Effects of Mu-opiate Receptor Engagement on Microbial Translocation and Residual Immune Activation in HIV-infected, ART Suppressed Opioid Use Disorder Patients Initiating Medication-assisted Treatment
Phase: PHASE2
Conditions: Opioid-use Disorder, HIV-1-infection, Immune Activation, Inflammation, Methadone, Buprenorphine, Naltrexone, Antiretroviral Treatment
Sponsor: University of Pennsylvania

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Protocol 
Effects of mu-opiate receptor engagement on microbial translocation and residual 
immune activation in HIV-infected, ART suppressed opioid use disorder patients 
initiating medication-assisted treatment 
 
 
 
Acronym 
AMOHI-1 (Anti-retroviral therapy, Medications for opioid 
use disorder, Opioids and HIV infection – Study 1) 
Principal Investigator 
Luis J. Montaner, D.V.M., M.Sc., D.Phil. 
Wistar Institute 
3601 Spruce Street, Philadelphia, PA 19104 
Phone: 215-898-9143 
Email: montaner@wistar.org 
Co - Principal Investigator  
David S. Metzger, PhD 
Department of Psychiatry, University of Pennsylvania 
HIV Prevention Research Division 
3535 Market Street, Suite 4000, Philadelphia, PA 19104 
Phone: 215-746-7346 
Email: dsm@pennmedicine.upenn.edu 
Site PI 
Van Hung, MD 
Vietnam CDC Ho Chi Minh City 
Funding Sponsor 
 
National Institute of Drug Abuse 
Regulatory sponsor 
University of Pennsylvania (David S. Metzger, PhD) 
NIH Grant Number 
 
NIDA R01 DA048728-01 
Investigational Product 
methadone, buprenorphine/naloxone, extended-release 
naltrexone 
IRB Number 
833720 
ClinicalTrials.gov Number 
Not yet applied 
 
 
Initial version: July - 17-2019 
V1.1: July-26-2019 
V1.2: Nov-01-2019 (correction of typos) 
V2.0: Feb-19-2020 
 
 

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Table of Contents 
 
LIST OF ABBREVIATIONS ................................................................................................................................................ 4 
1 
INTRODUCTION........................................................................................................................................................ 7 
1.1 
BACKGROUND AND RELEVANT LITERATURE ...................................................................................................................... 7 
1.2 
NAME AND DESCRIPTION OF THE INVESTIGATIONAL PRODUCT........................................................................................ 8 
1.2.1 
Clinical Data to Date..................................................................................................................................................................... 8 
2 
STUDY OBJECTIVES ................................................................................................................................................. 8 
2.1 
PRIMARY OBJECTIVE ............................................................................................................................................................. 8 
2.2 
SECONDARY OBJECTIVES ...................................................................................................................................................... 8 
3 
INVESTIGATIONAL PLAN ....................................................................................................................................... 9 
3.1 
GENERAL DESIGN .................................................................................................................................................................. 9 
3.1.1 
Screening Phase .............................................................................................................................................................................. 9 
3.1.2 
Study Intervention Phase ........................................................................................................................................................... 9 
3.1.3 
Follow Up Phase ............................................................................................................................................................................ 11 
3.1.4 
Allocation to Interventional Group ..................................................................................................................................... 11 
3.2 
STUDY ENDPOINTS ............................................................................................................................................................. 12 
3.2.1 
Primary Study Endpoint ........................................................................................................................................................... 12 
3.2.2 
Secondary Study Endpoints ..................................................................................................................................................... 12 
4 
STUDY POPULATION AND DURATION OF PARTICIPATION ....................................................................... 13 
4.1 
INCLUSION AND EXCLUSION CRITERIA ............................................................................................................................. 13 
4.2 
SUBJECT RECRUITMENT ..................................................................................................................................................... 14 
4.3 
DURATION OF STUDY PARTICIPATION .............................................................................................................................. 14 
4.4 
TOTAL NUMBER OF SUBJECTS AND SITES ........................................................................................................................ 14 
4.5 
VULNERABLE POPULATIONS: ............................................................................................................................................ 14 
5 
STUDY INTERVENTION ........................................................................................................................................ 14 
5.1 
DESCRIPTION ...................................................................................................................................................................... 14 
5.2 
RECEIPT OF MEDICATION .................................................................................................................................................. 14 
5.3 
STORAGE ............................................................................................................................................................................. 15 
5.4 
ADMINISTRATION AND ACCOUNTABILITY ........................................................................................................................ 15 
6 
STUDY PROCEDURES ............................................................................................................................................ 15 
6.1 
SCREENING.......................................................................................................................................................................... 15 
6.2 
STUDY INTERVENTION PHASE........................................................................................................................................... 16 
6.2.1 
Visit 1 (Baseline visit) ................................................................................................................................................................. 16 
6.2.2 
Visit 2 – Treatment induction ................................................................................................................................................ 17 
6.3 
FOLLOW UP PHASE OF THE STUDY ................................................................................................................................... 17 
6.3.1 
Monthly visit.................................................................................................................................................................................... 17 
6.3.2 
Visit week 4, 8, 12, 24, 36, 48 .................................................................................................................................................. 17 
6.3.3 
End of Study Visit .......................................................................................................................................................................... 17 
6.4 
SUBJECT WITHDRAWAL ..................................................................................................................................................... 17 
6.5 
EARLY TERMINATION VISITS ............................................................................................................................................ 18 
7 
STATISTICAL PLAN................................................................................................................................................ 18 
7.1 
PRIMARY ENDPOINT .......................................................................................................................................................... 18 
7.2 
SECONDARY ENDPOINTS .................................................................................................................................................... 18 
7.3 
SAMPLE SIZE AND POWER DETERMINATION ................................................................................................................... 19 
7.4 
STATISTICAL METHODS ..................................................................................................................................................... 19 
7.4.1 
Baseline Data.................................................................................................................................................................................. 19 
7.4.2 
Safety Analysis ............................................................................................................................................................................... 19 
7.5 
SUBJECT POPULATION(S) FOR ANALYSIS ......................................................................................................................... 20 

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8 
SAFETY AND ADVERSE EVENTS ......................................................................................................................... 20 
8.1 
DEFINITIONS ....................................................................................................................................................................... 20 
8.1.1 
Adverse Event ................................................................................................................................................................................. 20 
8.1.2 
Serious Adverse Event ................................................................................................................................................................ 20 
8.2 
RECORDING OF ADVERSE EVENTS .................................................................................................................................... 21 
8.3 
REPORTING OF ADVERSE EVENTS, ADVERSE DEVICE EFFECTS AND UNANTICIPATED PROBLEMS ............................. 21 
8.3.1 
Follow-up report ........................................................................................................................................................................... 21 
8.3.2 
Investigator reporting: notifying the study sponsor .................................................................................................. 21 
8.3.3 
Investigator Reporting: Notifying the Penn IRB........................................................................................................... 22 
8.4 
UNBLINDING PROCEDURES................................................................................................................................................ 22 
8.5 
MEDICAL MONITORING ..................................................................................................................................................... 22 
8.5.1 
Data and Safety Monitoring Plan ........................................................................................................................................ 22 
8.5.2 
Data Safety Monitoring Board .............................................................................................................................................. 22 
9 
STUDY ADMINISTRATION, DATA HANDLING AND RECORD KEEPING .................................................... 23 
9.1 
CONFIDENTIALITY .............................................................................................................................................................. 23 
9.2 
DATA COLLECTION AND MANAGEMENT ........................................................................................................................... 23 
• 
DATA ACQUISITION AND TRANSMISSION ................................................................................................................................ 23 
• 
DATA ENTRY METHODS ........................................................................................................................................................... 24 
9.3 
RECORDS RETENTION ........................................................................................................................................................ 25 
10 
STUDY MONITORING, AUDITING, AND INSPECTING .................................................................................... 25 
10.1 
STUDY MONITORING PLAN ................................................................................................................................................ 25 
10.2 
AUDITING AND INSPECTING ............................................................................................................................................... 25 
11 
ETHICAL CONSIDERATIONS ................................................................................................................................ 25 
11.1 
RISKS ................................................................................................................................................................................... 26 
11.2 
BENEFITS ............................................................................................................................................................................ 26 
11.3 
RISK BENEFIT ASSESSMENT .............................................................................................................................................. 26 
11.4 
INFORMED CONSENT PROCESS / HIPAA AUTHORIZATION ........................................................................................... 27 
12 
STUDY FINANCES ................................................................................................................................................... 27 
12.1 
FUNDING SOURCE ............................................................................................................................................................... 27 
12.2 
CONFLICT OF INTEREST ..................................................................................................................................................... 27 
12.3 
SUBJECT STIPENDS OR PAYMENTS .................................................................................................................................... 27 
13 
PUBLICATION PLAN .............................................................................................................................................. 27 
14 
REFERENCES ........................................................................................................................................................... 27 
15 
ADDENDUM ............................................................................................................................................................. 30 
SAMPLE COLLECTION ..................................................................................................................................................... 30 
Table 1: Total study sample collection for shipment .................................................................................................................... 30 
SAMPLE PROCESSING AND TRANSFER ...................................................................................................................... 30 
SAMPLE UTILIZATION .................................................................................................................................................... 30 
CELLULAR MEASURES OF RESIDUAL IMMUNE ACTIVATION AND SENESCENCE ON ART................................................................. 30 
IN-DEPTH IMMUNOFLUORESCENCE PROFILING ................................................................................................................................ 31 
SYSTEMIC PLASMA--BASED MEASURES OF RESIDUAL IMMUNE ACTIVATION/INFLAMMATION ON ART ...................................... 31 
MICROBIAL TRANSLOCATION AND DYSBIOSIS CAN BE ASSOCIATED WITH OPIOID USE IN PWIDS ................................................ 31 
REVERSAL OF THE ACTIVATION GENE EXPRESSION SIGNATURES IN MYELOID SUBSETS ................................................................ 31 
PERSISTENT HIV TRANSCRIPTION/REPLICATION ON ART ............................................................................................................. 31 
 
 
 
 
  
 

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List of Abbreviations 
 
 
 
ART: Antiretroviral therapy 
AE: Adverse event 
BUP/NX: Buprenorphine/naloxone 
MAT: Medication for opiate use disorder-Assisted Treatment 
MET: Methadone 
MOR: Mu Opiate Receptor 
MOUD: Medication for opiate use disorder  
SAE: Serious Adverse Event 
UDS: Urine Drug Screen 
XR-NTX: Extended-Release Naltrexone 
 
 

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Study Summary 
 
Title 
Effects of mu-opiate receptor engagement on microbial translocation 
and residual immune activation in HIV-infected, ART suppressed 
opioid use disorder patients initiating medication-assisted treatment 
Short Title 
AMOHI-1  
IRB Number 
833720 
Protocol Number 
NIDA R01 DA048728-01 
Phase 
Phase II  
Methodology 
3-arm randomized-controlled study 
Study Duration 
60 months  
Study Center(s) 
Go Vap clinic, Ho Chi Minh city, Vietnam 
Objectives 
Primary: 
• 
To compare plasma sCD14 concentration in HIV-positive opiate 
use disorder individuals who receive cART and methadone, 
buprenorphine/naloxone or XR-naltrexone over 48 months  
Secondary: 
• 
To evaluate the change in markers of cellular immune activation 
and 
senescence, 
systemic 
inflammation 
and 
bacterial 
translocation 
• 
To evaluate the effectiveness of the treatment administered 
towards a) reducing/suppressing opiate injection behavior and 
promoting adherence to the treatment schedule and b) 
suppressing HIV replication 
Number of Subjects 225 (75 per arm) 
Main Inclusion and 
Exclusion Criteria 
Inclusion criteria: 
• 
Age: male or female subjects 18- 65 years of age 
• 
Meet DSM-5 criteria for moderate to severe opiate use disorder 
(as determined by DSM-5 checklist) 
• 
Documented HIV-1 infection with CD4<350 cells/ μL and VL> 
10,000 copies/mL 
 
Exclusion criteria 
 
• 
Meet DSM-5 criteria for any other substance use disorder 
(except nicotine) 
• 
Already engage in opiate medication treatment at baseline 
(methadone, buprenorphine, buprenorphine/naloxone, 
naltrexone) 
• 
Advanced liver disease (FibroScan® METAVIR score F3-F4, 
liver elasticity >10kPa)   
 

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Investigational 
Product (drug, 
biologic, device, 
etc.) 
For Device include 
the planned use 
For Drug, food, 
cosmetic, etc. 
include the dose, 
route of 
administration and 
dose regiment 
Methadone: daily dosing, oral 
Buprenorphine/naloxone: daily dosing and thrice-weekly after 
stabilization, sublingual 
Extended-release naltrexone (Vivitrol®): monthly, depot injection 
Duration of 
administration (if 
applicable) 
12 months 
Reference therapy 
None (not intend to evaluate the efficacy of one treatment versus 
another but to evaluate change in markers of inflammation along a 
course of treatment) 
Statistical 
Methodology 
The primary outcome of interest is the change in sCD14 over 48 weeks 
following ART. This marker will be assessed over 7 consecutive time 
points (baseline, weeks 4, 8, 12, 24, 36, and 48). The primary analyses 
for this longitudinal data will use the mixed model regression model 
(MER) with restricted maximum likelihood 
Safety Evaluations  
• 
Liver function test 
• 
Adverse events and serious adverse events including 
hospitalization and overdose  
Data and Safety 
Monitoring Plan  
The study will be monitored by the PIs and co-investigators, 
University of Pennsylvania Institutional Review Board, Ho Chi 
Minh City CDC Institutional Review Board, Ho Chi Minh Pasteur 
Institutional Review Board, as well as by regulatory committees at the 
University of Pennsylvania (i.e., IRBs, OHR) and Center for Studies of 
Addiction Data Safety Monitoring Board (Penn DSMB). During the 
course of the study, safety and data quality monitoring will be 
performed on an ongoing basis by the Principal Investigators and the 
study staff. Dr. Charles O’Brien (Center for Studies of Addiction, 
University of Pennsylvania) will serve as the medical monitor for the 
study.   
 
 
 
 

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This document is a clinical research protocol. This study will be conducted in full accordance all applicable 
University of Pennsylvania Research Policies and Procedures and all applicable Federal and state laws and 
regulations including 45 CFR 46. All episodes of noncompliance will be documented. 
1 
Introduction 
1.1 
Background and Relevant Literature  
Chronic opiate use results in a range of adverse effects including increased susceptibility to infections and 
systemic immune activation. Opioid-induced over-expression of TOLL-like receptors (TLR) 2 and 4 in the gut 
mucosa leads to alterations in the epithelial tight junctions; in rodents this depends on the activation of myosin 
light chain kinase (MLCK), and results in bacterial translocation to the liver. Binding of opioids to cells of the 
enteric nervous system EMS plexa leads to decreased gut motility, resulting in chronic constipation and 
accumulation of bacterial products. Chronic opioid exposure results in changes in the gut microbiota 
(increase in Gram+ bacteria of the phylum Firmicutes, previously associated with systemic inflammation), 
which is TLR-2-dependent and may be related to acidification of the bile secretion [1-4]. Chronic opioid use 
has also been shown to effect innate immune functions, such as a reduction in NK cell activity through 
central dopaminergic signaling [5]. Thus, lack of immune clearance may also contribute to systemic immune 
activation. 
 
Three medications for opiate use disorder (MOUD) have been approved and showed efficacy in the 
treatment of opiate use disorder, all interacting with the mu opiate receptor (MOR) either as a full 
(methadone, MET) or partial (buprenorphine, BUP) agonists, or as an antagonist (naltrexone, NTX). The 
effect of persistent engagement of MOR on immune activation by MET remains controversial. Early reports 
indicated that production of IL-2, IL-10, IFN-g, but not IL-6, was normalized in subjects on MET. However, 
recent work evidenced high serum levels of IL-1b, IL-6, and IL-8 in MET users [6, 7].  On the other hand, 
MOR antagonist Naltrexone (and particularly its extended-release formulation, XR-NTX) has demonstrated 
efficacy in supporting abstinence in individuals with opioid and alcohol use disorders [8] [see also guidelines 
of the American Society of Addiction Medicine [9]], including HIV-1 infected individuals transitioning from 
the criminal justice system to the community [10]. In vitro, NTX exerted a direct anti-HIV effect by reversing 
the opioid-induced overexpression of the co-receptor CCR5 on peripheral blood mononuclear cell [11].  
Clinical use of NTX-based MOUD was also shown to improve HIV suppression on ART over placebo [12], 
mostly due to improved ART adherence, and was not associated with liver toxicity [13], even in the presence 
of HCV co-infection [14]. 
 
Worldwide, the HIV epidemic has involved three main transmission modalities: sexual transmission, vertical 
perinatal transmission, and by needle sharing during IV drug injections. Regarding the latter, Vietnam has 
a large population (currently about 271,000 individuals) of people who inject drugs (PWIDs), 85% of which 
inject opiates [15]. In 2014, approximately two-thirds of the reported 15,000 new HIV infections in Vietnam 
were attributable to injection drug use [15]. Since 2008, Vietnam has instituted outpatient MET-based MOUD 
programs.  Oral NTX is also currently available at selected addiction clinics (including the proposed study site: 
Thu Duc Addiction Treatment Center, Ho Chi Minh City, http://tuvancainghien.gov.vn). However, it is 
estimated that only 20-22% of individuals with opioid use disorder have access to MAT [16, 17]. Vietnam has 
also implemented a massive scale up of ART, but in 2015 only a third of HIV-positive individuals were on 
ART. The most common risk factor for late ART initiation is active drug-injecting behavior [18]. Integration of 
MOUD within HIV services has resulted in improved ART outcomes (e.g., enrollment, adherence, retention, 
and viral suppression) in multiple settings [19, 20], including Vietnam. However, there is less evidence to 
support that MAT decreases mortality on ART [21].  Vietnamese PWIDs have very high rates of co-infections 
with HCV (ranged from 89.8% and 98.5% [21, 22]), HBV (ranged from 20-40%, [23]), and tuberculosis (209 
cases per 100,000 inhabitants, [24]). HIV-HCV co-infection in PWIDs has been estimated as high as 98.5% 
[25], but unlike TB infection, it does not appear to significantly increase mortality in these settings [26, 27].  
This proposal takes advantage of an established collaboration with a large urban clinic providing ART and 
MOUD to HIV-infected PWIDs concurrently. Since November 2013, our group has implemented an 
integrated opioid use disorder and HIV-1 treatment program at Go Vap Clinic, Ho Chi Minh City, Vietnam, 
sponsored by a NIDA Grant (R01-DA033671). This program enrolled 448 heroin users with an average age 
of 32.4 years (SD= 5.2) and living with family (81%). The prevalence of HIV in the cohort was 34.2% (n=153). 
Participants received integrated treatment with either MET (n=268) or buprenorphine/naloxone (n=180) [28]. 
 

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1.2 
Name and Description of the Investigational Product  
The participants will be randomized in three arms: 
- 
Methadone (MET) is a full opiate agonist of the mu opioid receptor. Methadone has been used for 
decades to treat people who have opioid use disorder. When taken as prescribed, it is safe and 
effective. Methadone hydrochloride oral concentrate available in Vietnam is provided by 
Mallinckrodt PLC.  
- 
Buprenorphine/naloxone (BUP/NX) is a partial mu opiate receptor agonist and a kappa receptor 
antagonist. It has been approved for clinical use since October 2002 by the Food and Drug 
Administration (FDA). BUP/NX, in combination with counseling and behavioral therapies, provide a 
whole-patient approach to the treatment of opioid use disorder. When taken as prescribed, BUP/NX 
is safe and effective.  
- 
Extended-release naltrexone (XR-NTX) is a full mu opiate receptor antagonist. Naltrexone is a 
medication approved by the Food and Drug Administration (FDA) to treat opiate use disorder. The 
injectable extended-release form of the drug (Vivitrol) is administered at 380 mg intramuscular once 
a month. Naltrexone can be prescribed by any health care provider who is licensed to prescribe 
medications. To reduce the risk of precipitated withdrawal, patients are warned to abstain from 
illegal opioids and opioid medication for a minimum of 7-10 days before starting naltrexone. XR-
NTX is not yet available in Vietnam. For the study, we are using Vivitrol® provided by Alkermes, 
Inc.   
- 
All participants will receive combined antiretroviral therapy (cART) for HIV.  
- 
All participants will receive counseling sessions for 48 weeks. 
1.2.1 Clinical Data to Date 
The extent, change, and immunopathogenesis of residual immune activation in HIV-infected individuals 
receiving suppressive ART have important clinical repercussions as elevated expression of sCD14 
(positively correlated to microbial translocation) has been repeatedly reported to be associated with higher 
mortality and adverse metabolic outcomes in ART-suppressed individuals with chronic HIV-1 infection [29-
32]. In addition, T cell activation and particularly the expression of CD38 and HLA-DR on CD8+ T cells is 
associated with a lack of CD4+ T cell recovery on ART, and a loss of 35 CD4+ T cells/μl for each 5% increase 
in activation [33]. Thus, identifying the potential benefits of choosing MOR antagonists over MET after ART 
may better inform addiction treatment choices and public health policy.  
 
We have completed a pilot study assessing the expression of immune activation markers and soluble 
microbial translocation markers in a cross-sectional convenience cohort of HIV-1-infected PWIDs (and 
control, non-opioid injecting patients) receiving ART and MOUD at the Jonathan Lax Clinic/Philadelphia 
FIGHT (Philadelphia, PA). Within the limitations of a small convenience cohort, our pilot data strongly 
supports the hypothesis that in PWIDs receiving (or initiating) ART, continued MOR engagement through 
MET may result in impaired immune reconstitution outcomes as compared with NTX.  
 
2 
Study Objectives 
The overall objective of this study is to provide clinical evidence on the detrimental link between kinetics 
and characteristics of immune reconstitution (microbial translocation, residual immune activation, 
retained HIV expression) in HIV-1 infected people who inject drugs (PWIDs) due to a sustained 
interaction with the μ-opioid receptor (MOR) while on antiretroviral therapy (ART). 
2.1 
Primary Objective 
• To compare plasma sCD14 concentration in HIV-positive opiate use disorder individuals who 
receive cART and methadone, buprenorphine/naloxone or XR-naltrexone over 48 months. To 
this end, we will compare long-term changes (48 weeks) in a cohort of PWID with chronic HIV 
infection initiating ART and initiating either MET, BUP/NX or XR-NTX along with behavioral drug 
and risk counseling (BDRC) sessions. 
2.2 
Secondary Objectives  
• 
To evaluate the change in markers of cellular immune activation and senescence, systemic 
inflammation and bacterial translocation. 
 

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• 
To evaluate the effectiveness of the treatment administered towards a) reducing/suppressing 
opiate injection behavior and promoting adherence to the treatment schedule and b) 
suppressing HIV replication 
§ 
The effect of the intervention on CD4, adherence to ART, acceptability of MOUD, 
as well as retention in care 
§ 
The changes in HIV replication and latency measures (i.e., characterization of 
viral RNA and DNA in PBMC, persistence of cells latently infected with 
potentially replicative HIV in circulation) 
3 
Investigational Plan  
3.1 
General Design 
 
3.1.1 Screening Phase 
Participants will be recruited as they enter treatment at the Go Vap HIV and methadone clinic. Clinical staff 
at the HIV and addiction clinic will identify potential subjects and refer them to the research assistant (RA). 
The RA will describe the study and those who express interest will be referred to the study medical 
supervisor who will explain it in detail, answer questions, review the consent to screen, and have the subject 
sign it after confirming that he/she understands it. Tests to confirm eligibility for cART will be done on site. 
For those eligible, informed consent will be obtained in writing by the study medical investigator from all 
study participants prior to any procedure. Study personnel will first introduce potential participants to the 
study and explain the study procedures. A written informed consent form (ICF) translated in Vietnamese, 
the content of which will be approved by the local Ethics Committee according to local regulations, will also 
be provided to the candidates, who will be allowed time to read it and discuss it with the study personnel. 
ICFs will clearly state that participation in the research is completely voluntary and that there will be no 
consequence to the participant for declining participation or absences from study procedures. Candidates 
who consent to participate will sign and date the form; signed ICFs will be locked-stored at the Go Vap site 
according to local regulation and GCP directives. 
 
3.1.2 Study Intervention Phase  
All the participants will be evaluated at the clinic by the study medical director who will perform physical 
examinations and review laboratory work, document current opiate use and DSM-5 opiate use disorders, 
perform a clinical interview to rule out psychiatric disorders that exclude participation, review the ICF, and 
Refuse to 
participate / 
Meeting exclusion 
criterion 
- Blood sample: pre-cART 
biological variables 
 
Screening 
• 
>18 y.o. 
• 
HIV-1 infected 
• 
Opiate use disorder (DSM-5) 
• 
Seeking treatment for OUD and HIV 
Study enrollment / consent process 
Not eligible 
Referral to methadone, 
buprenorphine or 
naltrexone treatment and 
HIV clinic 
Randomization 
1:1:1 
n=225 
 cART + Methadone + BDRC 
n= 75 
cART + BUP + BDRC 
n= 75ç 
Post-study referral to 
methadone, 
buprenorphine or 
naltrexone treatment and 
HIV clinic 
* Anticipated dropouts: 
30 % in XR-NTX arm 
30 % in BUP arm 
15% in MET arm 
0 4 
8 12 
24 
36 
48 
- Clinical evaluation: 
physical, psychological, 
biological panel, 
Fibroscan® 
- Baseline assessment: 
sociodemographic, ASI, 
PHQ-9 
Continuous evaluation: substance use,  
treatment adherence, craving,  
psychological status, satisfaction with treatment 
 
Biological collection: 0, 4, 8,12, 16, 20, 24, 
36, 40, 44, 48 
Week 48 
End of intervention 
n= 150* 
cART + XR-NTX + BDRC 
n= 75ç 

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answer any remaining questions. A Fibroscan® will be performed for each potential participant, and those 
with a METAVIR score F3-F4 will be excluded from the study and will be referred to hepatitis C specialist 
for care.  
 
Following completion of informed consent procedures, a RA will complete baseline assessments, and 
randomize the participant to his/her medication condition. The study medical director will then coordinate 
the care at the clinic for cART (with HIV specialist) and methadone, buprenorphine or long-acting naltrexone 
treatment. A treatment plan with scheduled visits will be delivered to the participant.  
 
Treatment initiation 
• 
Methadone arm 
The induction on methadone will follow the Vietnamese guidelines. Basically, the dosing will commence with 
20 to 30 mg daily; thereafter, dose increases will be made following review of the participant’s response to 
their initial methadone dose. Assessments will review side effects, symptoms of withdrawal (suggesting not 
enough methadone) or intoxication (suggesting too much methadone or other drug use), ongoing cravings, 
and substance use. Generally, the doses are increased 5 to 10 mg every three to five days.  The dose will 
be gradually increased until a maintenance dose is reached, which consists of cessation (or marked 
reduction) of opioid use, alleviation of cravings and opioid withdrawal features between doses, whilst 
minimizing methadone side effects. Daily directly observed methadone dosage will be delivered along with 
weekly for 12 weeks, and then monthly counseling sessions up to 48-week follow-up.  
 
• 
Buprenorphine/naloxone arm 
The induction on buprenorphine/naloxone will follow the Vietnamese guidelines. Buprenorphine/naloxone 
tablets are administered sublingually, placed under the tongue until they are dissolved.  For doses requiring 
the use of more than two tablets, participants are instructed to either place all the tablets at once or 
alternatively (if they cannot fit in more than two tablets comfortably) place two tablets at a time under the 
tongue.  Either way, the participant should continue to hold the tablet under the tongue until they dissolve; 
swallowing the tablet reduces the bioavailability of the drug. 
 
To aid clinicians in determining the appropriate dose during the first two-to-three days, the study will use the 
Clinical Opiate Withdrawal Scale (COWS), an instrument measuring validated items of physical signs or 
symptoms of withdrawal, such as gooseflesh, vomiting, and sweating, to objectively assess withdrawal in 
both treatment arms [34].  The total score gives an index of the participant’s withdrawal intensity and can 
be administered over time to track changes and adjust dosing.  On day one, the clinician will wait until the 
participant shows at least mild opiate withdrawal by monitoring with the COWS.  If the score indicates at 
least mild withdrawal (the higher the score the more severe the withdrawal), the clinician will give 4 mg for 
the first dose.  COWS will be repeated in one hour; patients will receive further dosing as described below.   
Dosing will begin with a titration over a period of two to three days under supervision in the Go Vap clinic 
using the COWS as described above. On the first day of treatment, patients will initially receive a 4 mg dose 
of buprenorphine/Naloxone (expressed as the amount of buprenorphine) to be taken sublingually.  Most 
participants will begin with a total first day's dosage of 8 mg.  On Day 2, up to 16 mg may be given.  Up to 
32 mg may be given on Day 3 and thereafter until three-times-weekly dosing begins. The induction strategy 
is primarily dependent on three factors: 1) time since last opiate use; 2) type of opiate (e.g., long or short-
acting) used; and 3) degree of physical dependence.  Therefore, each dosing schedule will be tailored to 
the individual participant.  
 
Individuals will come to the study site daily for direct observation of dosing until they have stabilized (for up 
to three weeks).  After induction and stabilization, participants will be asked to come to the site for dosing 
three-times-weekly. The target dosage schedule for individuals whose daily dose was 16 to 24 mg/day is 
expected to be 32/32/48 mg administered on a three-times-weekly schedule (e.g., M/W/F); this is also the 
maximum three-times-weekly dosage. On rare occasions, for individuals who require more than 24 mg/day 
(i.e., 26, 28, 30, or 32 mg/day), it is unlikely that the 32/32/48 mg dosage schedule will be adequate.  For 
those individuals, as well as for others who received 24 mg or less per day but for whom the 32/32/48 mg 
three-times- weekly schedule is not adequate, dosing may be continued on a daily basis through Week 52 
of the study, with take-home doses administered for those days on which in-clinic dosing is not possible 
(e.g., 32 mg on M/Tu/W/Th/F/Sat with a take-home 32 mg dose on Sun).   
 
Daily or thrice-weekly buprenorphine dosage will be delivered along with weekly for 12 weeks and then 
monthly counseling sessions up to 48-week follow-up. 
 

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• 
Extended-release naltrexone (XR-NTX) arm 
The participants assigned to naltrexone arm will undergo supervised 2-week opioid inpatient detoxification 
at the Thu Duc Addiction Treatment Center (under the supervision of Dr. Do). The detoxification program 
follows Vietnamese guidelines (http://tuvancainghien.gov.vn). Adjunctive medications might also be used 
depending of the severity of the withdrawal symptoms, including benzodiazepines for anxiety and 
restlessness (e.g., oxazepam); low doses of sedating antidepressants (e.g., doxepin, trazodone) or 
zolpidem for insomnia; antiemetics for GI distress (e.g., prochlorperazine, ondansetron); and nonsteroidal 
anti-inflammatories for withdrawal-related aches. Withdrawal symptoms will peak at 24-48 hours after heroin 
cessation. Three counseling sessions will be scheduled during the detoxification, and participants will have 
access to counselors via telephone between sessions. The first XR-NTX will occur at day 12-14 of the 
detoxification with at least one day stay at the clinic before discharge and beginning of the outpatient follow-
up. Before injecting XR-NTX and as a confirmation of detox period, participants will receive a naloxone (fast 
acting opiate blocker) challenge. The challenge will be performed by slowly administering intramuscularly 
0.8-mg naloxone; if no withdrawal symptoms occur within 20 minutes, the absence of physiologic reaction 
is confirmed. Participants who experience withdrawal symptoms following the naloxone challenge will be 
treated symptomatically, observed until symptoms resolve, and will be offered counseling and invited to 2 
days of extended detoxification for a repeat naloxone challenge. Failure to pass the challenge on 3 
occasions disqualifies a patient from study participation, and a negative outcome will be considered an 
endpoint in our intention to treat analysis.  If a participant elects not to be part of the study for whatever 
reason, he/she will be strongly encouraged to receive opiate use disorder treatment and will be referred to 
methadone treatment or oral naltrexone treatment. The XR-NTX will be injected monthly by Dr. Trung 
Nguyen and Dr. Do Mahn who will receive training and supervision from Dr. George Woody (who has 
extensive experience in XR-NTX treatment in the USA and internationally).  
 
• 
cART (three arms) 
cART will follow Vietnamese guidelines for HIV treatment. We will look to coordinate to start methadone or 
XR-naltrexone within 2 weeks of cART, but starting cART will take priority over methadone/naltrexone in 
order to avoid losing patients due to the logistic issues in scheduling appointments.  To minimize the chance 
of developing resistance attributable to poor adherence from relapse in patients who happen to start cART 
before methadone or XR-naltrexone, we will start each treatment within 2 weeks of each other. Participants 
who change their mind or are found ineligible at any point will be referred to the usual addiction treatment 
and encouraged to continue with the HIV treatment program. Declining enrollment in the study will not affect 
eligibility for cART. 
 
 
• 
Counseling (three arms) 
The counseling session is modeled after the Behavioral Drug and Risk Counseling (BDRC) designed to 
address both addiction and HIV related behaviors [35, 36]. This model is rooted in cognitive behavioral 
therapy. Each session will assess the need for intervention in six areas of functioning:  1) adherence to 
treatments for Substance Use Disorder, HIV, TB; 2) continued drug use and related drug and sex risk; 3) 
cravings for drug use; 4) psychological status (depression, anxiety, symptoms of psychiatric disorder); 5) 
confidence in and satisfaction with MET, BUP/NX or XR-NTX treatment; 6) behavioral management 
strategies to be implemented until the next counseling session. Counseling sessions are delivered by trained 
and experienced staff guided by a counseling manual that has been developed for the NIDA-funded study 
at Go Vap clinic entitled “A Pilot Implementation Project of Methadone and Suboxone® for Injecting Drug 
Users in Ho Chi Minh City, Vietnam.” Counselors have worked in the current R01 study at Go Vap clinic and 
are trained and experienced in counseling heroin users.   
3.1.3 Follow Up Phase 
All participants will receive directly observed daily medication methadone, buprenorphine or monthly XR-
naltrexone injection and cART. All participants will also receive weekly structured manual-based counseling 
sessions for the first 3 months and monthly counseling sessions thereafter. Each session will last about 45 
minutes. Participants will be followed for 48 weeks.  
3.1.4 Allocation to Interventional Group  
The randomization schedule will be prepared using a randomize block strategy to ensure balance throughout 
the trial randomization will be performed on demand in real time using a dedicated CDETweb toolbox 
application, according to the schedule provided by the statistical team. 

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3.2 
 Study Endpoints  
3.2.1 Primary Study Endpoint 
The primary study endpoints will compare plasma sCD14 concentration in HIV-positive opiate use disorder 
individuals who receive cART and methadone, buprenorphine/naloxone or XR-naltrexone over 48 months  
• Plasma assessments (baseline, weeks 4, 8, 12, 24, 36, and 48) 
o sCD14 
3.2.2 Secondary Study Endpoints 
- 
To evaluate the change in markers of cellular immune activation and senescence, systemic 
inflammation and bacterial translocation. 
• 
Immune activation and senescence. These variables will be assessed by flow cytometry, ELISA 
or rtPCR on cryopreserved PBMC and plasma, at the indicated time points. 
• Flow cytometry (baseline, weeks 4, 8, 12, 24, 36 and 48) 
o CD38, HLA-DR and PD1 expression in C8+ T cells  
o Activation signature in monocytes 
o CD169 expression in monocytes 
• Plasma assessments (baseline, weeks 4, 8, 12, 24, 36, and 48) 
o 
sCD163 
• rtPCR-based assessments (baseline, weeks 12, 24, 36, and 48) 
o Type-I IFN signature in PBMC 
• 
Inflammation will be assessed using Luminex plexes on cryopreserved plasma at baseline, 
weeks 4, 8, 12, 24 and 48. 
• Acute phase reactants: Plasma hr-CRP and d-dimer 
• Cytokines and receptors: sTNFR-1, IL-6, IL-10, TGF-beta 
• 
Dysbiosis and bacterial translocation will be evaluated at baseline and at week 48 in plasma 
(ELISA) and stool samples (rtPCR). 
• Plasma based assessments: sCD14, LBP, LPS, endo-CAB, Intestinal fatty acid-binding 
protein (I-FABP), Zonulin-1 
• Stool-based assessments: s16 rDNA, bacterial Butyryl-CoA-CoA 
 
- 
To evaluate the effectiveness of the treatment administered towards a) reducing/suppressing opiate 
injection behavior and promoting adherence to the treatment schedule and b) suppressing HIV 
replication 
Clinical and virological outcomes that will allow us to assess the effectiveness of the treatment 
administered towards a) reducing/suppressing opiate injection behavior and promoting adherence 
to the treatment schedule and b) suppressing HIV replication. 
The clinical outcomes will be:  
• 
CD4 count (clinical indicator of successful viral suppression and immune reconstitution, 
assessed at baseline, weeks 4, 8, 12, 24, 36, 48) 
• 
Retention in care (% of completed medication visits) 
• 
cART adherence (% of pharmacy pickups) 
• 
Continued drug use (% of positive monthly urine opiate tests or positive self-report) 
• 
Acceptability of MOUD (qualitative assessment based on satisfaction questionnaire) 
• 
Virological outcomes (assessed in PBMC or plasma) 
• 
Viral load suppression (% of randomized individuals achieving VL <50 copies/ml; (% of 
exposed individuals achieving VL <50 copies/ml) 
• 
Cell-associated DNA, RNA species (poly-A, multi-sliced Tat-Rev), replicative index 
(linear measures indicating residual viral replication) 
 
 
 
 
 

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4 
Study Population and Duration of Participation  
4.1 
Inclusion and Exclusion Criteria 
 
Inclusion criteria 
Exclusion criteria 
Age: male or female subjects 18- 65 years of age 
Current cognitive impairment, schizophrenia, paranoid 
disorder, bipolar disorder not compatible with study 
procedure (assessed by the medical director of the 
study) 
Meet DSM-5 criteria for moderate to severe opiate use 
disorder (as determined by DSM-5 checklist) 
Known neurological, cardiovascular, renal, or other 
significant medical disorder that is likely to impair or 
make the individual’s participation hazardous 
Opiate use with a positive urine drug screen for heroin or 
other opiates (other than methadone, buprenorphine, 
buprenorphine/naloxone) at screening visit 
Active Tuberculosis or other symptomatic infectious 
disease 
Documented HIV-1 infection with CD4 less than 350 cells/ 
μL and VL more than 10,000 copies/mL 
AIDS-defining illness 
cART-naïve or or on cART no longer than 3 months if 
already started 
Current1 cancer or other malignancies 
Willingness to receive cART or on cART no longer than 
3 months if already started 
Advanced liver disease (FibroScan® METAVIR score 
F3-F4, liver elasticity more than10kPa) 
Willingness to be randomized to either daily methadone, 
buprenorphine/naloxone or monthly injection of extended-
release naltrexone treatment 
Use of immunomodulators 
Ability to understand and complete study procedures 
Meet DSM-5 criteria for any other substance use 
disorder (except nicotine) 
Provision of adequate locator information that lists all 
contact information a participant agrees that the research 
staff may use to reach him/her 
Engagement in opiate medication treatment at 
baseline (methadone, buprenorphine, 
buprenorphine/naloxone, naltrexone) 
All participants must be able to comprehend the purpose 
of the study and to provide informed consent 
Pending legal charges with likely incarceration within 
next 6 months 
Is, in the opinion of the study physician, in stable health as 
determined by pre-study physical examination, medical 
history, ECG, and laboratory evaluations and is likely to 
complete the study. 
 
Currently participating in another clinical trial  
Has a total body weight of more than 50 kg (110 pounds) 
and a body mass index (BMI) of more than 20 at 
screening. 
 
 
Female subjects: 
1. Cannot be pregnant 
2. Cannot be lactating 
3. Must be unable to conceive (i.e., surgically 
sterilized, sterile, or post-menopausal defined as 
1 year without bleeding or spotting) OR must 
agree to use an acceptable method of birth control 
(e.g., birth control pills, intrauterine device [IUD], 
or a double barrier method of birth control 
(condoms and spermicide together; or diaphragm, 
condom and spermicide together) 
 
 
  
                                               
1 Current is defined as in the last 5 years. 

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4.2 
Subject Recruitment  
The study will take place at the Go Vap HIV clinic in Ho Chi Minh City, Vietnam.  The Go Vap clinic 
opened in April, 2013 and provides outpatient treatment for opiate addiction, HIV treatment (antiretroviral, 
cART), and tuberculosis treatment. Currently, the clinic is treating 500 patients for opiate use disorder 
(methadone and BDRC-based counseling) with an 92% retention rate at 12-month and about 1,000 patients 
receiving antiretroviral (cART). The medical staff is comprised of 5 physicians, 6 nurses, 6 counselors, and 
4 pharmacists.  Participants in the proposed study will be HIV-infected, opioid dependent drug users 
(primarily heroin users who inject drugs).  Subjects will be recruited as they enter treatment for their HIV 
infection.  Intake staff will be trained on the basic inclusion criteria and will invite potential participants to 
meet with the research staff who will assess potential eligibility and interest in study participation.   
If necessary, we will expand the recruitment to other Ho Chi Minh City clinics with the help of the HIV AIDS 
Association (HAA).   
The participants assigned to XR-NTX arm will undergo supervised 2-week opioid inpatient detoxification at 
the Thu Duc Addiction Treatment Center (under the supervision of Dr. Do), before receiving their first 
injection of XR-NTX. After the first injection, follow-up will occur at Go Vap clinic, as outpatient visits, 
following the same scheduling than MET and BUP/Nx groups. 
4.3 
Duration of Study Participation 
The duration of the study subjects’ participation will be 48 weeks.  
4.4 
Total Number of Subjects and Sites  
It is expected that approximately 225 subjects will be enrolled in order to produce 158 evaluable subjects. 
All subjects will be enrolled at the Go Vap Clinic. 
4.5 
Vulnerable Populations:  
Children, pregnant women, fetuses, neonates, or prisoners are not included in this research study. 
 
5 
Study Intervention  
5.1 
Description 
All the participants will receive cART and counseling sessions.  
Depending on the arm, they will receive either: 
- 
Methadone hydrochloride oral concentrate available in Vietnam is provided by Mallinckrodt PLC.  
- 
Buprenorphine/naloxone sublingual tablets (Suboxone®) provided by Indivior, Inc.  
- 
Injectable extended-release naltrexone (Vivitrol®) administered intramuscular once a month. 
provided by Alkermes, Inc.   
 
5.2 
Receipt of Medication 
Methadone, and ART are already available in Vietnam and dispensed by the Go Vap clinic’s pharmacy.  
XR-NTX (Vivitrol®) is provided by Alkermes Inc. Alkermes will ship the medication to EMINENT Services 
Corporation (https://www.emiserv.com). EMINENT is a provider of Pharmaceutical, Biological and 
Information Technology Services for pharmaceutical and biotech Industry and Research Organizations 
including Federal Government. They have extensive experience with investigational drug formulation, 
development, manufacturing, testing, packaging and distribution. EMINIENT will receive Vivitrol® from 
Alkermes and will be in charge to package and to ship the medication to Ho Chi Minh City, Vietnam. The 
medication will be shipped to YTECO. YTECO will be in charge to receive the medication, to verify and to 
assure the quantity and quality of the medication.  YTECO will be in charge of the storage of the medication 
and will prepare the packaging and the shipping to the local clinic upon request by the clinic. 
The medications are stored in the clinic in controlled environment and temperature. The medications are 
locked and are dispensed only by pharmacist as per Vietnamese law. Participants will receive their 
medication at the clinic from the pharmacist, there are no take-home dose allowed in Vietnam. In the same 
building, another pharmacy is dedicated to infectious disease treatment (i.e., HIV, Hepatitis C, and 
tuberculosis). A pharmacist is responsible to deliver the cART medications. The pharmacists at the clinic 
are responsible upon receipt, to verify the medications, to assure their quantity and quality. The pharmacists 
are also responsible to log the medications and dose dispensed.  

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5.3 
Storage 
All medications will be stored in secured and locked, controlled environment (i.e., temperature, protection 
from light, sterile conditions).  
5.4 
Administration and Accountability    
MET and BUP/NX will be delivered under supervised dose administration by the clinic’s pharmacist following 
Vietnamese guidelines. The pharmacist will complete a daily log including: subject ID, date administered, 
amount administered, product remaining, product damaged/destroyed and date of damage/destruction, 
product returned and date of return.   
Monthly injection of XR-NTX will be done at the clinic by the medical supervisor. A log of date administration, 
amount administered and to which subjects, product remaining, product damaged/destroyed and date of 
damage/destruction, product returned and date of return will be completed by the clinic’s pharmacist.  
ART will be delivered monthly by the clinic’s pharmacist. The pharmacist will complete a log that gathers: 
subject ID, date of delivery, product delivered, amount delivered, product remaining, product 
damaged/destroyed and date of damage/destruction, product returned and date of return.   
 
6 
Study Procedures 
 
Study procedures timeline 
 
 
  
6.1 
Screening  
Clinical staff at the HIV and addiction clinic will identify potential subjects (those with HIV infection and opioid 
use disorder) and refer them to the research assistant (RA). The RA will describe the study and those who 
express interest will be referred to the study medical supervisor, or their designate, who will explain the 
purpose of the study and the procedures in detail, answer questions, and review the consent to participate.  
After confirming comprehension, the participant will be asked to sign the form.  
 
Informed consent procedure  
Study personnel will first introduce potential participants to the study and explain the study procedures. A 
written informed consent form (ICF) in Vietnamese, the content of which will be approved by the local Ethics 
Committee according to local regulations, will also be provided to the candidates, who will be allowed time 
to read it and discuss it with the study personnel. ICFs will clearly state that participation in the research is 
completely voluntary and that there will be no consequence to the participant for declining participation or 
absences from study procedures.  
 
Screening
Wk 
0
Wk 
1
Wk 
2
W
k 3
W
k 4
W
k 5
W
k 6
W
k 7
Wk 
8
Wk 
9
Wk 
10
Wk 
11
Wk 
12
Wk 
13
Wk 
14
Wk 
15
Wk 
16
Wk 
17
Wk 
18
Wk 
19
Wk 
20
Wk 
21
Wk 
22
Wk 
23
Wk 
24
Wk 
25
Wk 
26
Wk 
27
Wk 
28
Wk 
29
Wk 
30
Wk 
31
Wk 
32
Wk 
33
Wk 
34
Wk 
35
Wk 
36
Wk 
37
Wk 
38
Wk 
39
Wk 
40
Wk 
41
Wk 
42
Wk 
43
Wk 
44
Wk 
45
Wk 
46
Wk 
47
Wk 
48
Inclusion criteria
X
Blood test (screening)
X
Fibroscan®
X
DSM-5 SUD checklist
X
Urine drug screen
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Pregnancy test (women only)
X
Specific questionnaire - Baseline
X
Methadone induction
X
X
X
Methadone maintenance dose (daily)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Buprenorphine induction
X
Buprenoprhine maintenance dose
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Opiates detoxification (inpatient)
X
X
X
XR-NTX injection
X
X
X
X
X
X
X
X
X
X
X
X
Counseling sessions
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
HIV specialist - cART initiation
X
X
X
cART refill (pharmacist log)
X
X
X
X
X
X
X
X
X
X
X
Methadone adherence (pharmacist log)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Buprenorphine adherence (pharmacist 
log)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
XR-NTX adherence (pharmacist log)
X
X
X
X
X
X
X
X
X
X
X
X
X
Medical evaluation
X
X
X
X
X
X
X
X
X
X
X
X
X
Specific questionnaire - Follow-up
X
X
X
X
X
X
X
X
X
X
X
X
PHQ-9
X
X
X
X
X
X
X
X
X
X
X
X
X
Satisfaction with treatment
X
X
X
X
X
X
X
X
X
X
X
X
Labs (VL, CD4)
X
X
X
X
X
X
X
X
Blood sampling
X
X
X
X
X
X
X
Stool sampling
X
X

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Candidates who consent to participate will sign and date the form; signed ICFs will be locked-stored at the 
Go Vap site according to local regulation and GCP directives.  
 
Screening 
A clinic nurse will draw blood (phlebotomy) to measure HIV viral load and CD4 and to confirm eligibility for 
cART. Another blood sample will be collected for liver function test (measures of AST, ALT and total 
bilirubin). The measures will be performed at the Pasteur Institute of Ho Chi Minh City.  
All the participants will be evaluated at the clinic by the study medical director who will: 
- 
Conduct physical examinations 
- 
A urine pregnancy test will be performed for female subjects 
- 
Review laboratory work 
- 
Document current opiate use and DSM-5 opiate use disorders using the DSM-5 Substance Use 
Disorder Checklist. This form is a listing of the eleven DSM-5 criteria for substance use disorder. A 
score of 4-6 is considered moderate severity and from 7-11 is considered severe substance use 
disorder. An opioid use disorder score of 4 or greater will be required for study eligibility. We will 
screen for Opiates, Alcohol and benzodiazepine use disorder. Moderate to severe alcohol or 
benzodiazepine use disorder will be exclusionary.  
- 
Conduct a clinical interview to rule out psychiatric disorders that exclude participation.  
- 
Perform a Fibroscan® for each potential participant, and those with a METAVIR score F3-F4 will be 
excluded from the study and will be referred to hepatitis C specialist for care.  
6.2 
Study Intervention Phase 
6.2.1 Visit 1 (Baseline visit)  
After the enrollment process, including consent form and screening tests for inclusion/exclusion criteria, 
participants will be randomized 1:1:1 to MET, BUP/NX or XR-NTX. The randomization schedule will be 
prepared using a random-size block strategy to ensure balance throughout the trial. The randomization will 
be performed on demand in real time using a dedicated CDETweb toolbox application, according to the 
schedule provided by the statistical team.  
If the participant is assigned to a treatment that they refuse to enter after randomization, the participant will 
be allowed to switch to another arm upon consultation and approval by provider.  
If a participant switches immediately after randomization or after induction (detoxification or dose 
adjustment, depending on the arm), he/she will be kept on study, and will be treated as part of the switch 
arm in an “as treated” analysis (since there would be very little or no exposure to the original treatment), but 
will be failed in ITT analysis (cf. 8.5).  
 
The study medical director will then coordinate the care at the clinic for cART (with HIV specialist) and 
MOUD. A treatment plan with scheduled visits will be delivered to the participant.  
 
RA will complete baseline assessment that includes:  
• 
Locator Data Collection:  A basic philosophy of the research approach to be implemented is that 
follow-up begins at recruitment and enrollment. Thus, a detailed contact sheet (Locator Form) will 
be completed for all subjects as part of the baseline assessment. This locator form will be updated 
at each subsequent assessment visit. Participants in the research will also be asked to call or visit 
the research office in the clinic whenever they have questions about assessment schedules or have 
updates to provide regarding changes in contact information. Participants will be asked to provide 
contact information on themselves and three additional people with whom they are most likely to 
stay in contact. They will also be asked about other places where they might be able to be contacted 
in the future. This instrument has already been used by the investigators in Vietnam.  
• 
Study Specific Questionnaire:  This questionnaire has been developed and used in Vietnam in a 
previous R01 study. This questionnaire gathers demographic information (e.g. age, gender, race 
and ethnic identity, marital status, educational level, employment status, income and income 
sources) and include items from the Addiction Severity Index (ASI) [37] that gathers information on 
medical and psychiatric status, drug and alcohol use and history of treatment, legal status, 
family/social relations.  
• 
Risk Assessment Battery (RAB):  The RAB provides a brief self-report measure of drug use, 
injection-related risk behavior, and sexual risk and uses the preceding six months as the time 
interval of interest [38, 39].  The RAB has been translated into Vietnamese and has been used in 
the previous R01. 

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• 
Urine drug screen:  We will use the CLIA waived® ACON Dip-and-Read 8-panel test for THC, 
cocaine, opiates, amphetamines, PCP, methamphetamine, benzodiazepines, barbiturates, and 
buprenorphine. The urine sample will be provided using standard procedures with temperature 
monitoring to preclude tampering and dilution.   
• 
Patient Health Questionnaire (PHQ-9): Given the important role of depression in accessing and 
adhering to treatments for substance use disorder and other chronic medical problems, we will also 
assess severity of depression using the 9-item Patient Health Questionnaire (PHQ-9) [40]. This brief 
assessment asks participants to indicate the frequency of occurrence of each of the nine DSM-IV 
diagnostic criteria for depression during the previous two weeks.  
6.2.2 Visit 2 – Treatment induction 
The induction of all the treatments (dose adjustment for MET, BUP/NX, detoxification for XR-NTX) will occur 
over the following 2 weeks in conformity with international guidelines and applicable Vietnam policy.  
Participants assigned to the XR-NTX arm will be provided a two-week inpatient detoxification prior to the first 
injection of XR-NTX. Participants assigned to MET and BUP/NX will receive daily observed dose of treatment 
at the clinic.  
cART will be initiated according to current Vietnamese policies. Opiate addiction treatment and ART (both 
administered in same clinic) will be started within two weeks.   
On this visit, blood will be drawn by nurse at the clinic. The blood will be sent to the Pasteur Institute of Ho 
Chi Minh city for cell isolation, cryopreservation (cells, plasma), and target analyses of markers of cellular 
immune activation and senescence, systemic inflammation and virological markers (primary and secondary 
outcomes). A stool sample will be collected and send to the Pasteur Institute of Ho Chi Minh city for 
cryopreservation and analysis.  
 
6.3 
Follow Up Phase of the Study  
6.3.1 Monthly visit 
On week 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48, the RA will complete the follow-up specific 
questionnaire, RAB, PHQ-9 and a urine drug screen will be performed.  
 
6.3.2 Visit week 4, 8, 12, 24, 36, 48 
On these visits, blood will be drawn by nurse at the clinic. The blood will be sent to the Pasteur Institute of 
Ho Chi Minh city for cell isolation, cryopreservation (cells, plasma), and target analyses of markers of cellular 
immune activation and senescence, systemic inflammation and virological markers (primary and secondary 
outcomes).  
At week 48, a stool sample will be collected and send to the Pasteur Institute of Ho Chi Minh city for 
cryopreservation and analysis.  
Details on sample collection and utilization could be found on Addendum 1.  
 
6.3.3 End of Study Visit 
Counseling sessions will help participants plan for ongoing treatment after their participation in the research. 
Motivational interviewing strategies will be used to encourage participants to remain in treatment. 
Participants assigned to methadone or buprenorphine treatment will have the opportunity to continue 
receiving their treatment at the Go Vap clinic or to be referred to another clinic of their choice. Participants 
on XR-naltrexone arm who want to continue naltrexone treatment will be referred to the Thu Duc Addiction 
Treatment Center for outpatient oral naltrexone treatment in Ho Chi Minh City. If participants want to switch 
to methadone or buprenorphine treatment, referral will be made either at Go Vap clinic or at a clinic of their 
choice.   
6.4 
Subject Withdrawal  
Subjects may withdraw from the study at any time without impact to their care. They may also be 
discontinued from the study at the discretion of the Investigator for lack of adherence to intervention or study 
procedures or visit schedules. The Investigator may also withdraw subjects who violate the study plan, or 
to protect the subject for reasons of safety or for administrative reasons.  It will be documented whether or 

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not each subject completes the clinical study. Subjects who withdraw early will have one final visit to follow 
up regarding adverse events.  
6.5 
Early Termination Visits   
If a subject decided to leave the study early or is asked by the investigator to cease participation in the 
study. Motivational interviewing strategies will be used to encourage subjects to remain in treatment and 
appropriate referral will be made. 
7 
Statistical Plan 
7.1 
Primary Endpoint 
- 
The primary study endpoints will compare plasma sCD14 concentration in HIV-positive opiate use 
disorder individuals who receive cART and methadone, buprenorphine/naloxone or XR-naltrexone 
over 48 months  
• Plasma assessments (baseline, weeks 4, 8, 12, 24, 36, and 48) 
o sCD14 
7.2 
Secondary Endpoints  
- 
To evaluate the change in markers of cellular immune activation and senescence, systemic 
inflammation and bacterial translocation. 
• 
Immune activation and senescence. These variables will be assessed by flow cytometry, ELISA 
or rtPCR on cryopreserved PBMC and plasma, at the indicated time points. 
• Flow cytometry (baseline, weeks 4, 8, 12, 24, 36 and 48) 
o CD38, HLA-DR and PD1 expression in C8+ T cells  
o Activation signature in monocytes 
o CD169 expression in monocytes 
• Plasma assessments (baseline, weeks 4, 8, 12, 24, 36, and 48) 
o 
sCD163 
• rtPCR-based assessments (baseline, weeks 12, 24, 36, and 48) 
o Type-I IFN signature in PBMC 
• 
Inflammation will be assessed using Luminex plexes on cryopreserved plasma at baseline, 
weeks 4, 8, 12, 24 and 48. 
• Acute phase reactants: Plasma hr-CRP and d-dimer 
• Cytokines and receptors: sTNFR-1, IL-6, IL-10, TGF-beta 
• 
Dysbiosis and bacterial translocation will be evaluated at baseline and at week 48 in plasma 
(ELISA) and stool samples (rtPCR). 
• Plasma based assessments: sCD14, LBP, LPS, endo-CAB, Intestinal fatty acid-binding 
protein (I-FABP), Zonulin-1 
• Stool-based assessments: s16 rDNA, bacterial Butyryl-CoA-CoA 
 
- 
To evaluate the effectiveness of the treatment administered towards a) reducing/suppressing opiate 
injection behavior and promoting adherence to the treatment schedule and b) suppressing HIV 
replication 
Clinical and virological outcomes that will allow us to assess the effectiveness of the treatment 
administered towards a) reducing/suppressing opiate injection behavior and promoting adherence 
to the treatment schedule and b) suppressing HIV replication. 
The clinical outcomes will be:  
• 
CD4 count (clinical indicator of successful viral suppression and immune reconstitution, 
assessed at baseline, weeks 4, 8, 12, 24, 36, 48) 
• 
Retention in care (% of completed medication visits) 
• 
cART adherence (% of pharmacy pickups) 
• 
Continued drug use (% of positive monthly urine opiate tests or positive self-report) 
• 
Acceptability of MOUD (qualitative assessment based on satisfaction questionnaire) 
• 
Virological outcomes (assessed in PBMC or plasma) 
• 
Viral load suppression (% of randomized individuals achieving VL <50 copies/ml; (% of 
exposed individuals achieving VL <50 copies/ml) 
• 
Cell-associated DNA, RNA species (poly-A, multi-sliced Tat-Rev), replicative index 
(linear measures indicating residual viral replication) 

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7.3 
Sample Size and Power Determination 
Assessment of power and sample size was performed using program within PASS 15 for mixed effects 
models (PASS 15 Power Analysis and Sample Size Software (2017). NCSS, LLC, Kaysville, Utah, USA, 
ncss.com/software/pass), which uses simulation studies for the estimation of power of detectable 
differences.  
 
Comparing sCD14 levels between MET, BUP/NX and XR-NTX with a sample size of 50 per group, and 7 
measurements over time for each subject, with 80% power and a type 1 error rate of 5%, the detectable 
effect size for the main effect of group is 0.8, which corresponds to detecting a difference as small as 
255989.1. With the same parameters above, the detectable effect size for both the effect of time and 
interaction effect of group x time is 0.9, which corresponds to detecting a difference as small as 310386.9. 
The attainable sample size is 150 subjects, 75 per arm, with an expected minimum of 50 per arm retention 
(100 total) to the 48-week endpoint of the study. We anticipated a larger sample in MET arm (15% dropouts) 
than BUP/NX and XR-NTX arms (30% dropouts). However, the sample has been calculated to provide 
sufficient power to show any differences between the two arms. A conservative estimate of the detectable 
difference was based on 50 subjects per arm and does not consider the partial subject observations. As our 
analytical approach using Mixed Effect Regression models (MER) allows inclusion of partially observed 
subjects, our power calculation may be a slight underestimate for the detectable difference of our sample 
size. 
7.4 
Statistical Methods 
All analyses will be performed under the supervision of the PIs and Dr. Warren Bilker, Professor of 
Biostatistics at Penn in the Department of Biostatistics, Epidemiology, and Informatics. First, basic descriptor 
analysis, dropout handling, data missingness, normality, and adjustments for type 1 error analyses will be 
performed.  
 
The primary outcome of interest is the change in sCD14 over 48 weeks following ART. This marker will be 
assessed over 7 consecutive time points (baseline, weeks 4, 8, 12, 24, 36, and 48). The primary analyses 
for this longitudinal data will use the mixed model regression model (MER) with restricted maximum 
likelihood. The Mixed model analysis accommodates the non-independence of the multiple measures per 
subject. The models will include a random intercept to allow for different starting points for individuals. A 
compound symmetry (CS) covariance structure will be specified in the model. The basic models will contain 
variables for time (of the measurement), group, and a group by time or sex interaction. To further explore 
these relationships, a semiparametric trajectory clustering methodology will be applied, as implemented in 
SAS PROC TRAJ. Secondary variables include added immune and viral parameters measured in Aim 1 
and Aim 2 such as CD4 count, retention in care (% of participants), ART adherence (% of refills), continued 
drug use based on urine testing (% of positive tests), and acceptability of MOUD (Questionnaire scores). 
These will all be assessed at the same time points as the primary outcome unless stated otherwise 
(baseline, weeks 4, 8, 12, 24, 36, and 48). All analyses will assess equality of the measurements at specific 
time points across treatment groups (t-tests or Wilcoxon tests for continuous data such as CD4 and chi-
square Fisher’s exact test for proportions), as well as longitudinal trends in the measurements and how the 
trends differ between groups (MER with random intercepts) as described above. Binary data will use a mixed 
model version of longitudinal regression, which is available in STATA using xtmelogit and in SAS using 
PROC GLIMMIX. 
7.4.1 Baseline Data 
Baseline and demographic characteristics will be summarized by standard descriptive statistics including 
mean and standard deviation for continuous variables and standard percentages for categorical variables 
such as gender. 
7.4.2 Safety Analysis  
All subjects entered into the study and randomized at the baseline visit will have detailed information 
collected on adverse events for the overall study safety analysis. 
 
 
 
 

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7.5 
Subject Population(s) for Analysis 
Participants that switch immediately after randomization or after induction (detoxification or dose 
adjustment, depending on the arm) are kept on study, and we treat them as part of the switch arm in an “as 
treated” analysis (since there would be very little or no exposure to the original treatment), but we fail them 
in ITT analysis (See schema below). If they want to switch later, they get off study, but we still do the follow-
up as needed for safety. 
 
 
8 
Safety and Adverse Events 
8.1 
Definitions 
8.1.1 Adverse Event 
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in severity 
during the course of the study.  Intercurrent illnesses or injuries should be regarded as adverse events.  
Abnormal results of diagnostic procedures are considered to be adverse events if the abnormality: 
• results in study withdrawal 
• is associated with a serious adverse event 
• is associated with clinical signs or symptoms 
• leads to additional treatment or to further diagnostic tests 
• is considered by the investigator to be of clinical significance 
 
8.1.2 Serious Adverse Event 
Adverse events are classified as serious or non-serious. A serious adverse event is any AE that is:  
• fatal 
• life-threatening 
• requires or prolongs hospital stay 
• results in persistent or significant disability or incapacity 
• a congenital anomaly or birth defect 
• an important medical event 
All adverse events that do not meet any of the criteria for serious should be regarded as non-serious 
adverse events.  
 
Screen
Random.
MET
XR-NTX
BUP/NX
Dose adjust
Detox
Dose adjust
Treat
Treat
Treat
Analysis
Analysis
Analysis
ITT analysis
“As treated” 
(exposed) analysis*
Induction
ART
* Arm switching allowed only as an alternative to drop-out, and only within the first 5-8 weeks
FAIL
FAIL

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8.2 
Recording of Adverse Events 
At each contact with the subject, the investigator will seek information on adverse events by specific 
questioning and, as appropriate, by examination. Information on all adverse events will be recorded 
immediately in the source document, and also in the appropriate adverse event module of the case report 
form (CRF).   
 
All adverse events occurring during the study period will be recorded.  The clinical course of each event will 
be followed until resolution, stabilization, or until it has been determined that the study intervention or 
participation is not the cause.  Serious adverse events that are still ongoing at the end of the study period 
will be followed up to determine the final outcome.  Any serious adverse event that occurs after the study 
period and is considered to be possibly related to the study intervention or study participation will be 
recorded and reported immediately. 
 
Adverse events that occur at any point in the trial will be identified, managed, and documented. 
Medical and scientific judgment should be exercised in determining whether an event is an important 
medical event. An important medical event may not be immediately life-threatening and/or result in death or 
hospitalization.  However, if it is determined that the event may jeopardize the subject and/or may require 
intervention to prevent one of the other adverse event outcomes, the important medical event should be 
reported as serious.  
 
The relationship of each adverse event to the study procedures will be characterized. PIs and medical 
monitor will classify the AEs/ SAEs as definitely related, probably related, possibly related, unlikely or 
unrelated to the study. 
8.3 
Reporting of Adverse Events, Adverse Device Effects and Unanticipated Problems 
We will document all adverse events since the subject signs the Informed Consent to the last subject visit. 
All observed or volunteered adverse events regardless of treatment group or suspected causal relationship 
to the investigational product(s) will be reported as described in the following sections.  
 
A form will be designed so that there will be a distinction between an “adverse event”, a “serious adverse 
event” (such as hospitalization, or death), and an important medical event. All adverse events will be 
documented on this form (along with a description of measures taken in response to the event). 
 
Each report will contain at minimum the following information: 
• 
Study identifier 
• 
Study Center 
• 
Subject number 
• 
A description of the event 
• 
Date of onset 
• 
Current status 
• 
Whether study intervention was discontinued 
• 
The reason why the event is classified as serious 
• 
Investigator assessment of the association 
between the event and study intervention 
 
Each time an SAE or an important medical event is identified, the project staff member who identified the 
event will complete an SAE form. The report form will include date, description of the event, duration, 
severity, measures used to ameliorate effects of the event, and type of referral. The Project Coordinator will 
be notified immediately. The report form will be reviewed and signed by the Project Coordinator who will be 
responsible for ensuring that appropriate actions have been taken. 
 
8.3.1 Follow-up report 
If an SAE has not resolved at the time of the initial report and new information arises that changes the 
investigator’s assessment of the event, a follow-up report including all relevant new or reassessed 
information (e.g., concomitant medication, medical history) will be submitted to the IRB. The investigator is 
responsible for ensuring that all SAE are followed until either resolved or stable.  
8.3.2 Investigator reporting: notifying the study sponsor 
In the event of an SAE or an important medical event, the Principal Investigator (PIs) and the onsite PI will 
be notified via telephone or email within 8 hours of the event.  NIDA Program Officer (Tanya Ramey, MD, 

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PhD) must be notified within 72 hours since event occurrence. The Principal Investigator will report the event 
to each IRB within 72 hours of the event occurrence.  
 
8.3.3 Investigator Reporting: Notifying the Penn IRB  
Each time an adverse or serious negative event is identified, the project staff member who identified the 
event will complete an ANEF (Adverse and Negative Events Form). The report form will include date, 
description of the event, duration, severity, measures used to ameliorate effects of the event, and type of 
referral. The Project Coordinator will be notified immediately. The report form will be reviewed and signed 
by the Project Coordinator who will be responsible for ensuring that appropriate actions have been taken. 
In the event of an adverse event or a serious negative event, the PIs and the onsite PI will be notified via 
telephone or email within 8 hours of the event. The PIs will report the event to each IRB within 48 hours. If 
a serious adverse event occurs, the PI will inform and consult with the Institutional Review Board within 24 
hours of that event. 
8.4 
Unblinding Procedures 
Given the exploratory nature of the study, we do not intend to implement blinding across the entire clinical 
team and participants. However, to minimize bias, both the study Sponsors, all laboratory-based assays, 
and at least one member of the statistical team will remain blinded. Interim analyses (e.g., for IRBs/Ethics 
boards) will be structured as closed (unblinded, by-arm reports) and open (aggregated reports). Blinded 
team members will only have access to open reports.  
 
8.5 
Medical Monitoring 
The Project Director and the on-site coordinator in HCMC will monitor the regulatory requirements, safety 
events, and progress of the trial in country. Routine protocol monitoring will ensure that the research protocol 
specified and EC/IRB approved is being followed without unauthorized deviations. Special emphasis will be 
placed on ensuring that the consent process is being completed properly and that adverse events are being 
properly recorded and reported. Periodic assessments of the data quality and timeliness will be undertaken. 
Participant recruitment and retention will be reviewed. Scientific or therapeutic developments that may have 
an impact on the safety of the participants or ethics of the trial will be considered. With the assistance of the 
medical director and the PIs, the team will monitor the confidentiality of the trial data. 
 
Weekly meetings will be held by the study team as well as independent weekly remote meetings between 
Project Director and on-site staff to monitor the safety and progress of the trial. This will help to ensure 
standardized application of the protocol and will serve as an ongoing mechanism by which project staff and 
investigators will communicate to maintain consistently high-quality standards. Concerns identified will be 
addressed through personnel (re)training. In addition to the weekly monitoring schedule described above, 
the Project Director will make a formal confirmatory launch site visit (study initiation visit) to supervise staff 
training and to ensure that the protocol is being followed in all respects. Interview space will be confirmed 
as adequate, data collection storage capacity will be evaluated, sample route confirmed, and laboratories 
will be visited to ensure quality control.  
8.5.1 Data and Safety Monitoring Plan 
A Data and Safety Monitoring Plan describes the process to identify and mitigate potential risks to research 
subjects. 
• 
The Principal Investigators will monitor the study twice a year or more often if any issue arises 
• 
The Medical monitor will monitor the project on a quarterly basis or more often or more often if any 
issue arises 
• 
The Project Director will do a monitoring on-site visit twice a year or more often or more often if any 
issue arises 
• 
The Center for Studies of Addiction Data Safety and Monitoring Board (DSMB) at the University of 
Pennsylvania will review the procedure, recruitment and retention, AEs and SAEs every 6 months.  
8.5.2 Data Safety Monitoring Board  
The investigators recognize their responsibility to minimize risk to participants in this investigation and to 
conduct the study to the highest ethical standards. The project will be reviewed by the University of 

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Pennsylvania Data Safety and Monitoring Board. 
 
9 
Study Administration, Data Handling and Record Keeping 
9.1 
Confidentiality 
Information about study subjects will be kept confidential and managed according to the requirements of 
the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a signed 
subject authorization informing the subject of the following:  
• What protected health information (PHI) will be collected from subjects in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI  
 
Participation is voluntary, and individuals may withdraw from the study at any time. For those who meet 
inclusion criteria, detailed informed consent procedures will outline the nature of participation, all risks, and 
the schedule for data collection and compensation. Potential participants will then be given a detailed 
description of the research procedures, and all remaining questions will be answered by the research staff 
prior to completion of consent procedures and initiation of data collection. Subjects will be reminded that 
they are free to decline or stop participation at any time. All interviews will be conducted on the premises of 
the Go Vap clinic. 
 
Particular attention will be given to the protection of the confidentiality of any information shared by 
participants with study personnel. The study procedures are designed to prevent unintentional disclosure of 
personal information and loss of confidentiality. Staff will be trained on these procedures and on the priority 
of protection of privacy and confidentiality. Personal Health Information (PHI, as defined by HIPAA) will be 
collected locally with the purpose of identifying, scheduling and monitoring study participants, as well as for 
Pharmacy management purposes. Handling of PHI will be limited to the local study team: only partially 
deidentified limited dataset will be shared with the study Data Management and Statistical teams. If 
necessary, reidentification of study Participants will be performed by the local study team. Study-related 
limited datasets will be collected centrally by submitting electronic clinical report forms (eCRFs) available 
through our proprietary CDETweb Toolbox platform. This web-based platform is developed by Formed, Inc. 
(Philadelphia, PA) according to industry best practice, and has been audited for HIPAA and 21 cfr part 11 
compliance. Our web host (Rackspace, Inc., San Antonio, TX) is HITECH and HITRUST™ CSF certified for 
HIPAA compliance. 
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, retains 
the ability to use all information collected prior to the revocation of subject authorization.  For subjects that 
have revoked authorization to collect or use PHI, attempts should be made to obtain permission to collect 
at least vital status (i.e. that the subject is alive) at the end of their scheduled study period. 
9.2 
Data Collection and Management  
• 
Data acquisition and transmission 
All information will be derived from biological tests, standard clinical procedures, standardized interviews, 
and self-report questionnaires. All subjects will receive a unique participant number that will be kept separate 
from any personal information that includes identifying variables such as name, address, relatives, place of 
work, etc.  It is necessary for subject retention that personally identifying material be kept. 
All data collection will be conducted at the Go Vap clinic, Ho Chi Minh City, Vietnam under the direct 
supervision of the site Principal Investigator, who has extensive experience supervising research and 
implementation and data collection.  Protocol monitoring will ensure that the research protocol specified is 
being followed without unauthorized deviations. Special emphasis will be placed on ensuring that the 
Consent Process is being completed and documented properly, and that adverse events are being properly 
recorded and reported.  Ongoing assessments of the data quality and timeliness will be undertaken. 
Participant recruitment and retention will be reviewed formally on a monthly basis via conference call.  
Scientific or therapeutic developments that may have an impact on the safety of the participants or ethics of 
the trial will also be considered on an ongoing basis.   
 

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Weekly meetings will be held to monitor the progress of the trial. These meetings will involve the Project 
Director, Site coordinator and other study staff, as well as a representative of the Data management team.  
This will help to ensure standardized application of the protocol and will serve as an ongoing mechanism by 
which project staff and investigators will communicate in order to maintain a consistently high quality of 
study conduct.  Concerns identified will be addressed through training and retraining of personnel.  
 
• 
Data entry methods 
Dr. Azzoni (The Wistar Institute, Biomedical Research Support Core) will be responsible for providing and 
documenting appropriate user access to the study eCRFs and database (CDETweb Toolbox) and 
preventing data security problems, including unauthorized internal access to data, external access to data, 
and malicious intent to destroy data and systems. The Wistar BRS Core will ensure that only appropriate 
and authorized personnel are able to view, access, and modify trial data. Modifications to data will be audited 
though a continuous audit trail, which will document change, user signature, time and date, and reason for 
change. The BRS Core will also be responsible for optimizing database performance, reliability, and backup. 
 
External, unauthorized access to data will be prevented through cooperative efforts of the database 
administrator and network and systems administrators, and by active monitoring of account holders’ 
privileges. Standard measures will be employed through network firewall technologies to prevent 
unauthorized external access to data repositories. CDEweb Toolbox is regularly audited for HIPAA and CFR 
21 Part 11 compliance. 
 
• 
Electronic Data Management System: CDETweb Toolbox 
The CDETweb EDMS is a client-server web application. The architecture allows for multiple, asynchronous 
users. CDETweb modules and eCRFs are web pages and web forms that allow the user to input data and 
otherwise interact with the system. The system consists of a number of modules, some of which are 
standardized across all studies (e.g. Log-in and authority check module, patient registration module, etc.), 
while others are customized to specifically address individual trial and protocol requirements (e.g. Visit 
Schedule, Visit CRFs, Randomization module, etc.). Authorized users will sign onto the system from the 
CDETweb login portal.  The authentication module will validate the user’s credentials determining the user’s 
access-level by querying a central, BRSC-managed administration database.  The BRSC will customize 
core CDETweb modules in compliance with this Study protocol mandates, e.g. subject registration, visit 
management, and data capture, as well as any specially requested features (e.g. review pages, special 
reports etc.). 
 
CDETweb Toolbox addresses the following compliance features: 
• Validation. A risk-based validation is planned for selected elements of the tool. 
• Human Readable Copies. The system will be able to print readable version of all eCRFs 
• Protection and retention of records. The central database is hosted by a commercial host with 
outstanding reputation and HPAA compliant practices 
• Audit trails. Table-based audit trails are implements across the system 
• Access control. User must log into the system at the beginning of each session. User names and 
passwords are regulated 
• Authority checks. Role based, as well as granular CRUDAL permissions, are implemented. 
• Electronic signatures. Records are electronically signed (exclusively during logged-in sessions) at 
the time of locking. The signature is stored in the Audit trail, and consist of username and timestamp. 
To lock a record, the User must acknowledge their intention and responsibilities (i.e. they acknowledge 
that they are signing the form, and the content correspond to the source to the best of their knowledge) 
by confirming the entry on an overlay screen displaying this information: thus, intention and meaning 
of the signatures are implied. 
• Device checks. Access to CDETweb Toolbox by means of older, non-HTML-5 compliant browsers 
is not allowed. 
• Training. A manual will be provided to the User containing the basic information required to run the 
tool. Detailed one-on-one “hands on” training will be provided remotely 
• Written procedures. SOPs will be developed in collaboration with the Clinical sites to detail the data 
collection, entry and retrieval process 
• System documentation. Development documentation is maintained by our Web Developers 
(Formed, Inc.). Validation activities will be documented by the Wistar BRSC. 
 
Electronic Clinical Report Forms (eCRFs) will be developed in close consultation with the clinical study team 
to accurately reflect protocol data acquisition requirements. After testing and verification of product 

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specifications, the system will be validated according to the BRS Core SOPs. Site representatives (e.g. 
Study Team members) will be required members of the Validation team. 
 
Where possible, field-based checks will be implemented to improve data quality by checking input type, 
range etc. Similarly, eCRF sections will be displayed accordingly to prior entries as necessary. Finally, eCRF 
field completeness checks will be implemented before the user can lock an eCRF.  
Access to locked eCRFs will be administered by the Study Data manager in collaboration with the BRSC. 
CDETweb™ browser support is currently limited to fully HTML5-compliant browsers. 
 
All participants will be registered in CDETweb® and assigned a unique identifier. This identifier will be 
recorded in the participant’s clinical file, and will serve as the only link between HIPAA-protected information 
(stored exclusively at the clinical site as described below) and the electronic data, which will not include 
HIPAA-protected identifiers, with the exception of registration and visit/event dates. 
 
All data will be submitted to the central study database using CDETweb forms; the system will be available 
on a 24/7 basis for asynchronous data input (anticipated system uptime > 98%). Study coordinators at the 
clinical core will have separate accounts and will enter data according to the study Protocol and data 
management plan requirements. 
9.3 
Records Retention  
Questionnaires will be entered on the secured database on an ongoing basis. At the end of the subject 
participation and after verification of the data entry and QA/ Final QA, the paper copy of the evaluation will 
be destroyed.  Consent forms will be securely stored for 3 years after study completion.  
10 Study Monitoring, Auditing, and Inspecting 
10.1 Study Monitoring Plan 
• 
A Site Initiation Visit (SIV) will be conducted prior to site activation to confirm preparedness for 
protocol execution, satisfactory site facilities, clarify the applicable regulations and requirements of 
the protocol, carefully review the process of implementing the protocol at the site and conduct any 
necessary training prior to activating the site for enrollment.   
• 
Interim Monitoring Visits (IMVs) will be conducted to confirm participants’ rights are being protected; 
the study is being conducted according to the protocol and applicable regulations, including GCP; 
confirm accurate reporting of participant safety data and study endpoints.   
• 
For-cause visits (FCVs) are conducted to address any unanticipated issues that arise which require 
training, remediation or other situations in which the site requires assistance.   
• 
A Close-Out Visit (COV) will be conducted to ensure that all study data and other study 
documentation is complete and accurate and that all study records have been reconciled.   
• 
The study will be monitored on a semi-annual basis buy the DSMB of the Penn Center for Studies 
of Addiction. 
10.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the EC/IRB, the sponsor, 
government regulatory bodies, and University compliance and quality assurance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study data etc.).  
The investigator will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.). 
 
11 Ethical Considerations 
This study is to be conducted in accordance with applicable US government regulations and international 
standards of Good Clinical Practice, and applicable institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted independent Ethics 
Committee (EC) or Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study conduct.  The decision of the EC/IRB concerning the conduct of the study will be made 
in writing to the investigator and a copy of this decision will be provided to the sponsor before 
commencement of this study.  

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The Principal Investigators and Co-investigators have certifications of completion for required education on 
the protection of human research participants. Prior to recruiting human participants into the proposed study, 
the Project Director, Site/Field Coordinator, and personnel who have responsibility for the design and 
conduct of the study and/or direct contact with human study participants will complete certification 
requirements on ethical principles involved in the conduct of human subjects’ research by completing the 
online training module on the NIH website or its equivalent. Certificates of training completion will be kept 
on file in the project office. The PIs and Project Director will ensure that all project staff are oriented to the 
procedures during their training period. 
 
11.1 Risks 
The medications used in this study are FDA-approved, and commonly used for opiate use disorder treatment 
globally. MET, BUP/NX, and XR-NTX have a very favorable safety profile.  
 
For participants who continue to use opioids or other medications against medical advice, a risk of overdose 
exists with all three medications. These risks will be carefully explained to all participants as they enter the 
study and during weekly counseling sessions. These risks do not derive from the medications themselves, 
but from the continued use of opioids during treatment and after treatment ends. These risks will be clearly 
explained at enrollment and reinforced at each visit.  
 
 
Liver toxicity and possible medication interactions between MOUD and cART medications will be actively 
monitored through a combination of safety testing and clinical interactions (physical examinations, reported 
symptom assessment, etc.). When needed referrals to medical treatment or specialists will be made. 
 
The risks attributable to the behavioral assessments are considered minimal. Behavioral assessments may 
lead to embarrassment or discomfort for some subjects. The majority of this information is clinically relevant 
and could be considered as standard questions in many clinical settings.   
 
With regard to phlebotomy, risks are minimal and limited to soreness or bruising at the site where the blood 
is collected. Stool sampling present no additional risk. 
11.2 Benefits 
No benefits beyond those of the current standard of care in Vietnam (methadone) will be promised to 
participants. Subjects will receive a medication for opioid use disorder (MOUD) for opiate addiction. The 
discussions with his/her counselor may help him/her gain a better understanding of drug addiction. The 
counseling may also help him/her become more successful in reducing or stopping drug use and more 
successful in HIV treatment. Finally, the counseling sessions may give him/her some ideas on how to 
improve relationships with family and friends. 
 
All subjects will get appropriate HIV treatment at the Go Vap Clinic with enhanced monitoring of biological 
markers of progression of the disease.  
 
11.3 Risk Benefit Assessment 
The subject will receive MOUD for his/her opiate addiction. The discussions with his/her counselor may help 
him/her gain a better understanding of drug addiction. The counseling may also help him/her become more 
successful in reducing or stopping drug use and more successful in HIV treatment. Finally, the counseling 
sessions may give him/her some ideas on how to improve relationships with family and friends.  
 
All subjects will get appropriate HIV treatment at the Go Vap Clinic with enhanced monitoring of biological 
markers of progression of the disease. Participation in similar studies has been associated with improvement 
of health conditions and reductions in risky behavior for many subjects. 
In addition to potential individual benefits, the proposed study is expected to yield considerable information 
on biological and behavioral changes associated with the intervention that may inform the design of future 
treatment approaches, indirectly benefiting PWIDs.  
Overall, the potential benefits are expected to outweigh the minimal risks incurred through participation in 
this study. 

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11.4 Informed Consent Process / HIPAA Authorization  
Clinical staff at the HIV and addiction clinic will identify potential subjects and refer them to the research 
assistant (RA). The RA will describe the study and those who express interest will be referred to the study 
medical supervisor who will explain it in detail, answer questions, review the consent to screen, and have 
the subject sign it after confirming that he/she understands it. Tests to confirm eligibility for cART will be 
done on site. For those eligible, informed consent will be obtained in writing by the study medical investigator 
from all study participants prior to any procedure. Study personnel will first introduce potential participants 
to the study and explain the study procedures. A written informed consent form (ICF) in Vietnamese, the 
content of which will be approved by the local Ethics Committee according to local regulations, will also be 
provided to the candidates, who will be allowed time to read it and discuss it with the study personnel. ICFs 
will clearly state that participation in the research is completely voluntary and that there will be no 
consequence to the participant for declining participation or absences from study procedures. Monetary 
compensation for participation will also be fully described in the ICF. Candidates who consent to participate 
will sign and date the form; signed ICFs will be locked-stored at the Expertise France office near the Go Vap 
site according to local regulation and GCP directives. 
12 Study Finances 
12.1 Funding Source 
This study is financed through a grant from the US National Institute of Drug Abuse (R01 DA048728-01). 
Long-acting naltrexone study drug is donated by Alkermes, buprenorphine/naloxone study drug is donated 
by Indivior, whereas access to methadone and ART will be purchased from local pharmacies. 
12.2 Conflict of Interest 
All University of Pennsylvania Investigators will follow the University of Pennsylvania and Wistar 
investigators the Wistar Institute Policy on Conflicts of Interest Related to Research.  
12.3 Subject Stipends or Payments 
Participants will receive 120,000 VND (about $5.00) at each monthly assessment; this amount has proven 
acceptable to both participants and administrative staff in our current research and is not considered 
coercive. 
13 Publication Plan 
Generally, the University of Pennsylvania and the Wistar Institute recommend that its researchers share 
data through communication channels such as speaking engagements and publications, postings on 
laboratory or institutional webpages, or data archives or enclaves, as appropriate.  
 
It is anticipated that the results of this study will be presented at scientific meetings and/or published in a 
peer reviewed scientific or medical journal. The investigators involved in the proposed protocol will fully 
adhere to the timeline stipulated in the NIH policy of dissemination of clinical trial information and publicly 
disseminate the research observations within 30 months from completion of the study.  
 
14 References 
 
1. 
Banerjee, S., et al., Opioid-induced gut microbial disruption and bile dysregulation leads to gut 
barrier compromise and sustained systemic inflammation. Mucosal Immunol, 2016. 9(6): p. 1418-
1428. 
2. 
Khansari, M., M. Sohrabi, and F. Zamani, The Useage of Opioids and their Adverse Effects in 
Gastrointestinal Practice: A Review. Middle East J Dig Dis, 2013. 5(1): p. 5-16. 
3. 
Meng, J., et al., Morphine induces bacterial translocation in mice by compromising intestinal barrier 
function in a TLR-dependent manner. PLoS One, 2013. 8(1): p. e54040. 
4. 
Peng, J., S. Sarkar, and S.L. Chang, Opioid receptor expression in human brain and peripheral 
tissues using absolute quantitative real-time RT-PCR. Drug Alcohol Depend, 2012. 124(3): p. 223-
8. 
5. 
Saurer, T.B., et al., Morphine-induced alterations of immune status are blocked by the dopamine 
D2-like receptor agonist 7-OH-DPAT. J Neuroimmunol, 2004. 148(1-2): p. 54-62. 

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6. 
Chan, Y.Y., et al., Inflammatory response in heroin addicts undergoing methadone maintenance 
treatment. Psychiatry Res, 2015. 226(1): p. 230-4. 
7. 
Zajicova, A., H. Wilczek, and V. Holan, The alterations of immunological reactivity in heroin addicts 
and their normalization in patients maintained on methadone. Folia Biol (Praha), 2004. 50(1): p. 24-
8. 
8. 
Kunoe, N., et al., Injectable and implantable sustained release naltrexone in the treatment of opioid 
addiction. Br J Clin Pharmacol, 2014. 77(2): p. 264-71. 
9. 
Kampman, K. and M. Jarvis, American Society of Addiction Medicine (ASAM) National Practice 
Guideline for the Use of Medications in the Treatment of Addiction Involving Opioid Use. J Addict 
Med, 2015. 9(5): p. 358-67. 
10. 
Di Paola, A., et al., Design and methods of a double blind randomized placebo-controlled trial of 
extended-release naltrexone for HIV-infected, opioid dependent prisoners and jail detainees who 
are transitioning to the community. Contemp Clin Trials, 2014. 39(2): p. 256-68. 
11. 
Ho, W.Z., et al., Methylnaltrexone antagonizes opioid-mediated enhancement of HIV infection of 
human blood mononuclear phagocytes. J Pharmacol Exp Ther, 2003. 307(3): p. 1158-62. 
12. 
Springer, S.A., et al., Extended-Release Naltrexone Improves Viral Suppression Among 
Incarcerated Persons Living With HIV With Opioid Use Disorders Transitioning to the Community: 
Results of a Double-Blind, Placebo-Controlled Randomized Trial. J Acquir Immune Defic Syndr, 
2018. 78(1): p. 43-53. 
13. 
Tetrault, J.M., et al., Hepatic safety and antiretroviral effectiveness in HIV-infected patients receiving 
naltrexone. Alcohol Clin Exp Res, 2012. 36(2): p. 318-24. 
14. 
Mitchell, M.C., A. Memisoglu, and B.L. Silverman, Hepatic safety of injectable extended-release 
naltrexone in patients with chronic hepatitis C and HIV infection. J Stud Alcohol Drugs, 2012. 73(6): 
p. 991-7. 
15. 
UNAIDS. 
Country 
progress 
Report: 
HIV 
and 
AIDS 
estimates, 
Vietnam. 
2018; 
https://www.unaids.org/en/regionscountries/countries/vietnam]. 
16. 
Hoang, T.V., et al., Impact of a methadone maintenance therapy pilot in Vietnam and its role in a 
scaled-up response. Harm Reduct J, 2015. 12: p. 39. 
17. 
Vietnam Administration of HIV/AIDS Control, Methadone Maintenance Treatment Vietnam. 2017. 
18. 
Rangarajan, S., et al., Risk factors for delayed entrance into care after diagnosis among patients 
with late-stage HIV disease in southern Vietnam. PLoS One, 2014. 9(10): p. e108939. 
19. 
Low, A.J., et al., Impact of Opioid Substitution Therapy on Antiretroviral Therapy Outcomes: A 
Systematic Review and Meta-Analysis. Clin Infect Dis, 2016. 63(8): p. 1094-1104. 
20. 
Mlunde, L.B., et al., Association of opioid agonist therapy with the initiation of antiretroviral therapy 
- a systematic review. Int J Infect Dis, 2016. 46: p. 27-33. 
21. 
Sereno, L., et al., Epidemiology, responses, and way forward: the silent epidemic of viral hepatitis 
and HIV coinfection in Vietnam. J Int Assoc Physicians AIDS Care (Chic), 2012. 11(5): p. 311-20. 
22. 
Dunford, L., et al., Hepatitis C virus in Vietnam: high prevalence of infection in dialysis and multi-
transfused patients involving diverse and novel virus variants. PLoS One, 2012. 7(8): p. e41266. 
23. 
Nguyen, C.H., et al., Prevalence of HBV infection among different HIV-risk groups in Hai Phong, 
Vietnam. J Med Virol, 2011. 83(3): p. 399-404. 
24. 
World Health Organization, Global tuberculosis report 2014. 2014, WHO: Geneva, Switzerland. 
25. 
Quan, V.M., et al., Risks for HIV, HBV, and HCV infections among male injection drug users in 
northern Vietnam: a case-control study. AIDS Care, 2009. 21(1): p. 7-16. 
26. 
Quan, V.M., et al., Mortality and HIV transmission among male Vietnamese injection drug users. 
Addiction, 2011. 106(3): p. 583-9. 
27. 
Tanuma, J., et al., Incidence of AIDS-Defining Opportunistic Infections and Mortality during 
Antiretroviral Therapy in a Cohort of Adult HIV-Infected Individuals in Hanoi, 2007-2014. PLoS One, 
2016. 11(3): p. e0150781. 
28. 
Nguyen, T., et al., Change over 12-months of an Integrated Treatment Program for Opiate Addiction 
and HIV in Ho Chi Minh City, Vietnam: highlighting the role of counseling, in NIDA International 
Conference. 2017: Montreal, Canada. 
29. 
Hunt, P.W., et al., Gut epithelial barrier dysfunction and innate immune activation predict mortality 
in treated HIV infection. J Infect Dis, 2014. 210(8): p. 1228-38. 
30. 
Marchetti, G., et al., Microbial translocation is associated with sustained failure in CD4+ T-cell 
reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy. AIDS, 2008. 
22(15): p. 2035-8. 
31. 
Sandler, N.G., et al., Plasma levels of soluble CD14 independently predict mortality in HIV infection. 
J Infect Dis, 2011. 203(6): p. 780-90. 

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32. 
Sunil, M., et al., Unchanged Levels of Soluble CD14 and IL-6 Over Time Predict Serious Non-AIDS 
Events in HIV-1-Infected People. AIDS Res Hum Retroviruses, 2016. 32(12): p. 1205-1209. 
33. 
Hunt, P.W., et al., T cell activation is associated with lower CD4+ T cell gains in human 
immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral 
therapy. J Infect Dis, 2003. 187(10): p. 1534-43. 
34. 
Wesson, D.R. and W. Ling, The Clinical Opiate Withdrawal Scale (COWS). J Psychoactive Drugs, 
2003. 35(2): p. 253-9. 
35. 
Chawarski, M.C., M. Mazlan, and R.S. Schottenfeld, Behavioral drug and HIV risk reduction 
counseling (BDRC) with abstinence-contingent take-home buprenorphine: a pilot randomized 
clinical trial. Drug Alcohol Depend, 2008. 94(1-3): p. 281-4. 
36. 
Chawarski, M.C., W. Zhou, and R.S. Schottenfeld, Behavioral drug and HIV risk reduction 
counseling (BDRC) in MMT programs in Wuhan, China: a pilot randomized clinical trial. Drug 
Alcohol Depend, 2011. 115(3): p. 237-9. 
37. 
McLellan, A.T., et al., The Addiction Severity Index at 25: origins, contributions and transitions. Am 
J Addict, 2006. 15(2): p. 113-24. 
38. 
Metzger, D.S., et al., Human immunodeficiency virus seroconversion among intravenous drug users 
in- and out-of-treatment: an 18-month prospective follow-up. J Acquir Immune Defic Syndr, 1993. 
6(9): p. 1049-56. 
39. 
Navaline, H.A., et al., Preparations for AIDS vaccine trials. An automated version of the Risk 
Assessment Battery (RAB): enhancing the assessment of risk behaviors. AIDS Res Hum 
Retroviruses, 1994. 10 Suppl 2: p. S281-3. 
40. 
Spitzer, R.L., K. Kroenke, and J.B. Williams, Validation and utility of a self-report version of PRIME-
MD: the PHQ primary care study. Primary Care Evaluation of Mental Disorders. Patient Health 
Questionnaire. JAMA, 1999. 282(18): p. 1737-44. 

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15 Addendum 
 
Addendum 1 
Sample Collection 
The collection of biospecimens (a.k.a.: biological samples) from study participants is described in the table 
“Study procedures timeline” (rows labeled “blood sampling” and “Stool sampling”) in section 6 of the Study 
protocol 
A detailed assessment of the specimen collection timeline for each individual participant is provided in Table 
3; based on this assessment, we anticipate the following maximum total amount of individual vials 
(calculated based on 2 ml or 20x106 cells/vial) or boxes (calculated based on a 9x9 layout) 
Table 1: Total study sample collection for shipment 
Type of 
Sample 
Storage 
Per study subject 
total vials 
(see Table 3 
below) 
Total Study vials 
(225 participants 
max) 
Total 
shipment 
boxes  
(81 samples) 
Urine 
-70 
65 
14,625 
181 
PBMC 
Liquid 
Nitrogen 
35 
7,875 
98 
Plasma 
-70 
35 
7,875 
98 
Swabs 
-70 
4 
900 
12 
Serum 
-70 
7 
1,575 
20 
 
 
Grand totals 
32,850 
409 
 
Sample processing and transfer 
All samples will be collected at the point of care and processed at the Pasteur Institute – Ho Chi Minh City 
(blood separation and cryopreservation, initial Flow cytometry assessment on whole blood, sample aliquots 
and Swab processing).  
Cryopreserved samples will be transferred to the Wistar Institute for analysis. Shipments will be performed 
using cryogenic Liquid Nitrogen containers (“dry shippers”) using a cold chain commercial supplier (e.g. 
World Courier, Inc.). To maximize efficiency and reduce loss risk, samples will be sent in multiple batches 
throughout the course of the Study. 
Import and export permits will be obtained (or verified if already obtained) from the competent Country 
Authorities prior to each shipment. 
 
Sample utilization 
The samples collected will be necessary to perform the following assessment 
Cellular measures of residual immune activation and senescence on ART  
An initial analysis of cellular activation (e.g. expression of CD38 and HLA-DR on subsets of memory and 
naïve CD4+ and CD4+ T cells, as well as markers of activation on CD14+ and CD16+ monocytes) will be 
performed on 2 ml of fresh whole blood at the Pasteur Institute – Ho Chi Minh City, using 6-color flow 
cytometry. 

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In-depth Immunofluorescence profiling  
An in-depth secondary analysis will be based on two 16 color composite stains requiring 2 million PBMC 
each. We will prioritize analysis for pre-ART baseline, week 12, 24 and 48 (4 time points). Details are 
reported in the budget justification per fluorochromes and antibody clones to include activation changes in 
T cells and NK cells (PD-1, CD69, CD38, HLA-DR), NK (CD69, Perforin, CD57), monocyte 
(non-inflammatory: CD14+CD16-, inflammatory: CD14+CD16+) and monocyte/dendritic cell subset (PDL1, 
CD169, CD40). CD4 T cell HIV correctors will also be measured (CXCR4, CCR5). 
Systemic plasma--based measures of residual immune activation/inflammation on ART  
The effect of viral suppression (ART) and MAT on systemic activation and inflammation will be assessed 
measuring specific lymphocytes and acute phase reactants. To assess monocyte activation and microbial 
translocation, in addition to the PBMC-based stainings mentioned above, we will measure plasma levels of 
sCD163 and sCD14 using commercial ELISA kits. All time--points will be tested for sCD14 as the primary 
variable, whereas sCD163 will be tested at pre--ART baseline, week 12, 24, and 48 (4 time points). 
Microbial translocation and dysbiosis can be associated with opioid use in PWIDs  
Microbial translocation and dysbiosis will be assessed by three methods.  
First, plasma levels of LPS: LPS concentration will be determined by the Limulus Amebocyte assay 
according to the manufacturer’s protocol (Cambrex Bioscience, Walkersville, MD) in plasma samples 
diluted 1/100 with endotoxin-free water.  
Second, plasma bacterial 16S rDNA will be measured using droplet digital polymerase chain reaction 
(ddPCR) using the RainDrop system (RainDance Technologies), which allows significantly higher sample 
inputs than traditional quantitative PCR. DNA will be extracted from plasma using the DNeasy blood and 
tissue kit (Qiagen). The RainDrop Source instrument will be used with a microfluidic chip to generate a 
collection of uniformly sized (5 picoliter) aqueous droplets from each DNA sample mixed with assay 
reagents in a reaction volume of 50 μl with 25 μl of 2x TaqMan Universal Master Mix II, including UNG (Life 
Technologies), a final concentration of 450 nM for each primer and 150 nM for the probe, 2 μL 25x Droplet 
Stabilizer (RainDance Technologies). Droplets will be thermocycled at 50°C for 2 minutes, 95°C for 10 
minutes, then 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Single droplet fluorescence will be 
measured and data will be analyzed using the RainDrop Analyst software. Intra-assay variability is <5%. 
We will prioritize analysis for pre--ART baseline, week 12, 24 and 48.  
Third, butyryl-CoA transferase levels in the stool (a marker for low representation of butyrate--producing 
bacterial species associated with immune activation) will be assessed measuring Butyryl--CoA--CoA 
transferase transcripts by qPCR using SYBR green PCR reagent. These tests will be performed on DNA 
prepared on site from fresh stool samples (swabs) at baseline and at week 48 to maximize detectable 
change (2 time points). 
Reversal of the activation gene expression signatures in myeloid subsets  
Based on our prior characterization of monocyte gene expression changes in associated with HIV-mediated 
activation and monocyte apoptosis, including genes in the p53 (Rb1, MDM2), Bcl--2 (Mcl2, Bcl--w, Mcl--1, 
Bax, Bak1, Bik, CycS) and ISG (Mx1, IF6, IFI27, IFITM2) families, we will measure the expression of these 
genes at baseline and at week 48. This will provide an independent confirmation for the trajectory of 
changes between week 0 and 48 (primary end--point) and the inferences of retained/decreased innate cell 
immune activation between arms. Gene expression analysis (RT--PCR) will be performed in CD14+ cells 
purified from cryopreserved PBMC to greater than 98% purity by negative selection using magnetic beads 
(Miltenyi Biotech). Total RNA will be isolated and analyzed as described above for HIV measures. 
Persistent HIV transcription/replication on ART  
In addition to plasma viral load, we will assess the levels of HIV transcriptional activity index (defined as the 
ratio of cell--associated HIV RNA to cell--associated HIV total DNA). 1--2 million CD4+ T cells will be 
isolated by negative selection from 20 million PBMCS using EasySep Human CD4+ T cell enrichment kit 
(Stemcell Technologies). Cellular RNA and DNA will be purified from isolated CD4+ T cells using the AllPrep 
DNA/RNA kit (Qiagen). Absolute quantification of HIV total DNA, multi--spliced HIV RNA, and 

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polyadenylated HIV RNA, will be performed, in addition to TERT (telomerase reverse transcriptase; life 
technologies) as a cell counter, in duplex digital droplet PCR reactions 
using the RainDrop system and primers and probe sets as mentioned in the above references. For RNA, 
cDNA will be generated using SuperScript IV. The RainDrop Source instrument will be used as in Specific 
Aim 1. Droplet counts will be normalized to TERT counts. We will prioritize analysis for pre--ART, week 12, 
24, and 48. 
A summary of the specimen utilization is provided in table 2 below: of the anticipated 100 x 106 PBMC, we 
will use 60 x 106 in the tests below, and store 40 x 106 cells in case tests need to be repeated, or to 
compensate for insufficient cell recovery/loss of viability after thawing. 
 
Table 2: Sample utilization 
Specific 
Aim 
Description 
Tests 
Method 
Specimen 
Amount 
Comments 
1 
Cellular 
measures 
of residual immune 
activation 
and 
senescence on ART 
Immunostaining w/ 
6-color mAb panels 
6-color 
Flow 
cytometry 
fresh blood 
1 ml 
Pasteur 
Institute 
HCMC 
In-depth 
Immunofluorescenc
e profiling 
Immunostaining w/ 
additional 
mAb 
panels 
16-color 
Flow 
cytometry 
PBMC 
20 M 
n/a 
Systemic plasma--
based measures of 
residual 
immune 
activation/inflammat
ion on ART. 
Multiple 
analyte 
detection 
Luminex/ELISA/Meta
bolomics 
plasma 
1 ml 
n/a 
Microbial 
translocation 
and 
dysbiosis can be 
associated 
with 
opioid use in PWIDs 
LPS 
Biotest (limulus) 
plasma 
1 ml 
n/a 
Bacterial rDNA 
ddPCR 
plasma 
1 ml 
n/a 
Butyryl 
CoA-CoA 
transferase 
PCR 
rectal 
swabs 
1 ml 
University 
of CO 
  
Reversal 
of 
the 
activation 
gene 
expression 
signatures 
in 
myeloid subsets 
Rb1, MDM2, Mcl2, 
Bcl-w, 
Mcl-1, 
Bax, 
Bak1, 
Bik, 
CycS, 
Mx1, 
IF6, 
IFI27 and IFITM2 
RT-PCR 
PBMC 
20 M 
n/a 
2 
Persistent 
HIV 
transcription/replicat
ion on ART 
HIV DNA and RNA 
species on sorted 
PBMC subsets 
ddPCR 
PBMC 
20M 
n/a 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table 3: Biospecimen collection for each individual participant 
 
 
 
 
Sampling
Screen
wk0
wk1
wk2
wk3
wk4
wk5
wk6
wk7
wk8
wk9
wk10
wk11
wk12
wk13
wk14
wk15
wk16
wk17
wk18
wk19
wk20
wk21
wk22
wk23
wk24
wk25
wk26
wk27
wk28
wk29
wk30
wk31
wk32
wk33
wk34
wk35
wk36
wk37
wk38
wk39
wk40
wk41
wk42
wk43
wk44
wk45
wk46
wk47
wk48
per 
subject 
total
Urine
1
1
1
1
1
1
1
1
1
1
1
1
1
1
13
Blood Science
1
1
1
1
1
1
1
7
Swabs
1
1
2
serum
Volumes stored
VOL/n
Urine
10
10
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
0 10
0
0
130
Blood Science
100 100
0
0
0 100
0
0
0 100
0
0
0 100
0
0
0
0
0
0
0
0
0
0
0 100
0
0
0
0
0
0
0
0
0
0
0 100
0
0
0
0
0
0
0
0
0
0
0 100
700
Swabs (n)
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
4
serum
2
2
0
0
0
2
0
0
0
2
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
2
14
Vials
n
Urine (2 ml)
5
5
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
65
PBMC (20M/vial)
5
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
5
35
Plasma (2ml/vial)
5
5
0
0
0
5
0
0
0
5
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
5
35
Swabs (2ml)
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
4
Serum
1
1
0
0
0
1
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
7

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Page 34 of 37
 
 
 
VALIDATION SAMPLES 
CASE REPORT FORM 
 
PATIENT IDENTIFICATION 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Patient No.: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
In case of emergency, please contact: 
Institut Pasteur of Ho Chi Minh-City 
00 84 88 20 28 15 
00 84 90 38 17 317 
 
 

AMOHI-1 
 
Protocol 
 
 
AMOHI-1_Protocol_V2.0_Feb_19_2020 
 
Page 35 of 37
 
 
 
BIOLOGICAL SAMPLES  
 
1. 
Date of the sampling for investigation  
|__|__| / |__|__| / |__|__| 
2. 
blood sample taken 
£0 No 
£1 Yes 
2.1. 
Laboratory validation of the blood sample  
£0 No 
£1 Yes 
 
3. 
If blood sample is not validated, please comment (IN CAPITAL LETTERS) 
 
 
 
 
 
4. 
Urine sample taken 
£0 No 
£1 Yes 
4.1. 
Laboratory validation of the urine sample 
£0 No 
£1 Yes 
 
5. 
If the urine sample is not validated, please comment (IN CAPITAL LETTERS) 
 
 
 
 
 
6. 
Rectal swap sample taken 
£0 No 
£1 Yes 
6.1. 
Laboratory validation of the stool sample 
£0 No 
£1 Yes 
7. 
If the rectal sample is not validated, please comment (IN CAPITAL LETTERS) 
 
 
 
 
 
 
   D   D        M   M         Y    Y 

AMOHI-1 
 
Protocol 
 
 
AMOHI-1_Protocol_V2.0_Feb_19_2020 
 
Page 36 of 37
 
 
 
 
STUDY TERMINATION 
 
 
I have reviewed the data contained in this case report form and attest to the accuracy and 
completion thereof. 
 
8. 
Date 
 
|__|__| / |__|__| / |__|__|    
 
 
 
Signature of the principle investigator  
 
 
 
   D   D        M   M         Y    Y 

AMOHI-1 
 
Protocol 
 
 
AMOHI-1_Protocol_V2.0_Feb_19_2020 
 
Page 37 of 37
 
 
 
Declaration for Exportation of Biological Materials 
  
Material Description / Amount 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Shipment Method 
Shipment Date 
Does recipient require an import permit? 
Permit No. 
Ye
s 
No 
Source 
Name 
/ 
Address 
 
Recipient Name / Address 
 
 
 
 
 
Applicant Name 
Applicant E-Mail 
 
 
Applicant Signature / Date