol and other drugs on patients during TB treatment. 
 

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15 
4 
STUDY POPULATION 
We will recruit patients from the Worcester Community Day Centre (CDC) and Avian Park 
Clinic, two clinics in Worcester, the main town of the Breede Valley municipality in the Western 
Cape Province, South Africa. TB is endemic in this region, with an incidence of 1000 
cases/100,000 population, and the problem alcohol use rate is exceedingly high.4,50 This clinic 
provides free TB treatment services to patients residing within its catchment area, which 
includes the town of Worcester and the surrounding low socioeconomic status (SES) 
communities that are inhabited primarily by “Coloured” people (of mixed race ancestry), a 
population of ~150,000. In 2013, the TB clinic in the Worcester CDC started 664 patients on TB 
treatment, 75% of whom were >15 years (from the Electronic Tuberculosis Record or ETR.net, 
South Africa). 92% of adult TB patients had a documented HIV test and 30% were positive. 
Prior to initiating treatment, patients with suspected TB are screened for active TB disease by a 
symptom-based evaluation and microbiologic assay (Xpert MTB/RIF, AFB smears, and 
mycobacterial cultures). Patients are asked to return to the clinic 2-3 days later to receive 
results and initiate treatment if they test positive for active TB. Study participants will receive 
standard drug susceptible TB therapy from the clinic, which consists of 6 months of antibiotic 
treatment: a 2-month intensive phase of 4 drugs (INH, RIF, EMB, PZA) and then a 4-month 
continuation phase with 2 drugs (INH, RIF). Patients are given a 1-month take home supply of 
medication and return on a monthly basis to the clinic for monitoring and to receive their next 
month’s medication supply until the 6-month treatment period has been completed. Sputum 
samples are collected to document microbiologic cure at the 2- and 5-month treatment visits. In 
our preliminary work, we found that almost all patients attending this clinic speak Afrikaans 
and/or English (as do the clinic staff); as a result assessments will be offered in these 
languages. Patients who are isiXhosa speaking and who are not comfortable in speaking 
Afrikaans or English are generally treated at Empilsweni clinic and therefore would not be 
eligible for this study. In the rare event that a potential study participant speaks neither of these 
languages, they will not be enrolled in the study. Native Afrikaans speakers will translate the 
interviewer-administered questionnaires into Afrikaans using standard forward and back-
translation techniques used in other studies.51 
4.1 
Selection of the Study Population 
The participant population for this study will include all patients being treated for active 
pulmonary TB disease at Worcester CDC or Avian Park Clinic in Worcester, South Africa. 
Enrollment of participants will be completed in 3 years. We expect to recruit 20 patients per 
month and to reach our target sample of 438 participants over a 21-month period. On average, 
this clinic initiates 40 patients >15 years of age on TB treatment per month. Pregnant women 
will be excluded from enrollment and will therefore not be exposed to additional risk of chest x-
rays conducted for this study. Children under the age of 15 will not be enrolled in this study. 
Patients who are HIV positive will be enrolled in this study but will not be included in AIM 2 
because of the potential interaction of antiretroviral medicines with TB treatment drugs. AIM 2 
will include a subset of the total enrollment number. The qualitative component will recruit a 
subset of participants from the main cohort, as well as local TB service providers for individual 
interviews. 
 
During the study period, all patients initiating TB treatment who are identified through pre-
screening to meet the study’s eligibility criteria will be approached by study staff. If the patient is 
interested, the study staff will describe the study to the patient and obtain written informed 
consent. After which the study staff will screen the patient for final eligibility and potential 

 ____________________________________________________________________________________________ 
16 
enrollment. Patients who consent to participate and are enrolled will be asked to complete a 
locator form that will enable us to aid with tracking participants for daily medication monitoring 
and subsequent assessments. Enrollment will occur ideally on the same day as TB treatment 
initiation. Patients who do not meet this study’s clinical eligibility criteria but who are deemed to 
be capable and competent to provide informed consent will be asked about their interest in 
being part of other research studies conducted by the University of Cape Town’s South African 
Tuberculosis Vaccine Initiative’s (SATVI). If the patient expresses interest in participating in 
other studies and provides verbal consent for their contact details to be given to SATVI, they will 
be asked to provide their name and contact telephone number and this information will be 
passed on to SATVI.  SATVI will then make further contact with the patient in order to assess 
whether they are potentially eligible to participate in other studies. We will not consent patients 
on behalf of SATVI but will merely refer them on (with the patient’s verbal consent) for further 
assessment by the SATVI team for possible study eligibility. 
 
A. AIM 1. All participants enrolled in this study will contribute to Aim 1. 438 participants will be 
enrolled over 3 years and followed up through standard, 6-month TB treatment and then every 3 
months for 12 months after completion of treatment to evaluate post-treatment response and to 
perform behavioral assessments. Patients will get intensive follow-up weekly for the first 3 
months of treatment to evaluate treatment response. We anticipate that 30% of eligible 
participants will be HIV positive based on a review of the clinic's electronic health record system. 
All participants will be assessed at baseline and throughout the study for alcohol use using both 
self-report and biologic measurements. We estimate that 30% of all participants will meet 
criteria for problem alcohol use. This will allow for evaluation of association between alcohol 
consumption and TB treatment outcomes. 
 
B. AIM 2. A subset of the total enrolled participants, identified during screening and enrollment, 
will contribute to Aim 2. The first 200 participants who are eligible and agree to participate will 
be enrolled over 2 years. These participants will reflect the overall cohort of patients initiating TB 
treatment but will not include any HIV positive patients to remove potential drug interactions 
between TB drugs and antiretrovirals. These participants will contribute to information regarding 
TB drug PK. They will be asked to provide an 8-hour blood PK series one month into their TB 
treatment regimen. 
 
C. AIM 3. MTB isolates from participants previously and newly enrolled will be further 
interrogated using whole genome sequencing and drug susceptibility testing (MICs). 
 
D. AIM 4. Blood collected between 9 months and 18 months post-treatment from participants 
will be analyzed for HbA1c, glucose, stress and inflammatory biomarkers, and DNA epigenetic 
analyses of genes that may be associated with TB, HIV, and response to treatment. 
 
D. For the qualitative component we will select a subset of the total enrolled cohort and ensure 
that we have an equal representation of male and female participants. Focus groups will be 
separated by gender, and age (15-21 years old) will be in separate focus groups to adult 
participants (aged 21 years and older). After stratifying those who self-report alcohol use in the 
past year by gender and age, we will generate a random sample of 72 participants who will be 
eligible for the focus groups, with the goal of enrolling 48 of these participants into this component. 
These participants will contribute to us having a better understanding if and how participants 
perceive their alcohol use changes when they first become sick, through their course of TB 
treatment, as they start to feel well again, and beyond treatment. We also aim to improve our 
understanding of why participant alcohol use behavior changes, and participant readiness to 
partake in alcohol cessation programs or other alcohol use interventions. For individual interviews, 

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17 
we will select up to twelve service providers including TB nurses who provide care at the CDC 
and other key stakeholders that we have identified through our networks to better understand how 
they message alcohol use to newly diagnosed TB patients and throughout treatment, and their 
perceptions on the impact of alcohol use disorders (AUD) on patients during TB treatment. 
Inclusion of Women: Women will be included in this study. Pregnant women will be excluded 
from enrollment due to the additional risks associated with the diagnostic tests proposed 
(radiograph). We will do pregnancy testing on all women of reproductive age prior to enrollment. 
If a woman becomes pregnant after chest x-ray is performed and notifies study staff, this will be 
documented by the study but the woman will not be excluded from further participation. 
 
Inclusion of Minorities: Participants in this study will be of all races and ethnic groups 
represented by TB patients in Worcester, South Africa.  
 
Inclusion of Children: Children under the age of 15 will not be included in this study because 
drinking is not common under that age. The drinking age in South Africa is 18 but drinking 
practices are known to be common among adolescents under 18. Consent will be obtained from 
a legal guardian for children under the age of 18 and separate consent will be obtained from 
children age 15-17 years of age. We will include ages 15-17 years because this age group 
includes individuals with alcohol problems and adult-type TB disease that would benefit from 
findings of this study. Parents/guardians and adolescents will be informed about the proposed 
project by receiving consent forms that describe what is involved by participating in the study, 
confidentiality protection procedures of the project, the purposes of the project, and the benefits 
and risks associated with participation. To minimise risk of harm for adolescent participants, 
adolescent information on alcohol use will not be shared with their parents/guardian. Each party 
will be informed of this project policy during the consent process. 
4.2 
Inclusion/Exclusion Criteria 
 
 
To be eligible for this study, participants must meet all of the following inclusion criteria.  
Participants must be:  
(1) at least 15 years old  
(2) initiating TB treatment  
(3) expect to remain in the local area for the next 2 years  
(4) agree to comply with all study requirements, including provision of contact 
information and attendance at all study appointments 
(5) provide written, informed consent to participate in the study if ≥18 years of age or 
written individual consent and separate parental consent if <18 years. 
(6) has microbiologic confirmation (Xpert MTB/RIF, AFB smear, or mycobacterial 
culture) of MTB  
 
Participants will be excluded from this study if: 
(1) they have been treated for TB in the last 4 months (defined as cumulative treatment 
duration of one month or greater) 
(2) they have RIF-resistant TB (RIF resistance will be known at screening from Xpert 
MTB/RIF or drug susceptibility testing [DST] from culture) 
(3) they have a) only an extrapulmonary form of TB or b) pulmonary TB plus a treatment-
extending form of extrapulmonary TB 
(4) they have a contraindication to start on standard 4-drug therapy 

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18 
(5) they are pregnant at study enrollment 
(6) they do not have documented HIV status from the previous 6 months and refuse HIV 
testing 
(7) they are currently on treatment for epilepsy 
(8) For Aim 2 only: they are HIV seropositive or have been treated for TB in the past 2 
years 
(9) they do not speak English or Afrikaans 
Participants will be excluded if they have been treated for TB in the last 4 months to avoid false 
positive Xpert MTB/RIF results. We want all participants to be receiving the same standard 6-
month, 4 drug TB regimen, so both RIF-resistant TB and treatment-extending forms of 
extrapulmonary TB cases (i.e., TB meningitis, miliary TB, and skeletal TB) are excluded. Forms 
of extrapulmonary TB that will lead to withdrawal from the study are discussed in Section 7.9. 
Patients with these types of TB will continue to receive the standard of care applicable and 
appropriate to their type of TB.  Additionally, participants with only extrapulmonary TB (i.e., no 
pulmonary form of TB) will be excluded as we will capture sputum time to positivity for 
pulmonary treatment response and do not have the means to track an extrapulmonary 
treatment response. The exception will be patients who have pulmonary TB and also TB 
disease of the pleural lining of the lung (i.e., pleural effusion), which is considered an 
extrapulmonary form of TB, but is regarded as having more similar pathophysiology to 
pulmonary disease than other extrapulmonary forms, therefore, will not be excluded. The 
diagnosis of pleural effusion may be noted by the clinic at time of diagnosis, or may be later 
noted by the study staff when chest X-ray results are received. The local IRB of the SAMRC has 
approved the continued enrollment of patients with pulmonary TB and pleural effusion until this 
amendment can be formally approved by their ethics committee in early 2018. We also exclude 
patients who have other contraindications to start on standard therapy such as those currently 
being treated for epilepsy. Pregnant women are excluded due to minimal risk of performing 
chest X-ray on them. We are excluding candidates who do not have documented HIV status 
from the previous 6 months and are unwilling to take an HIV test, as this is important information 
for Aim 2 of our study. Lastly, for Aim 2 only, we will exclude candidates if they are HIV 
seropositive to avoid potential drug interaction effects between ART and TB medications 
measured during PK studies and exclude participants who have had TB in the past 2 years to 
avoid including participants relapsing from previous TB. 
 
Qualitative Component Inclusion/Exclusion Criteria 
 
For the Focus Groups the participant must have the following inclusion criteria: 
1. Be 15 years or older 
2. Be a current or former participant of the larger study, TRUST 
3. Have self-reported alcohol use on a TRUST interview 
4. And be willing to comply with study procedures, including the audio recording of the 
focus groups 
 
And exclusion criteria: 
 
1. Candidate must not have already participated in a TRUST focus group. 

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19 
 
For the Key Informant Individual Interviews the participant must have the following inclusion 
criteria: 
 
1. Be 18 years or older 
2. Work as a TB or substance use professional 
3. Be willing to comply with study procedures, including the audio recording of the focus 
groups 
 
And exclusion criteria: 
 
1. Candidate must not have already participated in a TRUST Individual Interview 
 

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20 
5 
STUDY PROCEDURES/EVALUATIONS 
5.1 
Study Procedures 
Enrollment and initial assessment procedures: Immediately following study enrollment, 
participants will complete an interviewer-administered questionnaire, a sputum sample will be 
collected to obtain a mycobacterial isolate for AFB smear, MGIT growth, WGS, and MIC 
determination. We will collect an additional baseline sputum sample on participants with positive 
Xpert Ultra test results, which will be used for sequencing of MTB DNA extracted directly from 
sputum. A urine sample will be collected to assess recent drug use, and blood will be drawn to 
test for recent alcohol use using the phosphatidylethanol (PEth) test, assess RNA signature of 
baseline immune function and biomarker predictors of treatment response (contingent upon 
available funding), and to assess baseline health with CBC, HbA1c, AST/ALT, Cr, INR/PTT, and 
albumin. The results from the blood analysis baseline health assessment (except PEth results) 
will be provided to the TB Program. HbA1C will be measured as a baseline indicator of diabetes 
which has low detection rates in health clinics and which due to impact on the immune system, 
affect TB treatment outcomes and is therefore important to document. Participants who report 
substance use will be offered a referral to a local substance abuse treatment service (Toevlug 
Centre for Alcohol & Drug Dependence) and/or the social worker associated with the TB clinic. 
Patients who report or test positive for serious depression will also be referred to the social 
worker associated with the TB clinic. If not already done by the TB program, a chest radiograph 
will be obtained on each participant at Worcester Mediclinic, which will be saved in an electronic 
version together with interpretation. If already done by the TB program, the radiograph 
interpretation will be recorded. The enrollment process and initial biobehavioral assessment 
should take no longer than two hours, plus an additional thirty minutes for a chest radiograph. 
These activities will take place in a private room within the clinic. Following this assessment, 
participants will continue with their TB treatment. Study staff will also extract data on the 
participant’s TB, HIV and physical health status, including known co-morbidities and active 
medications, from their clinic records. 
 
Behavioral assessments.  
Socio-demographic data will be collected on age, gender, race, residence, highest level of 
education completed, employment status, living situation, and current marital status. 
Alcohol use: The Alcohol Timeline Follow Back (TLFB) technique will be used to assess recent 
drinking behavior.52 The patient is asked to recall his/her daily alcohol consumption over a time 
period ranging from 7 days to 24 months prior to the interview. TLFB data can be used to 
assess frequency, quantity, volume, patterns and types of alcohol consumption over the 
specified time period. Studies have shown strong correlations between self-reported information 
on the TLFB and collateral reports and also excellent test-retest reliability for this measure.53 We 
will also use the 12-item Alcohol Use Disorders Identification Test (AUDIT) to assess hazardous 
and harmful patterns of alcohol use.54,55 We will inquire about age of initiation of alcohol use, 
and duration of regular and heavy alcohol consumption to assess the chronicity of alcohol use.  
Tobacco use: The Fagerström Test for Nicotine Dependence (FTND), a 6-item scale, will be 
administered to assess current tobacco use and dependence.56 Good correlations between 
FTND score and salivary cotinine have been found.57  
Illicit drug use: We will inquire about lifetime use of any illicit drugs, types of illicit drugs used in 
the past month, age of initiation, number of days each illicit drug was used in the month and 
mode of use. In addition, the 11-item Drug Use Disorders Identification Test (DUDIT) will be 
administered to assess for harmful patterns of illicit drug use and dependence.58,59 

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21 
Depression: The Center for Epidemiologic Studies Depression Scale (CES-D), a 20-item scale 
will be administered to measure depressive feelings and behaviors during the past week.60 
Food security: The Household Hunger Scale, a 3-item instrument, will be used to assess food 
deprivation and security. Internal, external and cross-cultural validity of this scale has been 
demonstrated in South Africa.61  
TB side effect screen: We will collect data on adverse reactions to first line drug regimens. 
Patients will be asked whether they have experienced any of the following since the last study 
visit: fevers, sweats, cough, rash, itching, phototoxicity, jaundice, nausea, vomiting, diarrhea, 
abdominal pain, loss of appetite, numbness/tingling of extremities, headache, dizziness, 
insomnia, joint pain, musculo-skeletal pain, or new infections.  In addition, patients will be asked 
whether they have had other symptoms that are not listed above. 
 
Mobile phone technology for monitoring of adherence to TB treatment. In South Africa, 
directly observed therapy, short course (DOTS) was adopted in 1995 as standard of care to 
ensure adherence to TB treatment; however the DOTS program has been inconsistently 
implemented and TB control remains poor.62,63 In discussions with PI Jacobson, the Worcester 
CDC staff expressed concern about their DOTS program. They lack clinic staff to conduct 
DOTS and often rely on family members to observe treatment. They have noticed 
inconsistencies between medication counts (returned pills) and DOTS cards. They do not 
receive information on DOTS until patients return for their monthly clinic appointments, which is 
too late to put strategies in place to reduce non-adherence. To provide accurate and complete 
recording of adherence, we will employ our own DOTS workers to conduct treatment 
monitoring, and we will use mobile phone technology to permit centralized daily review of each 
participant’s adherence to treatment. A GIS coordinate will be collected each time a DOTS 
worker logs a visit through the mobile phone app, allowing the study team to monitor their 
performance. If a participant is found to be adhering poorly, the TB Program will be informed. 
 
Additional assessments. Contingent upon available funding, a PEth test may be repeated at 3 
months of treatment, 6 months of treatment (i.e., end of treatment), and at 12 months post-
treatment follow-up to validate the bio-behavioral assessments and assess changes in alcohol 
consumption throughout treatment. At the end of each of the 6 active treatment months, 
participants will complete an additional bio-behavioral assessment in a private room in the clinic. 
These assessments, which will be timed to coincide with the patient’s regularly scheduled visit, 
will provide information about alcohol use since the last appointment and screen for TB 
treatment side effects. If there is a positive side effect screen at any assessment, we will check 
serum liver function (AST/ALT), creatinine, and CBC. If any results are outside a normal range 
or a patient has a significant new physical complaint, this information will be provided to the TB 
Program to be used at their discretion. In addition, we will extract data on weight and sputum 
smear results from participants’ clinical records to assess for TB treatment response. During the 
active TB treatment period, if a participant reports that he/she had an unscheduled visit to a 
health care provider for a reason other than routine health care, or if the study team finds out 
otherwise, a study staff member will go to the clinic or hospital the participant visited to extract 
basic information on this unscheduled visit from the medical record.  
 
End of treatment will be determined by the clinic, after which the post-treatment period will 
commence. For participants unenrolled from our study before treatment completion, we will look 
within their medical record after their estimated treatment completion date to abstract the clinic-
assigned treatment outcome (if available). This is important for the study with respect to 
secondary study question of alcohol use and risk of poor final TB treatment outcome (e.g., loss 
to follow-up, failure, death) (Section 11.1). This will be the only information we collect on 
participants after their withdrawal from the study. For all prospectively-enrolled participants, we 

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22 
will explicitly state this possibility during informed consent. Current participants will be re-
consented on the updated ICF.   
 
After the end of the treatment phase, participants will be contacted for interviews at 3, 6, 9, and 
12 months post-treatment, during which they will be screened for any re-occurrence of TB 
symptoms and for alcohol use. At the 6 and 12 month post-treatment visits, the AUDIT, DUDIT, 
Fagerstrom Test, Household Hunger Scale, and CES-D will be reassessed. Participants with a 
positive TB symptom screen will be referred back to the clinic for microbiologic assessment, and 
we will extract the results of these assessments from their clinical records. If a participant has a 
positive TB symptom screen, a positive sputum/and or culture from the TB clinic, and/or is 
started on treatment again during the post-treatment phase, we will collect an additional sputum 
specimen to compare the strain against that of the baseline specimen. If it is not possible to 
collect a sputum sample in time from the patient (within one week of treatment re-initiation) we 
will collect their clinic sample to compare the strain against that of the baseline specimen. We 
will collect additional information on the participant’s relapse/reinfection, including the type of 
test conducted to confirm TB and test results, drug susceptibility, cycle threshold, and outcome 
of the TB episode, from the participant medical record and/or the South Africa National Health 
Laboratory Services (NHLS) or Western Cape Data Bank. We will retrospectively collect 
information on the diagnosis, test results, and outcome from the medical record, NHLS, and/or 
Western Cape Data Bank on any COVID-19 diagnoses our participants experience during their 
18 months in the study. A subset of participants may be asked to provide a second chest 
radiograph during the treatment or post-treatment phase to evaluate for changes in the lung 
from baseline. We will collect up to 30 mL between 9 months and 18 months post-treatment for 
HbA1c, blood glucose, stress and inflammatory biomarker analyses and epigenetic analyses of 
genes associated with response to TB, HIV, and treatment. 
 
Qualitative Research Procedures. We will conduct six FGs among current participants from 
the parent cohort.  We will conduct up to 3 focus groups each with women and men who self-
report alcohol use, with one of each of these focus groups consisting of adolescent male and 
female participants. FGs will contain between 6-8 participants each. Focus groups offer a 
comprehensive, yet time- and cost-efficient way to gather information, and the format provides 
participants with a “permissive environment” allowing for the expression of a wide range of 
experiences and opinions. Participants’ comments are used to determine the flow of the 
discussion so that the ideas most important to the participants may emerge, particularly those 
unanticipated by the investigators. Focus groups will last up to an hour and a half and will be 
audio-recorded with written consent from the participants. These groups will be facilitated by a 
researcher who is trained in qualitative research methods. 
 
In order to obtain the service provider perspective, we will also conduct 6-12 individual interviews 
with TB nurses who provide care at the CDC and other stakeholders that provide TB or substance 
use services in the area. While we anticipate that this number will be sufficient for us to achieve 
thematic “saturation,” if we find we have not reached saturation, we will continue sampling until 
we have confidence that the sample is adequate for examining key topics of interest. Recordings 
of the interviews and focus groups will be professionally transcribed verbatim and translated by a 
professional transcription company used frequently by the SAMRC following a structured protocol 
including replacing names/identifiers with pseudonyms as needed. In line with current ethical 
standards, recordings will be destroyed following transcription validation.  

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23 
5.2 
Laboratory Evaluations 
5.2.1 Laboratory Evaluations/Assays 
 
Determination of bacillary kill rates and MIC. Following standard procedures, spontaneous 
sputum specimens will be collected weekly from 1-12 weeks of treatment. Specimens will be 
transported once per week by a designated study member or accredited courier to co-I Warren’s 
laboratory at the SAMRC. There they will be processed on liquid medium using BACTEC MGIT 
960 system (Becton Dickinson, Sparks, MD). Time to positivity (TTP) will be recorded in days. 
These data will be used to build a time-to-event model to describe treatment response in these 
patients. On baseline, pre-treatment isolates, INH, RIF, and EMB MICs will be determined using 
the microdilution plate method. MIC for PZA will be determined using the BACTEC MGIT 960 
PZA kit (Becton Dickinson Biosciences, Sparks) for patients co- enrolled in Aim 2; PZA DST will 
be performed on patients participating only in Aim 1. INH MICs will be completed while patients 
are still receiving therapy and any evidence of resistance will be transmitted to the clinical care 
team to decide if therapy will be adjusted based on their standard procedures. MICs and DST 
for the other 3 drugs will be batched and performed in years 1, 3, 4. Upon approval of Aim 3, 
baseline isolates will undergo expanded MIC testing for 13 drugs (INH, RIF, rifabutin, 
ethambutol (EMB), amikacin, kanamycin, levofloxacin, moxifloxacin, ethionamide, linezolid, 
delaminid, bedaquiline and clofazimine) using the CRyPTIC microdilution plate or a similar 
method that tests >3 (and up to 7) concentrations below the clinical critical concentration (CC) 
across the 13 drugs. This will be led by Prof Robin Warren and Dr. Maha Farhat at Harvard 
University, who has been added as a PI for this component of the study. Additional genetic 
sequencing of bacterial DNA will be done on the weekly isolates to detect mutations associated 
with resistance to the first-line TB drugs. DNA will be isolated at Warren’s lab and testing will be 
done at Stefan Niemann Laboratory at Institute Borstel, Germany and Mount Sinai Lab in New 
York City. All microbiologic data will be entered into a secure database from Warren’s laboratory 
to ensure immediate transmission to BMC. If a participant is identified as having relapsed within 
the one year post-treatment follow-up period, an additional sputum specimen will be collected to 
compare the strain of the infection to the baseline specimen.  
 
Biologic measurements. Sputum AFB smear will be recorded from clinical charts as well as 
verified in Warren’s lab in standard fashion (negative, scanty, +, ++, +++) and mycobacterial 
culture from MGIT will be recorded as TTP (days). These measures will be used to estimate 
mycobacterial burden of TB disease at baseline and response to treatment. MICs for INH, RIF, 
EMB, and PZA will be performed to determine MTB drug susceptibility. An established scoring 
system will be used to quantify cavitary and non-cavitary disease burden from CXR at baseline 
as well.64 Xpert MTB/RIF, AFB smear, and/or mycobacterial culture results will be recorded from 
the clinical chart documenting presence of TB and whether the rpoB gene is positive or negative 
for RIF resistance mutations or if there is evidence of RIF resistance via DST. Patients with RIF 
resistance will be excluded from this study but the number of patients with RIF resistance will be 
recorded to estimate incidence of resistance in this population. Biologic measures to capture 
end organ disease, including bone marrow suppression via white blood cell count, hemoglobin, 
platelets; liver function via ALT, AST, INR/PTT; renal function via Cr, as well as diabetes via 
HbA1c and blood glucose, will be recorded as continuous variables and dichotomized as normal 
or abnormal using standard cut-offs. Results will be shared with TB Program’s clinical staff to be 
used at their discretion. BMI and albumin will be used with standard cut-offs to evaluate macro-
nutritional status. Drug testing will be conducted at the baseline visit using urine dipstick tests 
and will provide indication of recent use of cannabis, methamphetamine, cocaine, opiates, 
amphetamines, and methaqualone (Mandrax). PEth testing will be conducted using dried blood 

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24 
spots (DBS) from venous blood. The testing will be performed at the United States Drug Testing 
Laboratories in Des Plaines, IL. The PEth tests will be done in batches throughout the study. 
PEth will be interpreted in a standard fashion using the 16:0/18.1 homologues.65 Drug 
concentrations will be measured in McIlleron’s laboratory by validated liquid chromatography-
tandem mass spectrometry (LC-MS/MS) methods. If a patient is found to have TB disease 
again, spoligotyping and targeted sequencing will be performed on baseline and repeat Mtb 
specimen to assess for evidence of relapse versus reinfection. We will also perform drug 
susceptibility tests (as outlined below) on the repeat specimen to assess for evidence of 
resistance development. With the post-treatment 9 or 12 month visit for currently and newly 
enrolled participants or 12-18 month post-treatment visit for previously enrolled participants, we 
will measure markers of stress (i.e. cortisol) and inflammation (i.e. IL6, TNF, CRP, adiponectin 
and insulin). 
 
Epigenetic Analyses. DNA will be extracted from cells stored from EDTA blood samples. 
Epigenetic modifications will be assessed at DNA sites known to be involved in TB risk and 
neutrophil, monocyte, T and B cell function, using a variety of techniques, including but not 
limited to, investigating differential methylation patterns between the groups, by bisulfite 
converting DNA for analysis on DNA methylation arrays available from Illumina Inc, as well as 
nanopore sequencing of native DNA to assess methylation and acetylation patterns. 
5.2.2 Special Assays or Procedures 
 
PK phlebotomy and determination. After 4 weeks of therapy (but no more than 8 weeks, or as 
long as the participant is still in the intensive phase of treatment) participants enrolled for AIM 2 
will be scheduled for an intensive phlebotomy day for PK sampling. Participants will be 
transported by research staff to Brewelskloof Hospital in Worcester. During that time the patient 
will have access to magazines, television, food, and water. One of the study nurses will manage 
and perform the procedures. Participants will be asked to arrive without eating and to not have 
taken their medication that morning; their TB drugs will be administered in person ideally under 
fasting conditions under observation. If a participant inadvertently eats or drinks the morning of 
PK, the nurse will ensure two hours elapse between time of last ingestion and when the first 
sample is taken and dose administered. The dosage times from the day before will be recorded 
from the study DOTS program. A breathalyzer test will be administered to record blood alcohol 
content that morning. Blood samples will be drawn by the study nurse immediately before the 
dose and then 1.5, 3, 5, and 8 hours after drug ingestion. Samples will be centrifuged and at 
least 1.2 ml of plasma will be stored in a -80° freezer on site within 30 minutes of collection. 
Samples will then be batch couriered on dry ice to the McIlleron’s laboratory at UCT where 
plasma concentrations of INH, RIF, PZA, and EMB will be measured. Individual patient steady 
state 24-hour area under curve (AUC) and peak concentration (Cmax) will be identified from PK 
models implemented in NONMEM (Ellicott City, Maryland). 
 
Assessment of biomarkers. Contingent upon available funding, participants may have blood 
drawn for transcriptomic and biomarker analysis during the baseline visit, at 2 weeks of 
treatment, and at 6 months of treatment (i.e., treatment completion). At each of these time 
points, approximately 3 ml of blood will be collected into Paxgene or Tempus tubes. They will be 
stored interim at room temperature for a maximum of five days before being moved to the 
Warren’s laboratory at Stellenbosch University to be stored at -80 C. Further analysis on these 
samples will be planned and performed based on funding availability. 
 

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25 
Whole Genome Sequencing (WGS). Whole genome sequencing of TB bacteria DNA will be 
conducted on 1-3 isolates collected from previously or newly enrolled participants. We plan to 
sequence the baseline and 8 week sample (or if culture negative by 8 weeks the last positive 
culture available ≥2weeks from treatment initiation). A third may also be sequenced if the 
participant continues to be culture positive ≥ 5 months or relapses after treatment completion. 
We will also select and re-culture 15 pairs of technical control archived isolates to help 
determine noise related to archiving and/or re-growth in vitro. No colony selection will be 
performed; isolates will be grown, or regrown, from their frozen/archived state, by a technologist 
in Prof. Warren’s laboratory using standard procedures in MGIT liquid culture. After culture, bulk 
aliquots will be lysed using lysozyme and proteinase K and DNA extracted using the 
CTAB/Chloroform method. Extracts will be shipped to a US and German lab identified using 
Genohub.com for library preparation (NexteraXT or latest) and sequencing HiSeq X or similar 
(Illumina, San Diego, CA), generating 150bp paired-end reads with target depth of 250x 
consistently across patient isolates. PacBio long-read sequencing: We will select 95 paired 
isolate populations and 110 additional baseline samples. In total we plan to perform PacBio 
sequencing on 300 samples. Following HMW DNA extraction, library preparation and SMRT 
sequencing will be performed according to manufacturer’s specifications at Mount Sinai 
Genomic Center with which we have a standing collaboration. Direct-from-sputum 
sequencing: For 150 newly enrolled participants who are Xpert Ultra positive, we will collect an 
additional baseline first morning sputum sample, 96 of which will be used for direct sequencing 
if DNA enrichment is successful. The sputum sample will be transported to Prof. Warren’s 
laboratory for processing where it will first be treated to deplete human/non-Mtb cells prior to 
DNA isolation. We will compare NaOH/NALC incubation and centrifugation (standard 
processing prior to Mtb culture) vs. NALC alone to liquefy, followed by separation of cellular 
material by size using a cheap (<1$) microfluidic unit to minimize loss of Mtb cells. 
Subsequently the sputum sample will be treated with MolYsis Basic kit (Molzym, Germany) for 
additional removal of human DNA. Homogenation and cell lysis of the remaining material with 
be performed with glass beads, lysozyme and proteinase K and DNA extracted using the CTAB 
/ Chloroform method. Mtb DNA capture and enrichment will be performed using Twist 
Biosciences baits that will be custom designed to capture prevalent Mtb lineages in Western 
Cape South Africa. We will follow manufacturer protocols for DNA capture using these probe. 
This will be followed by manufacturer recommended amplification (that will be standardized 
across samples to allow for bacterial burden comparisons). Negative controls will be run with 
each batch of experiments to confirm the lack of contamination. Samples that have sufficient 
DNA by Qubit or equivalent will undergo Illumina library preparation. DNA libraries will be frozen 
and transported to a Genohub.com designated lab for Illumina sequencing. Bioinformatic 
sequence analysis: Reads from each isolate population will be quality controlled to confirm 
mapping to Mtb complex using Kraken, Illumina reads will be trimmed and both Illumina and 
PacBio reads will be aligned to our graph reference genome using VG or MiniGraph. PacBio 
reads will be separately de-novo assembled (reference free) using Flye and the assembly 
polished with Illumina data using Pilon to build the within population reference. Fixed and micro-
variants will be identified using Pilon, Quiver and/or Minimap2. Expecting a coverage of 250x 
Illumina reads, we anticipate the ability to call variants down to 2% abundance in the source 
population (requiring ≥5 reads with base quality phred score >30 with expected false positive 
error of <10-15). For populations with both PacBio and Illumina data we will be able to detect 
variants at 0.5% or lower abundance. We will exclude regions of the genome not sufficiently not 
sufficiently covered with high quality reads across a specific pair and across participants. Our 
graph genome approach will enable calling not only SNSs but also insertions/deletion and more 
complex genomic variants changes especially for isolates with long read data. 
 
Specimen Shipping 

 ____________________________________________________________________________________________ 
26 
All specimens will be coded and have no personal identifiers.  
• 
Sputum specimens will be shipped to Rob Warren’s laboratory at Stellenbosch 
University, Tygerberg, South Africa 
• 
Bacterial DNA from sputum specimens will be shipped to the Stefan Niemann 
Laboratory at Institute Borstel, Germany and Mount Sinai Lab in New York City 
upon the execution of the appropriate MTAs and obtainment of a South African 
Department of Health Export Permit. 
• 
Blood specimens for PK testing will be shipped to Helen McIlleron’s laboratory at 
the University of Cape Town, Cape Town, South Africa 
• 
Blood specimens for stress and biomarker inflammatory analyses and epigenetic 
analyses will be transported to Anna Coussens’ Lab at the University of Cape 
Town, Cape Town South Africa. 
• 
Blood specimens for transcriptomic and biomarker analysis as well as SARS-
CoV-2 serology will be shipped to Rob Warren’s laboratory at Stellenbosch 
University, Tygerberg, South Africa.  
• 
Upon signing an MTA (Material Transfer Agreement) between the providers 
(SAMRC and BMC) and receiver (Ragon Institute) and obtaining the appropriate 
export permits, blood samples stored in PAXgene tubes will then be shipped to 
Dr. Amy Barzack at the Ragon Institute of MGH, MIT, and Harvard in Boston, 
MA. 
• 
Serum stored for SARS-CoV-2 serology will be stored at Rob Warren’s Lab and 
will be analyzed pending future funding. 
• 
Dried blood spots will be shipped to the United States Drug Testing Laboratories 
in Des Plaines, IL, USA for PEth testing 
• 
Blood specimens for clinical tests (CBC, liver function tests, creatinine, albumin, 
HbA1C, blood glucose) will be shipped to and performed by a certified local 
clinical laboratory near Worcester, South Africa 

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27 
6 
STUDY SCHEDULE 
6.1 
Pre-Screening 
We will review the medical records of patients after they have been evaluated for TB infection 
but before they return for potential TB treatment initiation in order to assess their eligibility for 
study inclusion based on clearly defined pre-screening criteria. To the degree possible, we will 
assess for the following criteria: 
• 
Age 
• 
Microbiologic confirmation of TB (e.g., positive Xpert MTB/RIF, AFB smear, 
mycobacterial culture) Location of TB disease (pulmonary vs extrapulmonary) 
• 
History of treatment for active TB in the previous 4 months 
• 
Pregnancy 
• 
HIV status 
• 
Epilepsy history  
 
Pre-screening data beginning from May 2017 will be retained in a de-identified manner to help 
describe the demographics of TB suspects at the CDC clinic.  
 
For the Qualitative Component Key Informant Individual Interviews, we will ask 3 pre-
screening questions before consent: 
1. Are you older than 18? 
2. Do you work as a TB or substance use professional 
3. Have you participated in a TRUST individual interview before 
 
If the participant is eligible they will be explained the study in full detail and if interested, 
consented prior to the investigators asking any additional demographic information and before 
conducting the focus groups. 
6.2 
Screening 
We will approach the potentially eligible participants identified through pre-screening during their 
Treatment Initiation visit with the TB program. During screening we will: 
• 
Approach potential participants, provide information about the study, answer any potential 
questions 
• 
Obtain and document consent for study participation. 
• 
Review of medical records and brief assessment of medical history to confirm eligibility 
based on inclusion/exclusion criteria. This will include abstracting from their medical 
records HIV status, AFB smear, mycobacterial culture and Xpert MTB/RIF results, 
concomitant medications, and pregnancy status. 
• 
Perform a urine pregnancy test (if patient is a woman of child-bearing potential)  
• 
Eligible participants will complete locator form. 

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28 
6.3 
Enrollment/Baseline Visit (Treatment initiation visit) 
• 
Interviewer-administered questionnaire to collect data about alcohol, tobacco, and illicit 
drug use, as well as participant demographics, depression, and food security 
• 
Review of medical records to obtain medical history, HIV status, history of TB disease 
and treatment, physical health, and any other medical information considered relevant.  
• 
Chest X-ray (if results are not available from the local TB program), 
• 
Collection of a blood sample (total volume up to 30 mL) for baseline health screening, 
PEth testing, and, contingent upon funding, transcriptomic and biomarker analysis, and 
SARS-CoV-2 serology. 
• 
Collection of urine sample for drug testing. 
• 
Collection of expectorated sputum sample for culture and if positive on Xpert Ultra, a 
second sample for DNA extraction from whole sputum. 
6.4 
Weekly Visits (Treatment Weeks 1-12) 
• 
Collection of expectorated sputum samples weekly to measure AFB smear status, time 
to positivity (TTP), and to assess rate of sterilization/culture and smear conversion. 
These will be collected by the DOTS workers at the patient home or other mutually 
decided location on weeks the patient is not attending clinic. On clinic visit weeks, these 
samples will be collected at the monthly clinic visit. 
• 
Collection of approximately 3ml of blood at week 2 for transcriptomic and biomarker 
analysis (contingent upon funding) 
6.5 
Daily Assessments (Treatment Months 1-6) 
• 
DOTS worker will go to the patient’s home or other mutually decided location to 
document TB drug ingestion 5 days/week (Monday to Friday) and obtain self-report 
information about weekend medication compliance 
• 
DOTS worker will transmit this information (witnessed medication ingestion yes/no; if no, 
reason did not occur) via a mobile app to a secure server.  
6.6 
Single PK/PD Visit (In Treatment Week 4 – Treatment Week 8 
window) 
• 
200 HIV-negative participants will participate in a single 8-hour TB drug PK testing 
• 
Will be performed at Brewelskloof Hospital in Worcester; participants will be transported 
by study car if needed 
• 
Five blood draws of up to 5 ml per draw during the 8 hour visit (baseline, 1.5, 3, 5, and 8 
hours) 

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29 
6.7 
Monthly Follow-Up Visits (Treatment Month 1-6) 
• 
Brief interviewer-administered questionnaire to collect data about alcohol use and TB 
drug side effects 
• 
At month 6, interview-administered questionnaire to collect data about alcohol, tobacco, 
and illicit drug use, as well as participant demographics, depression, and food security 
• 
Medical record review for participant’s weight and AFB smear results  
• 
At month 5, collection of expectorated sputum sample to confirm AFB smear result 
documented by program. If the clinic’s sputum culture is positive, the PI may request to 
obtain the clinic’s microbial culture to determine whether the bacterial strain is the same 
or different from the baseline strain. This is so we have two samples to analyze in case 
the month 5 sputum we collect does not grow or is contaminated. If the TRUST program 
culture is positive, we may request that the participant provide an extra sample at their 
month 6 visit. 
• 
Screen for TB drug side effects 
• 
Contingent upon funding, collection of a blood sample (up to 5mL at each time point) to 
assess PEth for recent alcohol consumption at month 3 and month 6  
• 
If positive on side effect screening, collection a blood sample (up to 10 mL) to test for 
CBC, ALT, AST, and Cr and refer back to clinic for additional assessment 
• 
Contingent upon funding, collection of a blood sample (approximately 3mL) for 
transcriptomic and biomarker analysis at month 6 
• 
We may ask for a 2nd follow up chest radiograph from a subset of non-pregnant 
participants to evaluate for persistent effects of TB on the lungs. 
6.8 
Post-Treatment Follow-Up (Post-treatment Months 3, 6, 9, 12) 
• 
Brief interviewer-administered questionnaire to collect data about alcohol use (months 3 
and 9) 
• 
Interviewer-administered questionnaire to collect data about alcohol, tobacco, and illicit 
drug use, as well as participant demographics, depression, and food security (months 6 
and 12) 
• 
TB symptom screen 
• 
If positive TB symptom screen, participants will be referred back to clinic for TB 
assessment, and results of the assessment will be extracted from the medical record 
• 
If the participant has a positive TB symptom screen, a positive sputum culture and/or 
smear from the clinic, and/or is initiated on treatment again (i.e., considered to have 
developed TB again), collection of expectorated sputum specimen to compare strain of 
infection to baseline specimen. We will also collect information on the relapse/reinfection 
episode from the medical record, NHLS, and/or Western Cape Data Bank. 
• 
Contingent upon funding, collection of a blood sample (up to 5mL) to assess PEth for 
recent alcohol consumption at month 12. 
• 
We may ask for an additional blood sample (up to 5 mL) to evaluate for potential RNA 
signatures that may indicate likelihood of TB/relapse reinfection 

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30 
• 
We will retrospectively collect information on any COVID-19 diagnoses during the 
participant’s time in the study including laboratory tests, treatment plan, and outcome 
from the medical record, NHLS, and/or the University of Cape Town Provincial Health 
Data Centre. 
6.9 
We will collect up to 30 mL of blood between the post treatment 
9-month visit and the participant being 18 months post 
treatment for HbA1c, blood glucose, stress and inflammatory 
marker analyses, and epigenetic analyses. Criteria for 
Discontinuation or Withdrawal of a Participant (or a Cohort), if 
applicable 
 
• 
Clinical reasons believed to be life-threatening by the physician or the investigators 
• 
Request by the participant to withdraw from study 
• 
Participant judged by the investigator to be at significant risk of failing to comply with the 
provisions of the protocol as to cause harm to self or seriously interfere with the validity 
of the study results 
• 
Participant moves and/or is transferred out to another clinic 
• 
Participant shows bacteriologic evidence of treatment failure on month 5 sputum 
specimen 
• 
Participant’s treatment regimen duration is extended beyond the standard 6 months 
• 
While on treatment, participant with pulmonary TB is also diagnosed with an 
extrapulmonary form of TB that leads to an extension of treatment duration, including TB 
meningitis, miliary TB, and skeletal TB. 
• 
Participant is lost to follow-up for 2 months or longer 
• 
Participant’s TB disease relapses/recurs in the 12 month post-treatment follow-up period 
and is re-initiated on TB treatment 
• 
At the discretion of the sponsor, IRB/REC or Office for Human Research Protections, 
investigators, or other government agencies as part of their duties to ensure that 
research participants are protected 
 
 

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31 
7 
ASSESSMENT OF OUTCOME MEASURES  
7.1 
Specification of the Appropriate Outcome Measures 
7.1.1 Primary Outcome Measures 
 
Aim 1: We will calculate the time to culture conversion, in patients with problem alcohol use 
compared to those without, at 12 weeks. This outcome has been shown to correlate well with 
poor treatment outcomes and to have detectable differences in alcohol-using populations.7,66,67  
 
Aim 2: We will calculate the rate of decline in slowly dividing MTB subpopulations (β-slope or 
sterilizing activity) in the PD model. We will also investigate the effect of problem alcohol use on 
the PK measures Cmax and area under the curve (AUC) and the effect on sterilizing activity. 
 
Aim 3:  
(A) We expect to see a clear signal of selective sweeps on candidate tolerance and resistance 
genes in host. By extrapolation from other pathogenic bacteria we expect selective forces on 
Mtb immune-regulator genes to generate more complex structural variation, possibly in a 
pattern of in host diversification. Immune selective forces are expected to be attenuated in HIV 
co-infected patients and potentially also in patients with problem alcohol use. 
 
(B) It is possible that retreatment patients will have different rates of pathogen diversity or a 
higher rate of mixed strain infections. If so, this will be an important finding and we will 
accordingly stratify the analysis by retreatment status. Our study is the first to propose a 
thorough examination of in host pathogen diversity as a predictor of treatment response. Based 
on preliminary data, we suspect participants with viable and resilient pathogen populations to 
have a higher degree of genetic diversity at baseline or manifest persistent diversity or selective 
sweeps of resistance variants overtime. We expect these same participants to be at much 
higher risk for delayed culture conversion and negative treatment outcomes 
 
(C) The primary outcome of this aim is generating an in silico MIC model with error metrics for 
predicting in vitro MIC and binary DST, and hazard ratios for association with delays in culture 
conversion. This model can be used to predict MICs from WGS for new observations and will 
capture a large number of genetic variants and the effect of hetero-resistance. 
 
Aim 4: A) Using HbA1c and blood glucose we will assess differences in rates of dysglycemia in 
PLWH and HIV-uninfected persons who have completed treatment for TB disease and assess 
how alcohol consumption modifies the relationship. B) We will assess if there are differences in 
stress and inflammation biological markers post TB treatment amongst PLWH and HIV-
uninfected groups and assess how alcohol use modifies the relationship. Epigenetic 
modifications will be assessed at DNA sites known to be involved in TB risk and neutrophil, 
monocyte, T and B cell function 
 
7.1.2 Secondary Outcome Measures 
 
We will calculate the risk of poor final TB outcomes, defined as treatment failure, death, and 
relapse. We will report on barriers to adherence and individual patient level challenges based on 

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32 
daily reports from DOTS workers. We will determine whether any individuals who report alcohol 
abstinence have positive PEth biomarkers and re-classify these patients in the model. We will 
estimate the percentage of patients who develop side effects to the TB drugs and if there are 
significantly more side effects in patients who are acutely ingesting alcohol or who have a longer 
history of problem alcohol use relative to patients who do not drink. Contingent upon funding, 
we will perform repeat biomarker evaluation to see how transcriptomic profiles change with 
treatment and if those changes are modified by a history of problem alcohol use. 

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33 
8 
SAFETY ASSESSMENT AND REPORTING 
ICH E6 and South African GCP guidelines define an Adverse Event (AE) as any untoward medical 
event occurring in participant during his or her participation in the study irrespective of any causal 
relationship to any study procedure.  An AE can therefore be any unfavorable and unintended 
sign (including abnormal laboratory finding), symptom or disease temporally associated with the 
administration of a study procedure (i.e., phlebotomy), whether or not related to the study 
procedure.  
This study is largely observational, and is very low risk to participants. The only study procedures 
than can result in medical or psychological harm to participants are the phlebotomy, exposure to 
a single chest X-ray, and the potential risk for breach of confidentiality.  
The occurrence of an AE may come to the attention of study personnel during study visits and 
interviews or by a recipient presenting for medical care.  Any medical condition that is present at 
the time that the patient is screened should be considered as baseline and not reported as an AE.  
8.1 
Definition of Adverse Event (AE) 
 
Adverse events will be monitored for each participant participating in the studies and attributed 
to the study intervention by the PIs according to the following categories: 
 
a) Definite: Adverse event is clearly related to the intervention. 
b) Probable: Adverse event is likely related to the intervention. 
c) Possible: Adverse event may be related to the intervention. 
d) Unlikely: Adverse event is likely not to be related to the intervention. 
e) Unrelated: Adverse event is clearly not related to the intervention. 
 
The following scale will be used in grading the severity of adverse events noted during the 
study: 
 
1. Mild adverse event 
2. Moderate adverse event 
3. Severe adverse event 
8.2 
Definition of Serious Adverse Event (SAE) 
 
In addition to grading the adverse event, the PI will determine whether the adverse event meets 
the criteria for a Serious Adverse Event (SAE). An adverse event is considered serious if it: 
 
1. is life-threatening 
2. results in in-patient hospitalization or prolongation of existing hospitalization 
3. results in persistent or significant disability or incapacity 
4. results in a congenital anomaly or birth defect 
5. results in death 
6. based upon appropriate medical judgment, may jeopardize the participant’s health and may 
require medical or surgical intervention to prevent one of the other outcomes listed in this 
definition, or 
7. adversely affects the risk/benefit ratio of the study 

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34 
 
An adverse event may be graded as severe but still not meet the criteria for a Serious Adverse 
Event. Similarly, an adverse event may be graded as moderate but still meet the criteria for a 
Serious Adverse Event. The PI and mentorship team will determine the grade of the event as 
well as its “seriousness.” 
8.3 
Reporting Procedures 
 
In the unlikely event that such events occur, serious and unanticipated and related adverse 
events or unanticipated problems involving risks to participants or others will be reported in 
writing within 48 hours to the respective site IRB/REC, the DMID Medical Officer and the DMID 
Program Officer. The PI will apprise the investigative team and study personnel of all adverse 
events that occur during the conduct of this research project through regular study meetings and 
via email as they are reviewed by the principal investigator. The protocol’s Program Officer and 
the appropriate IRB/RECs will be informed of moderate or greater in severity adverse events 
within three days of the event becoming known to the PI. 
8.3.1 Serious Adverse Event Detection and Reporting 
 
Serious adverse experiences (as defined in section 9.2 above) associated with the 
conduct of the study will be reported to the Ethics Committees in Boston and South 
Africa and the NIH (DMID Program Officer and DMID Medical Officer) in accordance 
with DHHS and international guidelines within three days of site awareness of their 
occurrence. Due to the laboratory/epidemiologic focus of the study, few serious adverse 
events related to study procedures are anticipated in this study. 
8.3.2 Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings 
We will report all laboratory results for routine blood tests (e.g., AST/ALT, Cr, albumin, 
complete blood count, HbA1c, INR/PTT) and INH MIC results to the TB treatment 
program in the clinic to interpret and use at their discretion. The clinic can then decide if 
it will take any steps to change clinical care based on those findings. 
8.3.3 Type and Duration of the Follow-up of Participants After Adverse 
Events 
AEs will be followed until resolution or stabilization even if this extends beyond the 
study-reporting period.  Resolution of an AE is defined as the return to pretreatment 
status or stabilization of the condition with the expectation that it will remain chronic. 

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35 
9 
CLINICAL MONITORING STRUCTURE 
9.1 
Site Monitoring Plan 
The study investigators are responsible for ensuring that, for this study:  
• 
human participants’ rights and well-being are protected; 
• 
data are accurate, complete, and verifiable from source documents; 
• 
the study complies with the protocol/amendment(s), sponsor requirements, ICH 
and South African Good Clinical Practice guidelines, and applicable regulatory 
requirements. 
Site monitoring is conducted to ensure that the human subject protections, study and laboratory 
procedures, study intervention administration, and data collection processes are of high quality 
and meet sponsor, ICH and GCP guidelines and applicable regulations, and that the study is 
conducted in accordance with the protocol, protocol-specific Clinical Quality Management Plan 
(CQMP), and applicable sponsor standard operating procedures. The study team will conduct 
quarterly audits on study documentation and procedures as detailed in the CQMP. 
If the Sponsor deems necessary, monitoring visits by a sponsor-designated professional or 
monitor may occur at scheduled intervals prior to, during, and at study completion. Monitoring 
visits may include, but are not limited to, the review of regulatory files, CRFs, informed consent 
forms, medical and laboratory reports, and protocol compliance. Study monitors will meet with 
investigators to discuss any problems and actions to be taken and document visit findings and 
discussions. The investigational site will provide direct access to all study-related sites, source 
data/documents, and reports for the purpose of internal monitoring and in the event of auditing 
by the sponsor and inspection by regulatory authorities.   
 

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36 
10 
STATISTICAL CONSIDERATIONS 
10.1 Study Outcome Measures 
 
Questions that will be addressed in Aim 1.  
 
Primary questions: 1) We will estimate the prevalence of problem alcohol use among patients 
starting TB treatment and will evaluate changes in behavior during the course of and 1-yr post-
treatment. 2) We will assess baseline TB disease characteristics associated with problem 
alcohol use, including TTP and CXR scores, and first line drug resistance prevalence. 3) We will 
calculate the time to culture conversion, in patients with problem alcohol use compared to those 
without, at 12 weeks. We will adjust for multiple potential confounders and mediators of these 
relationships as well, including tobacco and drug use, age, BMI/albumin, liver dysfunction, bone 
marrow suppression, HIV status, mycobacterial burden at baseline. We will stratify on 
adherence, defined as missing more or less than 20% of doses in the 12-week period.  
 
Secondary questions: 4) We will calculate the risk of poor final TB outcomes, defined as 
treatment failure, death, and relapse. 5) We will explore whether there is a threshold effect on 
treatment outcomes for alcohol use and assess for the presence of any interactions with 
gender. 6) We will build models to assess whether acute alcohol ingestion, based on recent 
reported use, better predicts TB treatment outcomes compared to measures of chronic 
ingestion, including self-report measures and biologic indicators of end organ damage. 7) We 
will report on barriers to adherence and individual patient level challenges based on daily 
reports from DOTS workers. 8) We will determine whether any individuals who report alcohol 
abstinence have positive PEth biomarkers and re-classify these patients in the model. 9) We will 
estimate the percentage of patients who develop side effects to the TB drugs and if there are 
significantly more side effects in patients who are acutely ingesting alcohol or who have a longer 
history of problem alcohol use relative to patients who do not drink. 10) Contingent upon 
funding, we will assess if transcriptomic profiles change through the course of treatment and if 
those changes are modified by a history of problem alcohol use. 
 
Questions to be addressed in Aim 2. 1) We will confirm preliminary findings of variability of 
specific drug PK among patients and the association with 12-week culture conversion and rate 
of decline in slowly dividing M. tuberculosis subpopulations (β-slope or sterilizing activity) in the 
PD model. 2) We will investigate the effect of problem alcohol use on the PK measures Cmax 
and area under the curve (AUC) and the effect on sterilizing activity, with adjustment for drug 
concentrations and mycobacterial drug MIC. 3) We will identify other potential modifiers of the 
alcohol-drug concentration-sterilization relationship, including BMI, gender, and age, and use 
these findings to build a prediction model of who is at highest risk of low PK with delayed PD. 4) 
We will also assess how these other modifiers explain variability in PK/PD seen in the control, 
non-problem alcohol population. 
 
Questions that will be addressed in Aim 3. (A) We will evaluate Mtb diversity in host, its 
dynamics overtime and in a specific set of DR, drug tolerance, virulence and immune regulator 
genes for evidence of directional and diversifying selection. (B) We will evaluate how Mtb 
diversity and genes under selection associate with time-to culture conversion (three consecutive 
weeks of negative growth) and negative treatment outcomes, adherence, HIV, diabetes mellitus 
(DM), and substance use. (C) We will leverage MIC and sequence data from TRUST. We will 
combine these with a large public Mtb MIC and WGS dataset enriched for high level antibiotic 

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37 
resistance generated by studies that include the NIAID funded Harvard TB CETR. We will train 
an in silico MIC predictor and probe interactions between mutations and the Mtb lineage on a 
genome-wide scale. These points will be studied to address the question of whether there is a 
metric that could indicate which patients need early adjustment of the standard first-line therapy, 
or which patients would have good outcomes with shortened duration of therapy. If so, this 
would be a major step forward toward more precision TB care and could reduce additional 
bacterial resistance generation.   
 
Questions that will be addressed in Aim 4. (A) Collecting follow-up HbA1c and blood glucose 
measures at participants’ 9-18 month post-treatment study visit provides an opportunity for us to 
assess differences in rates of dysglycemia in PLWH and HIV-uninfected persons who have 
completed treatment for TB disease and how alcohol use modifies the relationship. (B) 
Collecting follow-up samples to measure markers of stress (i.e. cortisol) and inflammation (i.e. 
IL6, TNF, CRP, adiponectin and insulin) at the post-treatment 9-18 month study visit will allow 
us to gather preliminary data to assess if there are differences in these biological markers post 
TB treatment amongst PLWH and HIV-uninfected groups and how alcohol use modifies the 
relationship.  
10.2 Sample Size Considerations 
We powered the study using a logrank test to estimate the power to detect the difference in time 
to positive sputum culture with and without problematic alcohol consumption. Fig. 2 shows the 
detectable hazard ratios (HR) with 291 participants (87 
patients being problem alcohol drinkers), assuming a 
5% drop-out rate at 12 weeks, and varying levels of 
survival among those without problematic alcohol 
consumption. In recent studies in this region, 20-30% 
of patients on standard therapy had mycobacterial 
growth in MGIT sputum cultures at 8 weeks of therapy 
(personal communication, Mark Hatherill, MD). If just 
15% of those without problematic alcohol have a 
positive culture at 12 weeks, then we will be able to 
detect a HR of 0.63, indicating that 0.3 in the alcohol 
group had a positive culture at 12 weeks. For Aim 1, 
we will inflate the 291 by 5% to be able to stratify on 
patients with poor adherence (not fully loss to follow-
up but take <80% of medications) and then inflate by 
an additional 30% for HIV positive patients. This would 
mean recruiting 438 patients for Aim 1 (307 HIV 
negative). For Aim 2, we will use the same model and 
recruit 200 participants from the 307 HIV negative, 
omitting any individuals who do not want the full day of phlebotomy or not able to participate due 
to their schedule. For this Aim, we will be able to detect a HR of 0.57, indicating 0.34 in the 
alcohol group having positive cultures at 12 weeks. 
 
For Aim 3, 
(A) All genes pre-specified for interrogation were identified to be under positive selection in 
previous work, hence these will be enriched to have high selection coefficients (i.e. high rates of 
mutation fixation often at ≥2 magnitude above the neutral rate. Previous work also suggests that 
a simple approximation for genome-wide mutation distributions due to genetic drift (i.e. neutral 
evolution or the Null) is a Poisson. Based on our preliminary longitudinal data, we estimate the 
Figure 2. Detectable hazard ratio (HR) at 80% power for 
varying assumptions of positive culture probability 

 ____________________________________________________________________________________________ 
38 
Poisson rate of variants occurring at >50% allele frequency to be 0.25 SNSs/patient/day or 13.4 
SNSs/patient/8weeks in non-PE/PPE regions. Using this rate and our previously developed 
power calculator in R70 for paired isolate genome-wide association, we expect >80% power to 
detect genes with 100 fold mutation rate assuming (1) average gene length of 1000bp across 
the 23 non known DR loci, (2) ≥10% observed SNSs in these genes among 330 patients with 
paired isolates and (3) Bonferroni corrected family wise error rate of 0.05. Similarly power will be 
>73% with a sample of ~200 patients expected to have HIV and/or problem substance use 
(60% of 330). 
 
(B) Among 160 TRUST-I participants with sufficient follow up to date, 28% converted their Mtb 
culture ≥12weeks. The main comparison will be time-to-culture conversion between participants 
with and without relevant micro-variation or high diversity. The power of the log-rank/survival 
analysis assumes 88% retention (N=400) and 30% of participants have identified micro-
variation/diversity signal. This is reasonable given preliminary data on diversity distributions in 
host. 
 
(C) We plan to synthesize all available WGS and Mtb data available globally to increase power 
and accuracy of our model. Given the performance of our existing models built using ~3500 
isolates we believe the model built here will only show superior performance. Synthesizing 
multiple MIC datasets will enrich in antibiotic resistance and better represent global MTB 
diversity producing a more generalizable MIC model.  
 
For Aim 4, This pilot study has a limited sample size. Currently, there are 67 (30%) PLWH 
(exposed) and 158 (70%) HIV-uninfected (unexposed) TB positive patients enrolled in the 
TRUST study. With a baseline risk of hyperglycemia in the unexposed group estimated at 
33%[30], our sample size of 225 patients (30% PLWH), would provide roughly 50% power to 
detect a 25% relative decrease in the risk of hyperglycemia (Table 2). Our sample would need 
to be twice as larger (167 exposed/ 333 unexposed) to have >80% power to detect this 
difference between groups, providing stronger evidence for our proposed future R01 aims. As 
such, the proposed analytical plan in this application is describing the assessment of simple 
associations/correlations between HIV groups. 
 
As a supplementary study component, we propose to conduct focus groups with a subset of the 
study sample (n=48) and up to 12 TB or substance use professionals to assess 1) participants’ 
perceptions of the relationship between alcohol use and TB disease and the reasons for changes 
in alcohol consumption during illness and throughout TB treatment and 2) readiness of 
participants who consume alcohol to partake in reduction or cessation programs during TB 
treatment.  
10.3 Participant Enrollment and Follow-Up 
In our projections, we have factored in that 3-5% of patients will have RIF resistant TB and that 
a small proportion will refuse to participate. Based on findings from previous studies of heavy 
drinkers in this population, we anticipate no more than a 5% attrition rate at the end of the 12-
week sputum sampling period. Myers and Parry have extensive experience in conducting 
longitudinal studies that involve tracking alcohol and other drug users.68,69 We will follow 
procedures that have been used to successfully limit attrition, including a dedicated team to 
actively track participants taking their daily medications and between appointments, mobile 
phone, verbal, and face-to-face appointment reminders, and incentives to attend follow-up 
appointments. If a participant misses an appointment or 3 days of medication, study staff will 
use all of the contact information contained in the locator form to contact him/her. 

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39 
10.4 Analysis Plan 
 
Aim 1. We will first use basic descriptive statistics to estimate the prevalence of problem alcohol 
use in this population, as well as, baseline characteristics associated with problem alcohol use. 
Consistent with our primary hypothesis, we will investigate the relationship between problem 
alcohol use and TB outcomes. Problem alcohol use will be assessed using both continuous and 
categorical measures from the follow-up visit data to assess the frequency of acute exposure 
and the regularity of alcohol ingestion. We will use basic bivariate tests to assess the 
association between alcohol use and the primary TB outcomes, as well as, the association 
between potential confounders and problem alcohol use. We will investigate the role of 
mediating factors by estimating the direct, indirect, and total effects of alcohol use on our 
outcomes of TTP and poor TB outcomes, using the classic approaches70 as well as newer 
innovations.70,71 We will control for HIV as a potential confounder and, if necessary, stratify our 
results by HIV status. We will model TTP using a poisson or negative binomial model, unless 
there is censoring, in which case we will use a Cox proportional hazards model. Poor TB 
outcomes will be modelled using logistic regression and Cox models, as appropriate. We will 
investigate potential threshold effects of alcohol by using a penalized spline for alcohol in the 
model and assessing nonlinear patterns graphically and with statistical tests. 
 
We will further investigate the distinction between acute versus chronic effects of alcohol by 
fitting three different models: 1) model with acute consumption patterns as the exposure, 2) 
model with chronic measures of alcohol (i.e., liver function, years of drinking), and 3) model with 
both acute and chronic alcohol exposures, including an interaction between these types of 
exposures. We will use fit statistics (i.e. AIC and BIC) to assess the best fitting model and 
examine statistical significance of the interaction terms to detect potential synergy between the 
two classes of exposure. The outcome in these models will be time to culture conversion. We 
will also model the use of alcohol during the course of the study as a function of TB disease 
severity using a repeated measures model based on self-reported alcohol usage at each time 
point. All models will assess goodness-of-fit. Analyses will be performed in R (r-project.org) and 
SAS (SAS Institute, Cary, NC). 
 
Aim 2. Our statistical modeler in Year 5 will build a time-to-event model to describe treatment 
response in patients as outlined in previous work by Co-Investigator McIlleron.59 In this model, 
the hazard of a positive culture result will be directly related to the bacillary load (measured 
using TTP data in sputum liquid cultures) predicted from the model. The nonlinear semi-
mechanistic model will describe the biexponential decline in bacillary load using the α- and β-
slopes. The β-slope is thought to reflect terminal sterilizing activity. Individual estimates of β-
slope will be generated by the model for evaluation of the effects of drug exposure on sterilizing 
activity, in a multivariate adaptive regression splines (MARS) analysis. MARS is a non-
parametric regression data analysis technique that combines recursive partitioning with fitting of 
splines to variables in the dataset. This method identifies the significant predictor variables, 
which enables splitting of the data into subregions of interest, while the spline fitting enables 
determination of relationships within the sub-regions. Drug interactions can be additive, 
synergistic or antagonistic. Potential predictors of the primary outcome (i.e. the β-slope) 
included in the MARS analysis will be problem alcohol use (versus non), AUC, Cmax, AUC/MIC, 
Cmax/MIC, individual drug MICs, the percentage of the 24-hour dosing interval that 
concentration exceeded MIC (%TMIC), gender, and BMI. Other variables found to significantly 
predict final outcomes in Aim 1 will also be considered for addition to this model. 
 
Aim 3: 

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40 
 
(A) Bacterial burden: We have designed the direct sequencing protocol to allow for comparisons 
of genome copy number, computed as average coverage along a unique and conserved 
Mtb sequence element, across patients. Diversity and selection: We will measure expected 
changes in allele frequency due to sequencing error, noise and in vitro expansion using the 
technical controls. We will compare raw diversity between direct and from-culture 
sequences. We will differentiate between diversity arising from random drift and/or 
demographic population changes and those due to selection using neutral evolutionary 
theory and simulation. We will examine different metrics to distinguish between populations 
under purifying selection and those under drift using the observed site frequency spectrum, 
estimates of effective population size (Ne), prior estimates of Mtb’s molecular clock rate µ84 
and relative abundance of non-synonymous and synonymous variants. We will examine 
evidence for directional and diversifying selection including a disproportionate rate of fixation 
and/or diversification in pre-specified genes relative to other sections of the genome and in a 
convergent manner across patients using models that incorporate expected population 
shrinkage under drug pressure. In a hypothesis generating, secondary analyses we will 
examine these forces on other genes and compare evidence for selection between patients 
with or without HIV or problem alcohol use.   
 
(B) We will test our hypothesis that in host population diversity allows for monitoring of treatment 
response by studying: (1) the association between resistance and tolerance micro-variation 
at baseline and time-to-culture conversion up to 12 weeks (primary analysis) as well as 
overall treatment outcome determined at 12 months after treatment (secondary analysis). (2) 
We will compare in host genome-wide population diversity at baseline, at 8 weeks, and the 
change between baseline and 8 week follow up. (3) We will assess if any of these three 
metrics can be better predictors of delays in culture conversion than other methods of 
bacterial burden measurement. (4) We will examine the relationship between diversity and 
each of the following: cavitary disease on CXR, smear grade, Xpert CT, emergence of 
resistance or tolerance variation overtime, treatment adherence, 
pharmacokinetics/measured drug levels, HIV, problem alcohol, and other substance use. 
We will dichotomize patients by identifying a threshold on the diversity metric (number of 
segregating sites) into high and low diversity groups based on the distribution of this metric 
alone without data on outcome so as not to bias our results. We will then use survival 
models (e.g. Cox) (for delays in culture conversion) and logistic regression models (for 
negative treatment outcomes) and adjust for multiple hypothesis testing in each case. We 
will also explore mediation analyses as bacterial population diversity are more likely 
mediators of delayed culture conversion or poor outcome than independent of or 
confounded by certain host level factors.  
 
We will combine data from Aim 1 with data from ~17,000 samples with Illumina WGS and MICs 
curated from our prior work and from public sources and use this data to build a machine-
learning MIC predictor. We will assess if MIC predictions improve the fit of the time-to-culture 
conversion survival model built from host level variables alone.    
 
Aim 4. (A) Collecting follow-up HbA1c and blood glucose measures at participants’ 9-18 month 
post-treatment study visit provides an opportunity for us to assess differences in rates of 
dysglycemia in PLWH and HIV-uninfected persons who have completed treatment for TB 
disease and how alcohol use modifies the relationship. For PLWH and HIV-uninfected 
individuals, we will also create a simple table populated with proportions and corresponding 
confidence intervals (CIs) displaying patient’s blood glucose levels at enrollment (categorized as 
diabetic (previous diagnosis), hypoglycemic, normal and hyperglycemic) stratified by their blood 

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41 
glucose levels at 15-months (hypoglycemic, normal and hyperglycemic). We will also stratify 
results by alcohol use, in addition to clinical and demographic risk factors for dysglycemia (e.g.  
age, gender, obesity), along with time on ART and specific drug classes in ART regimens for 
PLWH. Where possible, we will use regression models to assess all predictors of dysglycemia, 
however, the small sample size shown below may not make this possible. (B) Collecting follow-
up samples to measure markers of stress (i.e. cortisol) and inflammation (i.e. IL6, TNF, CRP, 
adiponectin and insulin) at the 15-month study visit will allow us to gather preliminary data to 
assess if there are differences in these biological markers post TB treatment amongst PLWH 
and HIV-uninfected groups and how alcohol use modifies the relationship. We will use chi-
square tests to assess differences in simple proportions. Median and interquartile ranges for 
each marker will be calculated according to HIV status. Mann-Whitney U test p-values will be 
calculated to evaluate the difference in levels of each marker by HIV status. Pearson Correlation 
test will be used to assess correlations between circulating levels of all makers and blood 
glucose levels stratified  by HIV status. We will also stratify results by alcohol use, in addition to 
clinical and demographic risk factors for diabetes (e.g. age, gender, obesity), along with time on 
ART and specific drug classes in ART regimens for PLWH. Where possible, we will use 
regression models to assess all predictors of stress, inflammation, and insulin levels. 
 
Qualitative Study Component. Transcripts will be coded and analyzed using the qualitative 
software, NVIVO. To code the data, passages referring to the different conceptual categories will 
be marked and identified by cover terms that reflect the conceptual category. The marked 
passages will be compared to enable the identification of similarities and differences and themes 
that emerge organically from the data. The initial transcripts will be coded by two of the South 
African investigative team (Prof. Myers and Dr. Carney), with the possible assistance of an expert 
in qualitative research at Boston Medical Center (where the PI is based) in order to create a 
codebook. Constant comparative coding will be employed so that all transcripts are coded with 
the full set of codes that emerges. According to this method, the initial conceptual categories are 
then applied to new data, and the categories revised to reflect the addition of the new data.  Upon 
completion of this process and review of all transcripts, we will have identified a series of 
categories that reflect participants’ experiences and perceptions.  

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42 
11 
ACCESS TO SOURCE DATA/DOCUMENTS 
All study-related records (case report forms and laboratory results) will be maintained at the 
strictest level of confidentiality by all participating investigators, their staff, and the Sponsor and 
their agents. Participant identifiers will be replaced with a unique study identification number in 
all records to maintain confidentiality. All participant case report forms will be maintained in 
locked study-dedicated file cabinets at the study site at Ukwanda Rural Clinical School. Medical 
records will be maintained per standard practice in the hospital and participating medical clinics. 
Electronic records will be maintained on study-specific computers with limited access and 
shared between BMC, SAMRC, and UCT only through secure file transfer. Participant data for 
the purpose of Aim 3 will be completely de-identified and shared via secure file transfer with the 
Harvard Medical School investigators upon the signing of the appropriate data transfer/data use 
agreements. Participant data for the purpose of Aim 4 will be completely de-identified and 
shared via secure file transfer with the Boston University and University of Cape Town 
investigators upon the signing of the appropriate data transfer/data use agreements.  
The audio recordings of individual interviews with be stored on a simple audio recording device 
until they have been transcribed and QA/QC procedures conducted and then destroyed within 
one month of finishing these procedures. The transcriptions of the audio recordings will be 
stripped of any identifiers. 
The Sponsor (or Sponsor representative), the IRB/REC or Regulatory Agency representatives 
may consult and/or copy study documents in order to verify case report form data. By signing the 
informed consent document, the participant agrees to this process. Any documents to be copied 
will identify the participant only by unique study ID number, except in the case of the mobile DOTS 
app which will capture participant first name and first initial of surname. These study documents 
include, but are not limited to medical records (office, clinic or hospital) for the participants in this 
study. The clinical study site will permit access to such records. 

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43 
12 
QUALITY CONTROL AND QUALITY ASSURANCE 
Following a written site CQMP, the participating site is responsible for conducting routine quality 
assurance (QA) and quality control (QC) activities to internally monitor study progress and 
protocol compliance. The site principal investigator will provide direct access to all study-related 
sites, source data/data collection forms, and reports for the purpose of monitoring and auditing 
by the sponsor and/or its designee, and inspection by local and regulatory authorities. The site 
principal investigator will ensure all study personnel are appropriately trained and applicable 
documentations are maintained on site. 
The investigators agree to be responsible for implementing and maintaining quality control and 
quality assurance systems with written SOPs to ensure that the proposed study is conducted 
and data are generated, documented, and reported in compliance with the protocol, accepted 
standards of Good Clinical Practice (GCP) and all applicable federal, state, and local laws and 
regulations relating to the conduct of this clinical study.   
Following written SOPs, the investigators and staff will verify that the clinical study is conducted 
and data are generated, documented (recorded), and reported in compliance with the protocol, 
GCP, and the applicable regulatory requirements. As required, the clinical sites at Worcester 
CDC, Avian Park Clinic, Ukwanda, Brewelskloof Hospital, SAMRC, UCT, SU, and BMC will 
provide direct access to all source data/documents and reports for the purpose of regular 
internal monitoring and auditing, monitoring by the sponsor (or designee), and inspection by 
local and regulatory authorities. 
The investigators will implement quality assurance (QA) and quality control (QC) procedures 
beginning with the completion and verification of data collection instruments. The data query 
and resolution process are outlined in the CQMP. QA/QC are essential elements in GCP 
guidelines regarding data management. These efforts run from data collection, laboratory 
conduct, and clinical conduct, to inventory maintenance and control. Automated queries via the 
database software will help the Research Coordinator identify potential problems (outliers, 
illogical errors, missing values, inconsistent responses, missing visit reports, etc.) with recorded 
data. Queries will be transmitted to the clinical sites for response by the clinical staff who can 
check the original source documents for correction/verification. Identified corrections will be 
made to the original CRF and to the database. Corrections will be made according to GCP.   

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44 
13 
ETHICS/PROTECTION OF HUMAN PARTICIPANTS 
13.1  Ethical Considerations 
Each Institution engaged in the research will hold a current Federal Wide Assurance (FWA) 
issued by the Office of Human Research Protections (OHRP) for human subjects research that 
is U.S. federally-funded. 
13.2 Declaration of Helsinki 
This study will be conducted in compliance with good clinical practice (GCP) as described in the 
International Conference on Harmonization (ICH) document “Guidance for Industry – E6 Good 
Clinical Practice: Consolidating Guidance,” dated April 1996 and the South African guidelines 
for Good clinical practice. The study will also be carried out in keeping with local legal and 
regulatory requirements. All study procedures will comply with Declaration of Helsinki’s Ethical 
Principles for Medical Research Involving Human Subjects.  
13.3 Institutional Review Board 
The protocol and informed consent forms that will be used must be approved by the Division of 
Microbiology and Infectious Diseases (the Sponsor), Boston University Medical Campus 
Institutional Review Board (U.S. IRB), the SAMRC Ethics Committee, the University of 
Cape Town Health Research Ethics Committee, and the Stellenbosch University Human 
Research Ethics Committee (South African RECs) before the study is initiated and before any 
proposed amendments are implemented. The SAMRC, University of Cape Town, and 
Stellenbosch University's Health Research Ethics Committees are registered with the National 
Health Research Ethics Council (NHREC) in South Africa and with OHRP. Ethical approval from 
the primary South African Institute (the SAMRC), Boston Medical Center, as well as Harvard 
Medical School for the Aim 3 analytical work must be received prior to commencement of the 
analytical work. 
Other investigator responsibilities relative to the requirements of the IRB/REC include: 
• 
Submit to the IRB/REC for review any advertisements that will be used to recruit 
patients. 
• 
During the conduct of the study, submit progress reports to the IRB/REC, and request 
re-review of the study at least once a year. 
• 
Report, in writing, to the IRB any serious adverse events (SAEs) that occur during the 
study. 
• 
Inform the IRB/REC of any amendments in the protocol and obtain documented 
IRB/REC approval of those amendments. 
13.4 Informed Consent Process 
The investigator is responsible for the content of the informed consent form, but the content 
must be approved by the IRB/RECs (listed above) and the Sponsor. The content of the consent 
document must comply with the ICH and the South African Good Clinical Practices guidelines. It 
must also include any additional information required by local laws. 

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45 
A designated study staff member is responsible for obtaining informed consent from each 
participant in the study. Consent forms will be translated into Afrikaans after IRB/REC approvals 
are obtained, after which a translator qualification form will be sent to the IRB/REC. Participants 
will be asked to read and review the Consent document. Upon reviewing the document, the staff 
member will explain the research study to the participant, describing in detail the study 
procedures and risks, and answer any questions that may arise. The participants will sign the 
informed consent document before any study-specific procedures are done. The participant may 
withdraw consent at any time throughout the course of the study. In case the participant is not 
able to read the consent form, the consent process will be performed in the presence of a 
literate witness unrelated to the study team. This witness will either be a family member or friend 
accompanying the patient or another person from the clinic who will be impartial and unrelated 
to the study. The witness will be asked to sign the consent form to verify that the consent 
process was properly performed and that the consent seemed to have been understood and 
given by free will. A copy of the informed consent document will be given to participants for their 
records. The rights and welfare of the participants will be protected by emphasizing to them that 
the quality of their medical care will not be adversely affected if they decline to participate in this 
study. 
 
Waiver of Informed Consent: A waiver of informed consent is requested to obtain and analyze 
bacterial (mycobacterium tuberculosis) DNA extracted from previously enrolled participant 
sputum for the Aim 3 analyses with the goal of understanding how TB bacteria responds to TB 
therapy. 
 
Justification for the waiver of consent is described below.  
  
• The research involves no more than minimal risk to subjects.  
  
The proposed study is minimal risk and has abundant mechanisms in place to ensure protection 
of patient confidentiality. The risk of breach of confidentiality is minimized through rigorous 
ethics training for staff and protection of participant data as described below. Samples will be 
labeled with a Specimen ID. The laboratory conducting analyses on these samples will have no 
access to participant data linked to that Specimen ID. 
  
• Waiving the requirements for informed consent will not adversely affect the rights and welfare 
of study subjects  
  
The samples the investigators are requesting access to under this waiver are already-collected  
sputum samples and thus pose no physical risk to participants. No human DNA or other 
biomarkers will be obtained from the samples. Only bacterial DNA will be extracted and 
sequenced. The DNA sequencing has no impact on individual participants. Results will not be 
reported in any way that identifies individuals and will therefore not adversely affect participants’ 
rights and welfare.  
  
• The research could not be carried out practicably without the waiver or alteration.  
  
In order to accurately study, determine, and propose a metric that could indicate which TB 
patients need early adjustment of the standard first-line therapy, or which patients would have 
good outcomes with shortened duration of therapy, which would be a major step forward to 
more precision TB care and could reduce additional bacterial resistance generation, we require 
as close to complete TB bacteria whole genome sequencing data during the study period as 

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46 
possible. Each bacterial DNA whole genome sequence obtained from TRUST specimens will be 
critical to informing the model. This model will then be further informed by epidemiologic 
information provided by consented participants. We will make every attempt to reach and 
obtain consent from our participants for this aim of the study; however, the waiver or 
alteration is critical for analyzing specimens from participants who may have passed 
away, moved, or are otherwise unreachable. 
  
• If the research involves using identifiable private information or identifiable biospecimens, the 
research cannot practicably be carried out without using such information or biospecimens in an 
identifiable format.   
 
All samples are labeled with a Specimen ID. Any third party contracted for sequencing of the TB 
bacteria will be fully blinded to any additional participant data except for the Specimen ID. 
  
• Whenever appropriate, the subjects or legally authorized representatives will be provided with 
additional pertinent information after participation.  
  
The investigators do not anticipate the TB bacteria whole genome sequencing to generate 
information that will be pertinent to study participants. There are no plans for providing 
additional information to participants regarding the study procedures occurring under waiver of 
informed consent, including results.   
13.4.1 Informed Consent Process 
Parental consent will be obtained for children <18 years of age enrolled in this study.  
Separate consent will be obtained from minor children aged 15 to 17 years.    
13.5 Exclusion of Women, Minorities, and Children (Special 
Populations) 
 
Special care will be taking with these populations and the consent forms will capture required 
elements according to 45 CFR 46 Subparts B and D.   
 
Pregnant women will be excluded from enrollment due to the additional risks associated with the 
diagnostic tests proposed (radiograph). We will perform a pregnancy test before the radiograph 
in all women of child bearing age. If a woman becomes pregnant after enrollment, she will be 
not be removed from this study. 
 
Participants in this study will be of all races and ethnic groups represented by tuberculosis 
patients in Worcester, South Africa. 
Children under the age of 15 will not be included in this study because drinking is not common 
under that age. 
13.6 Participant Confidentiality 
Participant records will be maintained at the strictest level of confidentiality by all participating 
investigators, their staff, and the Sponsor and their agents. Participant identifiers will be 

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47 
replaced with a unique study identification number in all records to maintain confidentiality. 
Participant identifiers collected in a pre-screening log will be blacked out immediately after a 
patient is deemed to be potentially eligible and approached for screening, or deemed to be 
ineligible. All participant case report forms will be maintained in locked study-dedicated file 
cabinets at the Ukwanda site.  Medical records will be maintained per standard practice in the 
medical clinic (Worcester CDC or Avian Park Clinic). Participant consent will include access to 
medical records maintained at these facilities. Electronic records will be password protected and 
maintained on study-specific computers with limited access. Participant records, not limited to 
medical records (office, clinic or hospital), will not be available for health insurance companies, 
employers, or to other individuals. Audio recordings will be stored on a simple recording device 
and when not in use, in a locked cabinet in a locked office at the SAMRC. Audio recordings of 
focus groups and individual interviews will be destroyed within one month of transcription and 
QA/QC procedures. The transcription of the audio recordings will be stripped of any identifiers 
and will be secured electronically following the same safety and confidentiality procedures as 
the study’s other electronic records. 
The Sponsor (or Sponsor representative), the IRB/REC or Regulatory Agency representatives 
may consult and/or copy study documents in order to verify case report form data.  By signing 
the informed consent document, the participant agrees to this process. Any documents 
including medical records (office, clinic or hospital) to be copied will identify the participant only 
by unique study ID number. The clinical study site will permit access to such records. 
This study has an NIH-issued Certificate of Confidentiality (CoC). The CoC protects the privacy 
of subjects by limiting the disclosure of identifiable, sensitive information. Under the new policy, 
disclosure is not up to the discretion of the investigator. Disclosure is only permitted in the 
following circumstances: 1) if required by other Federal, State, or local laws, such as for 
reporting of communicable diseases; 2) if the subject consents; or 3) for the purposes of 
scientific research that is compliant with human subjects regulations.  
All biological samples will be stored in South Africa under the supervision of the site PI. 
Samples will be stored in -80ºC freezers or liquid nitrogen indefinitely. MTB DNA extracted for 
Aim 3 will be sent to (to be determined) institutions for the sequencing work upon the signing 
and acquisition of the appropriate MTAs and Export Permits. To assure patient confidentiality, 
stored samples will be labeled using the study identification number. Patients will be 
encouraged to maintain their address/contact updated in order to allow future contact to inform 
them about relevant research information or to obtain a new specific informed consent for stored 
sample use in a new project.  
13.7 Future Use of Stored Specimens 
Biological samples (blood and sputum) will be banked indefinitely for this research or future 
research on tuberculosis or substance use disorders that that may not be planned yet, pending 
funding. The use of stored samples will never be for commercial purposes. Blood and sputum will 
be stored at Stellenbosch University. All samples will be de-identified with the only existing link to 
patient information existing on a secured and locked server at Stellenbosch University.  

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48 
14 
DATA HANDLING AND RECORD KEEPING 
It is anticipated that participants will have been recruited and completed the study in 
approximately 18 months from initial screening to final visit. Data will be collected, maintained, 
and analyzed by investigators at SAMRC, University of Cape Town, Stellenbosch University, 
Harvard Medical School, BU, and BMC. Files will be kept in dedicated locked file cabinets. 
Medical records will be stored at the clinical sites per standard operating practices. Source 
CRFs will be maintained at the Ukwanda site. 
Electronic data and files will be maintained on password protected computers. Archival 
protection of all electronic files (CRFs and electronic databases) will be maintained on servers 
at Boston University that meet HIPAA compliance related to protected health information (PHI). 
An automated audit trail will be maintained by REDCap. REDCap will automatically log and 
document all user activity (e.g., entering data, exporting data, modifying a field, running a report, 
or add/modifying a user) as well as queries and corrections made to every data point. If errors 
are found, corrections will be made according to GCP standards both to the original CRF as well 
as to the electronic database.  
14.1 Data Management Responsibilities 
 
It is the responsibility of the clinical sites and data clerks to enter completed CRFs into the 
REDCap database within five business days of verification by the field coordinator. Timely 
submission allows for prompt query generation and resolution and securing of data.  
Investigators and staff will manage data capture, management, query submission/resolution, 
quality control and assurance, and provide statistical analysis of all results. 
14.2 Data Capture Methods 
All study forms will be linked to each other by a unique participant number. The pen-and-paper 
forms will be stored in locked filing cabinets at the Ukwanda study site, with consent and locator 
forms being stored separately from questionnaires and biological screening forms. All forms will 
be transported from the clinical site at Worcester CDC and Avian Park Clinic back to the 
Ukwanda study site and secured in locked filing cabinets at the end of each working day. 
Quality control methods will be implemented consistently over time to ensure high quality of the 
incoming data stream and to avoid drift in data reporting/ascertainment practices. We will use 
REDCap (Research Electronic Data Capture) for data entry and database management, hosted 
on the Boston University Medical Center (BUMC) and maintained by study staff at Boston 
Medical Center. 
 
The MRC site is experienced in conducting clinical studies to international standards (R01 
HD058320; R21DA03054; 1R01DA032061-04-S1). UCT and the Warren laboratory at 
Stellenbosch University will transmit results following the same procedures. Any queries on data 
will be transmitted back on a weekly basis via REDCap; the field coordinator will then work to 
verify any discrepant data at the clinical site. All laboratory data queries will be submitted to 
McIlleron or Warren to review with their technician to assure validity. 
14.3 Types of Data 
Data collected for this study will include:  

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49 
1. Clinical questionnaire and evaluations*;  
2. Behavioral assessments; 
3. Observational Adherence data; 
4. Sputum from TB cases for AFB smear*, culture*, MIC testing*, Whole Genome 
Sequencing (WGS), and spoligotypingǂ;  
5. Chest X-ray from TB cases*;  
6. HIV serology from TB cases*; 
7. Serum for biologic measures; 
8. Serum for PK testing; 
9. Dried blood spot for PEth testing; 
10. Whole blood for RNA sequencing and biomarker analysisǂ  
11. Epigenetic analyses of genes associated with the bodies response to TB, HIV and 
treatment response.   
12. Urine for pregnancy and drug testing  
13. Audio recordings of focus group discussion and individual interviews 
14. De-identified transcriptions of the audio recordings 
 
*Procedures conducted all or in part by the National TB Program 
ǂContingent upon available funding 
14.4 Timing/Reports 
Reports on the enrollment and progress of study activities will be sent to NIAID as requested. 
As per all NIH funded genomic research we will deposit the Mtb sequencing data to the NIH 
National Center for Biotechnology Information (NCBI) and TB portal upon completion of our 
analyses. This data will not be linked to any participant information. Study Records Retention 
 
The investigator will make study data accessible to the Sponsor or its representatives, and to 
other Regulatory agency inspectors. All study-related records (patient charts, case report forms, 
and other study records) will be retained until disposal is authorized by NIAID and/or DBT. 
14.5 Protocol Deviations 
A protocol violation/deviation is any nonadherence to the clinical trial protocol, GCP, or Manual of 
Procedures requirements. Violations are deviations that threaten the integrity of the study, while 
deviations are those which do not. As a result of violations or deviations, corrective actions will be 
developed by the site and implemented promptly. Protocol violations should be reported within 7 
days to the local IRB/REC per the local IRB/REC guidelines. Any protocol deviation that meets 
reporting requirements of the local IRB/REC will also be reported with the same timeliness to the 
Boston Medical Center IRB and to the DMID Program Officer. All deviations from the Protocol 
must be addressed in the study participant source documents. The documentation should include 
the reason(s) for the deviation and all attempts to prevent or correct the deviation.  
Publication Policy 
 
Following completion of the study, the investigators plan to publish the results of this research in 
peer-reviewed scientific journals. Investigational plans, protocols, and data related to this study 

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50 
will be treated as confidential information. Authorship will be extended to the following 
individuals: study investigators, site PIs, and other individuals having major contribution to the 
study design, implementation, data analysis, and preparation of the written manuscript.  
  
 

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51 
15 
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58 
 
 
 
Follow-Up Schedule 
Procedures 
Pre-screening 
Screening 
Baseline 
Daily during 
treatment 
Weekly during 
treatment 
Monthly during 
treatment 
Aim 2 PK visit 
Post treatment 
Assessment of Eligibility Criteria 
X 
X 
 
 
 
 
 
 
Signed Consent Form 
 
X 
 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
Complete locator form 
 
X 
 
 
 
 
 
 
Participant interview 
 
X 
X 
 
 
X 
 
 
Medical records review 
 
X 
X 
 
 
X 
 
 
Biobehavioral screen  
 
 
X 
 
 
X 
 
X 
Chest radiograph 
 
 
X 
 
 
 
 
 
Drug side effect screen 
 
 
 
 
 
X 
 
 
Sputum collection 
 
 
X 
 
X2 
X3 
 
 
Blood collection 
 
 
X 
 
 
X4 
X 
X6 
Witnessed pill ingestion + count 
 
 
 
X 
 
 
 
 
Symptom screen and recent 
alcohol use questions5 
 
 
 
 
 
 
 
X 
2Weekly sputum collected only Weeks 1-12 
3Sputum will be collected at the month five study visit to confirm the results of the routine smear done at 
month five of treatment by the national TB program 
4Blood collection will occur if participant has TB drug side effect symptoms. After study visit, participant 
will be referred to TB program for additional assessment. Additionally, contingent upon funding, blood 
collection will occur at months 3 and 6 for repeat PEth testing and at week 2 and month 6 for biomarker 
analysis 
5Screening for TB symptoms and assessment of recent (previous 2 weeks) alcohol consumption will be 
done every three months after end of treatment for a total of 12 months (post treatment months 3, 6, 9, 
and 12) 
6Contingent upon funding, blood collection will occur for PEth testing at post treatment month 12. 
Between the post treatment 9-month visit and the participant being 18 months post treatment, blood 
collection will occur for HbA1c, blood glucose, stress and inflammatory marker analyses, and epigenetic 
analyses.