The Pharmacokinetic Parameters of Tenofovir (TFV) in Maternal Blood and Breast Milk in Women Treated With Daily Tenofovir Disoproxil Fumarate (TDF; 300mg) for Prevention of Mother to Child Transmission (PMTCT) of Hepatitis B Virus (HBV) Mono-infection
Phase: PHASE2
Conditions: Hepatitis B, Tenofovir, Pregnancy
Sponsor: University of Oxford

 
 
 
 
 
Title: The pharmacokinetic parameters of tenofovir (TFV) in maternal blood and 
breast milk in women treated with daily Tenofovir Disoproxil Fumarate (TDF; 
300mg) for prevention of mother to child transmission (PMTCT) of hepatitis B 
virus (HBV) mono-infection 
 
Short Title: Tenofovir in pregnancy to prevent mother to child transmission of Hepatitis B. 
Date and Version No: V.3.0, dated 28 October 2019 
Trial registration number: NCT04114890 
 
 
 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 1 OF 37 
 
 
Title: The pharmacokinetic parameters of tenofovir (TFV) in maternal blood and 
breast milk in women treated with daily Tenofovir Disoproxil Fumarate (TDF; 
300mg) for prevention of mother to child transmission (PMTCT) of hepatitis B 
virus (HBV) mono-infection 
 
Short Title:   Tenofovir in pregnancy to prevent mother to child transmission of Hepatitis B. 
OxTREC reference: 39-19 
Protocol no.: VIR19001 
Date and Version No: V.3.0, dated 28 October 2019 
 
 
Study sponsor:  University of Oxford 
Funder: iTPA Award 
 
 
 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 2 OF 37 
Name 
Department 
Role & Function in 
this project 
Email 
Rose McGready 
Professor tropical maternal and child health (Deputy 
Director SMRU) 
Principal Investigator 
rose@shoklo-unit.com  
Marieke Bierhoff 
Department of Internal Medicine and Tropical 
Diseases,  Academic Medical Center Amsterdam, The 
Netherlands 
Co-Principal 
Investigator 
mariekebierhoff@yahoo.com  
Podjanee Jittmala 
Department of Tropical Hygiene, Faculty of Tropical 
Medicine, Mahidol University Thailand 
Faculty Tropical 
Medicine Study 
Representative  
Podjanee@tropmedres.ac 
Joel Tarning 
Department of Pharmacology, Mahidol Oxford 
Tropical Medicine  Research unit, Mahidol University 
Co-investigator 
joel@tropmedres.ac  
Clare Ling 
Department of Microbiology, SMRU 
Co-investigator 
clare@tropmedres.ac 
Francois Nosten 
Director SMRU 
Co-investigator 
francois@tropmedres.ac  
David Burger 
Department of Pharmacy, Radboud University Medical 
Center, Nijmegen, The Netherlands 
Co-investigator 
David.Burger@radboudumc.nl  
 
Conflict of Interest 
The investigators declare no conflict of interest. 
 
Confidentiality Statement 
This document contains confidential information that must not be disclosed to anyone other than the Sponsor, 
the Investigator Team and members of the Research Ethics Committee. This information cannot be used for any 
purpose other than the evaluation or conduct of the clinical investigation without the prior written consent of the 
principal investigator.   
 
Signature…………………………………………………  Date …………………………….. 
Name ……………Rose McGready…………… 

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             PAGE 3 OF 37 
TABLE OF CONTENTS 
TABLE OF ABBREVIATIONS ........................................................................................................................................... 5 
1. SYNOPSIS .......................................................................................................................................................................... 6 
2. INTRODUCTION – BACKGROUND AND PRIOR RESEARCH .............................................................................. 7 
3. STUDY RATIONALE & JUSTIFICATION ................................................................................................................... 8 
4. STUDY OBJECTIVES AND OUTCOME MEASURES................................................................................................ 8 
5. STUDY DESIGN ................................................................................................................................................................ 9 
6 PARTICIPANT IDENTIFICATION AND RECRUITMENT ..................................................................................... 10 
6.1 STUDY POPULATION ..................................................................................................................................................... 10 
6.2 INCLUSION CRITERIA .................................................................................................................................................... 10 
6.3 EXCLUSION CRITERIA ................................................................................................................................................... 10 
6.4 CO-ENROLMENT GUIDELINES ....................................................................................................................................... 11 
6.5 STUDY SITE ................................................................................................................................................................... 11 
7. STUDY PROCEDURES .................................................................................................................................................. 11 
7.1 PRE-STUDY TRAINING ................................................................................................................................................... 11 
7.2 SUBJECT SCREENING AND ENROLMENT ......................................................................................................................... 11 
7.3 STUDY EVALUATION .................................................................................................................................................... 11 
7.3.1 Screening Visit, Day -7 ........................................................................................................................................ 11 
7.3.2 Study enrollment, Day 0 (Baseline) ..................................................................................................................... 12 
7.3.3 TDF administration ............................................................................................................................................. 12 
7.3.4 Evaluation of TDF regimen ................................................................................................................................. 12 
7.3.5 Follow up ............................................................................................................................................................. 13 
7.3.5.2 Delivery ............................................................................................................................................................. 13 
7.3.5.3 Post-partum follow up of mothers ..................................................................................................................... 14 
7.3.5.5 Post-partum follow up of infants ....................................................................................................................... 15 
7.3.6 Subjects transferred to another hospital .............................................................................................................. 15 
8. NOTES ON STUDY PROCEDURES / INVESTIGATIONS ....................................................................................... 15 
8.1 CLINICAL ...................................................................................................................................................................... 15 
8.2 BLOOD SAMPLE COLLECTIONS ...................................................................................................................................... 16 
8.2.1. Mother blood sample collections ........................................................................................................................ 16 
8.2.2. Cord blood sample collections ............................................................................................................................ 16 
8.2.3. Infant blood sample collections .......................................................................................................................... 16 
8.2.4 BREASTMILK SAMPLE COLLECTION ........................................................................................................................... 17 
8.2.4 Blood tests ............................................................................................................................................................ 18 
8.3 SAMPLE LABELLING ...................................................................................................................................................... 19 
8.5 SAFETY NET .................................................................................................................................................................. 19 
9. DRUG MANAGEMENT ................................................................................................................................................. 20 
9.1 SUPPLY STORAGE AND HANDLING ............................................................................................................................... 20 
9.2 DRUG ACCOUNTABILITY .............................................................................................................................................. 20 
9.3 CONCOMITANT MEDICATIONS ...................................................................................................................................... 20 
10. DISCONTINUATION/WITHDRAWAL OF PARTICIPANTS FROM STUDY .................................................... 20 
10.1 VOLUNTARY WITHDRAWAL OF CONSENT.................................................................................................................... 20 
10.2 PI WITHDRAWAL ......................................................................................................................................................... 20 
10.3 MANAGEMENT OF WITHDRAWN SUBJECTS .................................................................................................................. 21 
10.4 STUDY DISCONTINUATION.......................................................................................................................................... 21 
11. SUBJECT SAFETY MONITORING & ADVERSE EVENT REPORTING ........................................................... 21 
11.1 ADVERSE EVENTS....................................................................................................................................................... 21 

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11.2 GRADING OF AN ADVERSE EVENT .............................................................................................................................. 21 
11.3 RELATEDNESS OF ADVERSE EVENT TO TDF............................................................................................................... 22 
11.4 SERIOUS ADVERSE EVENT RECORDING ...................................................................................................................... 22 
11.5 SUBJECT MANAGEMENT OF ADVERSE EVENTS ........................................................................................................... 22 
11.6 SERIOUS ADVERSE EVENTS ........................................................................................................................................ 22 
11.7 REPORTING OF SERIOUS ADVERSE EVENTS ................................................................................................................ 23 
11.8 SAFETY REVIEW OF THE LABORATORY REPORT ......................................................................................................... 23 
12. STATISTICS .................................................................................................................................................................. 23 
12.1 THE NUMBER OF PARTICIPANTS ................................................................................................................................. 23 
12.2 STUDY ENDPOINTS ...................................................................................................................................................... 23 
12.2.1 Primary endpoint ............................................................................................................................................... 23 
12.2.2 Secondary endpoints .......................................................................................................................................... 23 
12.3 DATA ANALYSIS ......................................................................................................................................................... 24 
12.3.1 Continuous data ................................................................................................................................................. 24 
12.3.2 Pharmacokinetic data ........................................................................................................................................ 24 
12.3.3 Infant drug exposure .......................................................................................................................................... 24 
13 HUMAN SUBJECTS CONSIDERATIONS ................................................................................................................. 25 
13.1 ETHICAL REVIEW ....................................................................................................................................................... 25 
13.2 INFORMED CONSENT .................................................................................................................................................. 25 
13.3 RISKS .......................................................................................................................................................................... 26 
13.3.1 Risks of oral TDF ............................................................................................................................................... 26 
13.3.2 Risk of Phlebotomy ............................................................................................................................................ 27 
13.4 BENEFITS .................................................................................................................................................................... 27 
13.4.1 Benefits to the individuals .................................................................................................................................. 27 
13.4.2 Benefits to society .............................................................................................................................................. 27 
13.4.3 Future research & drug policy implications ...................................................................................................... 27 
13.5 ALTERNATIVES TO STUDY PARTICIPATION ................................................................................................................. 27 
13.6 COMPENSATION .......................................................................................................................................................... 27 
13.7 PARTICIPANTS CONFIDENTIALITY .............................................................................................................................. 28 
13.8 INSURANCE ARRANGEMENT ....................................................................................................................................... 28 
14. LABORATORY SPECIMENS & PRACTICE ........................................................................................................... 28 
14.1 LABORATORY SPECIMENS .......................................................................................................................................... 28 
14.2 QUALITY CONTROL AND QUALITY ASSURANCE ......................................................................................................... 29 
14.3 SPECIMEN STORAGE ................................................................................................................................................... 29 
14.4 SPECIMEN TRANSPORT ................................................................................................................................................ 29 
15. ADMINISTRATIVE PROCEDURES .......................................................................................................................... 29 
15.1 DATA MANAGEMENT ................................................................................................................................................. 29 
15.2 RECORD RETENTION ................................................................................................................................................... 30 
15.3 USE OF INFORMATION AND PUBLICATIONS ................................................................................................................. 30 
16. REFERENCES ............................................................................................................................................................... 31 
17. APPENDIX A: THAI NATIONAL GUIDELINE FOR PREVENTION OF MATERNAL-TO-CHILD 
TRANSMISSION OF HEPATITIS B ................................................................................................................................ 34 
18. APPENDIX B: STUDY FLOW ..................................................................................................................................... 35 
19. APPENDIX C: SCHEDULE OF STUDY PROCEDURES ........................................................................................ 36 
20. APPENDIX D: AMENDMENT HISTORY ................................................................................................................. 37 
 
 

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Table of Abbreviations 
ALT 
Alanine transaminase 
ARVs 
Antiretrovirals 
AUC 
Area Under Curve 
CRF 
Case Report Form 
DNA 
DeoxyriboNucleic Acid 
DOT 
Directly Observed Treatment  
EGA 
Estimated Gestational Age 
GCP 
Good Clinical Practice 
HB 
Hepatitis B 
HBeAg 
Hepatitis B e antigen 
HBIG 
Hepatitis B immunoglobulin 
HBsAg 
Hepatitis B s antigen 
HBV 
Hepatitis B virus 
HIV 
 Human Immunodeficiency Virus 
ICF 
Informed Consent Form 
MTCT 
Mother to child transmission 
OXTREC 
Oxford Tropical Research Ethics Committee 
PI 
Principal Investigator 
PIL 
Participant/ Patient Information Leaflet 
PK 
Pharmacokinetics 
PMTCT 
Prevention Mother to child transmission 
REC 
Research Ethics Committee 
SOP 
Standard Operating Procedure 
SMRU 
Shoklo Malaria Research Unit 
TDF 
Tenofovir disoproxil fumarate 
TFV 
Tenofovir  
 
 

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1. Synopsis 
Study Title 
The pharmacokinetic parameters of tenofovir (TFV) in maternal blood and breast milk 
in women treated with daily Tenofovir Disoproxil Fumarate (TDF; 300mg) for 
prevention of mother to child transmission (PMTCT) of hepatitis B virus (HBV) 
mono-infection. 
Internal ref.no. 
VIR19001 
Study Design 
Prospective descriptive dense pharmacokinetics study of Tenofovir (TFV) in 24 
migrant pregnant women treated with Tenofovir disoproxil fumarate (TDF) to prevent 
mother to child transmission of Hepatitis B. 
Study Participants 
Women with viable pregnancy with HBV mono-infection  
Planned Sample 
Size 
24 pregnant women – 12 will enroll in 2nd trimester (T2) and 12 in 3rd trimester (T3) 
Planned Study 
Period 
August 2019 -   August 2021 
 
Objectives 
Outcome Measures  
Primary 
To measure the dense PK parameters of 
TFV in a steady-state during three 
gestational periods in TDF treated HBV 
mono infection  
PK parameter profiles in maternal blood 
Secondary 
1. To investigate the TFV concentration 
, breastmilk and infants  
- Maternal breastmilk TFV concentrations 
- Milk to maternal plasma concentration 
ratios (M/P) 
- Infant plasma drug  concentration  
2. To investigate if/when HBV DNA is 
detected in blood and breastmilk after 
cessation of TDF. 
HBV DNA in blood and breastmilk 
 
 
 
 
 
 

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2. Introduction – Background and Prior Research 
Hepatitis B virus (HBV) can be asymptomatic for years but can also lead to chronic disease, cancer and hepatitis, 
hepatocellular carcinoma, and liver failure be eradicated with the current therapy. (1-3) Chronic maternal Hepatitis 
B virus (HBV) infection is an important source of perinatal transmission in regions of high HBV prevalence. In 
antenatal clinics at Shoklo Malaria Research Unit (SMRU), the Hepatitis B (HB) surface antigen (sAg) prevalence 
is 8.3% with a HB e-antigen (HBeAg) prevalence of 32.7% in those positive for HBsAg in 2012-2014.(4) Perinatal 
infection occurs in 70–90% of women with HBeAg positive chronic HBV compared with 0–30% in those with 
HBeAg negative chronic HBV (inactive carriers).(1) These infection rates reflect, in part, the failure of maternal 
and child health programs to prevent perinatal transmission with hepatitis B immunoglobulin (HBIG) and HB 
vaccines. Prevention of mother to child transmission (PMTCT) fails in an estimated 8–32% of cases with adequate 
preventive techniques.(5, 6) Antiretrovirals (ARVs), like Tenofovir Disoproxil Fumarate (TDF) are active against 
HBV and may reduce the risk of HBV transmission at birth if offered at the right time in pregnancy.(7-9)  
One of the major gaps in implementing this strategy is adequate pharmacokinetic (PK) data in pregnant women 
that informs correct dosing. One recently published population PK study in 154 women who provided single 
maternal blood samples (32 and 36 weeks of pregnancy, at delivery, and at 1 and 2 months postpartum) reported 
a tenofovir area under curve (AUC) 0–24 that was estimated to be 20% (95% CI, 19-21%) lower during pregnancy 
than during postpartum suggesting no dose adjustments are needed in 3rd trimester.(10) Most PK studies for TDF 
in pregnancy have been for Human Immunodeficiency Virus type 1 (HIV) infections. However, these patients 
often receive additional antiretroviral medications, preventing conclusions on PK parameters of Tenofovir (TFV) 
alone (TFV results following absorption after conversion of the oral prodrug TDF).(11) Doses that are optimal 
for  Human Immunodeficiency Virus (HIV) may not be appropriate for HBV. 
When TDF is administered during pregnancy and potentially during lactation, it is important to establish the infant 
drug exposure. Previous human studies have shown that antiretrovirals administered to lactating mothers are 
present in the breast milk and have detected a low TDF breast milk concentration representing 0.03% or less of 
the proposed infant dosage.(13)((14) However, there is no data on this subject  in therapeutic treatment of HBV 
infected women. In resource poor settings TDF administration will be ceased after 1 month post-partum and while 

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             PAGE 8 OF 37 
there is some data to understand what happens to viral load post cessation in non-pregnant, post-partum TDF 
cessation is less well understood but where immunity may differ critically.(12, 15) With breastmilk as the primary 
source of nutrition for babies in resource limited settings it is important to know the viral exposure from 
breastmilk, if any, as these settings may also have problems achieving birth dose, HBIG and the 3 dose vaccine 
completion.  
3. Study rationale & justification 
We propose a dense PK study of once daily TDF 300 mg during pregnancy given for PMTCT of HBV mono-
infection. Tenofovir PK will be measured in maternal blood samples in steady-state, in the 2nd and 3rd trimesters 
and post-partum. The presence of HBV DNA in blood and breast milk will also be explored while women after 
cessation of treatment until 6 months post-partum. 
4. Study Objectives and Outcome Measures 
Objectives 
Outcome Measures 
Timepoint(s) of evaluation of this 
outcome measure (if applicable) 
Primary Objective 
To measure the dense PK 
parameters of TFV in a steady-state 
during three gestational periods in 
TDF treated HBV mono infection 
 
PK parameter profiles s in maternal 
blood 
 
 
Blood sampling every month and 
dense sampling every gestational 
period  
Secondary Objectives 
To investigate the TFV 
concentration , breastmilk and 
infants 
 
 
 
 
 
 
 
To investigate if/when HBV DNA 
is detected in blood and breastmilk 
after cessation of TDF. 
- Maternal  breastmilk TFV 
concentrations 
- Milk to maternal plasma 
concentration ratios (M/P) 
- Infant plasma drug concentration  
HBV DNA in blood and breastmilk 
 
At delivery and 1 month post-partum 
 
 
 
 
 
 
 
 
 
 
Post-partum monthly maternal blood 
and breastmilk samples up to 6 
months. 
 

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5. Study Design  
Participants with HBV mono infection and a measurable HBV DNA viral load attending SMRU antenatal clinics 
(ANC) on the Myanmar-Thailand border will be invited if they have a gestational age of at least 20 weeks. A total 
of 24 women: 12 participants enrolled in 2nd trimester (EGA 20-<24 weeks) and 12 enrolled in 3rd trimester 
(EGA 28-<36) with complete samples are required. 
Women, identified by routine antenatal care screening for HBV will be invited to participate if they are HBsAg 
positive and meet the inclusion and exclusion criteria. Participants will be provided TDF 300mg daily and will 
continue TDF treatment until one month after delivery. The TFV concentrations will be measured monthly before 
delivery, at delivery, in cord blood and 1 and 2 months postpartum. Additional dense PK blood sampling will be 
done in the second and third trimester and postpartum. Breast milk samples timed with mother blood and infant 
blood samples will be included to measure drug concentrations in breastmilk as possible presence of HBV DNA 
viral in breastmilk following cessation of TDF.  
The dense sampling for pharmacokinetics assessments will occur at least after 2 weeks of TDF treatment in second 
trimester between 22–26 weeks gestation, in third trimester between 30–38 weeks gestation, and 4 weeks 
postpartum. The dense sampling will entail twelve blood samples in a 24 hour period (24ml of blood in total). 
Women will be counseled about the importance of adherence. Direct Observed Treatment (DOT) in the 3 days 
prior to the dense blood sampling will be performed. The woman can choose to be admitted for these days or if a 
home visitor lives close enough be supervised at home. 
The follow-up for women will be until 6 months post-partum, to check for flares. TFV concentrations will be 
collected by venous blood testing but an indwelling catheter can be used during the rich sampling 24 hour period. 
Infants will be followed up from delivery, and at 1, 2, 4 and 6 months of age for vaccination and growth. Infant 
drug exposure is measured at month 1 and HBV DNA will be measured at birth from cord blood and HBsAg by 
venepuncture at 2 months of age. The flow chart is available in Appendix B. 
 
 
 

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Study duration 
We will enroll volunteers into this study after ethical approval. Because we will enroll subjects in gestational 
windows we expect the recruitment and follow up to last at least 2 years. 
6 Participant identification and recruitment 
6.1 Study Population 
Pregnant women aged 16-49 years with confirmed HBV mono infection (identifiable from the routine care 
provided at SMRU clinics) will be included.   
6.2 Inclusion criteria 
All of the inclusion criteria must be answered as ‘yes’ to consider the participant eligible: 
 Single viable pregnancy at enrollment day 
 Estimated Gestational Age (EGA) 20-<24 weeks for 2nd trimester or EGA 28-<34 for 3rd trimester    
 Willing and able to give informed consent for participation in the study 
 Hepatitis B infected (HBsAg and HBeAg confirmed positive or HBsAg confirmed positive and HBV DNA 
detected in HBeAg negative)  
 Burmese and Karen female, 16-49 years (inclusive) 
 Willing to take TDF daily during pregnancy  
 Plans to deliver at SMRU clinics  
 
6.3 Exclusion criteria 
If any exclusion criteria are answered as ‘yes’ the subject may not enter the study: 
 Undetectable HBV DNA in HBeAg negative women 
 HIV infected or other chronic illness incompatible with the study requirements or receiving 
Immunosuppressive therapy 
 Creatinine at screening >1 mg/dL 
 Serum phosphate <2.4  mg/dL 
 History of chronic kidney disease  

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6.4 Co-Enrolment Guidelines 
Subjects can be enrolled into any other study while participating in this study as long as it doesn’t interfere with 
the inclusion criteria or study procedures.  
6.5 Study site 
The study will take place in the Shoklo Malaria Research unit (SMRU) clinics, Mae sot.  
7. Study Procedures  
7.1 Pre-study training 
Pre-study training is an essential part of this study to ensure this study is conducted according to Good Clinical 
Practice (GCP) and the approved version of protocol.  
7.2 Subject screening and enrolment 
Records of currently pregnant women attending SMRU antenatal care clinics (ANC) will be reviewed by the 
doctor and those who are HBV infected will be approached. For new pregnant women attending ANC, a routine 
part of antenatal care (not study related) is screening at the first visit for HBV and HIV. Samples collected from 
women identified as HBsAg positive by routine rapid detection kit Pacific Biotech Co Ltd. (2 drops of capillary 
blood) at the SMRU clinics will be sent to a certified laboratory in Thailand for confirmation of HBsAg and 
HBeAg status (routine). When the rapid test is found positive women will be invited to learn more about the study 
by discussion with a trained counsellor. 
After signing the informed consent form, all HBV infected subjects will be given a screening number at the time 
of screening. If they pass screening and are enrolled into the study, they will be given a subject number.  
7.3 Study Evaluation 
7.3.1 Screening Visit, Day -7 
At screening visits, the subjects who are tested HBV infected and have a viable singleton pregnancy will be 
required to undergo the following tests and procedures: 
 
Obtain informed consent 

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             PAGE 12 OF 37 
 
Obtain blood samples for laboratory tests; biochemistry (creatinine, phosphate, ALT), HBsAg, and 
HBeAg tests (and HBV DNA qualitative test if the mother if HBeAg negative) 
7.3.2 Study enrollment, Day 0 (Baseline) 
 
The participant comes to get an ultrasound to make sure there is a single viable pregnancy. The blood 
tests will be checked and if the participant is eligible, they can enroll in the study. If the criteria are not 
met, the participant can not enroll.  
 
Obtain demographic information 
 
Perform physical examination including vital signs, height and weight 
 
Provide medical history including history of all medications, vitamins, and allergies 
 
Provide obstetric data (Symphysis fundal height, fetal gestational age and heartbeat, details of any past 
pregnancies, complications, place of delivery and outcome will be recorded) 
 
7.3.3 TDF administration 
Generic GPO-TDF 300mg tablet will be used for this study. TDF 300mg will be administered daily and continue 
until 28 days post-partum.   
The subject will be counseled about the importance of adherence. For the dense PK sampling it is of great 
importance to be in a steady state (see 7.3.5.1 “Pharmacokinetic Evaluation). To ensure this, we will perform DOT 
in the 3 days prior to the dense blood sampling. The subject can choose to be admitted for DOT administration 
(see  8.2.4 “Blood tests”) 
Adherence survey and pill count will be performed monthly when the subject returns to the clinic to ensure 
compliance of TDF. 
 
7.3.4 Evaluation of TDF regimen 
Subjects will have creatinine and ALT concentrations checked every month. They will be asked about other side 
effects like nausea, vomiting or diarrhea.  

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7.3.5 Follow up  
7.3.5.1 Monthly follow up during pregnancy  
From the time TDF is started through the end of the study, the participants will return for monthly study visits. At 
each visit pregnancy will be checked for maternal and fetal well-being. Obstetric follow-up at each monthly visit 
will include the following: 
 
physical examination 
 
vital signs and weight 
 
symphysis fundal height measurement 
 
fetal heart beat 
 
concomitant medications 
 
blood sample for biochemistry (creatinine, ALT), and HBV DNA (See details in Appendix C). 
7.3.5.1.1 Pharmacokinetic Evaluation 
The dense PK sampling will occur after 2 weeks of TDF treatment in second trimester between 22–26 weeks 
gestation and in third trimester between 30–38 weeks gestation. The blood collections (approximately 2 ml) will 
be taken at the following times relative to the dose of TDF: pre-dose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 
hours post-dose. 
7.3.5.2 Delivery 
The following will be performed at delivery: 
 
Physical examination, vital signs, co-medication 
 
Pregnancy complications and mode of delivery will be recorded.  
 
Anthropometric measurements of the newborn will be done in the first hour of life and will include birth 
weight, length, head and arm circumference, by trained staff and according to work instructions and 
standard operating procedures.  
 
Vaccination standard HB vaccine at delivery (birth dose) and at 2, 4 and 6 months post-partum will be 
provided to newborns of mothers with chronic HBV infection.  

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             PAGE 14 OF 37 
 
HBIG will be provided to all children born to mothers in the study according to the Thai National 
Guidelines.  
 
Mother blood sample will be obtained for clinical laboratory assessments; biochemistry (creatinine, 
Phosphate, ALT), TDF concentration and HBV DNA.  
 
Cord blood sample will be obtained after the birth of the baby and before delivery of the placenta for HBV 
DNA and TDF concentrations, and for HBsAg and HBeAg testing. 
 
Breast milk collection (until 2 days post partum). 
 
Pill count and adherence survey  
 
AE and concomitant medications 
 
7.3.5.3 Post-partum follow up of mothers 
The subject will be followed up monthly until 6 month post-partum. The following procedure will be performed 
on the mothers: 
 
Physical examination and vital signs at each post-partum visit. 
 
TDF will be continued until one month post-partum to theoretically decrease the risk of flares as their 
immune system returns to baseline.  
 
The last dense PK sampling will occur at 4 weeks post-partum. 
 
At the 1 month post-partum follow up, the woman will again have dense PK sampling of her blood. A 
single breastmilk sample will be taken 4 hours after drug dosing.  
 
At the 1 month post-partum visit, Creatinine  will be measured, if Creatinine is increased (≥ 1.5 times 
from the baseline) this will be repeated according to routine care. 
 
The last blood sampling on PK (single blood sample) will be at the 2 month post-partum. 
 
At the 1 and 4 months post-partum visits, ALT will be measured (from 5ml and 2 ml respectively). If 
ALT is increased with a Grade 3 or higher (described in 13.3.1) but there is not a true flare, the ALT will 
be repeated at 6 months post partum. 

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 
 At each post-partum visit month 1 to month 6, breastmilk samples will be collectedand blood samples 
will be collected for HBV DNA tests  
 
7.3.5.4 Post partum follow up of mothers with flare 
The majority (95%) of the women will have the study endpoint at 6 months. However, if flare is detected, these 
women will be followed for a further 6-9 months and provided with drug therapy where monitoring shows it is 
required. 
 
7.3.5.5 Post-partum follow up of infants 
The following will be performed: 
 
Evaluation of growth  at 1, 2, 4 and 6 months 
 
Vaccinations at 2, 4, and 6 months 
 
 TDF drug concentration at 1 month  
 
Test for HBsAg at 2 months with a 3 mL venepuncture sample. 
7.3.6 Subjects transferred to another hospital 
If a subject needs to be referred to another hospital for an adverse event, such woman is still considered to be in 
the study and attempts will be made to obtain relevant data and follow them up after hospital discharge in 
accordance with the study schedule.  
8. Notes on study procedures / investigations  
8.1 Clinical 
On the days of the dense PK sampling, vital signs will be done just before the blood sample to obtain an accurate 
blood pressure measurement in the absence of pain/stress associated with taking the blood sample.  
A more detailed physical examination will be conducted if clinically indicated by the reported symptoms and the 
vital sign data. Further investigations will be done as clinically indicated. See on Adverse Events. 

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8.2 Blood sample collections 
The total blood volume of blood over the course of the study for the mother depends on the timing of enrollment 
and the details are provided in the sample collection table below. On average a woman who commences 
participation in trimester 2 will give 144 mL total while participation commencing in trimester 3 will provide 96 
mL total. 
 
8.2.1. Mother blood sample collections 
A 6 ml venipuncture sample of whole blood will be collected at baseline and during monthly follow up (Table 1). 
Three mL will be placed in EDTA, plasma separated, and stored in 2 separate Eppendorf tubes for later testing of 
HBV DNA concentration. Another 3 ml will be placed in a plain tube, serum separated and (HBsAg, HBeAg at 
baseline) ALT, creatinine, and serum phosphate will be measured. HBV DNA and TDF concentrations will be 
determined. Any leftover serum will be stored frozen at -80◦C. 
8.2.2. Cord blood sample collections 
Cord blood samples of 12 mL will be collected after the birth of the baby and before delivery of the placenta. The 
cord will be cleaned with a swab and a 23 g needle inserted into the umbilical cord to aspirate 12 mL with 6 mL 
into EDTA and 6 mL into a plain tube. The tubes will be centrifuged and resulting plasma will be aliquoted in 
two separate Eppendorf tubes and tested for HBV DNA and TDF concentrations. Plain tubes will be centrifuged 
after clot formation and serum aliquoted, and eppendorf tubes sent for HBsAg and HBeAg testing at Mae Sot, or 
if leftover sample, frozen at -80°C. 
8.2.3. Infant blood sample collections 
Infant blood sample of 3 mL at the 1 and 2 month visit will be drawn up and 1.5 mL placed into EDTA and 1.5 
mL into a plain tube. Resulting plasma from the 1 month EDTA blood sample will be aliquoted in two separate 
Eppendorf tubes and tested for HBV DNA and TDF concentrations.  Plain tubes will be centrifuged after clot 
formation and serum aliquoted, and Eppendorf tubes sent for HBsAg testing at Mae Sot, or if leftover sample, 
frozen at -80°C.  

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 17 OF 37 
8.2.4 Breastmilk sample collection 
At each sample time, staff and the participant will assess if she has adequate breast milk to collect a sample without 
adverse effect to the baby (eg. insufficient nutrition) or herself (eg. excessive squeezing). Participants will have 
the option to obtain breast milk by manual expression or using a (electronic) pump. If the woman has a cracked 
or bleeding nipples, this will be noted and appropriate care will be provided for the woman. If possible, milk for 
measurement of viral load will be collected from an intact nipple. 
A. Colostrum (Viral load): 
When the woman begins to have adequate colostrum production postpartum (day 1-2 on average), she will be 
asked to express 3 ml of colostrum into a sterile container.  The woman may express from either or both breasts.  
Use of a breast pump will be allowed but hand expression is expected to be most effective. 
B. 1 month breast milk (PK and viral load): 
At hour 4 post-dose the mother will be asked to express the entire contents of one breast (choose the more full 
breast) into one clean container. The breast milk will be measured, mixed and a minimum of 10 ml aliquot taken. 
Remaining milk will be given to the mother to feed back to her infant with spoon, cup, or syringe, according to 
her preference.  Infant feeding will be allowed to continue on demand (as is the cultural norm and recommended 
by the WHO) between the drug dose and the breast milk sample, and exposure to the infant will be considered 
essentially continuous.  
C. 2-6 month breast milk (viral load): 
At a convenient time during the visit, the mother will be asked to express a minimum of 5 ml into one clean 
container.  
Table 1. Sampling schedule 
Type of 
sample 
Timing of sample 
 
EGA 
20 
EGA
24 
EGA 
28 
EGA
32 
EGA 
36 
delivery 
PP 1 
month 
PP 2 
month 
PP 3,4,5,6 month 
Total 
Baseline 
 
 
 
 
 
 
 
 
 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 18 OF 37 
Type of 
sample 
Timing of sample 
 
EGA 
20 
EGA
24 
EGA 
28 
EGA
32 
EGA 
36 
delivery 
PP 1 
month 
PP 2 
month 
PP 3,4,5,6 month 
Total 
Mother 
blood 
6ml 6ml 
6ml 
6ml 
6ml 
6ml 
6ml 
6ml 
6ml 
72ml 
Dense 
PK 
 
24ml 
 
 
24ml 
 
24ml 
 
 
72ml 
Delivery 
cord 
 
 
 
 
 
12ml 
 
 
 
12ml 
Baby 
 
 
 
 
 
 
3ml 
3ml 
 
6ml 
Breastmilk  
 
 
 
 
colostrum 
3ml 
10ml 
mature 
milk 
5ml 
mature 
milk 
5ml 
mature milk 
38ml 
Total mother blood (if enrolled at EGA 20 weeks) 
144ml 
Total mother blood (if enrolled at EGA 36 weeks) 
96ml 
 
8.2.4 Blood tests 
Pharmacokinetic measures: We will measure TFV (and HBV DNA) concentrations every month but dense 
sampling will occur at three periods: in second trimester between 22–26 weeks gestation (if enrolled), in third 
trimester between 30–38 weeks gestation and 4 weeks post-partum. During dense PK sampling, blood will be 
drawn via an intravenous catheter inserted before the first sample. The exact time frames are shown in Table 2, 
where 0 is the sample before the first dose of TDF.  
Table 2. PK sampling times 
 
Blood samples Dense PK timing, at baseline and hours after drug intake 
0 (pre-
dose) 
0.5 
1 
1.5 
2 
3 
4 
6 
8 
12 
16 
24 
EGA 22-26 
2.0ml 
2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 
2.0ml 
EGA 30-38 
2.0ml 
2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 
2.0ml 
1 month post-
partum 
2.0ml 
2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 2.0ml 
2.0ml 
 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 19 OF 37 
PK: Whole blood into EDTA tubes will be taken and spun down at 1500-2000 ×g for 10 minutes to obtain plasma. 
The plasma will then be aliquoted in two separate cryovials, labelled and stored at minus 80⁰C until analysis in 
the laboratory of Prof. David Burger in Nijmegen, the Netherlands. This laboratory is accredited (NEN-EN-ISO 
15189) and uses an in house validated Liquid chromatography–mass or tandem-mass spectrometry (LC-MS/MS). 
Biochemistry: creatinine, ALT and phosphorus and HBV DNA (1st and delivery cycle): Another aliquot of whole 
blood 6 mL of blood will be drawn up and 3 ml put into EDTA and 3 ml into a plain tube. EDTA will be processed 
immediately. Blood will be centrifuged to separate plasma and serum for 5 minutes.  
Plasma will be used for HBV DNA quantification using the HBV DNA assay Fast Track Diagnostics 
(http://www.fast-trackdiagnostics.com/human-line/products/ftd-hepatitis-b-dna/). Remaining serum or plasma 
will be stored at -80°C in case any test result need clarification.   
8.3 Sample labelling 
Study samples, including stored samples, will be labelled with: 
 Subject number 
 Date of specimen collection  
 Time specimen collection  
 Type of specimen 
 
8.5 Safety net 
The net will include: 
 Pre-study training will be given to the study team with an emphasis on:  
o taking informed consent 
o performing  monthly clinical assessments  
o making it easy to ask for a consult by senior colleagues 
o explaining how to use the TDF stopping rule as a guide (eg. kidney failure or ALT rise due to TDF 
treatment, see 13.3.1) 
 check for hepatic flare (see 13.3.1) 

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9. Drug Management  
9.1 Supply Storage and Handling 
All TDF tablets in this study should be kept in a controlled room temperature (25°C or 77°F); excursions permitted 
between 15°C to 30°C (59°F to 86°F). 
9.2 Drug Accountability 
SMRU clinics will maintain a record of the numbers of all TDF tablets obtained, used by the subjects and returned 
to the pharmacy. These information will be recorded in the drug accountability record during the study. On study 
completion, a copy of the drug accountability record will be filed in the study folder. Unused TDF will be returned 
to the SMRU pharmacy.     
9.3 Concomitant Medications 
Subjects may develop minor illnesses during follow up. These will be treated by the research team.  
Drugs that may cause kidney failure or interfere with the metabolism of TDF must is prohibited throughout the 
study participation. (e.g. aminoglycosides (e.g., gentamicin), and high-dose or multiple NSAIDs.  
10. Discontinuation/Withdrawal of Participants from Study 
Each subject has the right to withdraw from the study at any time.  In addition, the Investigator may discontinue 
a subject from the study at any time if the Investigator considers it necessary for any reason. 
10.1 Voluntary withdrawal of consent 
Subjects may withdraw from the study at any time and for any reason.  
10.2 PI withdrawal 
A subject will be withdrawn by the PI from the study if: 
 Significant protocol deviation (for example not taking medication for >2 months) 
 Subjects experience adverse drug reaction that may affect their safety during the study as judged by the 
investigator 
 Significant non-compliance with treatment regimen or study requirements 
 Loss to follow up ( >3 visits) 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
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10.3 Management of withdrawn subjects 
The care of pregnant women that have been withdrawn from the study will continue through the delivery and post-
partum period irrelevant of the reason for withdrawal. It is important for pregnant women to receive proper 
antenatal, delivery and post-partum care. The data collected until the point of withdrawal will be included in the 
study and this is stated in the participant information sheet. The reason for withdrawal will be recorded in the 
CRF. If a subject withdraws after receiving TDF, Alanine transaminase (ALT) testing will be done (when possible) 
to monitor for hepatic flare. Every effort will be made to make sure that the subject understands this. Participants 
who withdraw will be replaced as complete sample sets are necessary for the analysis. 
10.4 Study Discontinuation 
The study may be discontinued by the ethics committee/s and/or the study sponsor. Reasons for terminating the 
study may include, but are not limited to, the following: 
▪   The incidence or severity of AEs indicating a potential health hazard to participants 
▪   Investigators do not adhere to the protocol 
11. Subject Safety monitoring & adverse event reporting  
This study will use the Common Toxicity Criteria v 5.0 for grading adverse events. It is available from this 
website:  
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/ctcae_v5_quick_reference_5x7.pdf 
11.1 Adverse Events 
From the time TDF is started through the end of the study, the subjects will return for monthly study visits (timed 
with routine ANC visits) where a case report form, an adverse events (AE) form, and an adherence form will be 
completed. Pre-specified AEs include headache, nausea, vomiting, and ALT flares after discontinuation of TDF 
will not be recorded as an AE. Any unexpected AEs will be documented according to standard definitions and 
procedures.     
11.2 Grading of an Adverse Event 
We will use the CTCAE as a reference for grading adverse events  

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             PAGE 22 OF 37 
11.3 Relatedness of Adverse Event to TDF 
In this study, the relationship between an AE and TDF will be reported as one of the following: 
 Not related 
 Unlikely related  
 Possibly related 
 Probably related or 
 Definitely related. 
11.4 Serious Adverse Event Recording 
All adverse events are recorded in the CRF. A serious adverse event (SAE) occurring to a subject should be 
reported to the EC that gave a favorable opinion of the study where in the opinion of the Chief Investigator the 
event was ‘related’ (resulted from administration of any of the research procedures) and ‘unexpected’ in relation 
to those procedures. Reports of related and unexpected SAEs should be submitted within 5 working days of the 
Chief Investigator becoming aware of the event, using the SAE form.        
11.5 Subject Management of Adverse Events 
All AEs will be treated as clinically indicated, and if concomitant treatment is given this will be recorded on the 
Concomitant Medication CRF. If necessary, participants will be referred for specialist care/advice.  
The subject will be followed and treated by the research team until the clinical or laboratory AE has resolved or 
stabilised. The physician should perform any tests that are clinically indicated. 
11.6 Serious Adverse Events 
A serious adverse event is any untoward medical occurrence that: 
 results in death 
 is life-threatening 
 requires inpatient hospitalization or prolongation of existing hospitalization 
 results in persistent or significant disability/incapacity 
 consists of a congenital anomaly or birth defect. 
Other ‘important medical events’ may also be considered serious if they jeopardize the subject or require an 
intervention to prevent one of the above consequences. 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 23 OF 37 
11.7 Reporting of Serious Adverse Events 
In this protocol, reporting means that SAEs must be reported by email by the study physicians to the study co-PI 
(mariekebierhoff@yahoo.com, 0987520822) within 24 hours of awareness using the SAE report form. The study 
co-PI (Marieke Bierhoff) will be responsible for reporting the SAEs to the Ethics Committee per required timeline.  
11.8 Safety Review of the Laboratory Report  
All laboratory data will be reviewed by the team physicians to determine if kidney failure or a laboratory AE is 
present. The physicians or the co-PI will sign the laboratory report to confirm the report has been seen. Safety 
information will be reviewed on a regular basis. 
12. Statistics  
12.1 The Number of Participants 
As this is a descriptive study of the PK, the sample size of 24 participants, who generate 60 dense PK curves, is 
assumed to describe the PK profile adequately and generate robust parameter estimates with reasonable precision.  
12.2 Study endpoints 
12.2.1 Primary endpoint 
The primary endpoint is the Dense TDF concentration for 24 participants repeated at 3 gestational periods (2nd 
and 3rd trimester of pregnancy and 1 month post-partum). The end of the study is the date of the last visit to the 
clinic by a study subject which is anticipated as when the last infant involved in the study reaches 6 months of 
age.  
12.2.2 Secondary endpoints 
These are: 
 Infant drug exposure comparing TDF concentrations in breastmilk samples, infant plasma and maternal 
plasma.  
 Presence or absence of HBV DNA in maternal blood and breastmilk particularly after TDF cessation  

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 24 OF 37 
 Proportion of infants that are positive for HBsAg and HBV DNA at birth (using cord blood), and HBsAg 
2 month of age 
12.3 Data Analysis 
Data cleaning will be performed prior to analysis to identify incomplete and incorrect data. All women will be 
censored at delivery for the analysis. Detailed statistical methods are described below.  
12.3.1 Continuous data 
These data will be summarized by medians (ranges) and means (standard deviations), as appropriate, and will 
include the biochemical parameters.  
12.3.2 Pharmacokinetic data 
Densely collected TDF samples will be analysed using a model-independent approach (i.e. non-compartmental 
analysis) with the help of pharmacokineticist specialists (prof David Burger and prof Joel Tarning). All collected 
samples will be pooled and analysed using nonlinear mixed-effects modelling to develop a detailed PK model that 
describes the concentration-time relationship and possible impact of clinical covariates, such as age and body 
weight. Particular focus will be on describing and quantifying potential PK differences during pregnancy (i.e. 
increasing gestational age) and compared to post-partum.  
12.3.3 Infant drug exposure 
Infant exposure to TFV will be estimated using drug concentration in maternal plasma, maternal breast milk, and 
infant plasma. Milk/plasma ratio will be calculated using the paired breast milk and plasma concentrations at 4 
hours after dosing. This may slightly overestimate the M/P ratio as determined by the ratio of the concentration 
AUC for breast milk and plasma, as hour 4 is expected to be the peak breast milk concentration, and plasma 
concentrations will be decreasing. This M/P ratio will be compared with published values from prior studies in 
women receiving using TDF in combination with other antivirals for treatment or prevention of HIV.  

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 25 OF 37 
13 Human subjects considerations  
13.1 Ethical Review 
All projects are discussed with the Tak Community Advisory Board (T-CAB) for relevance, advice and 
acceptability. The protocol, participant information sheet, informed consent form will be submitted to OxTREC 
and local ethics committees for written approval. The Investigator will submit and, where necessary, obtain 
approval from the above parties for all amendments to the original approved documents.  
13.2 Informed Consent 
Informed consent will be administered by the ANC counsellor trained in the procedure and fluent in the language 
of the participant. The subject (and guardian for minors) must personally sign and date the latest approved version 
of the informed consent form before any study specific procedures are performed. Illiterate participants will be 
accompanied by an independent literate witness throughout the procedure. 
Written version of the participant information and informed consent will be presented to the participants (and 
guardians for minors) detailing no less than: the exact nature of the study; the implications and constraints of the 
protocol; the known side effects and any risks involved in taking part. Moreover, it will inform about the total 
blood volume that will be taken throughout the study, the sampling procedure for the PK and the follow up of the 
child. It will be clearly stated that the subject is free to withdraw from the study at any time for any reason without 
prejudice to future care, and with no obligation to give the reason for withdrawal. If someone withdraws after 
starting on drug, we will monitor for flares for 3 months for the woman’s own safety. This is clearly stated in the 
information sheet. 
The subject (and guardian in minors) will be allowed as much time as wished to consider the information, and the 
opportunity to question the Investigator, midwife or other independent parties to decide whether they will 
participate in the study.  Written Informed Consent will then be obtained by means of subject (and guardian for 
minors) dated signature and dated signature of the person who presented and obtained the informed consent (or 
thumbprint in the case of non-literate participants). The person who obtained the consent will be suitably qualified 
and experienced, and have been authorised to do so by the Chief/Principal Investigator. A copy of the signed 
Informed Consent will be given to the participants. The original signed form will be retained at the study site. 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 26 OF 37 
The informed consent and assent forms are available as separate documents which have been written in English 
until their review by the Ethics Committees. This approved document will be translated to Karen and Burmese 
language and submitted to the Ethics Committees before the study commencement.  The Karen and Burmese 
version will be back translated to English for accuracy. 
13.3 Risks 
13.3.1 Risks of oral TDF 
TDF is globally being used in pregnant women for prevention of mother to child transmission of HIV. It is 
generally well tolerated with the most common adverse reactions identified from any of the large controlled 
clinical trials include rash, diarrhea, headache, pain, depression, asthenia, and nausea.(16, 17)  
Other side effects are: 
 
Kidney failure defined as:  
o A rise in serum creatinine ≥ 1.5 times from the baseline OR  
o An absolute rise in serum creatinine of ≥26.5 µml/L (≥ 0.3 mg/dl) within 48 hours  
 
ALT rise to Grade 3.  
Grade 3 or higher is >5x upper limit of normal (ULN) if baseline levels were below the ULN; or if baseline 
levels were >ULN and, then grade 3 or higher is >3.5x baseline value. The normal value of ALT will be 
defined as 0-31 U/L.(18) 
 
Hepatic flare 
A tangible adverse consequence that requires further study is the potential for hepatitis flare following 
discontinuation of therapy in the mother. All women will continue on study until the 4 month post-partum 
visit. At the 1 and 4 months post-partum visits ALT will be measured (from a 5ml and 2 ml blood sample 
respectively). If 3 months after stopping TDF, a flare is detected (ALT >5x baseline if this was increased 
at baseline or >10x the upper limit of normal with no alternative diagnosis) the woman will continue to 
be monitored until resolution of the flare. If the flare worsens or does not resolve they will be placed on 
anti-HBV therapy and be reviewed by a physician. If ALT is increased but there is not a true flare, 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 27 OF 37 
(ALT>3.5x baseline if this was increased at baseline or >5x the upper limit of normal), they will be 
followed for an additional 6-9 months and repeat ALT testing done at 6 months. 
13.3.2 Risk of Phlebotomy 
The primary risks of phlebotomy include local discomfort, occasional bleeding or bruising of the skin at the site 
of needle puncture, and rarely haematoma or infection. There is a small risk of infection with cannulation but 
these will be minimized by universal precautions and removing the cannula directly after the last sample. 
13.4 Benefits 
13.4.1 Benefits to the individuals 
 
Potentially treatment with TDF of the mother during pregnancy and post-partum will decrease the number of 
children infected with HBV though MTCT. 
13.4.2 Benefits to society 
This study will provide information on the PK of TFV following oral TDF for prevention of MTCT of HBV 
mono-infection, which is relevant to provision of correct dosing.  
13.4.3 Future research & drug policy implications 
With the recent shift in WHO policy and new guidelines for the Elimination of Hepatitis B. The issue of cessation 
of TDF in breast feeding mothers requires clarity and this study will provide additional insight in this area. Since 
TDF is not merely used in HBV PMTCT but also in HIV PMTCT, results of this study could potentially inform 
us about alternate regimens in this patient group as well.  
13.5 Alternatives to Study Participation 
Subjects are free to decline participation in this study and may withdraw from it at any time. 
13.6 Compensation  
In this study participants who get the dense PK sampling done will receive 300 Baht for that day. The participants 
who are admitted for DOT will receive additionally 100 baht per day admitted. Transportation costs for study 
related visits will be reimbursed. If the subject is withdrawn from the study before the study ends, the subject will 
be compensated for the time that the subject is actually enrolled in the study. In addition, a small gift valued 100 
Baht for the baby will be given at 2 months. 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 28 OF 37 
13.7 Participants Confidentiality 
All study related documents will be stored securely at the study site in locked filing cabinets in areas with access 
limited only to study staff. All laboratory specimens, including stored specimens, reports, study data collection, 
process, and administrative forms will be identified by a subject number.  
Forms, logbooks, appointment books, informed consent forms, and any other listings that link subject numbers to 
other identifying information will be stored in a separate, locked file in an area with limited access. All databases 
will be secured with password-protected access systems. Participant’s study information will not be released 
without the written permission of the participant, except as necessary for the independent monitoring. 
Representatives of sponsor, monitors, regulatory authorities, the ECs can request to see the source documents. 
The results of the research study will be published with subject details de-identified.  
Subject’s clinical data and results from blood analyses stored in our database may be shared with other researchers 
to use in the future. However, the other researchers will not be given any information that could identify the 
subject. 
13.8 Insurance Arrangement 
The University of Oxford has a specialist insurance policy in place, Newline Underwriting Management Ltd, at 
Lloyd’s of London, which would operate in the event of any subject suffering harm as a result of their involvement 
in the research. 
14. Laboratory specimens & practice  
14.1 Laboratory Specimens 
The study site will follow the principles of good clinical laboratory practice (GCLP) for clinical trials and local 
standard operating procedures (SOPs) and SOPs or work instructions specifically for this study for the collection, 
processing, labelling, transport and storage of all specimens.   
The following laboratory tests/procedures will be performed as part of this protocol: 
▪ routine biochemistry 
▪ HBV DNA qualitative testing 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 29 OF 37 
▪ HBV DNA quantitative testing 
▪ HBV serology (HBsAg and HBeAg) 
▪ TFV drug concentrations (in Nijmegen, the Netherlands) 
14.2 Quality Control and Quality Assurance 
The clinics have their own systems for quality assurance (QA) e.g. running internal quality control, machine 
calibrations, in accordance with the manufacturer’s instructions and relevant international guidelines. The PK 
laboratory at Nijmegen, the Netherlands has ISO accreditation for quality and competency and safety (NEN-EN-
ISO 15189) since 2016.  
14.3 Specimen Storage 
Study site staff will store specimens in appropriate, fully functioning freezers (e.g. DNA can be stored at -20 to 
80°C) so that the protocol related analyses can be performed. After these tests have been performed, blood will 
be kept for long term storage for 10 years in the first instance, if subjects give signed informed consent. Informed 
consent will specify that the stored blood samples may be used in the future for the analyses of hepatitis B related 
fields of research including genetics without participants identifier. 
14.4 Specimen transport 
After the last subject gave the ;ast PK blood sample, the specimens for TDF drug concentrations will be transferred 
from Mae Sot to Nijmegen, the Netherlands by air. In-house SOPs or work instructions will be followed for 
packing and transferring samples. The necessary IATA Regulations will be followed. The signed Material 
Transfer Agreement will be arranged and submitted to the local Ethics Committee.  
15. Administrative procedures 
15.1 Data Management 
Direct access will be granted to authorised representatives from the Sponsor and host institution for monitoring 
and/or audit of the study to ensure compliance with regulations. During the study, investigators from the above 
mentioned institutions, and their institutional review boards, may have access to study documents like informed 
consent forms to make sure that the study is properly conducted. After the study, data may be shared with other 
scientists only after identifiers (names, date of birth, residence, etc.) have been removed. 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 30 OF 37 
Only those staff with permission to review the files will enter data. All study data will be entered on a MACRO 
database at SMRU using SOP at SMRU. The MACRO database will be password protected and only people 
working on the study will have access to this database. The participants will be identified by a study specific 
participants number and/or code in any database. The name and any other identifying detail will NOT be included 
in any study data electronic file. Data will be stored securely at SMRU for 20 years. Records will be kept in locked 
cabinets unless they are in use by the study team. 
 
15.2 Record Retention 
The Investigator will maintain, and store in a secure manner, complete, accurate study records throughout the 
study. Study records include CRFs, consent forms, and all other essential documents including reports and 
correspondence relating to the study, minutes of team minutes, e-mail communication with the sponsor.   
All pertinent study documentation will be kept in the Investigator site file (ISF).    
According to the University of Oxford policy on retention period, subject records will be retained until the 
youngest child participating in the trial reaches 21 or five years following completion of the study, whichever is 
longer. The study database will be retained indefinitely. 
 
15.3 Use of Information and Publications 
The results of this study will be published following international guidelines and norms. Any data published in 
the peer-reviewed medical literature will protect the identity of the subjects. This trial will be registered in a web 
based protocol registration scheme. All those who have made a substantial contribution will be co-authors on 
publications. 
 
 

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             PAGE 31 OF 37 
16. References 
1. 
Hoffmann CJ, Thio CL. Clinical implications of HIV and hepatitis B co-infection in Asia and Africa. Lancet 
Infect Dis. 2007;7(6):402-9. 
2. 
Kourtis AP, Bulterys M, Hu DJ, Jamieson DJ. HIV-HBV coinfection--a global challenge. N Engl J Med. 
2012;366(19):1749-52. 
3. 
Tsuchiya N, Pathipvanich P, Rojanawiwat A, Wichukchinda N, Koga I, Koga M, et al. Chronic hepatitis B 
and C co-infection increased all-cause mortality in HAART-naive HIV patients in Northern Thailand. 
Epidemiol Infect. 2013;141(9):1840-8. 
4. 
Banks T, Kang J, Watts I, Tyrosvoutis ME, Min AM, Tun NW, et al. High hepatitis B seroprevalence and 
risk factors for infection in pregnant women on the Thailand-Myanmar Border. J Infect Dev Ctries. 
2016;10(4):384-8. 
5. 
Chen SC, Toy M, Yeh JM, Wang JD, Resch S. Cost-effectiveness of augmenting universal hepatitis B 
vaccination with immunoglobin treatment. Pediatrics. 2013;131(4):e1135-43. 
6. 
Jourdain G, Ngo-Giang-Huong N, Harrison L, Decker L, Khamduang W, Tierney C, et al. Tenofovir versus 
Placebo to Prevent Perinatal Transmission of Hepatitis B. New England Journal of Medicine. 
2018;378(10):911-23. 
7. 
Unal ER, Lazenby GB, Lintzenich AE, Simpson KN, Newman R, Goetzl L. Cost-effectiveness of maternal 
treatment to prevent perinatal hepatitis B virus transmission. Obstet Gynecol. 2011;118(3):655-62. 
8. 
O'Hara GA, McNaughton AL, Maponga T, Jooste P, Ocama P, Chilengi R, et al. Hepatitis B virus infection 
as a neglected tropical disease. PLoS Negl Trop Dis. 2017;11(10):e0005842. 
9. 
Siemieniuk RA, Foroutan F, Mirza R, Mah Ming J, Alexander PE, Agarwal A, et al. Antiretroviral therapy 
for pregnant women living with HIV or hepatitis B: a systematic review and meta-analysis. BMJ open. 
2017;7(9):e019022. 
10. Cressey TR, Harrison L, Achalapong J, Kanjanavikai P, Patamasingh Na Ayudhaya O, Liampongsabuddhi 
P, et al. Tenofovir Exposure during Pregnancy and Postpartum in Women Receiving Tenofovir Disoproxil 

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Fumarate for the Prevention of Mother-to-Child Transmission of Hepatitis B Virus. Antimicrob Agents 
Chemother. 2018;62(12). 
11. Kearney BP, Flaherty JF, Shah J. Tenofovir disoproxil fumarate: clinical pharmacology and 
pharmacokinetics. Clinical pharmacokinetics. 2004;43(9):595-612. 
12. Wang T, Wang M, Duan G, Chen X, He Y. Discrepancy in impact of maternal milk on vertical transmission 
between Hepatitis B virus and Human cytomegalovirus. International journal of infectious diseases : IJID : 
official publication of the International Society for Infectious Diseases. 2015;37:1-5. 
13. Benaboud S, Pruvost A, Coffie PA, Ekouevi DK, Urien S, Arrive E, et al. Concentrations of tenofovir and 
emtricitabine in breast milk of HIV-1-infected women in Abidjan, Cote d'Ivoire, in the ANRS 12109 
TEmAA Study, Step 2. Antimicrobial agents and chemotherapy. 2011;55(3):1315-7. 
14. Mugwanya KK, Hendrix CW, Mugo NR, Marzinke M, Katabira ET, Ngure K, et al. Pre-exposure 
Prophylaxis Use by Breastfeeding HIV-Uninfected Women: A Prospective Short-Term Study of 
Antiretroviral Excretion in Breast Milk and Infant Absorption. PLoS Med. 2016;13(9):e1002132. 
15. Cao J, Chi H, Yu T, Li Z, Hansen BE, Zhang X, et al. Off-Treatment Hepatitis B Virus (HBV) DNA Levels 
and the Prediction of Relapse After Discontinuation of Nucleos(t)ide Analogue Therapy in Patients With 
Chronic Hepatitis B: A Prospective Stop Study. J Infect Dis. 2017;215(4):581-9. 
16. Colbers AP, Hawkins DA, Gingelmaier A, Kabeya K, Rockstroh JK, Wyen C, et al. The pharmacokinetics, 
safety and efficacy of tenofovir and emtricitabine in HIV-1-infected pregnant women. AIDS (London, 
England). 2013;27(5):739-48. 
17. Chen JZ, Liao ZW, Huang FL, Su RK, Wang WB, Cheng XY, et al. Efficacy and safety of tenofovir 
disoproxil fumarate in preventing vertical transmission of hepatitis B in pregnancies with high viral load. 
Scientific reports. 2017;7(1):4132. 
18. Abbassi-Ghanavati M, Greer LG, Cunningham FG. Pregnancy and laboratory studies: a reference table for 
clinicians. Obstet Gynecol. 2009;114(6):1326-31. 
19. Ito S, Lee A. Drug excretion into breast milk--overview. Advanced drug delivery reviews. 2003;55(5):617-
27. 

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20. Ito S, Koren G. A novel index for expressing exposure of the infant to drugs in breast milk. British journal 
of clinical pharmacology. 1994;38(2):99-102. 
21. Hirt D, Urien S, Ekouevi DK, Rey E, Arrive E, Blanche S, et al. Population pharmacokinetics of tenofovir 
in HIV-1-infected pregnant women and their neonates (ANRS 12109). Clin Pharmacol Ther. 
2009;85(2):182-9. 
22. Mirochnick M, Capparelli E. Pharmacokinetics of antiretrovirals in pregnant women. Clinical 
pharmacokinetics. 2004;43(15):1071-87. 
23. Mirochnick M, Thomas T, Capparelli E, Zeh C, Holland D, Masaba R, et al. Antiretroviral concentrations 
in breast-feeding infants of mothers receiving highly active antiretroviral therapy. Antimicrobial agents and 
chemotherapy. 2009;53(3):1170-6. 
24. Mirochnick M, Taha T, Kreitchmann R, Nielsen-Saines K, Kumwenda N, Joao E, et al. Pharmacokinetics 
and safety of tenofovir in HIV-infected women during labor and their infants during the first week of life. 
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PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 34 OF 37 
17. Appendix A: Thai National Guideline for prevention of maternal-to-
child transmission of hepatitis B 
 (Pediatric Infectious Disease Society of Thailand) 
 
 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 35 OF 37 
18. Appendix B: Study flow 
 
 
 
 
 
Pregnant woman HBsAg positive at clinic
counseling about hepatitis B
if EGA ≽20 weeks, single viable pregnancy 
counseling TDF study, acquire informed consent
blood samples for CBC, HBsAg, HBeAg, ALT, Phosphorus, creatinine and HBV 
DNA
give results blood tests
ultrasound
in-exclusion criteria
Repeat counseling follow up 6 months post partum
start TDF
routine ANC care (e.g.physical examination)
ultrasound
Adverse events
adherence (pill count/questionnaire)
blood tests
new medication
Rich PK sampling at: 22-26 weeks and 30-38 weeks EGA
blood sample mother for HBsAg, HBeAg, ALT, Creatinine, Phosphorus
cord blood sample
record delivery
HBV vaccination +/- HBIG
newborn measurements
adherence (pill count/questionnaire)
Colostrum sample mother
ANC day 0
ANC day 7
Patient eligible
Monthly visit
Delivery
Post Partum
Month 1
Dense PK sampling at 4 weeks post partum
stop TDF, counsel follow up month 4
record baby measurements and feeding
Breastmilk sample and mother blood sample
Month 2
HBV vaccination
record baby measurements and feeding 
baby blood sample HBsAg, HBeAg
Mother blood and breastmilk sample
Month 3
Mother blood and breastmilk sample
Month 4
HBV vaccination 
record baby measurements and feeding
mother blood and breastmilk sample
Month 6
HBV vaccination 
record baby measurements and feeding
neurodevelopment test
Mother blood and breastmilk sample
Study End

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 36 OF 37 
19. Appendix C: Schedule of study procedures 
Activity 
Visits timing 
  
Day -7 Day 0 
Monthly 
pregnant 
visits 
Delivery 
1 mth 
post-
partum 
2 mth 
post-
partum 
3 mth 
post-
partum 
4 mth 
post-
partum 
5 mth 
post-
partum 
6 mth 
post-
partum 
Mother 
Study Drug Dispensing 
  
X 
X 
X 
 X 
  
  
  
  
  
Obtained Informed consent 
X 
  
  
  
  
  
  
  
  
  
Medical History 
 
X 
  
  
  
  
  
  
  
  
Physical Examination 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital Signs 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Measure Height and Weight 
 
X 
X 
  
  
  
  
  
  
  
Ultrasound 
  
X 
X 
  
  
  
  
  
  
  
Serology (HBsAg/HBeAg) 
X 
  
  
X/X3 
  
  
  
  
  
  
Eligibility checklist 
X 
X 
  
  
  
  
  
  
  
  
Enrollment 
  
X 
  
  
  
  
  
  
  
  
HBV DNA 
X 
  
X 
X 
X 
X 
X 
X 
X 
X 
Blood Chemistry 
Creatinine 
X 
  
X 
X 
X 
  
  
  
  
  
Phosphate 
X 
  
  
X 
  
  
  
  
  
  
ALT 
X 
  
X 
X 
X 
  
  
X 
  
X1 
TDF concentration 
  
  
X2 
X 
X2 
X 
  
  
  
  
Adherence survey 
  
  
X 
X 
X 
  
  
  
  
  
Pill count 
  
  
X 
X 
X 
  
  
  
  
  
Directly Observed Therapy 
  
  
X4 
  
X 
  
  
  
  
  
AE and Concomitant Medication 
Assessment 
  
  
X 
X 
X 
X 
X  
X  
X  
X  
Breastmilk sample collections 
  
  
  
X 
X 
X 
X 
X 
X 
X 
Infant 
Anthropometry: weight, length, 
head (and arm) circumference 
  
  
  
X 
X 
X 
  
X 
  
X 
Cord blood HBsAg/HBeAg/HBV 
DNA/TDF concentration 
  
  
  
X 
  
  
  
  
  
  
HBV vaccination 
  
  
  
X 
  
X 
  
X 
  
X 
Infant HBsAg 
  
  
  
  
  
X 
  
  
  
  
Infant TDF concentration 
  
  
  
  
X 
  
  
  
  
  
1 Follow-up will continue if suspected or confirmed flare 
 
 
2 The dense PK blood sampling is further shown in table 2.  
 
 
 
 
 
3 in Cord blood 
 
 
 
 
 
4 Before dense PK sampling  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PROTOCOL  V 3.0  DATED 28 OCTOBER 2019 
 
 
 
 
 
             PAGE 37 OF 37 
20. Appendix D: Amendment History  
Amendment No. 
Protocol 
Version No. 
Date 
issued 
Author(s) of changes 
Details of Changes made 
 
 
 
 
 
 
List details of all protocol amendments here whenever a new version of the protocol is produced.  This is not 
necessary prior to initial REC submission.