Clinical trial NCT02613169
Conditions: Tuberculosis
Keywords: M. tuberculosis infection isoniazid HIV-exposed infants
Sponsor: University of Washington
Start date: 2016-08-01; Completion date: 2020-12-01
Enrollment: 300 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Randomized controlled trial (RCT) of isoniazid (INH) vs. no INH to prevent Mycobacterium tuberculosis infection in HIV-exposed uninfected (HEU) infants.

Detailed description:
The purpose of this trial is to determine whether isoniazid (INH) reduces the risk of Mycobacterium tuberculosis (MTB) infection in HIV-exposed but uninfected (HEU) children, as well as to determine epidemiologic and immunologic correlates of MTB infection in HEU.

Arms: arm 1: Isoniazid (INH) \~10 mg/kg (7-15 mg/kg), will be administered once daily to infants in INH arm for 12 months. arm 2: No INH will be administered to this arm.
Interventions: intervention 1: Isoniazid
Intervention descriptions: intervention 1: HIV-exposed uninfected infants will be randomized to receive either INH or no INH daily for 12 months for the prevention of Mycobacterium tuberculosis (MTB) infection.

Protocol documents:
ICF_002.pdf: 
Infant TB Infection Prevention Study (“iTIPS”), RCT 
Consent, version  1.9 
 August 11, 2017 
1 
UNIVERSITY OF WASHINGTON (UW) and UNIVERSITY of NAIROBI (UoN) Collaborative Study Group 
 
CONSENT and PARENTAL PERMISSION FORM FOR RANDOMIZED TRIAL 
 
Preventing Mycobacterium tuberculosis Infection in HIV-Exposed Infants 
 
Short Title: Infant TB Infection Prevention Study (“iTIPS”) 
 
NCT: NCT02613169 
 
Document date: August 11, 2017 
 
 
 


Infant TB Infection Prevention Study (“iTIPS”), RCT 
Consent, version  1.9 
 August 11, 2017 
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UNIVERSITY OF WASHINGTON (UW) and UNIVERSITY of NAIROBI (UoN) Collaborative Study Group 
 
CONSENT and PARENTAL PERMISSION FORM FOR RANDOMIZED TRIAL 
 
Preventing Mycobacterium tuberculosis Infection in HIV-Exposed Infants 
 
Short Title: Infant TB Infection Prevention Study (“iTIPS”) 
 
Name 
Position  
Department 
Telephone Numbers 
Grace John-Stewart  
Protocol Co-Chair 
Global Health, UW 
+1-206-543-4278 
John Kinuthia 
Protocol Co-Chair 
Research & Programs, 
KNH 
+254-722-799-052 
Thomas Hawn 
Co-Principal Investigator 
Medicine, UW 
+1-206-616-4124 
Barbara Richardson 
Co-investigator 
Global Health, UW 
+1-206-744-2425 
Elizabeth  
Maleche-Obimbo 
Co-investigator 
Pediatrics, UoN 
+254-202-720-947 
Lisa Cranmer 
Co-investigator 
Pediatrics, Emory 
+1-404-727-2905 
Sylvia LaCourse 
Co-investigator 
Medicine, UW 
+1-206-616-7217 
Daniel Matemo 
Study Coordinator 
Obstetrics and 
Gynecology, UoN 
+254-722-322-378 
 
Emergency telephone number: Dr. John Kinuthia: +254-722-799-052 
  
1. Researcher’s Statement: 
 
We are asking you and your child to be in a research study. The purpose of this form is to give you the information 
you will need to help you decide whether you and your child will be in the study or not. Please read the form 
carefully. You may ask questions about the purpose of the research, what we would ask you to do, the possible 
risks and benefits, your rights as a volunteer, and anything else about the research or this form that is not clear. 
When we have answered all your questions, you can decide if you want to be in the study or not. This process 
is called “informed consent.” This form serves as a both as a record of your consent to be in the study and as a 
parental permission form.  We will give you a copy of this form for your records. 
 
The word “you” in this form refers to you and your child. 
  
2. What you should know about this study: 
• 
This form explains what would happen if you join this research study. 
• 
Please read it carefully. Take as much time as you need. 
• 
Please ask the research team questions about anything that is not clear. 
• 
You can ask questions about the study at any time. 
• 
If you choose not to be in this study, it will not affect any other care received at clinic. 
• 
If you say ‘Yes’ now, you can still change your mind later. 
• 
You can quit the study at any time. 
• 
You would not lose benefits or be penalized if you decide not to take part in the study or to quit the study 
later. 
  
3. What is the goal of this study? 
  
The goal of any research study is to answer questions. We (the research team listed on the front of this form and 
our staff) are doing this research study to answer the following question: 
  
- Does the medicine isoniazid (INH) decrease the risk of Mycobacterium tuberculosis (MTB) infection?  
 
MTB causes the disease called tuberculosis (TB). Once infected with MTB, some people go on to get TB disease.  
Young children, as well as people with HIV are more likely to get TB disease because their body defenses are 
sometimes weak. Children, even if they are not infected with HIV, are more likely to become infected with TB if 
someone else in their household is also infected with both HIV and TB.  INH has been successfully used to 


Infant TB Infection Prevention Study (“iTIPS”), RCT 
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decrease the chances of getting TB disease after MTB infection, but little is known whether it can prevent getting 
MTB infection in the first place. 
 
4. Why do I have the option of joining this study? 
  
You have the option to take part in this research study by being an HIV-infected mother and having a baby that 
was exposed to HIV. 
  
5. How many people will take part in this study? 
  
We think that about 300 mothers and their infants will take place in this research study at sites in western Kenya.  
  
6. If I agree to join this study, what would I need to do? 
  
STUDY PROCEDURES 
This is what will happen if you agree to participate in this study. We will ask you to read, discuss, and sign or 
make your mark on this form. After this form is signed or marked, the study staff will ask you questions about 
you and your child’s health, including HIV status, questions about your pregnancy, medications, and if you have 
been exposed to someone with TB. A study clinician will collect up to 5 mls of blood (teaspoon) from your infant 
on enrollment at 6 (+4) weeks of age, 10 weeks (+/- 4) weeks of age and 12 months after starting the study, and 
if your infant is diagnosed with TB disease during the study. We will also ask for a stool sample from your infant. 
You will also be asked to give 30 mls of breast milk and 5 mls of blood at the beginning of the study. These 
samples will be used to study the body’s defenses against TB.  
 
You will be placed into 1 of 2 groups. You cannot choose which group you will be placed in. You will be placed 
in a group by chance based on a number that has been assigned to you. The two groups you may be assigned 
to include either 1) INH or 2) no INH. Your child’s chance of getting INH or no INH is the same, just like flipping 
a coin. 
 
INH group - If your infant is assigned to the INH group, you will be asked to give your child INH daily for 1 year. 
Although INH is well tolerated in infants, it can be associated in very rare cases with tingling, burning, or 
numbness in the hands and feet. To prevent this, you will also be asked to give you child a vitamin called 
pyroxidine. At the beginning of the study, you will be asked how you would like to receive your child’s medication 
every month. You can choose to come to the clinic to pick it up or have a field worker bring it to your home. Also, 
if your child is in the INH group we will draw blood to measure your infant’s baseline liver function at enrollment 
(before staring INH) and after taking the medicine for 4-6 weeks. The liver is the main part of the body that filters 
this medication in the body.  
 
No INH group – If your infant is assigned to the no INH group, you will still have the same study procedures 
performed as the INH group, such as exams and blood draws, except your child will not be given INH or 
pyroxidine. 
 
These tests and exams help us find out if being in this study causes any effects that are important to know about. 
We use them to check on the safety of the people in this study. We also use them to learn if the experimental 
treatment is helping or not. 
 
You will be asked to bring your infant to clinic to be evaluated on enrollment at approximately 6 weeks of age 
(enrollment), 10 weeks, 14 weeks, 6 months, 9 months, and 12 months of age. These visits are aligned with 
the Kenyan recommended schedule of pediatric well child/immunization visits. Additionally, you will be asked to 
come to the last study visit at 12 months post-enrollment. It is very important to come to the last visit as this is 
the visit when we will draw blood to see if your infant has been infected with tuberculosis.  If your infant is in the 
INH group, you will be given enough INH and pyroxidine at each visit to last until the next visit. 
 
Tuberculin skin test (TST). A study clinician will a small needle to put some testing material, called tuberculin, 
just under the skin of your infant at the end of study visit and if your infant is diagnosed with TB. We will ask you 
to return to the clinic in 2-3 days to check the result by measuring if there is a reaction on your skin. TST is a test 
that is used to diagnose MTB infection, but does not necessarily mean you have TB disease.   


Infant TB Infection Prevention Study (“iTIPS”), RCT 
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These tests and exams help us find out if being in this study causes any effects that are important to know about. 
We use them to check on the safety of the people in this study. We also use them to learn if the experimental 
treatment is helping or not.  
 
Blood for genetic testing – Some of the blood drawn at the beginning of the study from your infant will be 
stored to do a test to check the genes (NAT2) that are related to how the body filters INH. The samples that will 
be tested will be chosen after the study is completed. You will not be told of the result for this test because it is 
for investigation only and will be done after the study is completed.  
 
Urine and hair for INH testing – For children in the INH group, we will collect urine at the 10 weeks, 14 
weeks, 6 months, 9 months, 12 months of age, and study endpoint visit (approximately 14 months of age). This 
urine will be used to test for INH in the clinic using a dipstick. We will also cut a small thatch of hair 
(approximately 30 strands) at the end of study visit.  This hair will be used to measure INH. You will not be told 
of the result for this test on hair because it is for investigation only and will be done after the study is 
completed. 
 
MEDICAL RECORD INFORMATION 
We will ask for access to your and your baby’s clinic and pharmacy records to find out more information about 
your pregnancy, delivery, and postpartum care. If you agree to give us access to your medical records, we will 
get information from the clinics where you received care before, during, and after delivery of your baby, 
including: any health problems, medication adherence and side effects, and your baby’s health information. We 
will also record laboratory test results, like your CD4 and HIV viral load tests, and infant’s HIV tests. 
 
7. How long would I be in this study? 
  
If you choose to take part in all the study visits, you and your infant would be in the study for 1 year. If you join 
this study, you can decide to stop at any time, for any reason. If you decide to stop you would need to talk with 
site investigators so you leave the study in a safe way. 
  
The research study clinicians could also decide to take you out of this study. This might happen if we find out 
that it is not safe for you to continue in the study. It may also happen if you cannot come to enough of the study 
visits. If we ask you to leave the study we would always explain why and this would not hamper other care 
received at the facility in any way. 
  
8. What are the potential harms or risks if I join this study? 
  
There are potential harms or risks if you take part in this study. Some are common and some are rare. They are 
described below. 
  
Potential Harms and Discomforts (from the most common, to the most rare): 
  
• 
Local irritation due to blood draw 
• 
Local irritation due to TST 
• 
Maternal breast discomfort due to self-expression of breast milk 
• 
Nausea, vomiting, stomach discomfort due to INH 
• 
Peripheral neuropathy (numbness, tingling of the nerves in your hands and feet) due to INH 
• 
Hepatitis (irritation of the liver which is the organ that filters the medicine INH) 
• 
Some people feel uncomfortable answering questions about their health and their baby’s health 
 
 Because this research study involves a medication that has been used primarily to treat TB disease or prevent 
TB disease in the past (not prevent MTB infection): we do know that in general INH is generally well tolerated by 
infants. 
 
A Data Safety Monitoring Board (DSMB) will review the information from this research study. This board is made 
up of a group of experts responsible for looking at how people in the research study are doing. If you take part, 
we would tell you about any new information we learn that might affect your health or your willingness to stay in 
the study. 
  


Infant TB Infection Prevention Study (“iTIPS”), RCT 
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9. What are the potential benefits if I join this study? 
 
Potential Benefits for You: 
  
Being in this study might benefit you in the following ways: 
• 
Participants will benefit from direct medical care in the long-term research group. 
 
Potential Benefits for Others: 
• 
All infants - those born to HIV infected mothers and those born to uninfected mothers - will benefit from 
a better understanding in how the body defenses protect against MTB infection  
• 
We hope to use information we gain in this study to benefit others in regions with high tuberculosis rates. 
 
10. What other options do I have? 
  
Whether or not you decide to participate in this research study, you can continue to receive your mother-child 
health care at this clinic. 
 
11. Who is funding this study? 
  
The study team and/or the University of Washington and Kenyatta National Hospital are receiving financial 
support from the Thrasher Research Foundation Health in the United States. 
 
12. How would you keep my information confidential? 
  
We will keep your identity as a research subject confidential. Your HIV test results, your infants MTB infection 
test results, medical records, and responses to questions will be kept private, and no identifying information of 
any kind will be released to any other person or agency that is not working on this study, without your permission 
in writing. We will not publish or discuss in public anything that could identify you. Any specimens you provide, 
and your medical information will be identified by a code number. All of your information, including the link 
between your name and code number will be kept in a secure location at the clinic only. Once the study is 
completed, we will maintain the link for 5 years, after this time we will remove your name and all identifying 
information from the study files. Any publication of this study will not use your name or identify you personally. 
However, study team may share identifiable information about you in the case the study team becomes aware 
of possible harm to yourself or others.  
 
Although we will make every effort to keep your information confidential, no system for protecting your 
confidentiality can be completely secure. It is still possible that someone could find out you were in this study 
and could find out information about you. Government or university staff may review studies such as this one to 
make sure they are being done safely and legally. If a review of this study takes place, your records may be 
examined. The reviewers will protect your privacy. The study records will not be used to put you at legal risk of 
harm. 
 
Study records may be reviewed by: 
• 
University of Washington, including the Institutional Review Board 
• 
Kenyatta National Hospital and University of Nairobi, including the Ethics and Research 
• 
Committee 
• 
Kenya Medical Research Institute (KEMRI)  
 
A description of this clinical trial will be available on http://www.clinical.trials.gov, as required by U.S. Law. This 
Web site will not include information that can identify you. At most, the Web site will include a summary of the 
results. You can search this Web site at any time. A copy of your consent form will be placed in your study record. 
 
13. Would it cost me money to be in this study? 
  
If you take part in this study there would be no cost to you. 
  
14. What if I were injured because I joined this study? 


Infant TB Infection Prevention Study (“iTIPS”), RCT 
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If you think you or your infant has a medical problem or illness related to this research, contact Dr. John Kinuthia: 
+254-722-799-052 right away. He will treat you or refer you for treatment. If your child is injured as a result of 
being in the study, you will be offered free care at the study clinic. If you require medical care that the study clinic 
cannot provide, we will refer you to the appropriate organizations to receive care for the injury. The costs of the 
treatment may be billed to you or the National Hospital Insurance Fund (NHIF) just like other medical costs, or it 
may be covered by the UW's discretionary Human Subject's Assistance Program (HSAP) depending on a 
number of factors. The researcher may request HSAP coverage by following established procedures.  If you 
wish to request HSAP coverage yourself, you may contact the researchers listed on the first page, or the UW 
Human Subjects Division at hsdinfo@uw.edu or +1-206-543-0098. Ask the researchers if you would like 
information about the limits and conditions of the HSAP.  The UW does not normally provide any other form of 
compensation for Injury.  However, the law may allow you to seek payment for injury-related expenses if they 
are caused by malpractice or the fault of the researchers.   You do not give up any legal rights by signing this 
consent form.  
 
15. Would I be paid of I join this study? 
  
Participants will be provided a stipend for travel. 
  
16. If I join the study, can I stop? 
  
Yes. Taking part in research is always a choice. If you decide not to be in the study, you can change your mind 
at any time. We ask that you tell Daniel Matemo who can be reached at +254-722-322-378. 
  
If you choose to leave the study, it will not affect your care at the study site. You will not lose any benefits or be 
penalized if you choose to leave the study. We may ask you to come for a visit if you leave the study early. At 
that visit we may ask to collect 5 mls of blood (teaspoon) and place a TST like we would at the end of the study 
visit. 
 
17. Will my samples be used after this study is done? 
 
We would like to save samples of your blood and breast milk and your baby’s blood,stool, and hair at the 
KEMRI/CDC, University of Nairobi, the University of Washington, the Fred Hutchinson Cancer Research Center, 
Emory University, or the University of California, San Francisco, for future HIV and/or TB related research and 
maternal and infant health. This may include testing for genes which may affect whether a person is more or 
less likely to get infections, or things that may affect infant and maternal health (mother’s health during 
postpartum period with special emphasis on HIV-related illnesses, infant health with special emphasis on HIV-
exposure, and TB exposure).  
 
Information we get from you, and your samples, may be shared with other investigators studying HIV, TB, or 
mother and child health. We will not share your name or any identifying information with them. An Institutional 
Review Board or Independent Ethics Committee, which looks at study application to ensure the safety and rights 
of research participants, must approve future research studies in which we will use your or your baby’s samples 
to obtain information about both of you. Permission from the University of Nairobi’s Ethics Committee will be 
sought before any of these samples are used for future research. These tests are for research and are not useful 
for your or your baby’s clinical care. Before your samples or your baby’s samples leave the clinic, they will be 
assigned a code and your name or your baby’s name will not be on them. We will store these samples for ten 
years after completion of the study. Storage of samples past this time period will only occur with approval from 
an Institutional Review Board and Ethics Committee. 
 
If you do not want to have your or your baby’s samples saved for future research, you can still be in this study 
and your or your baby’s samples will be destroyed once testing for the study is completed. If you agree to store 
your or your baby’s samples now, but change your mind before the end of the study, let the study staff know and 
we will make sure that your or your baby’s samples do not get stored for future research. We will not sell your or 
your baby’s samples. Tests done on your or your baby’s samples may lead to a new invention or discovery. We 
have no plans to share any money or other benefits resulting from any potential invention or discovery with you. 
 
 
 


Infant TB Infection Prevention Study (“iTIPS”), RCT 
Consent, version  1.9 
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CONSENT FOR STUDY PARTICIPATION 
Subject’s statement: 
 
This study has been explained to me. I volunteer to take part in this research. I have had a chance to ask 
questions. If I have questions later about the research, I can ask one of the researchers listed above. If I have 
questions about my rights as a research subject, I can call the Kenyatta National Hospital Ethics and Research 
Committee, at 2726300 Ext. 44102. I give permission to the researchers to use my medical records including 
my baby’s as described in this consent form. I will receive a copy of this consent form. 
 
 
 
____________________________________ ____________________________________ ___________ 
Printed name of subject 
 
 
 
Signature or thumbprint of subject 
 
Date  
 
____________________________________ ____________________________________ ___________ 
Witness Name (if caregiver illiterate)  
 
 
Witness Signature  
 
 
Date  
 
CONSENT FOR SAMPLE STORAGE FOR FUTURE STUDIES INCLUDING GENETIC TESTING 
Please mark, one option for each question below: 
YES ___NO___  You can store my samples for future research into HIV, TB or maternal child health  
YES ___NO___  You can store samples from my baby for future research into HIV, TB or maternal child 
health  
 
YES ___NO___  You can store my samples for future research into HIV, TB or maternal child health including 
genetic testing 
YES ___NO___  You can store samples from my baby for future research into HIV, TB or maternal child health 
including genetic testing 
 
____________________________________ ____________________________________ ___________ 
Printed name of subject 
 
 
 
Signature or thumbprint of subject 
 
Date  
 
____________________________________ ____________________________________ ___________ 
Witness Name (if caregiver illiterate)  
 
 
Witness Signature  
 
 
Date  
 
Who do I contact if I have problems or questions? 
If you ever have any questions about this study, or if you have a research-related injury, you should contact Dr. 
John Kinuthia. If you have questions about your rights as a research participant, you should contact the 
Kenyatta National Hospital Ethics and Research Committee, at 2726300 Ext. 44102. 
 
____________________________________ ________________________ ______________ 
Printed name of study staff obtaining consent    Signature                                  Date 
 
 
Copies to: Researcher, Participant 

Prot_000.pdf: 
 
Preventing Mycobacterium tuberculosis Infection in HIV-Exposed Infants 
 
Short Title: Infant TB Infection Prevention Study (“iTIPS”) 
 
NCT: NCT02613169 
 
Document date: April 1, 2020 
 
 


Infant TB Infection Prevention Study (“iTIPS”)  
Infant TB Infection Prevention Study (“iTIPS”), RCT 
Protocol, version 1.9 
 April 1, 2020 
2 
 
Preventing Mycobacterium Tuberculosis Infection in HIV-Exposed Infants  
 
Short title: Infant TB Infection Prevention Study (“iTIPS”) 
 
 
 
 
 
 
Sponsored by: 
 
Thrasher Research Fund 
 
 
Protocol Chair(s): 
Grace John-Stewart MD, PhD 
Departments of Global Health, Medicine, Pediatrics, Epidemiology 
University of Washington 
 
Protocol Co-Chair: 
John Kinuthia, MBChB, MMed, MPH 
Head, Research and Programs 
Kenyatta National Hospital 
 
Immunology Principal Investigator: 
Thomas R. Hawn MD, PhD 
Division of Allergy & Infectious Diseases 
Department of Medicine 
University of Washington 
 
Pediatric Clinical Tuberculosis Lead: 
Elizabeth Maleche-Obimbo MBChB, MMed, MPH, CPulm 
University of Nairobi 
Kenyatta National Hospital 
 
Version 1.9 
 
April 1, 2020 
 
 
 


Infant TB Infection Prevention Study (“iTIPS”)  
Infant TB Infection Prevention Study (“iTIPS”), RCT 
Protocol, version 1.9 
 April 1, 2020 
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TABLE OF CONTENTS 
 
 
LIST OF ABBREVIATIONS AND ACRONYMS .................................................................... 5 
PROTOCOL TEAM ROSTER ................................................................................................... 6 
SCHEMA ....................................................................................................................................... 8 
OVERVIEW OF STUDY DESIGN AND RANDOMIZATION SCHEME .......................... 10 
Enrollment ................................................................................................................................ 10 
Allocation.................................................................................................................................. 10 
Follow-Up ................................................................................................................................. 10 
1.0 
INTRODUCTION........................................................................................................... 11 
1.1 
Background and Significance......................................................................................... 11 
1.2 
Innovation....................................................................................................................... 15 
1.3 
Supportive Preliminary Data .......................................................................................... 18 
1.4 
Rationale......................................................................................................................... 23 
2.0 STUDY OBJECTIVES AND DESIGN ............................................................................... 24 
2.1 
Primary Objectives ......................................................................................................... 24 
2.2 
Secondary Objectives ..................................................................................................... 24 
2.3 
Exploratory Objectives:.................................................................................................. 25 
2.4 
Study Design .................................................................................................................. 25 
3.0 STUDY POPULATION ....................................................................................................... 28 
3.1 
Inclusion Criteria ............................................................................................................ 28 
3.2 
Exclusion Criteria ........................................................................................................... 28 
3.3 
Recruitment Process ....................................................................................................... 28 
3.4 
Co-Enrollment Guidelines.............................................................................................. 30 
3.5 
Participant Retention ...................................................................................................... 30 
3.6 
Participant Withdrawal ................................................................................................... 32 
4.0 STUDY TREATMENT ........................................................................................................ 32 
4.1 
Treatment Content .......................................................................................................... 32 
4.2 
Treatment Administration .............................................................................................. 33 
4.3 
Treatment Supply and Accountability............................................................................ 33 
4.4 
Adherence Assessment ................................................................................................... 33 
4.5 
Toxicity Management .................................................................................................... 34 
4.6 
HIV Seroconversion ....................................................................................................... 35 
4.7 
Active TB screening and diagnosis, TB exposure in infants ......................................... 35 
4.8 
Concomitant Medications .............................................................................................. 36 
5.0 
STUDY PROCEDURES ................................................................................................ 36 
6.0 
SAFETY MONITORING AND ADVERSE EVENT .................................................. 36 
6.1 
Safety Monitoring .......................................................................................................... 36 
6.2 
Adverse Event Definitions and Reporting Requirements .............................................. 37 
6.2.1 
Adverse Event ......................................................................................................... 37 
6.2.2 
Serious Adverse Event ............................................................................................ 38 
6.2.3 
Adverse Event Reporting ........................................................................................ 39 
7.0 
STATISTICAL CONSIDERATIONS .......................................................................... 40 
7.1 
Review of Study Design ................................................................................................. 40 
7.2 
Endpoints ........................................................................................................................ 40 
7.2.1 
Primary Endpoints .................................................................................................. 40 
7.2.2 
Secondary Endpoints .............................................................................................. 40 


Infant TB Infection Prevention Study (“iTIPS”)  
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7.2.3 
Exploratory Endpoints ............................................................................................ 41 
7.3 
Accrual, Follow-up, and Sample Size ............................................................................ 41 
7.4 
Random Assignment / Study Arm Assignment ............................................................. 42 
7.5 
Blinding .......................................................................................................................... 43 
7.6 
Data Analysis ................................................................................................................. 43 
7.6.1 
Primary Analyses .................................................................................................... 43 
7.6.2 
Secondary Analyses ................................................................................................ 44 
7.6.3 
Exploratory Analyses .............................................................................................. 47 
7.7 
Data Safety and Monitoring Board (DSMB) ................................................................. 48 
7.8 
Data Monitoring ............................................................................................................. 48 
8.0 
HUMAN SUBJECTS CONSIDERATIONS ................................................................ 49 
8.1 
Population to be enrolled and followed.......................................................................... 49 
8.2 
Study design ................................................................................................................... 49 
8.3 
Approvals ....................................................................................................................... 49 
8.4 
Informed consent ............................................................................................................ 50 
8.5 
Study Discontinuation .................................................................................................... 52 
9.0 
 LABORATORY SPECIMENS AND BIOHAZARD CONTAINMENT ................. 52 
9.1 
Laboratory Specimens .................................................................................................... 52 
9.2 
Quality Control and Quality Assurance Procedures ...................................................... 53 
9.3 
Specimen Storage and Possible Future Research Testing .............................................. 54 
9.4 
Biohazard Containment .................................................................................................. 54 
10.0 
ADMINISTRATIVE PROCEDURES .......................................................................... 54 
10.1 
Protocol Registration .................................................................................................. 54 
10.2 
Investigator's Records ................................................................................................. 55 
11. 
REFERENCES ................................................................................................................ 56 
12.0 
APPENDICES ................................................................................................................. 62 
I 
SCHEDULE OF STUDY VISITS AND PROCEDURES ........................................... 62 
II 
 ISONIAZID DOSING ................................................................................................... 72 
III 
 PYRIDOXINE DOSING ............................................................................................... 73 
IV 
SAE TABLES SPECIFIC TO PERIPHERAL NEUROPATHY ............................... 74 
V 
PARTICIPANT RETENTION OF PREVIOUS RCT STUDIES IN KENYA BY 
STUDY STAFF ........................................................................................................................... 76 
VI 
HAIR COLLECTION PROTOCOL FOR ANALYSIS OF INH EXPOSURE ....... 77 
VII 
COVID-19 RESPONSE PROCEDURES ..................................................................... 82 
 
 
 


Infant TB Infection Prevention Study (“iTIPS”)  
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LIST OF ABBREVIATIONS AND ACRONYMS 
 
 
AE 
 
 
adverse event 
AIDS  
 
Acquired Immunodeficiency Syndrome 
BCG  
 
Bacille Calmette Guerrin 
BMCs  
 
breast milk cells 
CDC  
 
Centers for Disease Control and Prevention 
DNA  
 
deoxyribonucleic acid 
DAIDS 
 
Division of AIDS 
DSMB   
 
Data Safety and Monitoring Board 
EAE 
 
 
expedited adverse event 
EC 
 
 
ethics committee 
FDA  
 
(United States) Food and Drug Administration 
HEU  
 
HIV-exposed uninfected  
HIV 
 
 
Human Immunodeficiency Virus 
HLA  
 
human leukocyte antigen 
IGRA  
 
interferon gamma release assays  
IPT 
 
 
isoniazid preventive therapy  
INH 
 
 
isoniazid 
IRB 
 
 
Institutional Review Board 
KRTC  
 
Kenya Research and Training Center 
LTBI  
 
latent tuberculosis infection  
LFT 
 
 
liver function test 
MCH  
 
maternal child health  
MOH  
 
Ministry of Health  
MTB  
 
Mycobacterium tuberculosis 
PBMCs 
 
peripheral blood mononuclear cell 
PCR 
 
 
polymerase chain reaction 
PMTCT 
 
prevention of mother to child transmission 
NIAID  
 
(United States) National Institute of Allergy and Infectious Diseases 
NIH 
 
 
(United States) National Institutes of Health 
QFT-Plus 
 
Quantiferon Plus 
RCT 
 
 
randomized controlled trial 
SAE 
 
 
serious adverse event 
SUSAR 
 
suspected unexpected serious adverse reaction 
TLR 
 
 
toll-like receptor 
TB 
 
 
tuberculosis  
TST 
 
 
tuberculin skin test 
UW 
 
 
University of Washington, Seattle 
 
 


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PROTOCOL TEAM ROSTER 
 
Grace John-Stewart MD, PhD 
Principal Investigator 
Department of Global Health 
University of Washington 
325 Ninth Avenue 
Box 359909 
Seattle, WA 98112 
gjohn@uw.edu 
(Tel) +1-206-543-4278 
 
John Kinuthia, MBChB, MMed, MPH 
Protocol Co-Chair 
Medical Specialist, Department of Obstetrics and Gynaecology, 
Head, Research and Programs 
Kenyatta National Hospital 
PO Box 20723, Nairobi, Kenya 
kinuthia@u.washington.edu 
(Tel) +254-722-799-052 
 
Thomas R. Hawn MD, PhD  
Immunology Principal Investigator 
Division of Allergy & Infectious Diseases 
Department of Medicine 
University of Washington 
750 Republican St. 
Box 358061 
Seattle, WA 98109 
thawn@uw.edu 
(Tel)  +1-206-616-4124 
 
Elizabeth Maleche-Obimbo MBChB, MMed, MPH, CPulm,  
Pediatric Clinical Tuberculosis Lead 
Department of Paediatrics and Child Health 
University of Nairobi 
Kenyatta National Hospital 
P.O. Box 19676, Nairobi 00202, Kenya 
eobimbo@hcc.or.ke 
(Tel) +254-202-720-947 
 
Lisa Cranmer MD, MPH, Co-investigator 
Division of Pediatric Infectious Disease 
Emory University School of Medicine 
2015 Uppergate Drive NE  
Atlanta, GA 30322  
lisa.cranmer@emory.edu 
(Tel) +1-404-727-2905 


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Sylvia LaCourse MD, MPH, Co-investigator 
Division of Allergy and Infectious Diseases 
Department of Medicine 
University of Washington 
325 9th Avenue  
Box 359931  
Seattle, WA 98104 
sylvial2@uw.edu 
(Tel) +1-206-616-7217 
 
Daniel Matemo MDL, HND, Research Administrator & Co-investigator 
Department of Obstetrics and Gynaecology,  
University of Nairobi, 
Kenyatta National Hospital,  
PO Box 19676, 00.00, Nairobi, Kenya 
daniel.matemo@uonbi.ac.ke 
(Tel) +254-722-322-378 
 
Barbara Richardson PhD Study Biostatistician & Co-investigator 
Department of Biostatistics 
Harborview Medical Center 
University of Washington 
Box 359909 
325 Ninth Avenue 
Seattle, WA 98104 
barbrar@uw.edu 
(Tel) +1-206-744-2425 
 
 
 
COLLABORATING INSTITUTIONS 
Kenyatta National Hospital 
University of Nairobi 
University of Washington 
Kenya Medical Research Institute (KEMRI) 
 
FUNDING AGENCY 
Funding type: Grant 
Name of Funding agency: Thrasher Research Fund 
Principal Investigator on Proposal: Grace John-Stewart 
Proposal Identification Number: N/A 
Title of Proposal: Preventing Mycobacterium Tuberculosis Infection in HIV-Exposed Infants 
Dates: 01/Sept/2015 – -31/Aug/2020 (no-cost extension) 
 
 


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SCHEMA 
 
Purpose:  
To determine whether isoniazid (INH) reduces the risk of Mycobacterium 
tuberculosis (MTB) infection in HIV-exposed but uninfected (HEU) 
children. To determine the epidemiologic and immunologic correlates of 
MTB infection in HEU. 
 
Design: 
 
Randomized controlled trial (RCT) of INH vs. no INH with nested sub-
studies to evaluate epidemiologic and immunologic correlates of MTB 
infection, and to explore the role of INH in prevention of active TB and 
mortality.  
 
Study Population: 
HIV-exposed uninfected (HEU) infants and their mothers 
 
Study Size: 
300 infants, 150 per arm   
 
Treatment Regimen: Isoniazid (INH) ~10 mg/kg (7-15 mg/kg), will be administered once daily 
to infants in INH arm for 12 months.   
 
Study Duration: 
3 years (12 months of follow-up for each participant) 
 
Primary Objectives:  
 
• AIM: 1 Among HEU infants enrolled at approximately 6 weeks of age, compare the risk of 
acquiring MTB infection during 1 year of follow-up in infants randomized to receive INH vs. 
no INH using an IGRA assay to determine MTB infection status. 
 
Secondary Objectives:  
 
● AIM 2: Determine epidemiologic correlates of MTB infection among infants enrolled in the 
RCT. 
 
• AIM 3: Determine immune correlates of risk of primary MTB infection and their potential 
interactions with INH. Assays will include infant peripheral blood BCG-specific T-cell 
responses at approximately 6 weeks post BCG vaccination, and maternal breast milk and 
peripheral blood MTB-specific T-cell responses at approximately 6 weeks postpartum. 
 
Exploratory Objectives: 
 
● Investigate the impact of IPT on a combined endpoint of MTB infection, TB disease, and death 
among HEU infants 
 
Study Sites: 
Maternal Child Health (MCH) clinics in Western Kenya (Kisumu County 
Hospital, Jaramogi Oginga Odinga Teaching and Referral Hospital, 
Lumumba Sub-County Hospital, Ahero and Bondo). 
 
 


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Figure 1: Aims of RCT to evaluate INH to prevent MTB infection in HEU infants 
 
 
 
BCG 
Aim 1:  RCT of IPT 
in HEU infants 
Infected M  
Aim 3:  Discover immune correlates of 
risk of MTB infection
MTB 
M FATE
KILLED 
GROWS
MTB
INFECTION 
NO 
HIV 
T cell
Randomize to 
INH vs  no INH 
MTB Exposure 
Risk
YES 
12 months 
Aim 2:  Discover 
epidemiologic risk 
factors
birth 
in utero
6 weeks
(ongoing)


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Screening and Enrollment Visit 
6+/- 4 weeks of age 
-Obtain informed consent 
-Determine Eligibility 
-Assess baseline condition 
Randomized N=300 
Enrollment 
12 month after enrollment visit 
-infant blood collection,6 TST placement 
-infant hair collection7 
6 weeks of age visit 
-maternal breastmilk collection1 
--maternal blood collection1 
-infant blood collection1 
-infant stool collection2 
Follow-up visits (10 weeks, 14 weeks, 6 
months, 9 months, 12 months of age, and 
12 months post-randomization) 
 -maternal/infant TB symptom screen, active 
TB screening3, assess TB exposures4 
-assess infant morbidity including SAEs 
using standardized questionnaire3 
-infant blood collection3 (10 weeks of age) 
 
12 month after enrollment visit 
-infant blood collection,6 TST placement 
Control Arm 
No INH N=150 
Experimental Arm 
INH N=150
6 weeks of age visit 
-initiate INH, pyridoxine 
-maternal breastmilk collection1 
-maternal blood collection1 
-infant blood collection1,2 
-infant stool collection3 
Allocation 
Follow-up visits (10 weeks, 14 weeks, 6 
months, 9 months, 12 months of age, and 
12 months post-randomization) 
-maternal/infant TB symptom screen, active 
TB screening4, assess TB exposures5 
-assess infant morbidity including SAEs 
using standardized questionnaire  
-assess adherence via questionnaire, urine 
INH testing 
-infant blood collection2,3  (10 weeks of age) 
Follow-Up 
OVERVIEW OF STUDY DESIGN AND RANDOMIZATION SCHEME 
 
 
 
 
Figure 2: Study schema 
1Aim 3: Immunologic correlates of MTB infection objective  
2LFT in INH arm to monitor for SAEs 
3Sample collection for future exploratory aims (infant gut microbiome, role of infant antibodies and infant MTB infection, role of IPT on BCG response) 
4Exploratory aim (active TB)  
5Aim 2: Epidemiologic correlates of MTB infection objective 
6Aim 1: Blood drawn for IGRA to ascertain MTB infection status at 12 months 
7for INH levels 
Excluded  (n=   ) 
   Not meeting inclusion criteria (n=  ) 
   Declined to participate (n=  ) 
   Other reasons (n=  ) 


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1.0 
INTRODUCTION 
1.1 
Background and Significance 
Pediatric Tuberculosis (TB) Burden and Pathogenesis: Pediatric TB represents a major cause 
of childhood morbidity and mortality worldwide.1,2 In a recent model from 22 high burden 
countries, it was estimated that in 2010 7.6 million children <15 years of age had Mycobacterium 
tuberculosis (MTB) infection, of whom over 650,000 developed active TB.3 Children have 
different disease presentation than adults; with paucibacillary disease, rare development of 
cavitation, and more frequent miliary disease and TB meningitis.4-7 Children have a higher rate of 
progression from infection to active disease than adults (50% <1 year, 20-30% at years 1-2, vs. 
10-20% >10 years).6,8 Pediatric TB disease occurs soon after primary exposure to MTB without 
pre-existing adaptive immune responses, and both innate and early adaptive immune responses 
may influence susceptibility.4-7 Virtually all childhood TB disease reflects primary disease, 
whereas a significant portion of adult disease is due to reactivation of latent TB infection (LTBI).2 
 
TB Risk and Outcomes in HIV-Infected and HIV-Exposed Uninfected (HEU) Children: In 
adults and children MTB infection is correlated with likelihood and intensity of exposure to an 
infectious TB case. Children living with HIV-infected household members are at increased risk of 
TB exposure.9 Among children, accelerated progression from latent TB infection to active TB 
disease is associated with immunosuppression and younger age.10 Kenya is one of 22 high TB 
burden countries with a generalized TB epidemic affecting young adults. A large observational 
study from AMPATH in Western Kenya longitudinally estimated TB disease incidence in HIV-
infected children and noted a staggeringly high 17.1% annual incidence of active TB in a cohort 
with a median age of 1.0 year at enrollment.11 TB disease prevalence was also high at 3.6% on 
enrollment.11 Other studies demonstrated similarly high rates of TB in HIV-infected children.12-14 


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In a South African trial, which excluded infants with known household TB exposure, 12.6% of 
HIV-infected infants developed protocol-defined TB disease, and among HEU infants, 7.4% 
developed TB disease.15 Despite exclusion of infants with known household TB exposure, risk of 
TB disease in HIV-infected and HEU infants is high, reflecting substantial community exposure 
to TB.  
 
Isoniazid Preventive therapy (IPT) and TB: IPT has been used since the 1960s to treat latent 
tuberculosis infection and prevent progression to active TB disease. The famous Bethel, Alaska 
study was a household randomized trial with 6064 individuals randomized to placebo versus IPT. 
The NH recipients had a 55% reduction in active TB disease incidence with a benefit that persisted 
for >19 years.16,17 Other studies have found similar levels of efficacy to prevent active TB.18 
Despite this demonstrated efficacy of INH to prevent active TB disease, a recent cluster-
randomized trial of INH treatment for latent TB infection in adults in the South African gold mines 
did not demonstrate efficacy to prevent active TB.19 Variable efficacy of IPT has also been 
observed in children. In a randomized trial of IPT in HIV-infected without reported TB exposure 
(N=548) and HEU (N=804) infants (enrolled at 91-120 days of life) in South Africa and Botswana, 
INH (given for 96 weeks) did not prevent TB disease in either group after 96-108 weeks of follow 
up.20 Furthermore, in the HEU group, INH did not prevent MTB infection as measured by a single 
tuberculin skin test (TST) at week 96. In contrast, an RCT in South Africa randomized HIV-
infected children >8 weeks (N=263) to INH vs. placebo (independent of reported TB exposure) 
and found that INH prevented TB disease and decreased mortality.15 However, MTB infection was 
not assessed as an endpoint. In summary, IPT is partially effective in adults and variably 
effective for preventing TB disease in HIV-infected and HEU children. The reasons for the 
partial and variable efficacy of IPT are not known. A recent study from Botswana indirectly 


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suggests that IPT may prevent MTB infection among HIV-infected adults.21 In this study, TST 
negative HIV-infected adults were demonstrated to receive benefit from IPT in prevention of 
active TB, suggesting a potential effect of IPT in preventing acquisition of MTB infection as well 
as prevention of progression to TB disease among those already with MTB infection. The effect 
of IPT on preventing MTB infection has only been addressed in only one pediatric study with use 
of a single TST as an endpoint.20 The lack of baseline measurement of MTB infection in these 
studies means that MTB infection identified at study endpoint could represent either prevalent 
MTB infection acquired prior to IPT provision or incident infection occurring after IPT initiation. 
Because interferon gamma release assays (IGRA) offer higher specificity and potentially 
higher sensitivity for detection of MTB infection in the presence of recent Bacille Calmette 
Guerrin (BCG) vaccine, it is plausible that using IGRAs as an endpoint would enhance 
ability to detect potential preventive effect on MTB infection.  
 
BCG Efficacy in HIV-Infected and HEU Children: BCG vaccine has been used in humans since 
1921 and administered to >1 billion people globally, including millions of infants. Meta-analyses 
have noted benefits in preventing pediatric disseminated and meningeal TB disease.22,23 However, 
estimates of BCG efficacy are highly variable and may be influenced by BCG strain, 
environmental mycobacteria, or host factors.24 Until recently, it was believed that BCG did not 
prevent primary MTB infection. Intriguingly, over the past decade, an emerging body of 
evidence suggests that, in fact, BCG may prevent primary MTB infection. In several 
retrospective studies comparing TB-contacts with and without prior BCG, those with BCG were 
less likely to have latent TB infection as detected by IGRA. A recent meta-analysis demonstrated 
an overall protective efficacy of 19% among 14 studies and 3855 participants.25 New prospective 
studies of BCG immune responses and influence on MTB infection could reveal key BCG 


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protective immune phenotypes. Incidence of MTB infection in HEU infants as measured by IGRA 
is ~10-20% during the first year of life, providing sufficient statistical power for immunologic 
studies comparing early BCG-specific T-cell responses in infants who do versus do not develop 
MTB infection.26 
 
Impact of HIV Exposure on Mortality & Immune Response to BCG and MTB: In comparison 
to unexposed infants, HEU infants have higher overall mortality and an altered immune response 
to BCG vaccination and infection from several pathogens.27-32 The altered immune response 
includes increased T-cell proliferation in response to BCG, but decreased polyfunctionality of T-
cell responses to both BCG and Bordetella pertussis.27 Although these studies indicate that 
BCG-induced immune responses are altered in HEU infants, no studies have addressed 
whether BCG-induced immune responses are associated with a clinically relevant endpoint 
such as MTB infection or TB disease.33-39 
 
The Innate Immune and the Macrophage Response to MTB in HEU: From recognition to 
killing, the macrophage plays a central role in MTB pathogenesis.40-54 The quality or function of 
early, non-specific innate immune responses in HEU children could be influenced by a hyper 
inflammatory intrauterine milieu and also affect the immune response to BCG vaccination. Few 
longitudinal studies have been performed that measure innate, macrophage, or adaptive immune 
responses in HEU before MTB infection. 
 
Absence of an Efficacious TB Vaccine and New Strategies: Although vaccination with BCG 
offers some protection against childhood TB disease and possibly protection against adult MTB 
infection, its efficacy is not adequate for disease control and the correlates of protection are not 


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known. Development of a more effective vaccine is a global priority and depends on a thorough 
understanding of the host response to MTB infection. Given rapid progress in the field of innate 
immunity, new generation vaccine adjuvants are becoming available to stimulate tailored immune 
responses.55-57 To strategically inform TB preventive studies, molecular epidemiology studies are 
important to: 1) identify immunologic factors that increase risk of MTB infection; and 2) lead to 
innovative strategies for immune modulation through drugs or vaccines based on insights into the 
mechanisms of immune response identified. 
 
1.2 
Innovation 
 
Our project has several innovative features that include:  
HIV-TB Exposure and Co-Infection Research Infrastructure in Kenya with Longitudinal 
Cohorts: Our investigative team is uniquely poised to examine INH and MTB infection at Kenyan 
sites with experience in conducting epidemiologic, immunologic, and genetic studies for >25 
years. Importantly, these studies included HEU infants with serial peripheral blood mononuclear 
cell (PBMC) banking and immunologic analyses, including maternal infant TB IGRA studies.  
Examination of Pediatric MTB Infection in HEU Children: The population of HEU infants is 
growing as PMTCT programs succeed in preventing mother-to-child HIV transmission, and HEU 
infants have high risk of TB.  
Immune Profiling: We propose to examine mechanisms of immunity to MTB with multi-
parameter flow cytometry immunologic techniques.  
Evaluation of BCG-Induced Immune Responses in HIV-Exposed Infants and Correlates of 
Risk of MTB Infection: By measuring immune responses to BCG vaccination after perinatal 
exposure to HIV, we have an opportunity to examine the immune response to a standardized in 


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vivo stimulus AND determine whether these responses are associated with developing MTB 
infection. Previous studies have documented that HIV exposure alters infant immune responses to 
vaccination, but have not correlated these responses with an important longitudinal outcome such 
as MTB infection. 
IPT & Prevention of MTB Infection: Currently, we do not know why IPT has variable and partial 
efficacy in children. A prospective birth HEU birth cohort can provide an efficient approach to 
probe this question. 
Infant gut microbiome and risk of MTB infection and BCG response: We will also be 
collecting stool samples for cryopreservation for potential future studies evaluating the relationship 
between infant gut microbiome and risk of MTB infection and BCG response. 
Infant antibodies and risk of MTB infection and BCG response: We will also be collecting 
infant PBMC and plasma samples for cryopreservation for potential future studies evaluating the 
relationship between titer and effector function of infant mycobacterial antibodies and to evaluate 
if early use of isoniazid preventive therapy modifies infant innate and adaptive immune responses 
to BCG.  
INH metabolism and acetylation transferase 2 (NAT2) status: INH is primarily metabolized 
in the liver primarily through acetylation of NAT2, which is further converted by oxidation by 
cytochrome P450 2E1 to hepatotoxic metabolites. Genetic polymorphisms of NAT2 can 
associated with fast, slow, and intermediate phenotypes of acetylation. Fast acetylators can 
convert approximately 90% of INH to acetylisoniazid compared to 67% among slower 
acetylators.  The relationship between acetylation status and INH hepatotoxicity is not clear. 
Initially it was assumed that rapid acetylators may have higher risk of hepatoxicity due to greater 
conversion of INH to hepatotoxic metabolites.  However, in some series, slow acetylators have 


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higher risk of hepatotoxicity. Acetylation status could potentially affect the efficacy of INH in 
terms of Mtb infection prevention. Faster clearance of INH could be associated with lower 
protection from Mtb infection. We will use already collected samples to ascertain NAT2 
polymorphisms. 
Hair analysis as an objective measure of INH exposure: Many drugs are incorporated from the 
systemic circulation into hair as it grows, and the concentration of medications in hair reflects drug 
uptake from the systemic circulation over weeks to months.58 Our collaborators have developed 
methods to extract and analyze prevalent-use ARVs from hair, and demonstrated hair 
concentrations of ARVs are stronger predictors of treatment outcomes compared to self-reported 
aherance.59-62 Only a small thatch of hair is required (approximately 30 strands), and rates of 
acceptability and feasibility of collecting hair samples for hair ARV monitoring in African and 
Asian settings have been high  (>95%).63,64 They have recently expanded their hair analysis 
expertise to assess INH concentration in hair,65 including among children initiating TB treatment.66 
The assay has been validated over the linear dynamic range of 0.5–100 ng INH/mg of hair utilising 
20–30 strands of human hair (~1–3 mg). Unlike phlebotomy, hair collection is noninvasive and 
does not require specific skills, sterile equipment, or specialized storage conditions.60 The 
avoidance of phlebotomy in assessing drug adherence may be particularly desirable in pediatric 
populations.64,66 Hair sample collection merely requires a pair of scissors and storage is at room 
temperature. Additionally segmental analysis of hair samples allows for the assessment of 
adherence at various time points over the past few months since distance along the hair shaft serves 
as a marker of time.67 Drug levels in hair can provide a more objective measure of adherance than 
self-report alone,59,64,68 and information regarding adherance over longer time periods without the 
collection and storage issues associated with plasma, PBMCs, or dried blood spots.69-72 


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1.3 
Supportive Preliminary Data 
UW-Kenya Research Training Center (KRTC): Our UW-KRTC has successfully conducted 
collaborative HIV research in women and children for >25 years. This has included enrolment of 
>3,000 mother-infant pairs in longitudinal studies, numerous pediatric cohorts with detailed 
virologic and immunologic data, and studies of genetic markers including toll-like receptors 
(TLRs) and human leukocyte antigens (HLA) and their influence on HIV transmission and 
progression. Cohorts have had excellent retention (>90%), serial clinical evaluation by study 
pediatricians, and storage of PBMCs and DNA for molecular epidemiology studies. Studies from 
UW-KRTC have had translational impact in defining HIV transmission epidemiology and 
pathogenesis and have yielded >500 publications on HIV transmission or progression in high 
impact journals including JAMA, Lancet, J Infectious Diseases, New England Journal of Medicine, 
AIDS, and Clinical Infectious Diseases. Our team includes clinical researchers, immunologists, 
virologists, and molecular epidemiologists, with a focus on bench-to-bedside translational 
research. 
 
 


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Prospective Studies of TB IGRAs In HIV-Infected Mother-Infant Cohort In Kenya Using 
Repository: Among 333 HIV-infected pregnant 
women, we utilized cryopreserved PBMCs to 
conduct IGRAs and detected positive TB-specific 
IGRA responses in 42.7% of women. Women with 
positive IGRAs had significantly higher baseline 
median CD4 cell count (478 vs. 396 cells/mm3, 
p=0.03). Positive T.SPOT.TB IGRAs were associated with increased likelihood of subsequent 
active TB (aOR 4.8 95%CI 1.2-19.7, p=0.03) and with infant TB or mortality.73 Serial assays 
during pregnancy showed modest decline in magnitude of responses during pregnancy with stable 
responses postpartum.74 Among 6-month old infants born to HIV-infected mothers, we noted 
that 10.9% of 128 infants were IGRA positive.26 This suggests a cumulative incidence of TB 
infection of 20.6% among HIV-exposed infants.26 
These studies demonstrate high prevalence of latent TB 
among HIV-infected women in Kenya and high 
incidence of MTB infection and disease among infants 
born to HIV-infected women during the first year of 
life. While infants had lower phytohaemagglutinin 
(PHA) 
responses, 
representing 
lower 
mitogen 
responses than mothers, MTB-specific responses were 
of comparable magnitude in infants and mothers.  
MTB-specific IFN-γ breast milk responses: We 
recently examined MTB-specific T-cell responses in 
breast milk of HIV-infected mothers using the T-
1
10
100
1
10
100
1000
1
10
100
1000
1
10
100
1000
Log10 SFCs in PBMCs 
Log10 SFCs in PBMCs 
Log10 SFCs in BMCs 
Log10 SFCs in BMCs 
PBMCs
BMCs
PBMCs
BMCs
0
50
100
200
400
600
800
IFN-g SFCs per 2.5 x 105 cells
ESAT-6
CFP-10
p=.02 
p=.02 
Key 
A. 
B. 
r=.91 
p=.005 
r=.86 
p=.01 
Figure 2: Magnitude and correlation of IFN- response 
to ESAT-6 and CFP-10 in Maternal BMCs and PBMCs. 
The T-SPOT.TB assay was performed on maternal 
PBMCs and BMCs. A. SFCs per 2.5 x 105 cells in 
response to antigens ESAT-6 (closed circles) and CFP-
10 (open circles) are shown after subtraction of 
background in the nil control. Spearman’s correlation of 
BMC and PBMC IFN- responses to ESAT-6 (closed 
circles) and CFP-10 (open circles) were assessed. 


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SPOT.TB IGRA.75 HIV-infected women in Nairobi, Kenya were enrolled during pregnancy in 
2002 and mother-infant pairs followed monthly for one year postpartum.76 Breast milk and 
peripheral blood were collected at 1 month postpartum and breast milk cells (BMCs) and PBMCs 
were isolated and cryopreserved. Among 7 mothers with paired breast milk and blood assays, 
MTB-specific IFN- responses were higher in breast milk compared to blood (Fig. 2). The 
magnitude of IFN- responses in maternal breast milk and blood were correlated. Together, these 
data suggest that MTB-specific T-cell responses exist in BMCs. We will test whether these 
maternal responses are associated with protection from infant MTB infection in the current 
proposal. 
Active TB Screening in HIV-Infected Mothers: Our study team, in collaboration with Drs. 
David Horne and John Kinuthia, has also established studies to screen mothers for active TB using 
maternal WHO symptom screening, culture, AFB microscopy, GeneXpert, and urine LAM 
(LaCourse, Cranmer, Horne, IJTLD Barcelona 2014, IDSA 2014). During a one-year period 
between July 2013 to July 2014, 306 HIV-infected women were enrolled at the Bondo and Ahero 
Maternal Child Health (MCH) sites, of which 288 had at least one adequate sputum culture. The 
median age was 26 years and 9% reported prior TB disease. Prevalence of culture-confirmed 
pulmonary TB was 2.4% (95% CI 0.98-4.9%) among the 288 women, irrespective of symptoms. 
Correlates of culture confirmed TB included cough >2 weeks (OR 8.9, 95% CI 1.6-51), household 
member with positive WHO TB symptom screen (OR 23, 95% CI 4.4-116), and TST >5 mm (OR 
7.1, 95% CI 1.4-37). Overall, the sensitivity of symptom screen (43%) smear (0%), Xpert (43%), 
and LAM (0%) for pulmonary TB were low compared to culture. Among women with TST placed 
and who returned for reading, 12% were positive (95% CI 8-17%). Correlates of latent TB included 
age (OR 1.8 per 5 years, 95% CI 1.2-2.6), employment outside the home (OR 2.6, 95% CI 1.1-


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6.3), and prior TB disease (OR 7.8, 95% CI 2.9-21). This study illustrates the persistent burden 
of maternal and household TB to which HEU children are exposed.  
  
BCG-induced T-cell Responses in Infants Vaccinated 
with BCG: We and others previously discovered and 
characterized common TLR1, TLR5, and TLR6 non-
synonymous coding region polymorphisms which 
regulate IL-6 secretion in monocytes after receptor 
stimulation.46,77-82 These polymorphisms genetically 
define TLR1, TLR5, TLR6, and CD1A-deficient 
individuals.83 These data illustrate that common innate 
immune deficiencies exist and can be used to examine the role of these genes in regulation of 
human innate and adaptive immune responses. We currently collaborate with Dr. Thomas Scriba 
at the South African TB Vaccine Initiative at the University of Cape Town (consultant on this 
grant) who is an expert on examining the immune response to BCG and MTB. Dr. Scriba and 
SATVI investigators (originally Drs. Hanekom, Hussey, Mahomed; currently Drs. Scriba and 
Mark Hatherill) established a study to discover BCG-induced immune correlates of risk for 
developing TB disease. A cohort of infants were vaccinated at birth with BCG (N=5650), had 
blood drawn at 10 weeks of age which was stimulated with BCG, and were followed for 2 years 
to determine who developed TB disease.84,85 We examined whether genetic variation in the innate 
immune response was associated with BCG-induced T-cell immune responses. We discovered and 
published that individuals who are deficient in TLR1/6 signaling in myeloid cells have increased 
TH1-type T-cell responses after in vivo BCG vaccination.86 To our knowledge, this was the first 
description of polymorphisms in innate pathway genes that affect the adaptive response to in vivo 
 
Figure 3:  BCG-induced T-cell Responses 
in South African Infants.  A whole blood 
cytokine assay was performed on 10 week old 
infants as described in the text.  An 11 color 
flow cytometry panel with intracellular cytokine 
staining was performed with gating on CD4 
and CD8 cells (A).  Representative IL-2 & IFN-
 ICS plots depicted (B, C) along with 
frequencies of the CD4 and CD8-specific 
cytokines in 95 samples (D,E with median and 
IQR  plotted). 
IFN-
CD-4
CD-8
IFN-
BCG CD8+ T cell Memory
IL2
IFNg
TNF
IL17
IL22
0.001
0.01
0.1
1
10
% Cytokine
BCG CD4+ T Cell Memory
IL2
IFNg
TNF
IL17
IL22
0.001
0.01
0.1
1
10
% Cytokine
IL-2
IL-2
B. Media
C. BCG
A. 
D. 
E. 


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vaccination against a bacterial pathogen in humans. We recently extended these studies to examine 
whether innate immune variation is associated with a broader repertoire of T-cell cytokine 
responses measured by intracellular cytokine staining. Using an 11-color flow panel (CD3, CD4, 
CD8, CD14, IFN-γ, IL-2, TNF, IL-4, IL-22, IL-17, CD154), we measured BCG-induced T-cell 
responses in blood samples obtained 10 weeks after vaccination. Frequencies of various BCG-
specific cytokines are depicted in Figure 3. We are currently examining whether innate immune 
gene variants are associated with these T-cell responses. We are examining which genes regulate 
macrophage responses to MTB infection, by knocking down gene expression with siRNA, 
infecting macrophages with live MTB, and measuring binding, uptake, phagosome maturation, 
cytokine secretion by ELISA and replication.46,77,78,81,86,87 Together, these data demonstrate that 
BCG-induced T-cell responses are detectable with a variety of techniques currently in use in our 
laboratory that will be used within the proposed project. 
Summary of Preliminary Data: We have established a collaborative research site in Kenya that 
has been productive for >25 years with studies of HIV in women and children. We (GJS) 
broadened our research scope to include studies of TB over the past 5 years and have documented 
high rates of MTB infection in infants during the first year of life, MTB-specific IFN-γ breast milk 
responses, and potential TLR9 variants associated with MTB-specific T-cell responses. We (TRH) 
have also examined macrophage responses to MTB infection and the role of the innate immune 
response to BCG vaccination in other cohorts. Drs. John-Stewart (expertise in HIV epidemiology 
& clinical trials with women and children) and Hawn (expertise in innate immunity & 
immunogenetics of BCG vaccine responsiveness) have initiated collaborative studies to 
investigate the role of HIV and MTB during the immune response to BCG vaccination. 


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1.4 
Rationale 
HIV-exposed uninfected infants (HEU) in HIV/TB endemic settings have a high risk of MTB 
infection and TB disease, even in the absence of known MTB exposure. Because infancy is a time 
in which there is rapid progression from primary to active TB, it is important to define where, how, 
and when TB preventive interventions exert their effect and to build new strategies that adapt or 
extend approaches used in adults. Protecting HEU infants during this vulnerable, yet temporary, 
period of immunodeficiency may provide long term immunologic and mortality benefits. The 
primary goal of this proposal is to determine whether INH prevents primary MTB infection 
in HEU infants. Additionally we will examine cofactors of primary MTB acquisition in the 
first year of life, and examine the role of immune protective mechanisms in this cohort.  
Among HIV-infected infants, 2 randomized control trials (RCTs) yielded conflicting data about 
whether IPT prevents TB disease and/or mortality. Only one of these evaluated HEU infants, and 
found no protective effect of IPT in decreasing TB disease. While previous IPT RCTs have focused 
on prevention of active TB disease, there are scant data regarding the impact of IPT on primary 
MTB infection. We recently found that, in 6-month old HEU infants, over 10% had evidence of 
MTB infection as detected by IGRAs, corresponding to a 20% annual cumulative incidence of 
infection. This suggests that HEU infants have a substantial incidence of MTB infection related to 
community and household TB exposure. There are no published prospective longitudinal studies 
of evaluating the role of INH to prevent MTB infection among HEUs using IGRA testing. Unlike 
TSTs, IGRAs can detect MTB infection and distinguish it from immune response to recent BCG 
vaccination. A prospective birth HEU cohort using IGRAs to detect MTB infection can provide 
an efficient approach to probe determinants of MTB infection, more rapidly accruing endpoints 
(MTB infection) than studies of TB disease and this study design can contribute unique insights 
regarding mechanisms of prevention of primary MTB infection.  


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Current World Health Organization (WHO) guidelines recommend that all HIV-infected adult and 
adolescents living with HIV should be screened for TB with a clinical algorithm and those who do 
not report any one of the symptoms of current cough, fever, weight loss or night sweats are unlikely 
to have active TB and should be offered IPT.88 Children with HIV >12 months of age with who 
are unlikely to have active TB based on symptom-based screening, and have had no contact with 
a TB case should receive six months of IPT (10 mg/kg/day) as part of a comprehensive package 
of HIV prevention and care services as well. However the current WHO recommendations for IPT 
do not recommend routine IPT for children <12 months of age with HIV due to the previously 
mentioned conflicting data in children < 12 months, and remain silent regarding the role of IPT in 
HIV-exposed but uninfected children. Given there is equipoise in whether INH prevents MTB 
infection, and whether it would prevent MTB infection specifically in among HEU children, a 
RCT design would provide important information regarding the efficacy of INH in preventing 
MTB infection in this population. 
2.0 STUDY OBJECTIVES AND DESIGN 
2.1 
Primary Objectives 
• AIM 1: Among HEU infants enrolled at approximately 6 weeks of age, compare the risk of 
acquiring MTB infection during 1 year of follow-up in infants randomized to receive INH vs. 
no INH using an IGRA assay to determine MTB infection status. 
2.2 
Secondary Objectives 
• AIM 2: Determine epidemiologic correlates of MTB infection among infants enrolled in the 
RCT. 
• AIM 3: Determine immune correlates of risk of primary MTB infection and their potential 
interactions with INH. Assays will include infant peripheral blood BCG-specific T-cell 


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responses at approximately 6 weeks post BCG vaccination, and maternal breast milk and 
peripheral blood MTB-specific T-cell responses at approximately 6 weeks postpartum. 
2.3 
Exploratory Objectives: 
● Investigate the impact of INH on a combined endpoint of MTB infection, TB disease, and 
death among HEU infants. 
2.4 
Study Design 
2.4.1 Participating Study Sites: 
This study will be conducted in our collaborative maternal child health (MCH) research sites in 
western Kenya (Kisumu County Hospital, Jaramogi Oginga Odinga Teaching and Referral 
Hospital, Lumumba Sub-County Hospital, Ahero, and Bondo). We have enrolled pregnant HIV-
infected and uninfected women and their infants in longitudinal studies at these sites for more than 
4 years. The study sites are embedded in the public sector routine MCH clinics. We have 
collaborated with CDC-Kenya Medical Research Institute (KEMRI) for TB microbiologic and 
IGRA studies at these sites for more than 4 years. HIV-infected mothers in Kenya are followed as 
part of the national PMTCT program and currently receive Option B+ triple antiretroviral therapy. 
Rates of mother-to-child 
HIV transmission at 6 
weeks of age range from 
<1 to 10% in public MCH 
clinics 
that 
implement 
PMTCT screening and 
antiretroviral 
therapy 
administration. A recent 
Figure 4: Overall Study Strategy 
Study Design: 
Non-blinded randomized control trial 
Intervention: 
Intervention: Infant INH for 12 months  
Control group: No INH 
Primary 
Outcomes: 
Aim 1: MTB infection in HEU infants at 12 months post 
enrollment as measured by IGRA (QFT-Plus) 
Aim 2: Epidemiologic correlates of infant MTB infection 
Aim 3: Immunologic correlates of  infant MTB infection 
Population: 
HEU infants ~6 weeks of age and their HIV-infected mothers  
Exclusions: 
• Infants with known exposure to active TB in household 
• Positive HIV DNA at 6 weeks 
• Premature and/or < 2.5 kg 
Target 
enrollment: 
300 HEU infants and their HIV-infected mothers  
(150 each arm) 
Sampling 
framework: 
Consecutive enrollment of HEU infants and their HIV-
infected mothers at MCH/PMTCT clinics, Nyanza region of 
Western Kenya 


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estimate from Western Kenya of MTCT was 3% at 6 weeks of age. Our collaborative research 
team has conducted studies in Western Kenya sites for over 4 years and has extensive experience 
in recruitment of pregnant women and children into HIV research studies including RCTs with 
high rates of retention (Appendix V). Sites have defined TB referral clinics on the same campuses 
of the MCH clinics for women and children with suspected active TB.  
2.4.2 Schedule of Study Visits and Procedures: 
The study population will consist of HIV-exposed infants 6 weeks of age (within +/- 4 weeks), not 
premature and over 2.5 kg and their HIV-infected mothers. Infants with known exposure to 
household contacts with active TB at enrollment will be excluded from participation. In routine 
clinical care in Kenya, the majority of infants, including HEU infants, receive BCG vaccination 
(Tubervac-SII Russia strain, Serum Institute of India) at birth through the national immunization 
program. Documentation of BCG vaccination is provided on routine MCH immunization cards. 
Infants and mothers are then seen at routine postnatal visits, including at approximately 6 weeks 
postpartum. The proposed study will enroll and randomize infants to INH versus no INH at or 
close to the 6-week postpartum visit. Infants will be followed longitudinally for one year with 
clinical follow-up to assess for development of MTB infection. For the efficacy study and endpoint 
determination, 5 ml blood will be drawn at 12 months following enrollment to determine infant 
MTB infection by an IGRA assay. QuantiFERON-TB Gold (QFT) is a specific IGRA which 
measures the amount of interferon-gamma (INF-γ) released by primarily CD4+ T helper 
lymphocytes after stimulation with TB-specific antigens (ESAT-6, CFP-10 and TB7.7) to measure 
MTB infection. We will also assess of the presence of MTB infection at the time of TB diagnosis 
of disease using QFT-Plus (same assay used to identify MTB infection status for the primary 
endpoint) and TST.  Recently developed, QuantiFERON-TB Gold Plus (QFT-Plus) measures INF-


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γ released by CD8+ cytotoxic T lymphocytes as well, after stimulation with the same TB-specific 
antigens, which may have increased sensitivity in populations with lower CD4 counts including 
HIV.89 In the event blood collected is insufficient blood volume (<4ml) for QFT-Plus, the blood 
collected be processed for a different test for M. tuberculosis infection. TST will also be placed 
when is blood is drawn as an additional measure of M. tuberculosis infection status. Infants will 
be screened at scheduled study visits that correspond with the Kenyan MOH visits (10 and 14 
weeks of age, 6, 9, and 12 months of age) for any new known TB contacts, development of SAEs 
as well as symptoms concerning for active TB disease as part of our secondary and exploratory 
objectives. Liver function tests will be drawn at 6 and 10 weeks of age (at baseline and 1 month 
after INH initiation in the INH arm). For the immunologic studies, 5 mls of infant blood will be 
obtained at enrollment (approximately 6 weeks after BCG vaccination) and 1 month post-
enrollment (10 week of age visit) as well as 30 mls of maternal breast milk and 5 ml of maternal 
blood on enrollment. Additionally we will collect stool samples from infants at enrollment. We 
will collect hair samples from children in the INH arm at the study endpoint visit 12 months post-
randomization to measure INH exposure. We will measure infant immune responses in peripheral 
blood and maternal immune responses in peripheral blood and breast milk as outlined below.  Our 
primary analytic goal of these immunologic exploratory analyses is to determine which of these 
responses are associated with acquisition of MTB infection during 1 year of follow-up.  
Extended follow-up at 24 months of age: After completion of the primary trial, participants will 
be invited to return for an extended follow-up visit at 24 months of age for a blood draw and 
TST placement. 5-10 mls of blood will be obtained and will be used to determine MTB infection 
status and measure infant immune responses. Standardized questionnaires used during the parent 
trials will be used to evaluate interim infant and maternal health at the extended follow-up visit. 


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3.0 STUDY POPULATION 
HIV-exposed uninfected (HEU) infants and their HIV-infected mothers will be included in this 
study. Participants will be selected for the study according to the criteria in Section 3.1 and 3.2 
[and the guidelines in Section 3.4]. They will be recruited, screened, and enrolled as described in 
Section 3.3 [and assigned to the intervention or control group as described in Section 7.4]. Issues 
related to participant retention and withdrawal from the study are described in Sections 3.5 and 
3.6, respectively. 
3.1 
Inclusion Criteria 
HEU infants who meet all of the following criteria are eligible for inclusion in this study: 
● 
Aged 6 weeks within (+/- 4 weeks) 
 
● 
Born to HIV-infected mothers 
 
● 
Not premature and over 2.5 kg 
 
3.2 
Exclusion Criteria 
HEU infants who meet any of the following criteria will be excluded from this study: 
● 
Infants with known exposure to active TB in household 
● 
Premature and < 2.5 kg 
3.3 
Recruitment Process 
Recruitment: HIV-infected mothers with HEU infants will be informed about the study starting 
from 2 weeks postpartum and will be invited to enroll their infant in the study. We will recruit 
eligible HIV-infected mothers and their infants from MCH/PMTCT sites between 2-10 weeks after 
birth and enroll and randomize HEU children to INH vs. no INH at 6 (+/- 4) weeks of age. We 
anticipate the majority of infants will be recruited during their routine 6 week immunization visits, 


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but have allowed an additional 4-week window for infants presenting early/late for this visit. HIV-
exposed infants are routinely tested for HIV at ~6 weeks of age, however results of that test may 
not be available for a few weeks. 
Study staff will work in conjunction with antenatal and pediatric staff at the MCH clinics to 
aid in our ability to identify potential participants. Interested mothers of infants will have the study 
explained to them, will have their questions answered, and will be asked to provide written consent 
(or thumbprint in the case of illiteracy). We have successfully recruited mother-infant pairs at these 
MCH/PMTCT sites for >4 years. 
Study staff will help any women or infants with suspected tuberculosis to access care at TB 
clinics, as well as HIV care clinics if necessary. This insures that care for all women and children 
in this maternal and child health setting is not compromised by the presence of the study, and 
should ensure that subjects do not feel pressure to participate in the study to receive any postnatal, 
pediatric, TB or HIV-related services. Study staff have been working side by side with clinic staff 
at these sites for the last 4 years. Study staff are well trained in recruitment and are knowledgeable 
in recruitment without persuasion/coercion.  
Enrollment: On enrollment, a study nurse will administer a standardized questionnaire that 
addresses sociodemographic, clinical, obstetric and HIV-related factors, TB exposure and history, 
and ascertains current maternal TB symptoms (using WHO symptom screen) and household 
symptoms. Mothers with suspected TB by WHO screen will be referred to the TB program for 
sputum TB screening and if found to have active TB, will be ineligible for participation and infants 
will receive INH for known active TB exposure per current Kenya national guidelines. Mothers 
will undergo physical examination with weight, height and BMI estimation; medical records will 
be used to abstract data on ART regimen, other medications, maternal HIV viral load and CD4 
cell counts. Infants will be undergo physical examination and medical records and MCH cards will 


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be used to abstract maternal prior PMTCT ART, infant PMTCT prophylaxis, birth weight, BCG 
vaccination date, and intercurrent illnesses and vaccines. At enrollment, infants will be examined 
and growth measures (weight, length, head circumference), mid-upper arm circumference, and 
presence of BCG scar will be determined. A questionnaire will address infant feeding and 
symptoms (including cough and fever). Infant blood will be collected for baseline PBMC 
separation and IGRA assay. Maternal breastmilk and maternal peripheral blood will be collected. 
Additionally infant stool will be collected for cryopreservation for future gut microbiome studies. 
At enrollment, among mothers who consent, household locator information, HIV care medical 
identification number, and cell-phone contacts will be obtained to facilitate tracing. 
Randomization: Block site-stratified randomization will be used to allocate infants 1:1 to INH or 
no INH trial arms. Randomization numbers will be generated at UW prior to study start (under 
leadership of Dr. Richardson and with the UW CFAR Biostatistical Core). 
3.4 
Co-Enrollment Guidelines 
Infants should not be enrolled in other TB prevention or TB vaccine studies because they might 
affect ascertainment of primary and secondary endpoints. For example an infant enrolled in a 
vaccine or other TB prevention trial may affect that infant’s risk of MTB infection irrespective of 
INH or no INH administration. 
3.5 
Participant Retention 
Once a participant enrolls in this study, the study site will make every effort to retain him/her for 
12 months of follow-up in order to minimize possible bias associated with loss-to-follow-up. 
Participant retention procedures will be established such that loss rates do not exceed the incidence 
rate of the primary study outcome. Study site staff are responsible for developing and 


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implementing local standard operating procedures to target this goal. Components of such 
procedures include: 
● Thorough explanation of the study visit schedule and procedural requirements during the 
informed consent process and re-emphasis at each study visit. 
 
● Thorough explanation of the importance of the study treatment group to the overall success of 
the study. 
 
● Collection of detailed locator information at the study Enrollment Visit, and active review and 
updating of this information at each subsequent visit. 
 
● Use of appropriate and timely visit reminder mechanisms including cell phone SMS. 
 
● Follow-up on missed visits. 
 
● Mobilization of trained outreach workers or “tracers” to complete in-person contact with 
participants at their homes and/or other community locations. 
 
● Mothers of infants who miss their monthly study visit will be contacted by phone or home visit 
and encouraged to continue follow-up, particularly for the 12 month IGRA visit (primary 
endpoint). 
 
● Caregivers will be counseled on importance of INH adherence and adherence will be assessed 
using pill counts at monthly re-fill visits.  
 
● Study visits are aligned with routine medical care (child immunization and maternal ART 
visits). We anticipate following mother-infant pairs at visits aligned with routine 
immunization, pediatric, and maternal ART visits. 
 
● Travel reimbursement. 
 
● Participants who discontinue treatment shall be maintained in follow-up as originally 
scheduled whenever possible. 
 
● Extended follow-up at 24 months: for those participants who may have exited the study prior 
to consenting for a 24 month visit, participants will be re-contacted and given the option to 
consent for another follow-up visit at 24 months of age. 


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3.6 
Participant Withdrawal 
Regardless of the retention methods, participants may voluntarily withdraw from the study for any 
reason at any time. Participants also may be withdrawn if the study sponsor, government or 
regulatory authorities, or site IRBs/ECs terminate the study prior to its planned end date. 
Every reasonable effort will be made to complete a final evaluation (Appendix I) of participants 
who terminate prior to the final study visit, including measuring M. tuberculosis infection status 
at the time of study exit. Study staff will record the reason(s) for all withdrawals from the study in 
participants’ study records. 
4.0 STUDY TREATMENT 
4.1 
Treatment Content 
Isoniazid ~10 mg/kg (7-15 mg/kg) will be administered once daily to infants in the INH arm for 
12 months. WHO dosage for INH is 7-15 mg/kg and CDC recommends 10-15 mg/kg; the South 
Africa/Botswana RCT used 10-20 mg/kg dosing. The Kenya Ministry of Health (MOH) 
recommends ~10 mg/kg and has standardized weight-based dosing (by weight band using 100 mg 
scored tablets) which correspond to WHO dosing recommendations90,91 (APPENDIX II) and will 
provide INH for the study. Infants assigned to the control arm will not receive INH. Current 
Kenyan guidelines recommend IPT (isoniazid preventive therapy) for all TB-exposed children <5 
years of age and for all HIV-infected children >1 year of age. The guidelines illustrate the 
uncertainty regarding IPT for <1 year olds with HIV infection, following the RCT from South 
Africa/Botswana that failed to demonstrate IPT effectiveness in <1 year olds. However, we 
speculate that among HEU children exposed to community TB or unperceived household TB, INH 
may prevent MTB infection as detected by IGRA. Although data are conflicting, some adult 
studies noting benefit of longer periods of IPT (36 months versus 6 months) and demonstrating 


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IPT benefit in TST negative HIV-infected adults suggest that IPT may confer protection from 
primary MTB infection. Pyridoxine will be provided children to decrease the risk of INH-
associated peripheral neuropathy in the INH arm using Kenyan MOH weight-based dosing (5-7 
kg ¼ 50 mg tab, 8-14 kg ½ 50 mg tab) (APPENDIX III) and will be provided by the MOH.  
4.2 
Treatment Administration  
Participants in the experimental arm will be given their daily INH and pyridoxine by caregivers 
for 12 months. Caregivers of infants in the experimental arm will be given 1 month supplies of 
INH and pyroxidine at monthly med pick up visits. 
4.3 
Treatment Supply and Accountability 
The Kenya Ministry of Health will provide INH and pyroxidine for the RCT. Study staff will 
maintain complete records of all study drugs received and subsequently dispensed to study 
participants. All unused meds will be returned to the Kenyan MOH after the study is completed or 
terminated. 
4.4 
Adherence Assessment 
Caregivers will be counseled on importance of INH adherence and adherence will be assessed 
using pill counts at monthly re-fill visits. In addition, we will assess isoniazid in urine at follow-
up visits using in-house urine test strips which are inexpensive (1.5 cents per strip) and have high 
sensitivity and specificity for detection of isoniazid in African adult and pediatric populations.92,93 
We will also collect hair at 12 months post-enrollment at the study endpoint to assess for isoniazid 
levels as a more objective measure of adherence over time. 


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4.5 
Toxicity Management 
INH is well tolerated in pediatric populations. INH is metabolized in the liver and excreted 
primarily through the kidneys. Hepatotoxic effects are rare in children but can be life threatening. 
In children given recommended doses, peripheral neuritis or seizures caused by inhibition of 
pyridoxine metabolism are rare, and most do not need pyridoxine supplements. Pyridoxine 
supplementation is recommended for exclusively breastfed infants and for children and 
adolescents on meat- and milk-deficient diets; children with nutritional deficiencies, including all 
symptomatic HIV-infected children; and pregnant adolescents and women. In this study all infants 
in the INH arm will be provided with pyridoxine. For infants and young children, isoniazid tablets 
can be pulverized. IPT has been safe in prior RCTs and is administered routinely to TB-exposed 
infants. Routine liver function monitoring is not recommended during INH in children, however 
baseline liver function tests will be drawn at enrollment (6 weeks of age) and at 10 weeks of age 
(1 month after INH initiation) in those infants randomized to INH. 
If toxicity is suspected, study administered drug will be immediately discontinued and in the case 
of concern for hepatoxicity, liver function tests (LFTs) will be performed. For this study, we will 
use the NIH Division of AIDS (DAIDS) Table for Grading the Severity of Pediatric Adverse 
Events to screen for eligibility and to grade clinical and laboratory toxicities and can be found at 
http://rsc.tech-
res.com/Document/safetyandpharmacovigilance/DAIDS_AE_GRADING_TABLE_v2_NOV201
4.pdf. If there is an increase of LFTs to > Grade 3, we will follow the study participant until 
resolution of the toxicity to ≤ Grade 2. For > Grade 3 hepatic abnormality supported by repeat 
laboratory tests: we will hold INH for two weeks and recheck LFTs in 2 weeks. If a > Grade 3 
abnormality is still present after withholding INH for two weeks: continue to hold INH and recheck 
LFTs in 2 weeks. 


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4.6 
HIV Seroconversion 
HIV-exposed infants in Kenya are routinely tested at ~6 weeks of age using HIV DNA PCR.  On 
enrollment, we may not yet have the results of the 6 week testing. Infants found to be HIV-infected 
from their 6 weeks of age testing will be followed throughout the study period, but will be excluded 
from the primary analysis. A recent estimate from Western Kenya of MTCT was 3% at 6 weeks 
of age. 
Infant HIV status will be determined at exit using repeat HIV PCR DNA testing at KEMRI/CDC 
laboratories to confirm that infants remain HIV uninfected. Infants found to be HIV-infected at 
study exit will also be excluded from the primary analysis. All infants found to be HIV-infected 
will be immediately referred to the on-site pediatric HIV care clinics. 
4.7 
Active TB screening and diagnosis, TB exposure in infants 
At scheduled follow-up visits intercurrent infant morbidity will be evaluated using standardized 
questionnaires. Both mothers and infants will be evaluated with standard TB screening questions regarding 
their own and household TB exposures. Any mother or infant with suspected active TB will be referred for 
TB microbiologic testing and X-rays, and these results will be abstracted to the Study database. Mothers 
with suspected active TB will be offered sputum AFB and GeneXpert testing consistent with Kenyan 
Ministry of Health guidelines. Infants with suspected TB will have chest X-ray, gastric aspirate testing by 
GeneXpert, and clinical review and classification as definite, probable or possible TB using Graham and 
NIH/WHO 2014 criteria.94 Infants with report of close contact with TB case will be undergo evaluation for 
active TB. If active TB is ruled out, infants who are in the control (no INH arm) will be referred to the 
MOH TB programme clinic for IPT evaluation per Kenyan guidelines. We will also assess of the presence 
of MTB infection at the time of TB diagnosis of disease.  


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4.8 
Concomitant Medications 
Enrolled study participants may continue use of all concomitant medications — except those listed 
under criteria for exclusion or treatment discontinuation — during this study.  
All concomitant medications [taken or received by participants within the 2 weeks prior to study 
enrollment] will be reported on applicable study case report forms. In addition to prescribed and 
over-the-counter medications, other traditional preparations will be recorded. Medications used 
for the treatment of AEs that occur during study participation also will be recorded on applicable 
study case report forms. 
5.0 
STUDY PROCEDURES 
An overview of the study visit and procedures schedule is presented in Appendix I.  
6.0 
SAFETY MONITORING AND ADVERSE EVENT 
6.1 
Safety Monitoring  
Close cooperation between the Protocol Chairs, Investigators, study biostatistician, DSMB, and 
other study team members will be necessary in order to monitor participant safety and respond to 
occurrences of toxicity in a timely manner. Before the study begins, the team will decide on a 
schedule to hold regular conference calls during the period of study implementation, and additional 
ad hoc calls will be convened if required. 
The Protocol Chairs/Investigators are responsible for continuous close monitoring of all adverse 
events (AEs) that occur among study participants, and for alerting the rest of the protocol team if 
unexpected concerns arise. A decision to stop the trial may be made by the protocol team at this 
time, or at any such time that the team agrees that an unacceptable type and/or frequency of AEs 
has been observed. 


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6.2 
Adverse Event Definitions and Reporting Requirements 
6.2.1 Adverse Event 
An adverse event (AE) is defined as any untoward medical occurrence in a clinical research 
participant administered an investigational product and which does not necessarily have a causal 
relationship with the investigational product. As such, an AE can be an unfavorable or unintended 
sign (including an abnormal laboratory finding, for example), symptom or disease temporally 
associated with the use of an investigational product, whether or not considered related to the 
product. The most frequent adverse events observed with INH are peripheral neuropathy and 
hepatotoxicity. For hepatotoxicity we will use the DAIDS Table for the Grading Severity of 
Pediatric Adverse Experiences (also referred to as the “Toxicity Table”) which is available on the 
RSC 
website: 
http://rsc.tech-
res.com/Document/safetyandpharmacovigilance/DAIDS_AE_GRADING_TABLE_v2_NOV201
4.pdf. For peripheral neuropathy, we will use the tables in Appendix IV regarding the measurement 
of peripheral neuropathy by grade as well as age appropriate measures of peripheral neuropathy. 
Study participants will be provided a 24-hour telephone number and instructed to contact the study 
clinician to report any AEs they may experience, except for life-threatening events, for which they 
will be instructed to seek immediate emergency care. Where feasible and medically appropriate, 
participants will be encouraged to seek evaluation where the study clinician is based, and to request 
that the clinician be paged or otherwise contacted upon their arrival. With appropriate permission 
of the participant, whenever possible records from all non-study medical providers related to AEs 
will be obtained and required data elements will be recorded on study case report forms. All 
participants reporting an AE will be followed clinically, until the AE resolves (returns to baseline) 
or stabilizes. 


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Study site staff will document on study case report forms all AEs reported by or observed in 
enrolled study participants regardless of severity and presumed relationship to study product. All 
AEs will be graded using the DAIDS Table for the Grading Severity of Pediatric Adverse 
Experiences (also referred to as the “Toxicity Table”) which is available on the RSC website: 
http://rsc.tech-
res.com/Document/safetyandpharmacovigilance/DAIDS_AE_GRADING_TABLE_v2_NOV201
4.pdf. The investigator or designee will assess the relationship of all AEs to the study product 
based on the Investigator’s Brochure, Package Insert, DAIDS Drug Risk List, and his/her clinical 
judgment. 
These 
documents 
are 
all 
available 
at 
http://rsc.tech-
res.com/Document/safetyandpharmacovigilance/. 
6.2.2 Serious Adverse Event  
For the purposes of this study, serious adverse event (SAE) will be defined as an AE occurring 
that: 
● Results in death  
● Is life-threatening 
● Results in persistent or significant disability/incapacity 
● Requires inpatient hospitalization or prolongation of existing hospitalization  
This includes important medical events that may not be immediately life-threatening or result in 
death, or hospitalization but may jeopardize the patient or may require intervention to prevent one 
of the outcomes listed above. 
Hospitalization itself is not an adverse event, but is an outcome of the event. The following types 
of hospitalization as related to our study do not require expedited reporting: 


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● Any admission unrelated to an AE (e.g. for administrative, or social admission for 
temporary placement for lack of place to sleep) 
● Admission for diagnosis or therapy of a condition that existed before receipt of study 
agent(s) and has not increased in severity or frequency as judged by the clinical investigator 
6.2.3 Adverse Event Reporting 
6.2.3.1 Adverse Event Reporting to DSMB 
For the purposes of this study we will use the definitions of Adverse Events (AEs) are outlined in 
DAIDS EAE Manual, which is available on the NIH RSC website at http://rsc.tech-
res.com/Document/safetyandpharmacovigilance/DAIDS_AE_GRADING_TABLE_v2_NOV201
4.pdf. Although the study is not sponsored by NIH, we will use their recommendations on 
reporting and grading adverse events. Adverse events will be reported to DSMB. 
6.2.3.2 Reporting Requirements for this Study 
In addition to the EAE Reporting Category identified above, other AEs that must be reported in an 
expedited manner are: hepatic failure, peripheral neuropathy, diagnosis of active TB, diagnosis of 
HIV.  
6.2.3.3 Grading Severity of Events 
The most current Division of AIDS Table for Grading the Severity of Pediatric Adverse Events 
(DAIDS AE Grading Table) is used and is available on the RSC website at http://rsc.tech-
res.com/Document/safetyandpharmacovigilance/DAIDS_AE_GRADING_TABLE_v2_NOV201
4.pdf . 
6.2.3.4 Expedited AE Reporting Period 
The expedited AE reporting period for this study is 6 month post study completion. After the 
protocol-defined AE reporting period, unless otherwise noted, only suspected unexpected serious 


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adverse reaction SUSARs as defined in the EAE Manual will be reported to the DSMB if the study 
staff become aware of the events on a passive basis (from publicly available information). 
7.0 
STATISTICAL CONSIDERATIONS 
7.1 
Review of Study Design 
Using a non-blinded randomized control study design, we will compare the risk of acquiring MTB 
infection among HEU infants enrolled at approximately 6 weeks of age during 1 year of follow-
up between infants receiving INH vs. no INH using an IGRA assay to determine MTB infection 
status. 
7.2 
Endpoints 
7.2.1 Primary Endpoints 
Consistent with the primary study objective to compare the incidence of primary MTB infection 
in HEU infants randomized to receive INH vs. no INH, the following endpoint will be assessed: 
• MTB infection as detected by interferon gamma release assays (IGRA) at 12 months post-
enrollment.  
7.2.2 Secondary Endpoints 
 
Consistent with the secondary study objective to investigate epidemiologic correlates of MTB 
infection (AIM 2), the following correlates will be assessed: 
• 
Maternal HIV viral load and CD4 cell counts, maternal prior PMTCT/ART regimens, infant 
PMTCT prophylaxis 
• 
Birth weight, BCG vaccination status and timing, and intercurrent illnesses and vaccines 


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• 
Growth measures (weight, length, head circumference, mid-upper arm circumference)  
• 
Infant feeding and symptoms (including cough, fever, weight loss, or growth faultering) 
• 
Infant HIV status at study enrollment and study end 
• 
Report of known TB exposure, maternal IPT status 
Consistent with the exploratory study objective to determine the immunologic correlates of MTB 
infection (AIM 3), the following correlates will be assessed: 
• 
Infant peripheral blood BCG-specific T-cell responses at approximately 6 weeks post BCG 
vaccination 
• 
Maternal breast milk and peripheral blood MTB-specific T-cell responses at approximately 6 
weeks postpartum. 
7.2.3 Exploratory Endpoints 
Consistent with the exploratory study objective to investigate the impact of INH on a combined 
endpoint of MTB infection, TB disease, and death the following events will be assessed: 
• 
MTB infection as measured by IGRA at 12 months post-enrollment 
• 
TB disease including microbiologically confirmed (culture or Xpert positive), or probable TB 
(clinical diagnosis). 
 
•    Death of HEU infant  
7.3 
Accrual, Follow-up, and Sample Size 
Table 1: AIM 1 Sample Size Estimates (Gray shaded 
close to study target) 


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Sample Size Estimations Aim 1: 
Number needed per arm to detect a 
maximum HR benefit of INH to 
decrease 
MTB 
infection 
as 
measured by IGRA positive at 12 
month follow-up. With 125 infants 
in each arm we would have power 
to detect at least a 65% decrease in 
MTB infection in INH arm vs. 
control if cumulative incidence of 
positive IGRA in the control arm at 12 months follow-up is 0.2, or to detect a 70-80% or higher 
(HR 0.3-0.2) decrease if the cumulative incidence of positive IGRA in control arm is 0.15 or 0.1 
(Table 1).  We will increase sample size by 20% to account for loss to follow-up, non-
adherence, and isoniazid resistance, enrolling 300 mother-infant pairs (150 per arm). 
We have estimated a substantive INH effect (65% decrease), which is consistent with IPT literature 
for active TB but completely undefined for MTB infection risk and one that may be persuasive for 
implementation. A larger sample size may be useful if prevalence of IGRA positivity is lower than 
we anticipate or if INH is less effective in prevention of MTB infection.  
7.4 
Random Assignment / Study Arm Assignment 
Site-stratified randomization will be used to allocate infants 1:1 to INH or no INH trial arms. We 
will use block randomization with block size of 4. Randomization numbers will be generated at 
UW prior to study start (under leadership of the Study Biostatistician Barbra Richardson and with 
CFAR Biostatistical Core). 
Power 80%,  
2-sided p 0.05 
1 year follow-
up 
Maximum 
HR for IPT 
detectable 
Risk of 
positive 
IGRA at 
m12 
Numbe
r per 
arm 
0.2 risk IGRA 
positive after 
12 mos follow-
up 
0.5 
0.2 
220 
0.4 
0.2 
150 
0.35 
0.2 
120 
0.32 
0.2 
100 
0.2 
0.2 
55 
0.15 risk IGRA 
positive after 
12 months 
follow-up 
0.5 
0.15 
300 
0.4 
0.15 
180 
0.35 
0.15 
150 
0.31 
0.15 
120 
0.2 
0.15 
75 
0.10 risk IGRA 
positive after 
12 months 
follow-up 
0.5 
0.1 
420 
0.4 
0.1 
270 
0.35 
0.1 
220 
0.3 
0.1 
180 
0.2 
0.1 
110 


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7.5 
Blinding 
We have designed the study to be a non-blinded RCT to enable prompt clinical management of 
children in each arm, given an understanding of drugs that they are receiving. With non-blinded 
trials there are concerns about differential reporting and clinical management; however, our 
endpoint (IGRA status) will be assessed in the KEMRI CDC laboratory, which will be blinded to 
INH status of the participant. This endpoint is robust and not influenced by unblinded trial design. 
7.6 
Data Analysis  
7.6.1 Primary Analyses 
The primary analysis will be comparison of proportion IGRA positive at 12 months following 
enrollment between INH and no INH arms among HEU infants. Baseline maternal and infant 
characteristics, including maternal CD4, ART regimen, age, TB exposure, reported TB household 
symptom screen, maternal TB history prior to enrollment, employment and infant birth weight, 
gestational age, and weight and height z-scores (WAZ, HAZ) and MUAC at enrollment, will be 
compared between randomization arms to assess adequacy of randomization. The primary analysis 
will be a comparison of proportion of infants in INH vs. control arm with IGRA positive assays 
using Chi square tests using an intent-to-treat analysis. Infants who are found to be HIV DNA 
positive at enrollment or at study end will excluded from this ITT analysis of our primary endpoint. 
We do not anticipate baseline immune responses to TB antigens at enrollment at ~6 weeks of age. 
However, if we find baseline responses, we will conduct additional modified intent-to-treat 
analyses, incorporating data from baseline assays (that utilize cryopreserved samples) to exclude 
any infant with evidence of immune responses to ESAT-6 or CFP-10 at enrollment visit. Thus, the 
modified intent-to-treat analysis will compare incidence of new MTB infection subsequent to 


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randomization in INH versus no INH trial arms among HEU infants. All missing data will be 
assumed as missing completely at random. 
7.6.2 Secondary Analyses 
AIM 2: Exposures to be evaluated as potential correlates of MTB infection: There are scant 
data on determinants of MTB infection in HEU infants. A combination of household and 
community TB exposures may contribute to infant risk. The RCT offers an opportunity to explore 
contributors to MTB acquisition in nested case-control studies overall and in the control arm alone. 
Variables to be considered include those associated with increased likelihood of infant exposure 
to active TB and those that reflect infant host susceptibility factors. In terms of potential TB 
exposure, mothers with low CD4 count, paternal positive HIV status, maternal employment, 
crowding, and household TB symptoms may be associated with likelihood of TB exposure.  
Potential infant determinants of MTB susceptibility include infant growth (WAZ, HAZ, WHZ) 
with indication of underweight (WAZ <-2) and stunting (HAZ<-2) at enrollment at 6 weeks or 
over the course of follow-up. Alternatively, growth deficits may accompany nutritional deficits, 
which could compromise immune responses to TB antigens and fail to reveal a positive assay 
despite MTB infection. 
 
Sample size considerations: 
For the nested studies to 
determine 
correlates 
of 
incident MTB infection, we 
anticipate 34 cases with all 
other infants without evidence of MTB infection as potential controls. In sample size estimates 
assuming at least 3 controls in comparison group, there is between 73% to >90% power to 
Table 2. Aim 2 Sample size estimates (Gray shaded 
adequate power) 
Case-control (1:3, 
34 cases: 102 
controls) Alpha 
0.05 
Power if prevalence of exposure in 
controls is: 
20%  
40%  
50%  
OR 2 
36% 
41% 
39% 
OR 3 
73% 
78% 
74% 
OR 4 
91% 
93% 
90% 


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determine ORs of 3 to 4 if prevalence of exposure in controls is between 20 to 50% as in Table 2 
above. 
 
AIM 2 Analyses: To identify epidemiologic correlates of MTB infection among infants enrolled 
in the study, cryopreserved specimens from the baseline visit will be assessed for responses to TB 
antigens concurrent with other assays outlined in immune correlates in the next paragraph. Any 
infant with positive IGRA at baseline will be excluded from cofactors analyses. We anticipate 25 
MTB infections in the control arm and ~9 in the INH arm (assuming INH is 65% effective). In 
nested case-control analyses, infants who had IGRA conversion suggestive of new primary MTB 
infection will be compared to those with valid negative assays at 12 months. Univariate and 
multivariate logistic regression will be used to compare mother-infant characteristics of infants 
with and without IGRA positive assays at month 12. We will initially build nested case-control 
studies incorporating all MTB infections from both arms of the RCT and then conduct stratified 
analyses in each trial arm to evaluate potential cofactors that are modified by INH. 
Aim 3. Determine immune 
correlates of risk of primary 
MTB infection and their 
potential interactions with 
INH. Assays will include 
infant peripheral blood BCG-
specific T-cell responses 6 
weeks post-vaccination and 
maternal peripheral blood and 
breast milk MTB-specific T-cell responses at 6 weeks postpartum. 
 
Fig. 5: Model of HIV exposure & effects on adaptive immune responses 
to BCG and risk of MTB infection: BCG vaccination in HEU infants 
activates macrophages and dendritic cells which prime T cells and the 
adaptive immune response to activate macrophages to kill MTB and 
orchestrate clinical outcomes. DC, dendritic cell; M, macrophage.  
CD8 T
MTB
Outcome 
in M
MTB
Mor 
DC
NO
Infection
Clinical
Outcome
Killed
MTB
Infection
Not Killed
MTB
Exposure
Cytokines
Vaccination
BCG
TLR
Mor 
DC
HIV in utero
CD4 T
Initiation, 
proliferation, & 
durability of CD4 & 
CD8 response
TH subset polarization 
& memory maturation
CD4 T
Aim 3A:Infant BCG-specific T-
cell responses & 
Association with MTB infection
Aim 3B: Maternal breast milk 
MTB-specific T-cell responses & 
Association with MTB infection
Randomize:  
INH vs placebo
Cytokines


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Rationale and conceptual framework for immunologic hypotheses: A major goal of our 
application is to evaluate immune response to BCG and susceptibility to MTB infection in HEU 
children. In studies of adults, acute HIV is associated with a rapid decrease in MTB-specific TH1 
cells.33 MTB-specific CD4 T-cells are preferentially infected with HIV and depleted.34 Studies 
suggest that BCG-specific T-cell responses are lower in HIV-infected infants, but not older 
children.35,36 Intrauterine exposure to HIV may alter immune responses in HEU infants.29,95,96 A 
recent study found that HEU infants have higher BCG-induced T-cell proliferation, but a lower 
percentage of polyfunctional T-cells compared to unexposed infants.27 However, there are no 
studies addressing whether BGC-immune responses in HEU are associated with MTB 
infection. To address these gaps, we will determine whether infant peripheral blood and 
maternal breast milk and peripheral blood immune responses are associated with MTB 
infection in infants. We hypothesize that intrauterine HIV exposure compromises the adaptive 
immune response to BCG vaccination with development of less effective BCG-specific memory T-
cell responses that are associated with increased susceptibility to MTB infection. We will examine 
early CD4 & CD8 responses, TH1 & TH17 CD4 T cell polarization, and memory T cell maturation. 
We also hypothesize the breast milk from HIV-infected mothers with a prior history of MTB 
infection will have MTB-specific immune responses that confer protection for infants for future 
MTB infection. To determine the immune correlates of risk of MTB infection and the effects of 
HIV exposure on response to BCG vaccination, we will examine infant BCG-specific T-cell 
responses and maternal breast milk MTB-specific T-cell responses.  
 
Immunologic Study Design and Primary Analytic Goals: After routine BCG vaccination at 
birth, eligible HEU infants who were enrolled and randomized at age 6 weeks to INH vs. placebo, 
will be followed longitudinally to assess for development of MTB infection. For the efficacy study 


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and endpoint determination, MTB status will be assessed by a blood draw at 12 months after 
randomization by QFT-Plus. For the immunologic studies in Aim 3, 5 mls of blood will be obtained 
at enrollment into the study (which is approximately 6 weeks after BCG vaccine). We will measure 
infant adaptive immune responses and maternal breast milk responses as outlined below. Our 
primary analytic goal for this aim is to determine which of these responses are associated with 
susceptibility to MTB infection.  
Aim 3 Analysis: To identify infant and maternal (including peripheral blood and breast milk) 
immune correlates of risk of primary MTB infection and assess their potential interactions with 
INH, magnitude of immune measures (BCG-specific T-cell responses measured by ICS, 
cytokine/chemokine levels in peripheral blood or breast milk at 6 weeks of age and maternal 
peripheral blood at enrollment will be compared between cases (who subsequently acquire MTB 
infection) and controls (who do not) using GEE with Gaussian link to account for clustering. We 
will use methods for normal data and then use non-parametric approaches or dichotomize and use 
binomial link GEE if data are highly skewed and cannot be transformed into a normal distribution. 
Please see Appendix I regarding details of the procedures and protocols as related to the infant and 
maternal breast milk immunologic studies. 
7.6.3 Exploratory Analyses 
We will compare combined infant endpoints by RCT arm as follows: Exploratory endpoint 
analysis #1: Infant MTB infection (IGRA positive) or active TB (probable or definite). 
Exploratory endpoint analysis #2 will combine MTB infection (IGRA positive), active TB, or 
death. The proportion of infants reaching the combined endpoints in each arm will be compared 
using chi 2 test. In addition we will compare median magnitude of IGRA responses using non-
parametric tests among all infants and among the su t with positive IGRA responses. 


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7.7 
Data Safety and Monitoring Board (DSMB) 
Our study will be monitored by the DSMB. The DSMB review will address the efficacy of 
isoniazid (INH) as described in the primary objectives, as well as the safety of INH, as described 
in Section 6. The DSMB will consist of experts in pediatric TB, biostatistics and RCT trial design. 
7.8 
Data Monitoring 
As noted above, the study will be reviewed by the DSMB for safety and efficacy. The interim 
efficacy analyses will be conducted and reviewed by the DSMB at 25%, 50%, and 75% of expected 
total number of primary endpoint events, which are 10, 20, and 30 MTB infections.  
Early stopping rules for efficacy will be guided by the O’Brien-Fleming symmetric group 
sequential boundaries. The Lan and DeMets implementation of the boundaries will be used to 
define proper nominal significance levels at the interim and final efficacy analyses (a total of 4 
looks). Unless there are safety concerns, significant differences (as defined by the stopping 
boundaries) on primary efficacy endpoints will be required in order to terminate the study early. 
In addition, monthly blinded safety monitoring reports will be sent to the medical officers, and 
protocol chairs and co-chairs for review which will include all adverse events of Grade 3 and 
above.


 
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8.0 
HUMAN SUBJECTS CONSIDERATIONS 
8.1 
Population to be enrolled and followed  
The study involves two vulnerable groups: HIV-infected women and their infants. It is important 
to address research questions to find interventions for these vulnerable populations. The study 
directly addresses a research need specifically relevant to HEU infants – with the aim of 
optimizing prevention of TB in these infants. 
8.2 
Study design 
We propose a randomized clinical trial of INH versus no INH. Currently, there is equipoise 
regarding use of IPT in HEU with conflicting results of prior IPT studies in infants, including 
HEU infants. IPT is not recommended during the first year of life for prevention of active TB 
following lack of benefit in a large RCT in South Africa. However, it is not known if INH could 
prevent MTB infection. The latter question is addressable in a Phase II RCT to evaluate impact 
of INH on MTB infection during the first year of life with IGRA assays to detect MTB infection. 
8.3 
Approvals 
The study will be reviewed at Institutional Review Board at University of Washington and at the 
Ethical Review Committee at Kenyatta National Hospital prior to any human subjects 
participation. In addition, we will obtain approval from the Kenyan Ministry of Health and 
National Pharmacy and Poisons Board. 


 
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8.4 
Informed consent 
 Study nurses will discuss the study rationale, design, and risks and benefits. Eligible women will 
be provided written informed consent prior to the participation of themselves and their infants in 
this study. 
Risks:  
INH: The study will involve provision of INH to HEU infants in the INH arm of the RCT. INH 
has been safe in prior RCTs and is administered routinely to TB-exposed infants. There have 
been concerns regarding hepatotoxicity with INH but this is a rare outcome and routine liver 
function monitoring is not recommended during INH in Kenya.  
Phlebotomy: All infants will have blood drawn on enrollment at 6 (+/- 4) weeks of age, after 1 
month of enrollment (10 +/- 4 weeks of age) and at one year following enrollment (5 mls per 
blood draw). Infants will also have blood drawn at 24 months of age as part of extended follow-
up (5-10 mls). Infants randomized to INH will also have blood drawn for LFTs on enrollment 
and at 1 month after INH initiation. All mothers will have blood drawn on enrollment (5 mls). 
Venipuncture can cause bruising, pain, and discomfort. Blood will be drawn by an experienced 
phlebotomist. The amount of blood volume and frequency of blood draws is relatively small. 
Breastmilk collection: Mothers will be asked to provide self-expressed breast milk at the 
enrollment visit. Self-expression of breast milk can cause mild breast discomfort. 
Confidentiality: All mothers participating in the RCT are HIV-infected and will already be in 
the PMTCT program. Study data and information about HIV status will be kept confidential. 
Data will be stored in a locked cabinet and identifiers will not be on study records or database. 
Specimen Storage for Future Studies: Specimens collected (infant blood, stool, hair, and 
maternal blood and breast milk) will be stored for future studies. Samples to be used in future 


 
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studies will be labeled with a de-identified patient number. Samples will be kept for 10 years 
after the study is complete. We will ask the Kenyatta National Hospital/University of Nairobi 
Ethical Review Committee and the University of Washington IRB to use of these samples in 
future studies prior to any new sample analysis. Caregivers who do not want to have their or their 
child’s samples stored for future research, can still be in this study. And their samples will be 
destroyed once testing for this study is completed. 
Benefits 
Participants will benefit from direct medical care in the longitudinal research cohort. 
If INH prevents MTB infection, infants in that trial arm will benefit from INH. 
• The general population of HEU and infants in general will benefit from insights gained 
into the tolerability and risk of SAEs associated with INH in the context of use of INH to 
prevent MTB infection. 
• The general population of HEU and infants will benefit from insights gained into 
mechanisms of immune protection that can inform new prophylaxis or vaccine strategies. 
Monitoring Plan  
We will convene an external Data Safety and Monitoring Board (DSMB) which will be 
convened prior to study initiation and at 25%, 50%, 75% and 100% of expected study endpoints. 
If accrual is slower or the event rate of MTB infection is lower than expected, the DSMB will 
meet at 6 months post study initiation to review data in open and closed report, and to identify 
whether changes will need to be made to the recruitment plan. The DSMB will include an expert 
in pediatric TB, statistician, and clinician. The study will include weekly summary and expedited 
reporting of severe adverse events.  


 
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Extended follow-up at 24 months of age: Participants in the study will go through an additional 
consent for a follow-up visit at 24 months of age. If participants have exited the study prior to 
consent, they will be re-contacted and given the option to consent to extended follow-up at 24 
months. 
8.5 
Study Discontinuation 
The study also may be discontinued at any time by the Thrasher Research Fund, Data Monitoring 
Board, and/or site IRBs/ECs.  
9.0 
 LABORATORY SPECIMENS AND BIOHAZARD CONTAINMENT 
9.1 
Laboratory Specimens 
 
Infant blood samples and maternal breast milk and peripheral blood will be collected at enrollment. 
These samples will transported via portable incubator to either KEMRI/CDC or CRC/CDC lab at 
Kisumu. These samples will be for be stimulated via the SATVI protocol and then cryopreserved. 
After cryopreservation, samples will be transported to Dr. Hawn’s laboratory in Seattle. Dr. 
Hawn’s laboratory will perform assays for detection of BCG-stimulated T-cell responses. A subset 
of samples will be transported to Dr. Cranmer at Emory University for future ancillary studies on 
immune responses to BCG vaccine. Stool will be collected from infants on enrollment via swab 
for cryopreservation at KEMRI-CDC for potential future infant microbiome studies. Blood will be 
drawn from infants at enrollment (6 weeks age) and 1 month post-enrollment (10 weeks of age 
visit) for plasma and PBMC separation. Blood will be drawn at baseline (6 weeks of age), and 1 
month post INH initiation (10 weeks of age) for LFTs in infants randomized to receive INH. 
Baseline samples will also be used to determine INH acetylator status  by testing for NAT2 
genotypes. Blood will be drawn from mothers on enrollment for PBMCs and plasma separation. 
Maternal breast milk will be collected at enrollment for breast milk cell (BMC) and supernatant 


 
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separation. A small thatch of hair (approximately 30 strands) will be cut and collected for INH 
exposure assessment. Hair analysis will occur at the University of California at San Francisco Hair 
Analytical Laboratory (HAL) (http://test.hairlab.ucsf.edu/). The infant 5 ml blood 12-month 
specimen will be collected for IGRA into a single blood collection tube containing lithium heparin. 
These blood collection tubes will be maintained at room temperature and transported by courier to 
the KEMRI/CDC lab for further processing. At the KEMRI/CDC lab this blood will be transferred 
into QuantiFERON-TB Gold In-Tube Plus (QFT-Plus) assay collection tubes (nil, mitogen, TB 
antigen 1 [ESAT-6 and CFP-10 CD4 peptides], TB antigen 2 [ESAT-6, CFP-10 CD4 and CD8 
peptides]) and processed per manufacture recommendations.89 Blood will be drawn from infants 
at the time of any TB disease diagnosis, or at the time of study withdrawal to assess MTB infection 
status using the same QFT-Plus assay. In the event blood collected is insufficient blood volume 
(<4ml) for QFT-Plus, the blood collected will be processed for a different test for M. tuberculosis 
infection.   Urine will be collected throughout the study from infants in the INH arm to test for 
INH metabolites as a measure of adherence.  
Extended follow-up at 24 months: Blood will be drawn from infants at 24 months of age for 
plasma and PBMC separation to assess MTB infection status. 
Each study site will adhere to standards of good clinical laboratory practice, and local standard 
operating procedures for specimen management including proper collection, processing, labeling, 
transport, and storage of specimens. 
9.2 
Quality Control and Quality Assurance Procedures 
The KEMRI/CDC lab has an internationally accredited TB immunology lab that undergoes routine 
monitoring for QA purposes. The CDC/CRC lab also has international accreditation and undergoes 


 
Infant TB Infection Prevention Study (“iTIPS”), RCT 
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routine monitoring for QA purposes. The Hawn lab has extensive experience in processing human 
samples for immunology studies including international trials. 
9.3 
Specimen Storage and Possible Future Research Testing 
The KEMRI/CDC or CDC/CRC lab will store all samples while in Kenya for the duration of the 
study. After shipment to the US, samples will either be stored at the University of Washington, 
Fred Hutchinson Cancer Research Center, Emory University, or the University of California San 
Francisco. If patients consent at the beginning of the trial, samples will be stored after study 
completion for potential further study and analysis. Samples of patients who do not consent for 
long-term storage and additional analysis will be destroyed at study completion. 
9.4 
Biohazard Containment 
As the transmission of HIV and other blood-borne pathogens can occur through contact with 
contaminated needles, blood, and blood products, appropriate blood and secretion precautions will 
be employed by all personnel in the drawing of blood and shipping and handling of all specimens 
for this study, as currently recommended by the United States Centers for Disease Control and 
Prevention. All infectious specimens will be transported in accordance with United States 
regulations (42 CFR 72). 
10.0 
ADMINISTRATIVE PROCEDURES 
10.1 
Protocol Registration 
Prior to implementation of this protocol, and any subsequent full version amendments, the protocol 
and the protocol consent form(s) will be approved by the UW IRB and University of 


 
Infant TB Infection Prevention Study (“iTIPS”), RCT 
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Nairobi/Kenyatta Hospital ERC. The study will be registered with ClinicalTrials.gov and with the 
Kenyan Pharmacy and Poisons Board. 
10.2 
Monitoring Plan 
We will convene an external Data Safety and Monitoring Board (DSMB) which will be convened 
prior to study initiation and 6-monthly to review data in open and closed report.  The DSMB will 
include an expert in pediatric TB, statistician, and clinician. The study will include weekly 
summary and expedited reporting of severe adverse events. 
10.2 
Investigator's Records 
The study site investigator will maintain, and store in a secure manner, complete, accurate, and 
current study records throughout the study. The investigator will retain all study records for at least 
three years after submission of the study results. Study records include administrative 
documentation — including protocol registration documents and all reports, and correspondence 
relating to the study — as well as documentation related to each participant screened for and/or 
enrolled in the study — including informed consent forms, locator forms, case report forms, 
notations of all contacts with the participant, and all other source documents. 
 
 


 
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65. 
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12.0 
APPENDICES 
I 
SCHEDULE OF STUDY VISITS AND PROCEDURES 
Table 3: Brief overview of study visits and procedures  
Table 3: Overview of Study Visits and planned procedures 
 
 
6 weeks 
postpartum 
Follow-
up visit* 
12 months 
post 
enrollment 
TB 
Diagnosis 
24 months 
of age 
HIV testing (per 
MOH) 
x 
x** 
x 
 
 
Enrollment 
x 
 
 
 
 
Sociodemographic 
survey 
x 
x 
x 
 
 
 
Health history 
x 
x 
x 
 
x 
Physical exam 
x 
x 
x 
 
x 
TB (infant and 
maternal) 
symptom screen 
x 
x 
x 
 
x 
SAE assessment 
 
x 
x 
 
 
Adherence 
assessment via 
questionnaire, 
urine INH 
testing*** 
 
x 
x 
 
 
TB exposure 
assessment 
x 
x 
x 
 
x 
Infant blood draw 
x 
x**** 
x***** 
x***** 
x 
Maternal blood 
draw 
x 
 
 
 
 
Maternal 
breastmilk 
collection 
x 
 
 
 
 
Infant stool 
collection 
x 
 
 
 
 
Infant TST 
placement 
 
 
x***** 
x***** 
x 
Infant hair 
collection*** 
 
 
x 
 
 
* Follow up visits will occur at 10 and 14 weeks of age, and 6, 9, and 12 months 
of age.  
** Infant DNA PCR will be drawn a 6 weeks of age and HIV antibody test will be 
drawn at 12 months of age per Kenyan MOH guidelines. 
*** For infants randomized to INH  


 
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****For all infants blood will be drawn for PBMCs and plasma at the 10 week of 
age visit. For infants randomized to INH arm, LFTs will be drawn at baseline (6 
weeks) and 10 weeks of age (1 month post INH inititation).  
***** Blood will be drawn to assess the presence of Mtb infection at study 
endpoint,time of TB diagnosis, and in the event of study withdrawal using QFT-
plus.TST will also be placed and read within 48-96 hours. If blood volume is 
insufficient for QFT-Plus (<4ml), blood will be processed for for a different test for 
M. tuberculosis infection. 
  
 
 


 
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Table 4: List of study visits and procedures (details on laboratory assays, infant adaptive immune 
response assays, and maternal breast milk TB specific cellular immune responses assays will be 
detailed below the table) 
 
Table 4: Detailed study visits and procedures 
Visits and procedures 
Details 
2 weeks 
postpartum 
Informed and 
invited 
HIV-infected mothers with HEU infants will be informed about the study 
starting from 2 weeks postpartum and will be invited to enroll their infant in 
the study. 
2 - 10 weeks 
Recruitment 
Written informed consent will be obtained before any study procedure. 
6 (+/- 4) weeks 
of age 
Enrollment, 
screening, and 
randomization 
Enrollment and screening: 
• 
HIV DNA PCR testing will be used to confirm HIV negative status. 
• 
Infant blood (5 ml) will be collected for baseline PBMC separation and 
WBA assays, and NAT2 genotype polymorphisms associated with INH 
acetylation phenotypes. 
• 
Maternal breast milk (30 ml) will be collected. 
• 
Maternal peripheral blood (5 ml) will be collected. 
• 
Growth measures, mid-upper arm circumference, infant feeding, and 
symptoms (including cough and fever), and presence of BCG scar will 
be assessed. 
• 
Infant stool will be collected for cryopreservation for future infant 
microbiome studies 
• 
Household locator information, HIV care medical identification number, 
and cell-phone contacts will be obtained to facilitate tracing. 
• 
Infant adaptive immune response and maternal breast milk TB specific 
cellular immune responses will be determined (detailed below). 
Randomization: 
• 
Block site-stratified randomization will be used to allocate infants 1:1 to 
INH or no INH trial arms. Randomization numbers will be generated at 
UW prior to study start (under leadership of the Study Biostatistician 
and with CFAR Biostatistical Core). 
Daily 
Intervention 
Isoniazid ~10 mg/kg (7-15 mg/kg) and pyroxidine (1-2 mg/kg) will be 
administered once daily to infants in INH arm for 12 months. 
 Follow-up 
visits (10 
weeks, 14 
weeks, 6 
months, 9 
months, 12 
months of age)  
 
 
 
 
 
 
 
 
INH pick-up; 
assessment of 
infant 
morbidity and 
adherence; 
TB symptom 
screening of 
mother and 
infant. 
 
 
 
 
 
 
• 
At follow up visits intercurrent infant morbidity will be evaluated using 
standardized questionnaires. 
• 
Both mothers and infants will be evaluated with standard TB screening 
questions regarding their own and household TB exposures. Any 
mother or infant with suspected active TB will be referred for TB 
microbiologic testing and X-rays, and these results will be abstracted 
to the study database. Mothers with suspected active TB will be 
offered sputum AFB and GeneXpert testing consistent with Kenyan 
Ministry of Health guidelines. Infants with suspected TB will have chest 
X-ray, gastric aspirate testing by GeneXpert, and clinical review and 
classification as definite, probable or possible TB using Graham and 
NIH/WHO 2014 criteria. 
• 
Adherence will be assessed using maternal report, pill counts at re-fill 
visits, urine INH dipstick testing (for infants in INH arm) 


 
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Follow-up 
visits (cont) 
 
 
 
 
 
Maternal ART 
visits. 
• 
Blood will be drawn from all infants for PBMCs and plasma at the 10 
week of age visit. 
• 
LFTs will be drawn at baseline (6 weeks) and 1 month post initiation 
(10 weeks)   for infants INH randomized to receive INH 
 
 
Maternal medical records will be abstracted to obtain data on maternal 
ART regimen, cotrimoxazole status, CD4 and viral load (if available). 
Anticipated maternal regimen will be tenofovir, efavirenz, emtricitabine (or 
lamivudine) with 6 weeks of infant nevirapine postpartum. 
At 12 months 
post 
randomization 
 
• 
Infant blood will be drawn for IGRA to ascertain potential MTB 
infection. 
• 
Infant HIV status will be determined at exit using repeat HIV DNA PCR 
testing at CDC-KEMRI laboratories to confirm that infants remain HIV 
uninfected. 
• 
TST will be placed and read within 48-96 hours 
• 
Infant hair will be collected at the study endpoint for INH levels for 
children in the INH arm 
 
At 24 months 
of age 
 
• 
Infant blood will be drawn for plasma and PBMC separation 
• 
Infant adaptive immune responses will be determined (detailed below) 
and will include ascertainment of potential MTB infection 
• 
TST will be placed and read within 48-96 hours 
• 
Intercurrent infant morbidity will be evaluated using standardized 
questionnaires. 
 
Laboratory Assays: At baseline, infant blood will be collected and stimulated using SATVI 
protocol and transported in a portable incubator to the CDC-KEMRI lab in Kisumu prior to 
cryopreservation and transport to Dr. Hawn’s laboratory for detection of BCG-stimulated and 
ESAT-6 and CFP-10 stimulated responses. This SATVI protocol was developed for use in this 
type of clinic, which is linked to a centralized laboratory. Blood will be drawn on enrollment from 
mothers on enrollment for PBMCs and plasma. Maternal breast milk will be collected at 
enrollment for BMCs and supernatant. All infants will have blood drawn at enrollment and at the 
10 week of age visit for plasma and PBMCs. Infants that are randomized to receive INH will have 
blood drawn on enrollment and 1 month post INH initiation for LFTs. Baseline samples will also 
be used to determine INH acetylator status by testing for NAT2 genotypes. The 12-month infant 
5 ml blood specimen will be collected into a single lithium heparin blood collection tube and kept 


 
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at room temperature until transported to KEMRI/CDC. At KEMRI/CDC blood from the single 
collection tube will be transferred for IGRA directly into QFT-Plus assay collection tubes (nil, 
mitogen, TB antigen 1, TB antigen 2) and processed per manufacture recommendations.89 The 
assay measures the amount of interferon-gamma (INF-γ) released by primarily CD4+ T helper 
lymphocytes after stimulation with TB-specific antigens (ESAT-6, CFP-10 and TB7.7) to measure 
MTB infection as well as the INF-γ released by CD8+ cytotoxic T lymphocytes after stimulation 
with the same TB-specific antigens. A response of ≥0.35 IU/ml to the TB antigens in either TB 1 
or TB 2 (with Nil < 8 IU/ml and positive mitogen control) will be considered a positive result. 
Blood for QFT-Plus assay will also be drawn in the event of an infant TB diagnosis, or study 
withdrawal. In the event blood collected is insufficient blood volume (<4ml) for QFT-Plus, the 
blood collected will be processed for a different test for M. tuberculosis infection. A small thatch 
of hair (approximately 30 strands) will be collected from children in the INH arm at the 12 months 
post randomization study endpoint visit (approximately 14 months of age) to measure INH levels. 
 
Infant Adaptive Immune Response Assays: We will determine several T-cell characteristics as 
follows: 
A. Frequency, cytokine profile, and effector/memory/homing/activation phenotype of 
mycobacterial-specific CD4+ and CD8+ T cells after short term incubation with a flow-cytometric 
intracellular cytokine assay. 
We will use multi-parameter flow cytometry with ICS using whole blood from the baseline 
enrollment time-point in cases and controls. We will use a short-term assay (7 & 12 hours) with 
200 ul of blood per condition with several stimuli including: 1. live BCG; 2. peptide pools of MTB 
antigens CFP-10/ESAT-6 (to exclude individuals with immune responses to MTB infection rather 


 
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than BCG); and 3. controls (medium alone and PHA). This assay was developed at SATVI and 
utilizes a 37°C incubator which can be deployed at clinic sites and used for transport to the 
reference laboratory. This protocol was used successfully with field site utilization for processing 
5,724 samples in an infant BCG trial 69. Tubes are pre-coated with anti-CD28 and anti-CD49d, 
incubated with blood for 7 hours with removal of supernatant, incubated an additional 5 hours with 
Brefeldin-A before harvesting and fixing with FACS lysis buffer and cell cryopreservation. 
 
Immune Measures: Frequency of single and combined expression of IFN-γ, IL-2, TNF-α,IL-17 
and IL-22 in viable CD4 and CD8 T cells; expression pattern of HLA-DR, CD38, CD45RA, 
CCR7, CXCR3, α1 and β4 in viable cytokine+ (i.e., specific) CD4 and CD8 T cells; and expression 
patterns of PD-1, CTLA-4, CD160, and FoxP3 on/in these cells. 
 
B. CD4+ and CD8+ T cell proliferation, survival, and differentiation and expression of cytotoxic 
markers after longer term incubation: PBMCs, pre-stained with Ki-67, will be incubated with 
BCG, CFP-10/ESAT-6, and control conditions for 3 or 6 days, and then fixed and stained for CD3 
and CD8. The short-term incubation (7 hours for secreted cytokine and 12 hours for ICS) measures 
a quantitative ex vivo snapshot of immunity, before cells are able to proliferate. In contrast, the 
longer term assays may evaluate distinct aspects of immunity (e.g., central memory cells), and 
allow detection of some markers not optimally measurable in the mycobacterial system with short 
term assays (e.g., cytotoxic markers, type 2 cytokine responses). Ki-67 is an excellent marker of 
specific cells in these assay systems, and its expression on day 6 correlates with traditional markers 
of proliferation such as BrdU and CFSE dilution79. The pattern of expression of cytotoxic markers 
may correlate with distinct cellular functional attributes. Further, direct measurement of 


 
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cytotoxicity is impractical, given available PBMC numbers. We will use several stimuli including: 
1. live BCG; 2. Peptide pools of MTB antigens CFP-10/ESAT-6 (to exclude individuals with 
immune responses to MTB infection rather than BCG); and 3. Controls (medium alone and PHA). 
 
Immune Measures: On day 3, frequency of single or combined expression of granulysin, granzyme 
B and perforin in viable, Ki67+ (i.e., antigen-specific, proliferating) CD4 and CD8 T cells. On day 
6, absolute numbers and frequency of viable CD4 and CD8 T cells; frequency of viable Ki67+ 
CD4 and CD8 T cells; expression pattern of IFN-γ, IL-2, TNF-α, IL-17, IL-4, IL-13 and IL-10 in 
viable Ki67+ CD4 and CD8 T cells. 
 
C. Secreted T cell cytokines in stimulated whole blood: We will assess soluble production of T 
cell cytokines at 7 hours and at 6 days, focusing on cytokines not readily detectable by the assay 
systems above, in supernatants, with bead arrays. Immune Measures: We will measure levels of 
29 cytokines/chemokines measured by multiplex bead array technology (which includes IL-2, IL-
4, IL-5, IL-10, IFN-γ, TNF-α, and TGF-β). 
Hair analysis for INH exposure: A small thatch of hair (approximately 30 strands) will be cut 
from the occiptital region close to the scalp, place in tin foil, sealed inside a plastic bag containing 
desiccant, and then stored at room temperature before being shipped to the UCSF Hair Analytical 
Laboratory (HAL). INH will be extracted from hair cut samples via methanol/water solution (v/v, 
8/2) containing 1% hydrazine dehydrochloride, followed by evaporation and reconstitution prior 
to separation by liquid chromatography/tandem mass spectrometry. Extracted sample analysis will 
be performed by mass spectrometer using positive ionisation. The assay has been validated over 


 
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the linear dynamic range of 0.5–100 ng INH/mg of hair utilising 20–30 strands of human hair (~1–
3 mg). 
Table 5: Sample Collection Schedule & Volumes 
Time 
Blood 
Breast 
milk 
Stool 
Hair 
Assay 
6 wk 
Infant: 5 mls 
(immunologic 
assays) 
+ 
2 mls 
(LFTs)  
 
Mothers: 5 mls 
30 mls 
< 5ml 
(swab) 
NA 
BCG induced T-cell profiling, PBMCs and plasma for innate 
& T-cell assays; ESAT-6 and CFP-10 IGRA in PBMCs and 
breast milk cells. Assays will include a whole blood cytokine 
assay performed at the time of the blood draw.  In addition, 
PBMCs and plasma will be cryopreserved and examined 
later with assays that include stimulation with BCG and 
CFP10/ESAT6 with analysis by flow cytometry, as well as 
determination of NAT2 genotype for acetylator phenotype. 
Stool collected by swab will be cryopreserved for potential 
future infant gut microbiome studies. 
 
For infants randomized to receive INH, blood will be drawn 
for LFTs at baseline. 
10 wk 
5 mls 
(immunologic 
assays) 
+ 
2 mls (LFTs) 
NA 
NA 
NA 
PBMCs + plasma for storage for future exploratory studies 
including role of antibodies and infant MTB infection, role of 
IPT on BCG response 
 
For infants randomized to receive INH, blood will be drawn 
for LFTs at 1 month post INH initiation. 
12 mo post 
randmoniz
ation 
5 mls 
NA 
NA 
Approx 
30 
strands 
Infant IGRA using QFT-Plus 
 
For infants randomized to receive INH,hair analysis for INH 
exposure 
 
TB 
diagnosis, 
Study 
withdrawal 
5 mls 
NA 
NA 
NA 
Infant IGRA using QFT-Plus 
(In the event blood collected is insufficient blood volume 
(<4ml) for QFT-Plus, the blood collected will be processed 
for a different test for MTB infection.) 
 
24 mo 
5 mls 
NA 
NA 
NA 
PBMCs and plasma for innate & T-cell assays; ESAT-6 and 
CFP-10 IGRA in PBMCs. 
 
Analysis of Adaptive Immune Measures and Association with MTB infection: Using the immune 
measures outlined in A-C above, BCG-specific immune measures at the enrollment visit will be 
compared in HEU infants who later develop MTB infection and those who remain MTB uninfected 
at 12 months following enrollment. For these comparisons, infants with evidence of baseline CFP-
10/ESAT-6 responses will be excluded. We will examine whether BCG-specific cytokine 
expression (by ICS or ELISA) is associated with the development of MTB infection. We will 
assess several T-cell characteristics including: 1. T-cell subtype (CD4 vs CD8), 2. 


 
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polyfunctionality of CD4 effector phenotype (assessing IFN-γ, IL-2, and TNF), 3. TH subset 
polarization with assessment of at least TH1 and TH17 subsets, and 4. central and effector memory 
subtypes (CD45RA/RO, CCR7). In addition, we will assess a recently described memory T-cell 
phenotype (CXCR3+CCR6+) that was found to be a dominant MTB-specific phenotype in the 
genome wide screen of CD4 epitopes in LTBI subjects.97 A priori we hypothesize that 6-week 
BCG-specific CD4 IFNγ, CD8 IFNγ, and/or polyfunctional responses will be less frequent, and 
that there will be fewer central memory (CD45RA-CCR7+) cells among infants who later acquire 
MTB infection than in those with no evidence of MTB infection, matching for age at assessment 
of IGRA status, and any reported TB exposure. To assess whether INH modifies BCG responses, 
we will compare BCG immune responses at 12 month follow-up in a randomly selected subset of 
20 infants from each trial arm. 
  
Maternal Breast Milk TB Specific Cellular Immune Responses 
In addition to testing infant peripheral immune responses to BCG, we will examine whether 
maternal immune responses in breast milk are associated with infant protection from MTB 
infection.  In a Canadian cohort, breastfed infants had significantly enhanced cell-mediated 
responses to BCG compared to formula fed infants.38 Similar to peripheral blood, there are several 
types of protective immune responses that could be present in breast milk including innate and 
MTB-specific T-cell responses. The MTB-specific T-cells could be present from prior maternal 
MTB infection or TB disease. The presence of these T-cells could provide protection for the infant. 
There are also unique molecules (e.g. lactoferrin) and potential for different cellular trafficking 
and differentiation within breast milk. With our preliminary data that MTB-specific T-cell 
responses are present in BMCs and of higher magnitude than peripheral responses, we will 


 
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examine whether these responses are associated with protection from infant MTB infection. To 
accomplish this sub aim, we will measure MTB-specific T-cell responses in maternal breast milk 
cells and peripheral blood: breast milk (30 mls) and peripheral blood (5ml) will be collected at 
enrollment and BMCs and supernatant will be isolated and cryopreserved. Cells will be thawed 
and stimulated with 1. Peptide pools of MTB antigens CFP-10/ESAT-6; and 2. controls (medium 
alone and PHA). We will use flow cytometry and ICS to measure a panel of CD4 and CD8 T-cell 
responses as described above. 
 
 


 
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II 
 ISONIAZID DOSING 
Table 6: Weight-based dose of isoniazid to be used in study using based on WHO and Kenya 
MOH national guidelines.  
 
Table 6: Dose of Isoniazid (INH) for Isoniazid preventive Therapy (IPT) in children 
Weight (kg)  
Daily Dose in mg  
Number of 100 mg tablets  
<5  
50  
½  
5.1 – 9.9  
100  
1  
10-13.9  
150  
1½  
14-19.9  
200  
2  
20-24.9  
250  
2½  
>25  
300  
3*  
 
*For children more than 25 kg, one can use 1 adult tablet of INH (300mg) once daily 
(max 300 mg/day) 
 
(Source: Kenya Ministry of Health. National Guidelines on Management of Tuberculosis in 
Children, Second Edition. August 2013. Division of Leprosy, Tuberculosis and Lung Disease.) 
 
 


 
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III 
 PYRIDOXINE DOSING 
Table 7: Weight-based dose of pyridoxine to be used in study using based on Kenya MOH 
national guidelines.  
 
Table 7: Dose of pyridoxine to be used with INH administration 
Weight (kg)  
Daily Dose in mg 
Number of 50mg tablets 
5-7  
12.5 
1/4  
8-14  
25 
1/2  
15 and above  
50  
1 
 
(Source: Kenya Ministry of Health. National Guidelines on Management of Tuberculosis in 
Children, Second Edition. August 2013. Division of Leprosy, Tuberculosis and Lung Disease. 
 
 
 


 
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IV 
SAE TABLES SPECIFIC TO PERIPHERAL NEUROPATHY 
Table 8: Supplemental toxicity table for grading severity of peripheral neuropathy in children  
 
GRADE  
SYMPTOM  
Grade 2 
Unable to do one or more upper or lower extremity age-appropriate task 
on truncated Denver Developmental test  
 
OR  
 
Conveys that there is mild pain or burning sensation in hands or feet by 
expressing discomfort on touch or pressure of extremity or by refusal to 
use extremity, but has normal ankle and knee reflexes, muscle bulk, 
tone and strength.  
Grade 3 
Unable to do any upper extremity or lower extremity age-appropriate 
tasks on truncated Denver Developmental test  
 
OR  
 
Conveys pain or burning sensation in hands or feet by expressing 
discomfort on touch or pressure of extremity or by refusal to use 
extremity  
 
AND  
 
Ankle reflexes are hypoactive or absent but knee reflexes are normal  
Grade 4 
Unable to do any upper extremity or lower extremity-age appropriate 
tasks on truncated Denver Developmental test  
 
OR  
 
Conveys that pain or burning sensation exists in hands or feet by 
expressing discomfort on touch or pressure of extremity or by refusal to 
use extremity  
 
AND  
 
Either:  
(1) ankle and knee reflexes are hypoactive or absent, or  
(2) muscle bulk, tone or strength is decreased, or  
(3) foot drop is present 
 
 
 


 
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Table 9: Supplemental toxicity table for grading severity of peripheral neuropathy in children  
Evaluation of peripheral Neuropathy using truncated Denver Developmental test. Participants 
should be able to pass age-appropriate evaluations listed below. Performance rated as “Yes, No, 
or Unable to assess (subject not cooperative).” 
 
AGE  
EVALUATION  
3-4 months  Tests for peripheral neuropathy in upper extremities:  
• Grasp rattle  
• Put hands together  
 
Tests for peripheral neuropathy in lower extremities 
• Bear weight on legs 
6 months  
Tests for peripheral neuropathy in upper extremities:  
• Pass a cube from hand to hand  
• Rake a bead  
 
Tests for peripheral neuropathy in lower extremities 
• Bear weight on legs  
9 months  
Tests for peripheral neuropathy in upper extremities:  
• Thumb finger grasp  
• Bang two cubes together  
 
Tests for peripheral neuropathy in lower extremities 
• Stand holding on  
12 months  
Tests for peripheral neuropathy in upper extremities:  
• Put block in cup  
• Bang two cubes held in hands  
 
Tests for peripheral neuropathy in lower extremities 
• Stand two seconds 
 
 
 


 
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V 
Participant retention of previous RCT studies in Kenya by study staff 
Table 10: Previous RCTs conducted by study team 
Study, Lead Investigators 
(publications) 
Cohort size 
Retention 
%, 
duration 
Finding 
1. Breast versus formula feeding 
Nduati, Kreiss (JAMA 2000, 2001, 
Lancet 2000) 
425 m-i pairs 
94% 2 
year 
Risk of breastmilk HIV 
transmission 
2. Rapid testing for PMTCT 
Malonza, John-Stewart (AIDS 2003) 
1249 preg 
women 
NA 
Rapid HIV test superior  
3. PMTCT compliance 
Kiarie, John-Stewart (AIDS 2003) 
139 m-i pairs 
84% 6 wk 
Comparable SC PMTCT 
adherence  
4. ZDV effect on genital HIV 
Mbori-Ngacha, John-Stewart (J Virol 
2003) 
42 preg women 
100% 1 
wk 
Rapid decline in genital HIV 
RNA 
5. NVP vs. ZDV PMTCT 
Chung, John-Stewart (AIDS 2005) 
66 m-i pairs 
85% 6 wk 
NVP longer HIV suppression 
in BM 
6. ZDV/NVP vs. HAART PMTCT 
Chung, 
John-Stewart 
(Antiviral 
Therapy 2006) 
58 m-i pairs 
85% 1 yr 
BM RNA but not DNA 
decline 
7. Valacyclovir to decrease HIV 
Drake, Farquhar (J Infect Dis 2012) 
148 m-i pairs 
94%, 12 
mos 
VCV decreases plasma HIV 
RNA 
8. Diary for pediatric adherence 
Wamalwa, John-Stewart (JIAS 2009) 
90 children 
84%, 15 
mos 
Diaries do not improve 
adherence 
9. ART counseling vs. alarm 
Chung, 
John-Stewart 
(PLoS 
Med 
2011) 
400 
87%, 18 
mos 
Counseling superior  
10. Partners HSV Study PI Celum; 
Nairobi Site PIs: John-Stewart, Kiarie, 
Farquhar (NEJM 2010, Lancet 2011) 
3408 couples  
416 Nairobi 
84% uninf, 
92% inf, 2 
yr 
HSV-suppression does not 
decrease HIV transmission 
11. Partners PrEP PI Baeten, Celum; 
Nairobi Site PIs: John-Stewart, Kiarie, 
Farquhar (NEJM 2012) 
4747 couples,  
485 in Nairobi 
96%, 2 
years 
PrEP decreases sexual 
transmission 
12. Albendazole in HIV, helminth-
infected adults Walson, John-Stewart 
(AIDS 2009) 
208 
97%, 3 
month 
Increased CD4 post-
albendazole in ascaris 
13. Program Helminth Eradication  
Walson, John-Stewart (Lancet Inf Dis 
2012) 
948 
95.5%, 2 
years 
Deworming does not change 
HIV progression 
14. Optimizing pediatric ART 
Wamalwa, John-Stewart (CROI 2012) 
42 
98%, 18 
months 
Early ART with PI safe but 
not durable 
15. CTX cessation post-ART 
Polyak, John-Stewart (CROI 2014) 
500 
98%, 1 
year 
CTX cessation increased 
malaria 
16. Partner HIV testing in PMTCT 
Osoti, Farquhar  (AIDS 2013) 
300 
99%, 6 
weeks 
Home-based partner testing 
effective 
17. Pediatric HIV vaccine Hanke, 
Jaoko, John-Stewart (Vaccine 2014) 
72 
99%, 48 
weeks 
MVA-HIVA safe but not 
immunogenic 
18. Mobile WACh for MCH 
Unger, John-Stewart 
300 
Ongoing 
Ongoing, 300 enrolled; in 
follow-up 
19. 
Urgent 
ART 
for 
hospitalized 
children 
Wamalwa, John-Stewart 
360 
Ongoing 
Ongoing, >120 children 
enrolled 
 


 
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VI 
HAIR COLLECTION PROTOCOL FOR ANALYSIS OF INH EXPOSURE 
Hair collection protocol 
 
Materials required: Scissors, piece of tin foil, patient 
labels (2), ziplock bag, alcohol swabs, and desiccant 
pellet  
Suggest making these “hair kits” ahead of time 
 
 
 
 
 
 
Step 1:  Clean the blades of a pair of scissors with 
an alcohol pad and allow blades to completely dry 
Clean off blades of scissors between patients 
 
 
 
 
 
Step 2: Lift up the top layer of hair from the occipital region of the scalp. Isolate a small thatch of 
hair (~30 fibers of hair) from underneath this top layer  
Can use hair clip to keep top layer of hair away if easier 
 
Step 3: Cut the small hair sample as close to the scalp as possible 
 
 
 
 
 
 
 
 
STRAIGHT HAIR 


 
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CURLY HAIR 
SHORT HAIR 
Can let hair fall directly into piece of tin foil when very 
short/cropped (no need to label end since too short) 


 
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BRAIDED HAIR 
Cut hair thatch from in-between braids or dread locks 


 
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Step 4:  Keep your fingers on the part of the hair that 
was FURTHEST away from the scalp and put the hair 
sample down on an unfolded piece of tin foil 
 
 
 
 
 
Step 5:  Put a thin label over the end of the hair sample 
that was FURTHEST away from the scalp 
If hair very short just let it fall into the piece of tin foil 
and no need to label the distal end 
 
 
 


 
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Step 6:  Refold the foil over to completely enclose 
the hair and place a study ID label on the folded 
piece of foil 
 
 
 
 
 
 
Step 7: Place the folded piece of foil inside the plastic 
(e.g. Ziplock®) bag (desiccant pellet in the bag is 
optional) and seal the bag;  
 
 
  
 
 
 
 
 
 
 
 
Hair samples should be kept at room temperature and in a dark place at 
each site prior to batch shipment (without biohazardous restrictions) to our hair laboratory 
at UCSF.   
Good collection: Distal end (side 
farthest from scalp) labeled 
Bad collection: Distal end could 
have been labeled (long enough) 
but not  
Okay not to label because too 
short 


 
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VII 
COVID-19 RESPONSE PROCEDURES 
In response to the COVID-19 pandemic and policy changes issued by the Kenyan Ministry of 
Health, we are transitioning to telephone follow-up visits. Participants typically attend in-person 
visits that coincide with routine clinic visits. However, the Kenyan Ministry of Health clinics 
have been advised to reduce clinic attendance and drug pick-up frequencies. Thus, to protect the 
safety of our participants and clinic staff, visits will be conducted via telephone. Sample 
collection may be delayed if clinic visits remain difficult for clients to attend. Procedures for 
telephone visits, including efforts to maintain confidentiality, are outlined below. 
Telephone Follow-up Visit Procedures: 
1. Clinic staff will only contact participants who meet the following criteria: 
a. Participant’s contact information is in the Link Log 
b. Participant has agreed that we can contact them (also in the Link Log) 
2. Find a quiet place away from others 
3. Using a study phone, call the telephone number on record in the Link Log for the participant 
a. Request to speak to the iTIPS participant on file by name. If someone other than the 
participant answers the phone, and the participant is unavailable, state that you are calling 
on a personal matter and will call later when the participant is available. No other 
message will be left. If questioned about the nature of the call, state that you are unable to 
disclose any additional information. No messages will be left on mobile phone voice 
mail. 
b. Read the following introduction statement to the participant: “Previously, you were asked 
to volunteer for a research study, agreed, and were enrolled. As a result of the spread of 
COVID-19 globally, we are no longer doing follow-up visits in person. We are now 


 
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conducting follow-up visits by telephone and we are asking you to participate. As in the 
in-person visits, this telephone visit will include several surveys. I will read the questions 
and answer choices out loud. You will tell me your answer and I will mark it down. 
Please be as honest as possible. You may skip a question that you do not wish to answer 
by saying “no answer.” You can stop the survey or visit at any time. You will be 
identified on these surveys only by a number. We will not link your name to your 
responses. We will not share your answers with anyone outside the Study Team.” 
4. Ask the following series of questions: 
a. “Do you agree to take part in the follow-up visit by telephone?” 
i. 
If ‘yes’: 
1. Assure them of confidentiality and ask them to find a quiet place away from 
others so you can conduct the visit without interference. 
2. Ask the language of preference to conduct the visit (English, Luo, Swahili) 
ii. 
If ‘no’: 
1. Thank the participant for their time and ask if they would like to participate 
when they return to regularly scheduled visits to the facility. 
b. “Is right now a good time, or would you like me to call back at another date/time that is 
convenient for you?” 
i. 
If ‘yes’ proceed with the visit 
ii. 
If ‘no’ ask when would be a good time to call back 
c. “Does your phone have enough battery to speak for about 30 minutes?” 
i. 
If ‘yes’ proceed with the visit 
ii. 
If ‘no’ ask if they are able to plug in the phone or if you should call back 


 
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d. “Are you in a quiet and private place right now to begin the interview?” 
e. Record answers in a call log 
5. Ask the following questions about COVID-19: 
a. “Have you heard of COVID-19?” 
i. 
Yes 
ii. 
No 
b. “In what ways has COVID-19 impacted your life?” Possible answers: 
i. 
My school has been closed 
ii. 
I can no longer work 
iii. 
I can no longer go to the health facility 
iv. 
I can no longer get medication refills 
v. 
I can no longer go outside 
vi. 
Other: ______________________ 
c. Do you have any questions about COVID-19? (Staff adds comments in additional 
comments section of CRF) 
6. At the end of the visit: 
a. Thank them for their time 
b. In the event that the interview is stopped midway due to network issues or 
communications as a result of noise or not hearing well, ask the participant to complete 
the interview at a later date or time that is convenient for them 
Data Management: 
1. In terms of REDCap, the process for follow-up surveys by phone will be identical to that of 
in-person follow-up surveys, with one exception: 


 
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Protocol, version 1.9 
 April 1, 2020 
85 
 
a. In the Maternal Interim Health CRF, for the question, “Is this visit being conducted in 
person or via phone?”, select “Via phone” and add to the Additional Comments section 
as needed 
2. Disconnected call: In the event that the line is disconnected while halfway through a CRF, 
save and exit the CRF. 
a. Attempt to call the participant back immediately to complete the CRF and visit 
b. Note that the call was disconnected in a call log and how you plan to complete the CRF 
and visit in the future 
c. Upload data to REDCap, per usual 
 

SAP_001.pdf: 
 
 
Infant TB Infection Prevention Study (“iTIPS”)
 
SAP October 22,2019 Version 3.0 
Page 1 of 16 
Preventing Mycobacterium tuberculosis Infection in HIV-Exposed Infants 
 
Short Title: Infant TB Infection Prevention Study (“iTIPS”) 
 
NCT: NCT02613169 
 
Document date: October 22, 2019 
 
 


 
 
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Preventing Mycobacterium Tuberculosis Infection in HIV-
Exposed Infants  
 
Short title: Infant TB Infection Prevention Study (“iTIPS”) 
 
 
 
Statistical Analysis Plan  
 
 
TABLE OF CONTENTS 
 
1. STUDY SUMMARY AND AIMS ....................................................................................................... 3 
2. STUDY END-POINTS ..................................................................................................................... 4 
3. SAMPLE SIZE CONSIDERATIONS ................................................................................................ 5 
4. STATISTICAL ANALYSES ............................................................................................................. 5 
4.1 Intention to treat ....................................................................................................................... 5 
4.2 Study accrual............................................................................................................................ 5 
Figure 2a – Detailed trial profile  .................................................... Error! Bookmark not defined. 
Figure 2b – Detailed Enrollment and Follow-up (Open report) ....... Error! Bookmark not defined. 
Figure 3 – Graph depicting loss to follow-up by arm (Closed report) ............................................. 8 
4.3 Baseline Characteristics .......................................................................................................... 8 
Table 1a Baseline characteristics: shell table (Closed report) ....................................................... 8 
Table 1b Baseline characteristics: shell table (Open report) .......... Error! Bookmark not defined. 
4.4 Analysis of study end-points ................................................................................................ 10 
Table 2a. Primary Outcome shell table by intervention group. (Closed report) ............................ 12 
Table 2b. Exploratory Outcome shell table by intervention group. Exploratory combined endpoint 
of MTB infection, TB diagnosis, and death by study arm. (Closed report) ................................... 12 
Table 2c. Proportion of exploratory outcomes due to MTB infection, TB diagnosis or Death (first 
event only) (Open report) ............................................................................................................ 13 
5.0 QUARTERLY SUMMARIES ........................................................................................................ 13 
6.0 INTERIM ANALYSIS ................................................................................................................... 13 
7.0 SAFETY MONITORING .............................................................................................................. 13 
Table 3a. Adverse event reporting by study arm shell table (Closed report) ............................... 14 
Table 3b. Adverse event reporting overall shell table (Closed report) ......................................... 15 
8.0 REFERENCES ............................................................................................................................ 16 
 


 
 
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1. STUDY SUMMARY AND AIMS 
Rationale: HIV-exposed uninfected infants (HEU) in HIV/TB endemic settings have a high risk of 
Mycobacterium tuberculosis (MTB) infection and TB disease, even in the absence of known MTB 
exposure. [1, 2] Because infancy is a time in which there is rapid progression from primary to active 
TB[3], it is important to define where, how, and when TB preventive interventions exert their effect and 
to build new strategies that adapt or extend approaches used in adults. Protecting HEU infants during 
this vulnerable, yet temporary, period of immunodeficiency may provide long term immunologic and 
mortality benefits. The primary goal of this proposal is to determine whether isoniazid preventive 
therapy (IPT) prevents primary MTB infection in HEU infants, to determine timing and cofactors 
of primary MTB acquisition in the first year of life, and to examine the role of immune protective 
mechanisms in this cohort.  
Among HIV-infected infants, 2 randomized control trials (RCTs) yielded conflicting data about whether 
IPT prevents TB disease and/or mortality.[4, 5] Only one of these evaluated HEU infants, and found no 
protective effect of IPT in decreasing TB disease.[4] While previous IPT RCTs have focused on 
prevention of active TB disease, there are scant data regarding the impact of IPT on primary MTB 
infection. We recently found that, in 6-month old HEU infants, over 10% had evidence of MTB infection 
as detected by interferon gamma release assays (IGRAs), corresponding to a 20% annual cumulative 
incidence of infection.[6] This suggests that HEU infants have a substantial incidence of MTB infection 
related to community and household TB exposure. There are no longitudinal studies of MTB infection 
among HEUs using serial IGRA testing. Unlike tuberculin skin testing (TST), IGRAs can detect MTB 
infection and distinguish it from immune response to recent BCG vaccination. A prospective birth HEU 
cohort using IGRAs to detect MTB infection can provide an efficient approach to probe determinants of 
MTB infection, more rapidly accruing endpoints (MTB infection) than studies of TB disease and this 
study design can contribute unique insights regarding mechanisms of prevention of primary MTB 
infection. 
 
Design: A 2-arm, non-blinded randomized clinical trial (RCT) comparing the efficacy of a 12 month 
course of isoniazid (INH) vs. no INH to prevent M. tuberculosis infection among HIV-exposed uninfected 
Kenyan children aged enrolled at 6 weeks of age.  
 
Additional nested sub-studies will evaluate epidemiologic and immunologic correlates of MTB infection, 
and to explore the role of INH in prevention of active TB and mortality. 
  
Population: HIV-exposed uninfected (HEU) infants and their mothers 
 
Sites: Kisumu County Hospital (KCH) in the Nyanza province of Kenya with additional sites in Ahero 
and Bondo if necessary for recruitment. 
 
Sample Size: 300 children will be randomized (150/arm) 
 
Duration of follow-up: 12 months (enrollment at 6 (+ 4) weeks of age, 10 & 14 weeks of age, 6 & 9 & 
12 months of age, and 12 months post study enrollment) 
 
 
 


 
 
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Primary Objective: 
AIM 1: Among HEU infants enrolled at approximately 6 weeks of age, compare the risk of acquiring 
MTB infection during 1 year of follow-up in infants randomized to receive INH vs. no INH using an IGRA 
assay or tuberculin skin test (TST) to determine MTB infection status. 
 
Secondary Objectives:  
AIM 2: Determine epidemiologic correlates of MTB infection among infants enrolled in the RCT. 
 
AIM 3: Determine immune correlates of risk of primary MTB infection and their potential interactions 
with INH. Assays will include infant peripheral blood BCG-specific T-cell responses at approximately 6 
weeks post BCG vaccination, and maternal breast milk and peripheral blood MTB-specific T-cell 
responses at approximately 6 weeks postpartum. 
 
Exploratory Objectives: 
Investigate the impact of IPT on a combined endpoint of MTB infection, TB disease, and death among 
HEU infants. 
 
Figure 1: Aims of RCT to evaluate INH to prevent MTB infection in HEU infants 
2. STUDY END-POINTS 
Primary study end-points: 
1. MTB infection will be assessed by interferon gamma release assays (IGRA, QFT-Plus) or TST 
at 12 months post-enrollment. QFT-Plus assay will be run at KEMRI/CDC Kisian TB immunology 
lab. 
 
Secondary end-points: 
2. Severe Adverse Events (SAE): number of infants with grade 3 or higher treatment-related 
adverse events as assessed by DAIDS Table for the Grading Severity of Pediatric Adverse 
Experiences.  
 
3. Combined outcome of MTB infection including IGRA, TST, and additional interferon-
gamma-independent immune markers in QFT-Plus supernatants.  Combined outcome will 
be defined as positive if IGRA OR TST OR interferon-gamma-independent marker is positive 
and combined outcome will be defined as negative if none of these is positive (if children do not 
have all three markers the definition will hold for available markers). 
 
4. Epidemiologic correlates of MTB infection will be assessed via structured questionnaire and 
medical record review, by collecting information on the following: 
BCG 
Aim 1:  RCT of IPT 
in HEU infants 
Infected M  
Aim 3:  Discover immune correlates of 
risk of MTB infection 
MTB 
M  FATE
KILLED 
GROWS
MTB
INFECTION 
NO 
HIV 
T cell
Randomize to 
INH vs  no INH 
MTB Exposure 
Risk
YES 
12 months 
Aim 2:  Discover 
epidemiologic risk 
factors
birth 
in utero 
6 weeks
(ongoing)


 
 
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• 
Maternal HIV viral load and CD4 cell counts, maternal prior PMTCT/ART regimens, 
infant PMTCT prophylaxis 
• 
Birth weight, BCG vaccination status and timing, and intercurrent illnesses and vaccines 
• 
Growth measures (weight, length, head circumference, mid-upper arm circumference)  
• 
Infant feeding and symptoms (including cough, fever, weight loss, or growth faultering) 
• 
Infant HIV status at study enrollment and study end 
• 
Report of known TB exposure, maternal IPT status 
 
5. Immunologic correlates of MTB infection will be assessed via: 
• 
Infant peripheral blood BCG-specific T-cell responses at approximately 6 weeks post 
BCG vaccination 
• 
Maternal breast milk and peripheral blood MTB-specific T-cell responses at 
approximately 6 weeks postpartum. 
 
Exploratory end-points: 
1. A combined endpoint of MTB infection, TB disease, and death will be assessed via: 
• 
MTB infection as measured by IGRA or TST at 12 months post-enrollment 
• 
TB disease including microbiologically confirmed (culture or Xpert positive), or probable 
TB (clinical diagnosis) 
• 
Death of HEU infant 
3. SAMPLE SIZE CONSIDERATIONS 
The primary endpoint will be a MTB 
infection measured by IGRA (QFT-
Plus). 
Assuming 
alpha=5%, 
power=80%, 2-sided test, and a 1:1 
allocation ratio, with 125 infants in each 
arm we would have power to detect at 
least a 65% decrease in MTB infection 
in INH arm vs. control if cumulative 
incidence of positive IGRA in the control 
arm at 12 months follow-up is 0.2, or to 
detect a 70-80% or higher (HR 0.3-0.2) 
decrease if the cumulative incidence of 
positive IGRA in control arm is 0.15 or 
0.1 (Table 1).  We will increase sample 
size by 20% to account for loss to 
follow-up, non-adherence, and isoniazid 
resistance, enrolling 300 mother-infant 
pairs (150 per arm). 
 
4. STATISTICAL ANALYSES 
4.1 Intention to treat 
Analysis of primary outcomes will be by intention-to-treat (all HIV-exposed uninfected participants).  
4.2 Study accrual 
Per CONSORT guidelines, we will report the number of individuals who (See Figure 2a): 
1. Underwent screening 
2. Met inclusion criteria  
Table 1: AIM 1 Sample Size Estimates (Gray shaded close to 
study target) 
Power 80%,  
2-sided p 0.05 1 
year follow-up 
Maximum 
HR 
for 
IPT 
detectable 
Risk 
of 
positive IGRA 
at m12 
Number 
per arm 
0.2 
risk 
IGRA 
positive after 12 
mos follow-up 
0.5 
0.2 
220 
0.4 
0.2 
150 
0.35 
0.2 
120 
0.32 
0.2 
100 
0.2 
0.2 
55 
0.15 risk IGRA 
positive after 12 
months follow-up 
0.5 
0.15 
300 
0.4 
0.15 
180 
0.35 
0.15 
150 
0.31 
0.15 
120 
0.2 
0.15 
75 
0.10 risk IGRA 
positive after 12 
months follow-up 
0.5 
0.1 
420 
0.4 
0.1 
270 
0.35 
0.1 
220 
0.3 
0.1 
180 
0.2 
0.1 
110 


 
 
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3. Did not meet inclusion criteria (and reasons) 
4. Enrolled in the study and were randomized 
5. Treated as per study protocol (received at least one of study drug in INH arm, and did not 
receive INH in the No INH arm) 
The number of individuals enrolled and randomized including visit completion will be described by arm. 
No formal statistical testing will be performed at interim DSMB reviews for enrollment or randomization.  
Loss to follow-up by arm will be presented by arm for the closed DSMB report. 
 
Figure 2a – Detailed trial profile  
 


 
 
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Excluded  (n=   ) 
   Not meeting inclusion criteria (n=  ) 
   Declined to participate (n=  ) 
   Other reason (n=  ) 
Randomized  
(N=  ) 
Completed Enrolment 
6 weeks of age 
(n=   ) 
Completed Visit 7 
12 months post enrolment 
(n=   , Expected n=   ) 
Completed Visit 2 
10 weeks of age 
(n=    , Expected n=   ) 
Assessed for eligibility 
(N=  ) 
Figure 2b – Detailed Enrollment and Follow-up (Open report) 
Completed Visit 3 
14 weeks of age 
(n=   , Expected n=   ) 
Completed Visit 4 
6 months of age 
(n=   , Expected n=   ) 
Completed Visit 5 
9 months of age 
(n=   , Expected n=   ) 
Completed Visit 6 
12 months of age 
(n=   , Expected n=   ) 
Primary Analysis 
(n=   ) 
Excluded  (n=   ) 
   Reason (n=  ) 
Excluded  (n=   ) 
   Reason (n=  ) 
Excluded  (n=   ) 
   Reason (n=  ) 
Excluded  (n=   ) 
   Reason (n=  ) 
Excluded  (n=   ) 
   Reason (n=  ) 
Excluded  (n=   ) 
   Reason (n=  ) 
Excluded  (n=   ) 
   Reason (n=  ) 
Excluded  (n=   ) 
   Reason (n=  ) 


 
 
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Figure 3 – Histograms depicting loss to follow-up by arm (Closed report) 
A histogram depicting proportion of participants retained in the study at each visit will be provided. 
4.3 Baseline Characteristics 
We will describe the distribution of baseline characteristics, using summary statistics appropriate for 
the measurement scale. For the Closed report, this will be presented by blinded intervention groups 
(X,Y) (Table 1a). For the Open report, this will be presented in aggregate.  
 
Table 1a Baseline characteristics: shell table (Closed report) 
 
 
 
All Participants 
N= 
 
INH 
N= 
 
No INH 
N= 
 
Characteristic 
N 
Median (IQR) 
or No. (%) 
N 
Median (IQR)  
or No. (%) 
N 
Median (IQR)  
or No. (%) 
 
Infant characteristics 
 
 
 
 
 
 
 
Sociodemographic  
 
 
 
 
 
 
 
 
Infant age (weeks) 
 
 
 
 
 
 
 
 
Weight-for-age Z score  
 
 
 
 
 
 
 
 
Underweight (WAZ < -2)  
 
 
 
 
 
 
 
 
Weight-for-age Z score 
among underweight  
 
 
 
 
 
 
 
 
Male 
 
 
 
 
 
 
 
Breastfeeding 
 
 
 
 
 
 
 
 
Ever breastfed 
 
 
 
 
 
 
 
 
Breastfed on enrollment 
 
 
 
 
 
 
 
Infant PMTCT/BCG 
 
 
 
 
 
 
 
 
Infant received ARVs for 
PMTCT 
 
 
 
 
 
 
 
 
Infant BCG vaccination 
 
 
 
 
 
 
 
Maternal characteristics 
 
 
 
 
 
 
 
Sociodemographic  
 
 
 
 
 
 
 
Maternal age 
 
 
 
 
 
 
 
Secondary education started 
 
 
 
 
 
 
 
Parity 
 
 
 
 
 
 
 
Maternal HIV/TB 
 
 
 
 
 
 
 
 
Maternal partner HIV status 
 
 
 
 
 
 
* 
 
Positive 
 
 
 
 
 
 
 
 
Negative 
 
 
 
 
 
 
 
 
Unknown 
 
 
 
 
 
 
 
 
Maternal HIV diagnosis 
 
 
 
 
 
 
 
 
Before pregnancy 
 
 
 
 
 
 
 
 
During pregnancy 
 
 
 
 
 
 
 
 
After delivery 
 
 
 
 
 
 
 
 
Maternal ARVs  
 
 
 
 
 
 
 
 
Initiated before 
pregnancy  
 
 
 
 
 
 
 
 
Initiated during 
pregnancy  
 
 
 
 
 
 
 
 
Initiated after pregnancy 
 
 
 
 
 
 
 
 
Maternal ARV regimen  
 
 
 
 
 
 
 


 
 
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Efavirenz-based regimen 
 
 
 
 
 
 
 
 
Nevirapine-based 
regimen 
 
 
 
 
 
 
 
 
Protease inhibitor-based 
regimen 
 
 
 
 
 
 
 
 
Maternal CD4 (cells/mm3) 
 
 
 
 
 
 
 
 
Maternal HIV RNA 
(copies/ml) 
 
 
 
 
 
 
 
 
HIV viral load >1000 
 
 
 
 
 
 
 
 
Maternal history of TB 
 
 
 
 
 
 
 
 
Maternal history of IPT 
 
 
 
 
 
 
 
 
Initiated before 
pregnancy  
 
 
 
 
 
 
 
 
Initiated during 
pregnancy  
 
 
 
 
 
 
 
 
Initiated after pregnancy 
 
 
 
 
 
 
 
Residential characteristics 
 
 
 
 
 
 
 
Crowding 
 
 
 
 
 
 
 
 
Persons in household 
 
 
 
 
 
 
 
 
Rooms in household 
 
 
 
 
 
 
 
Housing 
 
 
 
 
 
 
 
 
Toilet type in household 
 
 
 
 
 
 
 
 
None 
 
 
 
 
 
 
 
 
Pit latrine  
 
 
 
 
 
 
 
 
Flush 
 
 
 
 
 
 
 
 
Roof type 
 
 
 
 
 
 
 
 
Thatched 
 
 
 
 
 
 
 
 
Iron sheet 
 
 
 
 
 
 
 
 
Electricity in home 
 
 
 
 
 
 
 
 
Running water in home 
 
 
 
 
 
 
 
 


 
 
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4.4 Analysis of study end-points 
Note: Analysis focused on primary and safety end-points (Aim 1) 
 
Modified intention to treat: The primary modified intention-to-treat analysis will exclude infants found 
to be HIV DNA positive at enrollment or during the study. At the guidance of the DSMB TST was 
added in combination with IGRA for the primary endpoint of MTB infection. 
 
Primary endpoint: MTB infection. We will use a Chi square (2) test to compare the prevalence of 
infant Mtb infection (by IGRA or TST) at 12 months following enrollment between INH and No INH arms 
in an intent-to-treat analysis (ITT). If the baseline assessment of randomization (Table 1a) reveals an 
imbalance in characteristics between the treatment groups, we will evaluate these variables as potential 
confounders in a sub-analysis secondary to the ITT. Potential baseline confounders will evaluated 
individually to see if adjustment changes the odds ratio by more than 10%. Collinearity between 
potential confounders will be evaluated. After excluding collinear variables, these potential confounders 
will be added to a multivariable logistic model.  
 
We will also evaluate the cumulative incidence of Mtb infection (By IGRA or TST) and compare the 
hazard ratio of MTB infection between arms. Since infants are only tested once at the end of the study 
and the timing of actual Mtb infection is unknown, infants with Mtb infection assumed to have become 
positive at end of 6 months; infants without Mtb infection at end of study censored at completion of 12 
months and assumed to be negative at time of censor  
 
Per protocol: We will also evaluate our primary outcome by a per protocol analysis, considering only 
HEU infants who took at least one dose as taking INH vs. infants who did not take any INH. We 
anticipate future sensitivity analyses using IPT adherence and continuation data as exposure of interest 
and Mtb infection as outcome. 
 
Exploratory endpoint: combined endpoint of MTB infection, TB diagnosis, and death. Time to 
combined endpoint-free survival will be compared between randomization groups using Kaplan-Meier 
(K-M) survival analysis and a log-rank test in an ITT analysis. 
 
Table 2a demonstrates how primary endpoint data will be reported. Table 2b demonstrates how 
secondary MTB infection endpoint data will be reported. Table 2c demonstrates how exploratory 
endpoint data will be reported. 
 
Secondary endpoints:  
Safety outcomes: We will compare proportions of participants with severe adverse events (Grade >3 
per DAIDS Grading Severity of Pediatric Adverse Experiences) by arm using either Chi-squared or 
Fisher’s exact tests as appropriate for > grade 3 serious adverse events. 
 
Secondary epidemiologic and immunologic endpoints will evaluated after the end of the study, and will 
not be part of DSMB review. An analysis plan for these secondary endpoints is included below for 
reference only. 
 
In addition, we will conduct secondary analyses using an expanded Mtb infection definition including a 
positive TST, QFT-Plus, or IFN-γ-independent immune markers in QFT-Plus supernatants. 
 


 
 
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Epidemiologic correlates of MTB infection. To identify epidemiologic correlates of MTB infection 
among infants enrolled in the study, cryopreserved specimens from the baseline visit will be assessed 
for responses to TB antigens concurrent with other assays outlined in immune correlates in the next 
paragraph. Any infant with positive IGRA at baseline will be excluded from cofactors analyses. We 
anticipate 25 MTB infections in the control arm and ~9 in the INH arm (assuming INH is 65% effective). 
In nested case-control analyses, infants who had IGRA conversion suggestive of new primary MTB 
infection will be compared to those with valid negative assays at 12 months. Univariate and multivariate 
logistic regression will be used to compare mother-infant characteristics of infants with and without 
IGRA positive assays at month 12. We will initially build nested case-control studies incorporating all 
MTB infections from both arms of the RCT and then conduct stratified analyses in each trial arm to 
evaluate potential cofactors that are modified by INH. 
 
Immunologic correlates of MTB infection. To identify infant and maternal (including peripheral blood 
and breast milk) immune correlates of risk of primary MTB infection and assess their potential 
interactions with INH, magnitude of immune measures (BCG-specific T-cell responses measured by 
ICS, cytokine/chemokine levels in peripheral blood or breast milk at 6 weeks of age and maternal 
peripheral blood at enrollment will be compared between cases (who subsequently acquire MTB 
infection) and controls (who do not) using GEE with Gaussian link to account for clustering. We will use 
methods for normal data and then use non-parametric approaches or dichotomize and use binomial 
link GEE if data are highly skewed and cannot be transformed into a normal distribution. 
 
 
 


 
 
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Table 2a. Primary Outcome - Prevalence of MTB infection at endpoint (mITT) 
 
X group 
(N= ) 
Y group 
(N= ) 
Effect Estimate 
Outcome 
n 
% 
n 
% 
RR 
(95% CI) 
p-value 
 
 
 
 
 
 
 
MTB infection* 
 
QFT-positive 
 
TST positive 
**Either QFT or TST positive 
 
Table 2b. Primary Outcome - Cumulative incidence of MTB infection at endpoint (mITT) 
 
Cumulative incidence of Mtb infection over 12 months* 
Primary 
Outcome 
Overall 
(N=) 
X group 
 (N=) 
Y group 
 (N=) 
HR 
(95% CI) 
p 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
MTB 
Infection  
 
 
 
 
 
 
 
 
 
 
 
 
Overall 
(N=) 
X group 
 (N=) 
Y group 
 (N=) 
HR 
(95% CI) 
p 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
QFT 
positive 
 
 
 
 
 
 
 
 
 
 
 
 
Overall 
(N=) 
X group 
 (N=) 
Y group 
 (N=) 
HR 
(95% CI) 
p 
 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
Events 
Child-
years 
Incidence per 
100 CY  
(95% CI) 
TST 
positive 
 
 
 
 
 
 
 
 
 
 
 
 
Table 2c. Exploratory combined endpoint of MTB infection, TB diagnosis, and death by study 
arm.  
 
X group 
(N= ) 
Y group 
(N= ) 
Effect Estimate 
Outcome 
n Person-time Incidence rate 
(/100) 
n 
Person-time Incidence rate 
(/100) 
Hazard 
ratio 
(95% CI) 
p-value 
 
 
 
 
 
 
 
 
 
MTB infection, TB 
disease or Death 
 


 
 
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Table 2d. Proportion of exploratory outcomes due to MTB infection, TB diagnosis or Death 
(first event only) (Open report) 
 
Overall 
(N= ) 
Outcome 
n 
% 
 
 
 
 
MTB infection  
 
TB diagnosis 
 
Possible 
 
Probable 
 
Confirmed 
 
Death 
 
5.0 QUARTERLY SUMMARIES 
Quarterly summaries will be circulated to study co-investigators, study monitors, and the DSMB. Open 
reports will include data aggregated over both arms (Figure 2, Table 1b). Closed reports with data 
separated by arm will only be made available to the study statistician and DSMB members. The 
summary will include a summary of adverse events and severe adverse events by study arm for the 
Closed report (Table 3a) in aggregate for the Open report (Table 3b). At the request of the DSMB, 
summaries will be sent to the DSMB by the study statistician. We anticipate DSMB meetings to occur 
at study initiation, 50% enrollment, 25% study endpoint ascertainment. At the guidance of the DSMB 
quarter summaries were reduced to approximately every 6 months. 
6.0 INTERIM ANALYSIS AND ANTICIPATED DSMB MEETING 
Interim analyses for the risk of MTB infection and the exploratory endpoint will be performed when 25%, 
50%, and 75% of expected enrolled children have reached 12 months.  O’Brien-Fleming boundaries for 
benefit and harm will be used for interim monitoring and these boundaries will be provided by the study 
statistician in the closed reports.  The DSMB will review the interim reports and assess the study in 
terms of operational aspects, safety, and effectiveness and will make recommendations regarding 
continuation or modifications of the study. Futility will not be a basis for stopping rules because of the 
trials’ value in understanding epidemiologic and immunologic correlates of MTB infection in HEU infants. 
We anticipate DSMB meetings to occur at study initiation, 50% enrollment, and at 25%, 50%, 75% and 
100% of study endpoint ascertainment. At the guidance of the DSMB the number of formal interim 
analyses of the primary endpoint were reduced to one formal DSMB meeting at approximately 
50% enrollment, with administrative report reviews reduced to approximately Q 6 months. 
7.0 SAFETY MONITORING 
 
Adverse and severe adverse events (SAEs) will be monitored by the DSMB. SAE summaries of severe 
adverse events (SAEs) will be sent to the DSMB members during DSMB review and individual-child 
SAE forms sent to the safety officer per request. Each SAE will be assigned the plausibility of 
relatedness to study drug which will be assigned by study PIs. The data will not be presented in these 
reports by intervention group unless requested by DSMB. These reports will be descriptive (no statistical 
analyses). At the DSMB meetings to review interim analyses the SAE data will be presented by arm for 
review and the DSMB will make recommendations regarding any imbalances in safety outcomes. At 
the guidance of the DSMB, TB was removed from the SAE reporting. 
 


 
 
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Table 3a. Adverse event reporting by study arm shell table (Closed report) 
 
 
Overall 
(N=) 
X group 
 (N=) 
Y group 
 (N=) 
SAE 
n 
% 
n 
% 
n 
% 
Total participants with SAEs* 
 
 
 
 
 
 
Total SAEs 
 
 
 
 
 
 
Deatha 
 
 
 
 
 
 
Hepatic Failure 
 
 
 
 
 
 
HIV infection 
 
 
 
 
 
 
Hepatotoxicity*  
 
 
 
 
 
 
Peripheral neuropathy*  
 
 
 
 
 
 
Hospitalizationa,b,c 
 
 
 
 
 
 
Pneumonia/URTI/flu 
 
 
 
 
 
 
Gastroenteritis 
 
 
 
 
 
 
Malaria 
 
 
 
 
 
 
Otherd 
 
 
 
 
 
 
 
 


 
 
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Table 3b. Severe Adverse event reporting overall shell table (Grade 3 or above) (Open report) 
 
Overall 
(N=) 
Characteristic 
n 
% 
Total participants with SAEs* 
 
 
Total SAEs 
 
 
Deatha 
 
 
Hepatic Failure 
 
 
HIV infection 
 
 
Hepatotoxicity*  
 
 
Peripheral neuropathy*  
 
 
Hospitalizationa,b,c 
 
 
Pneumonia/URTI/flu 
 
 
Gastroenteritis 
 
 
Malaria 
 
 
Otherd 
 
 
 
 
 


 
 
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8.0 REFERENCES 
1. 
Cotton MF, Schaaf HS, Lottering G, Weber HL, Coetzee J, Nachman S. Tuberculosis exposure 
in HIV-exposed infants in a high-prevalence setting. Int J Tuberc Lung Dis 2008,12:225-227. 
2. 
Madhi SA, Nachman S, Violari A, Kim S, Cotton MF, Bobat R, et al. Primary isoniazid prophylaxis 
against tuberculosis in HIV-exposed children. N Engl J Med 2011,365:21-31. 
3. 
Marais BJ, Gie RP, Schaaf HS, Hesseling AC, Obihara CC, Starke JJ, et al. The natural history 
of childhood intra-thoracic tuberculosis: a critical review of literature from the pre-chemotherapy 
era. Int J Tuberc Lung Dis 2004,8:392-402. 
4. 
Madhi SA, Nachman S, Violari A, Kim S, Cotton MF, Bobat R, et al. Primary isoniazid prophylaxis 
against tuberculosis in HIV-exposed children. N Engl J Med 2011,365:21-31. 
5. 
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